Theranostics Based on Naturally Occuring Components

**45**

**Chapter 3**

*Arghya Sett*

**1. Introduction**

**Abstract**

Aptamers: Magic Bullet for

Aptamers are a short polymer of oligonucleotides (natural or modified) that can bind to its cognate target (small molecules to large macromolecules like proteins, cells, microorganisms etc.) with high affinity and selectivity. They can fold into unique secondary and tertiary conformation in solution (pH, ionic concentration) and bind to their targets in a specific manner (binding constants in sub-nano to pico molar range). They rival the monoclonal antibodies and other specific biological ligands with respect to affinity, stability, robustness, non-immunogenicity and facile to synthesis. Nucleic acid aptamers are selected from an oligonucleotide library by an iterative process called SELEX (**S**ystematic **E**volution of **L**igands by **Ex**ponential Enrichment Analysis). These aptamers are compatible to any kind of chemical modification, conjugation and functionalization. Briefly, this chapter discusses about the

Aptamers (Latin word *aptus* means 'to fit' and Greek meros meaning 'part')

are single stranded oligonucleotides, which act as synthetic ligands for its cognate target molecules. [1, 2] These molecules show high target specificity, selectivity and affinity, which resemble 'monoclonal antibody'. Similar to antibodies, aptamers also have immense potential to interact with their targets by structural recognition and thus they are termed as 'chemical antibodies'. [3] Different conformations allow aptamers to bind specifically with their target by 'lock and key' model. This hypothesis of binding mechanism is driven by the secondary and tertiary structure of aptamers in-bound state with their targets. Adopting various structures like hairpin loops, bulges, stem-loop, quartets, G-quadruplex, pseudo knots, aptamers can fit into the binding region of the target. [4] Intra and inter molecular interactions like hydrogen bonding, Vander Waals force, hydrophobic interaction, electrostatic forces play major crucial role in aptamer-target interaction. [5] However, the aptamers are primarily synthetic molecules and naturally occurring ribozymes are single stranded RNA molecules, which also have similar recognition domain acting in a similar manner. [6, 7] Aptamers are capable of forming various stable three-dimensional structures in physiological solution. The folding process in solution and the ligand-induced conformational switch is strongly dependent on the presence of divalent cations (magnesium, potassium etc.). [8] There are plethora of computer algorithms

Theranostic Applications

diagnostic and therapeutic application of aptamers.

**Keywords:** aptamers, SELEX, theranostics, chemical antibodies

#### **Chapter 3**

## Aptamers: Magic Bullet for Theranostic Applications

*Arghya Sett*

#### **Abstract**

Aptamers are a short polymer of oligonucleotides (natural or modified) that can bind to its cognate target (small molecules to large macromolecules like proteins, cells, microorganisms etc.) with high affinity and selectivity. They can fold into unique secondary and tertiary conformation in solution (pH, ionic concentration) and bind to their targets in a specific manner (binding constants in sub-nano to pico molar range). They rival the monoclonal antibodies and other specific biological ligands with respect to affinity, stability, robustness, non-immunogenicity and facile to synthesis. Nucleic acid aptamers are selected from an oligonucleotide library by an iterative process called SELEX (**S**ystematic **E**volution of **L**igands by **Ex**ponential Enrichment Analysis). These aptamers are compatible to any kind of chemical modification, conjugation and functionalization. Briefly, this chapter discusses about the diagnostic and therapeutic application of aptamers.

**Keywords:** aptamers, SELEX, theranostics, chemical antibodies

#### **1. Introduction**

Aptamers (Latin word *aptus* means 'to fit' and Greek meros meaning 'part') are single stranded oligonucleotides, which act as synthetic ligands for its cognate target molecules. [1, 2] These molecules show high target specificity, selectivity and affinity, which resemble 'monoclonal antibody'. Similar to antibodies, aptamers also have immense potential to interact with their targets by structural recognition and thus they are termed as 'chemical antibodies'. [3] Different conformations allow aptamers to bind specifically with their target by 'lock and key' model. This hypothesis of binding mechanism is driven by the secondary and tertiary structure of aptamers in-bound state with their targets. Adopting various structures like hairpin loops, bulges, stem-loop, quartets, G-quadruplex, pseudo knots, aptamers can fit into the binding region of the target. [4] Intra and inter molecular interactions like hydrogen bonding, Vander Waals force, hydrophobic interaction, electrostatic forces play major crucial role in aptamer-target interaction. [5] However, the aptamers are primarily synthetic molecules and naturally occurring ribozymes are single stranded RNA molecules, which also have similar recognition domain acting in a similar manner. [6, 7] Aptamers are capable of forming various stable three-dimensional structures in physiological solution. The folding process in solution and the ligand-induced conformational switch is strongly dependent on the presence of divalent cations (magnesium, potassium etc.). [8] There are plethora of computer algorithms

enable sequence based modeling of secondary structure of the oligonucleotide aptamers which actually strengthen the predictability of strongest binders with lowest free energy. [9, 10] Aptamers fold into tertiary conformations and bind to their targets through shape complementarity at the aptamer-target interface. [11] An aptamer binds to a protein can modulate protein functions by interfering with protein interaction with natural partners. Similar to antibodies, aptamers can enter to specific target cells via receptor-mediated endocytosis upon binding to cell surface ligands. [12] Importantly, aptamers can penetrate into tumor cores much more efficiently than antibodies due to their ~20–25-fold smaller sizes compared with full sized monoclonal antibodies. [13, 14]


#### **Table 1.**

*Comparison between Monoclonal antibody vs. Aptamer:*

#### **Figure 1.**

*Publication trend for Search strings: "Aptamers as diagnostics" and "Aptamers as therapeutics" (Source: Scopus).*

**47**

(**Figures 1** and **2**).

**Figure 2.**

**2. Basics of SELEX screening process**

throughput sequencing is performed.

sugar moieties [32] lipids, [33] and even whole cells. [34, 35]

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

Compared to antibodies, aptamers can be produced using cell-free chemical synthesis and are therefore less expensive for large-scale manufacture. Aptamers exhibit extremely low variability between batches and have better controlled post-production modification, they are minimally immunogenic, and are small in size. (**Table 1**) The rapidly growing aptamer industry was predicted to reach US \$244.93 million by 2020. [22] Presently more than 40 companies are actively engaged in diagnostics and therapeutics research to commercialize these "magic bullets" globally (EU countries, Asia, USA, UK etc.). [23] The largest company is "SomaLogic" (company based on SOMAmer- a patented "Slow Off-rate Modified Aptamer) founded by Prof. Larry Gold at Colorado, USA. Since the advent of aptamers scientists and researchers exploit different applications of aptamers that reflects the following trends in the publications.

*Publication trend for Search strings: "Aptamers as theranostics" (Source: Scopus).*

Back in 1990, two individual groups Prof. Larry Gold and Craig Tuerk from University of Boulder, USA and Prof. Jack Szostak and his student A.D. Ellington from Havard University, USA discovered the evolution process to obtain the oligonucleotide binders and they coined the term 'Aptamer' and the process as 'SELEX'. [24, 25] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a common screening process by which aptamers can be selected from an aptamer library which consist of 1024–25 number of various sequences. The method attempts to isolate an aptamer of interest from a pool of randomized library by an iterative cycle of incubation with the target, partitioning and amplification, until the pool of aptamers enriched enough to fit with the target. The SELEX procedure iterates over five basic steps- incubation of aptamer pools with the target, binding, partitioning and washing (to get rid of non-binders which are loosely bound with the target), then elution of positive target-bound aptamers and amplification of enriched pools. Traditionally, the positive pool eluted from last round is being analysed, and high-

An array of different RNA and DNA aptamers were isolated against a vast array of targets: ions, [26] low molecular weight metabolites, [27, 28] proteins, [29–31]

*Theranostics - An Old Concept in New Clothing*

compared with full sized monoclonal antibodies. [13, 14]

**Monoclonal Antibody Aptamer**

**Stability**: Monoclonal antibodies require refrigeration to avoid denaturation. Limited

**Production:** laborious, expensive, high batch-

**Size:** Larger in size, they can resist filtration by the kidneys, long half-lives. However, their size

**Ability to modification**: Antibodies cannot accommodate conjugates without negative consequences such as reduced activity.

*Comparison between Monoclonal antibody vs. Aptamer:*

**Immunogenicity**: They can cause immunogenic response. [17]

prevents access to smaller areas. [19]

shelf life. [15]

to-batch variation.

**Table 1.**

enable sequence based modeling of secondary structure of the oligonucleotide aptamers which actually strengthen the predictability of strongest binders with lowest free energy. [9, 10] Aptamers fold into tertiary conformations and bind to their targets through shape complementarity at the aptamer-target interface. [11] An aptamer binds to a protein can modulate protein functions by interfering with protein interaction with natural partners. Similar to antibodies, aptamers can enter to specific target cells via receptor-mediated endocytosis upon binding to cell surface ligands. [12] Importantly, aptamers can penetrate into tumor cores much more efficiently than antibodies due to their ~20–25-fold smaller sizes

**Stability:** Aptamers do not require refrigeration.

**Immunogenicity:** Aptamers are non-immunogenic. [18]

**Production:** simpler and controlled chemical reactions, little to no variation, automated, chemical synthesis, no

**Size:** Aptamers are small molecules. They are especially subject to kidney filtration, resulting in short half-lives. Compared to antibodies, aptamers can bind to smaller

**Ability to modification:** Easy to modify, modifications can also be incorporated during synthesis to prevent

Indefinite shelf life. [16]

contamination.

targets. [20]

*Publication trend for Search strings: "Aptamers as diagnostics" and "Aptamers as therapeutics" (Source:* 

kidney filtration. [21]

**46**

**Figure 1.**

*Scopus).*

**Figure 2.** *Publication trend for Search strings: "Aptamers as theranostics" (Source: Scopus).*

Compared to antibodies, aptamers can be produced using cell-free chemical synthesis and are therefore less expensive for large-scale manufacture. Aptamers exhibit extremely low variability between batches and have better controlled post-production modification, they are minimally immunogenic, and are small in size. (**Table 1**) The rapidly growing aptamer industry was predicted to reach US \$244.93 million by 2020. [22] Presently more than 40 companies are actively engaged in diagnostics and therapeutics research to commercialize these "magic bullets" globally (EU countries, Asia, USA, UK etc.). [23] The largest company is "SomaLogic" (company based on SOMAmer- a patented "Slow Off-rate Modified Aptamer) founded by Prof. Larry Gold at Colorado, USA. Since the advent of aptamers scientists and researchers exploit different applications of aptamers that reflects the following trends in the publications. (**Figures 1** and **2**).

#### **2. Basics of SELEX screening process**

Back in 1990, two individual groups Prof. Larry Gold and Craig Tuerk from University of Boulder, USA and Prof. Jack Szostak and his student A.D. Ellington from Havard University, USA discovered the evolution process to obtain the oligonucleotide binders and they coined the term 'Aptamer' and the process as 'SELEX'. [24, 25] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a common screening process by which aptamers can be selected from an aptamer library which consist of 1024–25 number of various sequences. The method attempts to isolate an aptamer of interest from a pool of randomized library by an iterative cycle of incubation with the target, partitioning and amplification, until the pool of aptamers enriched enough to fit with the target. The SELEX procedure iterates over five basic steps- incubation of aptamer pools with the target, binding, partitioning and washing (to get rid of non-binders which are loosely bound with the target), then elution of positive target-bound aptamers and amplification of enriched pools. Traditionally, the positive pool eluted from last round is being analysed, and highthroughput sequencing is performed.

An array of different RNA and DNA aptamers were isolated against a vast array of targets: ions, [26] low molecular weight metabolites, [27, 28] proteins, [29–31] sugar moieties [32] lipids, [33] and even whole cells. [34, 35]

#### **3. Library selection**

To select highly selective, specific aptamers, design of the initial aptamer library is the first and foremost step. In case of determination of the length of the random region researchers should consider the sequence space and structural diversity. The complexity of the initial aptamer library depends on the length of the random window of the aptamer library (If the random window is 40 and if we consider DNA aptamer library, so the complexity of the library is: 4^40 that equals to 1024–25). [36]

Special libraries would consist of specifically designed flanking sequences directing the aptamers to form a specific secondary structure, or include modified nucleotides. In capture SELEX, there is unique docking sequence (12–14 nucleotides long) which enables the library in such a way, that highly sensitive aptamers can be fished out against small molecules. [37, 38] The extended genetic alphabets or combination of artificial xeno nucleic acids (XNA) greatly broaden the diversity of sequences and can influence the properties of the aptamers, such as their in vivo stability or nuclease resistance. [39–42] Modified nucleotides can be introduced either during the library synthesis or in the post-selection optimization.

In a review article, Maria *et al.* summarized all key features of designing nucleic acid libraries for SELEX like nature, composition of the library (RNA, DNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. Different randomization strategies and computer algorithms of library designs were also discussed. [43]

#### **4. Various SELEX processes**

Specific aptamers are screened by the iterative processs of SELEX from a highly diverse pool of oligonucleotides. [44–46] After the incubation of the random aptamer pool with the target followed by the removal of non- binding aptamers, the bound aptamer species are recovered. These recovered nucleic acid sequences are amplified with PCR (in the case of DNA aptamer) or RT-PCR (for RNA aptamers). In addition to selection against a purified target molecule, SELEX process can be performed against live bacterial cells and even in mammalian cell lines to isolate cancer cell specific aptamers and furthermore it can lead to the identification of novel biomarkers. [47, 48]

**49**

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

methods for aptamer selection has been inevitable.

cost-effective and considerably less time-consuming.

**5. Modifications of naturally occuring aptamers**

enable the aptamers for biological applications.

enhances biostability of the modified candidates. [54]

**5.1 Aptamers in Drug development pipeline**

(U), guanine (G), cytosine (C).

A giant advancement of SELEX technology has been made since its discovery in 1990. Conventional SELEX is a well-established and effective method but due to its immense time- and labor-consumption, continuous improvement of alternative

High throughput SELEX (HT-SELEX), Functional screening (Microfludics or Flow cytometry based SELEX), Cross-over SELEX (where the target is alternatively changing from proteins and cells), (**Figure 3**) *in-vivo* SELEX, Spiegelmers selection, de-convolution SELEX are few examples of modern-era screening strategy of aptamers. [49] Cutting-edge functional screening process, the chemical modifications, Next-generation sequencing (NGS technology) enable SELEX more efficient,

DNA is the backbone of central dogma of our life cycle. Moreover, any form of nucleic acids play a crucial role in our genetic codon. DNA/RNA is an essential biomacromolecule consist of nucleotide bases such as adenine (A), thymine (T), uracil

There are various types of modifications (nucleotide base modifications, phosphate backbone modifications, peptide mimic oligonucleotides PNA etc.) available which can prevent aptamers from nuclease degradation. Locked nucleic acid (LNA) is one among them where 2′-oxygen has been linked to the 4′-carbon of the ribose sugar by a methylene bridge, thus completely locking the sugar into a 3′-endo conformation. LNAs increase the thermodynamic stability, binding affinity, and enable the oligonucleotides to prevent serum degradation. [50–52] These modifications

Compared to LNAs, the unlocked nucleic acid (UNA) is an acyclic ribose deriva-

Aptamers have been incorporated in drug development pipeline as they have the capacity to block the downstream signalling (phosphorylation of kinases etc.) of different biomolecules. They can play an important role to regulate various cellular crosstalks. To screen therapeutic aptamers either DNA aptamers or 2′-fluoro modified RNA, a combination of 2′-fluoro pyrimidines and 2′-hydroxyl purines (fYrR) are of major interest. fYrR is the "nuclease stable RNA" and can be easily generated by Y639F modified T7 RNA polymerase. Fovista, an anti-platelet derived growth factor (PDGF) aptamer, was previously DNA aptamer but later modified to augment the stability with the addition of backbone modifications. [55] As with the 2′-fluoro modification, the 2'-OMe modifications adopt a C3'-endo conformation. US FDA approved the first aptamer (Macugen®, pegaptanib sodium) in 2004 against vascular endothelial growth factor for the treatment of age-related macular degeneration. [56] This aptamer was modified with 2′-fluoro-pyrimidines and 2'-O-methyl-purines. The stability of the small aptamer was a critical factor but later which can be circumvented with a 3′-cap and a polyethylene glycol

tive that has increased flexibility. UNAs do not consist the C2'-C3' bond, which confers the flexibility observed in this modified nucleotide. [53] LNAs increase the melting temperature of the nucleotide by 1–10°C per LNA insertion but UNAs reduce the melting temperature by 5–10°C retaining the nuclear resistance. In case of, Peptide nucleic acid (PNA) in which sugar-phosphate backbone is modified by short stretch of N-(2-aminoethyl)-glycine units connected by peptide bonds,

**Figure 3.**

*Typical schema of Cross-over SELEX process.*

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

*Theranostics - An Old Concept in New Clothing*

To select highly selective, specific aptamers, design of the initial aptamer library is the first and foremost step. In case of determination of the length of the random region researchers should consider the sequence space and structural diversity. The complexity of the initial aptamer library depends on the length of the random window of the aptamer library (If the random window is 40 and if we consider DNA aptamer library, so the complexity of the library is: 4^40 that equals to 1024–25). [36] Special libraries would consist of specifically designed flanking sequences directing the aptamers to form a specific secondary structure, or include modified nucleotides. In capture SELEX, there is unique docking sequence (12–14 nucleotides long) which enables the library in such a way, that highly sensitive aptamers can be fished out against small molecules. [37, 38] The extended genetic alphabets or combination of artificial xeno nucleic acids (XNA) greatly broaden the diversity of sequences and can influence the properties of the aptamers, such as their in vivo stability or nuclease resistance. [39–42] Modified nucleotides can be introduced

either during the library synthesis or in the post-selection optimization.

In a review article, Maria *et al.* summarized all key features of designing nucleic

acid libraries for SELEX like nature, composition of the library (RNA, DNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. Different randomization strategies and computer algorithms of library

Specific aptamers are screened by the iterative processs of SELEX from a highly diverse pool of oligonucleotides. [44–46] After the incubation of the random aptamer pool with the target followed by the removal of non- binding aptamers, the bound aptamer species are recovered. These recovered nucleic acid sequences are amplified with PCR (in the case of DNA aptamer) or RT-PCR (for RNA aptamers). In addition to selection against a purified target molecule, SELEX process can be performed against live bacterial cells and even in mammalian cell lines to isolate cancer cell specific aptamers and furthermore it can lead to the identification of novel biomarkers. [47, 48]

**3. Library selection**

designs were also discussed. [43]

**4. Various SELEX processes**

**48**

**Figure 3.**

*Typical schema of Cross-over SELEX process.*

A giant advancement of SELEX technology has been made since its discovery in 1990. Conventional SELEX is a well-established and effective method but due to its immense time- and labor-consumption, continuous improvement of alternative methods for aptamer selection has been inevitable.

High throughput SELEX (HT-SELEX), Functional screening (Microfludics or Flow cytometry based SELEX), Cross-over SELEX (where the target is alternatively changing from proteins and cells), (**Figure 3**) *in-vivo* SELEX, Spiegelmers selection, de-convolution SELEX are few examples of modern-era screening strategy of aptamers. [49] Cutting-edge functional screening process, the chemical modifications, Next-generation sequencing (NGS technology) enable SELEX more efficient, cost-effective and considerably less time-consuming.

#### **5. Modifications of naturally occuring aptamers**

DNA is the backbone of central dogma of our life cycle. Moreover, any form of nucleic acids play a crucial role in our genetic codon. DNA/RNA is an essential biomacromolecule consist of nucleotide bases such as adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C).

There are various types of modifications (nucleotide base modifications, phosphate backbone modifications, peptide mimic oligonucleotides PNA etc.) available which can prevent aptamers from nuclease degradation. Locked nucleic acid (LNA) is one among them where 2′-oxygen has been linked to the 4′-carbon of the ribose sugar by a methylene bridge, thus completely locking the sugar into a 3′-endo conformation. LNAs increase the thermodynamic stability, binding affinity, and enable the oligonucleotides to prevent serum degradation. [50–52] These modifications enable the aptamers for biological applications.

Compared to LNAs, the unlocked nucleic acid (UNA) is an acyclic ribose derivative that has increased flexibility. UNAs do not consist the C2'-C3' bond, which confers the flexibility observed in this modified nucleotide. [53] LNAs increase the melting temperature of the nucleotide by 1–10°C per LNA insertion but UNAs reduce the melting temperature by 5–10°C retaining the nuclear resistance. In case of, Peptide nucleic acid (PNA) in which sugar-phosphate backbone is modified by short stretch of N-(2-aminoethyl)-glycine units connected by peptide bonds, enhances biostability of the modified candidates. [54]

#### **5.1 Aptamers in Drug development pipeline**

Aptamers have been incorporated in drug development pipeline as they have the capacity to block the downstream signalling (phosphorylation of kinases etc.) of different biomolecules. They can play an important role to regulate various cellular crosstalks. To screen therapeutic aptamers either DNA aptamers or 2′-fluoro modified RNA, a combination of 2′-fluoro pyrimidines and 2′-hydroxyl purines (fYrR) are of major interest. fYrR is the "nuclease stable RNA" and can be easily generated by Y639F modified T7 RNA polymerase. Fovista, an anti-platelet derived growth factor (PDGF) aptamer, was previously DNA aptamer but later modified to augment the stability with the addition of backbone modifications. [55] As with the 2′-fluoro modification, the 2'-OMe modifications adopt a C3'-endo conformation. US FDA approved the first aptamer (Macugen®, pegaptanib sodium) in 2004 against vascular endothelial growth factor for the treatment of age-related macular degeneration. [56] This aptamer was modified with 2′-fluoro-pyrimidines and 2'-O-methyl-purines. The stability of the small aptamer was a critical factor but later which can be circumvented with a 3′-cap and a polyethylene glycol

molecule, the half-life of Macugen® was extended to 131 hours at max. [57, 58] Anti-vascular endothelial growth factor (VEGF 165) aptamer Macugen, and an anti-Factor IXa aptamer REG1 were both selected from fYrR libraries, and subsequently 2′-O-methyl nucleosides have been incorporated in order to increase serum stability. [57]

There is a plethora of polymerase enzymes like KOD, Pwo, Phusion, Superscript III, vent (exo-), T7 polymerase have all been shown to be capable of incorporating modified triphosphates into DNA and RNA strands, which open up a new opportunities in aptamer selection strategies. [59] The use of Pfx DNA polymerase allows amplification of Ds-Px base pair in Ex-SELEX protocol where extended genetic alphabets were included in complexity of nucleic acid library. [60]

Several limitations of aptamers should be considered in the process of *in-vivo* applications of nucleic acid aptamers. Being polynucleotides, nucleic acid aptamers are naturally susceptible to enzymes degradation by exo and/or endo-nucleases, leading to a reduced *in vivo* circulatory half-life. This drawback can be alleviated by side chain chemical modifications to aptamers, incorporating unnatural nucleotide bases (locked and unlocked nucleic acids) and capping the aptamer ends, thus minimizing the susceptibility to endonuclease and exonuclease attack. [50, 51] Short blood residence time is another challenge with in vivo aptamer applications, which is due to fast removal of aptamer from the circulation by renal filtration as most aptamers have a size smaller than the renal filtration threshold of 40 kDa. [31] To achieve desired serum half-life, aptamers can be engineered by conjugation with a terminal polyethylene glycol (PEG), although this may compromise the extent of tumor penetration [61]. In some cases, post-SELEX modifications following the selection of aptamers may alter the 3-D structure of the aptamers, leading to the lost or altered binding affinity and specificity. Such problems can be prevented by using random aptamer pools containing modified nucleotides during the SELEX process. [62, 63] In addition, the ability of aptamers to interact with cells may decrease due to repulsion of nucleic acids by negatively-charged cell membranes. This can be refuted by increasing the binding affinity and specificity of aptamers toward their cell surface receptors to trigger receptor-mediated endocytosis.

In the field of oncology, two aptamers, namely, AS1411 and NOX-A12, have entered clinical trials. [45, 64] AS1411 (formerly ARGO100; Antisoma) is a guanine quadruplex aptamer obtained from a guanine-rich oligonucleotide library in the anti-proliferation screen, which is not a typical SELEX process. [65] The guanine quadruplex structure benefits AS1411 because it is resistant to nuclease degradation and enhances cell uptake. In *in-vitro* validations, AS1411 inhibits more than 80 types of cancer cell lines. In addition, the target of AS1411 has been verified to be nucleolin, and the relevant mechanism and specificity against cancer cells have also been described. In preclinical tests, AS1411 shows efficacy toward xenograft models, including non-small cell lung, renal, and breast cancers. It entered clinical trials in 2003 and passed phase II trials for acute myeloid leukemia. A subsequent phase II trial for renal cell carcinoma started in 2008 (clinical trial ID NCT00740441). [66] NOXA12 (Olaptesed pegol; Noxxon) is an L-form RNA aptamer known as Spiegelmer and is used for cancer therapy. NOX-A12 can bind to its target chemokine CXCL-12 and blocks its interaction with its receptor. [67] This disrupts the tissue gradient of CXCL-12 and reduces the cancer cell homing that might lead to tumor metastasis and drug resistance. [68] Currently, phase II clinical trials for NOX-A12 are underway for the treatment of chronic lymphocytic leukemia and refractory multiple myeloma (clinical trial IDs NCT01486797 and NCT01521533). [67] Aptamer based cancer therapeutics have immense potential for precise and less toxic treatment for cancer patients. [46]

**51**

**Figure 4.**

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

Aptamers can be used *in-vitro* and *in-vivo* as well. [69] In terms of *in vivo* diagnostics, 'escort' aptamers can be implied as vehicles for a detectable molecules, such as radionuclides, fluorophores etc. [70–72] The development of new agents like radio-pharmaceuticals is challenging. There are some important factors such as efficiency of the radiolabeling process, specific activity (radioactivity per moles e.g. Ci/μmol), chemical purity, radiochemical and chemical stability and shelf life of the final product. [73] Mostly, radiolabeling strategies for aptamers are similar as for proteins, or antibodies. Aptamers can be easily chemically modified at its 5′ or 3′

Radiohalogens (fluorine-18, bromine-76, iodine-125 etc.) are the most commonly used for radiolabelling oloigonuclotides which are often accompanied with prosthetic groups. Recently, click-chemistry for radiofluorination was demonstrated on antisense oligonucleotides and siRNAs. [74, 75] Another report used photoconjugation as strategy for the radiofluorination of an aptamer. [76] Oligonucleotides have also been radiolabeled with the radiohalogens such as bromine-76 for PET imaging and iodine-123 for SPECT (Single photon emission computed tomography) imaging. In addition, iodine-125 has been used to radiolabel antisense oligonucleotides, aptamers and spiegelmers for theranostic applications. Due to the harsh and non-aqueous reaction conditions usually needed to radiolabel prosthetic groups, it

end with a desired functional group for radiolabeling (**Figure 4**).

is performed before the conjugation process to the oligonucleotide. [73]

*Aptamers modified with radiolabelled molecules for disase diagnosis (Figure adapted from Gijs* et al*) [73].*

**6. Aptamers as diagnostic agents**

#### **6. Aptamers as diagnostic agents**

*Theranostics - An Old Concept in New Clothing*

serum stability. [57]

molecule, the half-life of Macugen® was extended to 131 hours at max. [57, 58] Anti-vascular endothelial growth factor (VEGF 165) aptamer Macugen, and an anti-Factor IXa aptamer REG1 were both selected from fYrR libraries, and subsequently 2′-O-methyl nucleosides have been incorporated in order to increase

alphabets were included in complexity of nucleic acid library. [60]

There is a plethora of polymerase enzymes like KOD, Pwo, Phusion, Superscript III, vent (exo-), T7 polymerase have all been shown to be capable of incorporating modified triphosphates into DNA and RNA strands, which open up a new opportunities in aptamer selection strategies. [59] The use of Pfx DNA polymerase allows amplification of Ds-Px base pair in Ex-SELEX protocol where extended genetic

Several limitations of aptamers should be considered in the process of *in-vivo* applications of nucleic acid aptamers. Being polynucleotides, nucleic acid aptamers are naturally susceptible to enzymes degradation by exo and/or endo-nucleases, leading to a reduced *in vivo* circulatory half-life. This drawback can be alleviated by side chain chemical modifications to aptamers, incorporating unnatural nucleotide bases (locked and unlocked nucleic acids) and capping the aptamer ends, thus minimizing the susceptibility to endonuclease and exonuclease attack. [50, 51] Short blood residence time is another challenge with in vivo aptamer applications, which is due to fast removal of aptamer from the circulation by renal filtration as most aptamers have a size smaller than the renal filtration threshold of 40 kDa. [31] To achieve desired serum half-life, aptamers can be engineered by conjugation with a terminal polyethylene glycol (PEG), although this may compromise the extent of tumor penetration [61]. In some cases, post-SELEX modifications following the selection of aptamers may alter the 3-D structure of the aptamers, leading to the lost or altered binding affinity and specificity. Such problems can be prevented by using random aptamer pools containing modified nucleotides during the SELEX process. [62, 63] In addition, the ability of aptamers to interact with cells may decrease due to repulsion of nucleic acids by negatively-charged cell membranes. This can be refuted by increasing the binding affinity and specificity of aptamers toward their cell surface receptors to trigger receptor-mediated

In the field of oncology, two aptamers, namely, AS1411 and NOX-A12, have entered clinical trials. [45, 64] AS1411 (formerly ARGO100; Antisoma) is a guanine quadruplex aptamer obtained from a guanine-rich oligonucleotide library in the anti-proliferation screen, which is not a typical SELEX process. [65] The guanine quadruplex structure benefits AS1411 because it is resistant to nuclease degradation and enhances cell uptake. In *in-vitro* validations, AS1411 inhibits more than 80 types of cancer cell lines. In addition, the target of AS1411 has been verified to be nucleolin, and the relevant mechanism and specificity against cancer cells have also been described. In preclinical tests, AS1411 shows efficacy toward xenograft models, including non-small cell lung, renal, and breast cancers. It entered clinical trials in 2003 and passed phase II trials for acute myeloid leukemia. A subsequent phase II trial for renal cell carcinoma started in 2008 (clinical trial ID NCT00740441). [66] NOXA12 (Olaptesed pegol; Noxxon) is an L-form RNA aptamer known as Spiegelmer and is used for cancer therapy. NOX-A12 can bind to its target chemokine CXCL-12 and blocks its interaction with its receptor. [67] This disrupts the tissue gradient of CXCL-12 and reduces the cancer cell homing that might lead to tumor metastasis and drug resistance. [68] Currently, phase II clinical trials for NOX-A12 are underway for the treatment of chronic lymphocytic leukemia and refractory multiple myeloma (clinical trial IDs NCT01486797 and NCT01521533). [67] Aptamer based cancer therapeutics have immense potential for precise and less

**50**

toxic treatment for cancer patients. [46]

endocytosis.

Aptamers can be used *in-vitro* and *in-vivo* as well. [69] In terms of *in vivo* diagnostics, 'escort' aptamers can be implied as vehicles for a detectable molecules, such as radionuclides, fluorophores etc. [70–72] The development of new agents like radio-pharmaceuticals is challenging. There are some important factors such as efficiency of the radiolabeling process, specific activity (radioactivity per moles e.g. Ci/μmol), chemical purity, radiochemical and chemical stability and shelf life of the final product. [73] Mostly, radiolabeling strategies for aptamers are similar as for proteins, or antibodies. Aptamers can be easily chemically modified at its 5′ or 3′ end with a desired functional group for radiolabeling (**Figure 4**).

Radiohalogens (fluorine-18, bromine-76, iodine-125 etc.) are the most commonly used for radiolabelling oloigonuclotides which are often accompanied with prosthetic groups. Recently, click-chemistry for radiofluorination was demonstrated on antisense oligonucleotides and siRNAs. [74, 75] Another report used photoconjugation as strategy for the radiofluorination of an aptamer. [76] Oligonucleotides have also been radiolabeled with the radiohalogens such as bromine-76 for PET imaging and iodine-123 for SPECT (Single photon emission computed tomography) imaging. In addition, iodine-125 has been used to radiolabel antisense oligonucleotides, aptamers and spiegelmers for theranostic applications. Due to the harsh and non-aqueous reaction conditions usually needed to radiolabel prosthetic groups, it is performed before the conjugation process to the oligonucleotide. [73]

#### **Figure 4.**

*Aptamers modified with radiolabelled molecules for disase diagnosis (Figure adapted from Gijs* et al*) [73].*

Till date, a plethora of aptamers have been modified or labelled with radioactive molecules. Aptamers against several important biomarkers like PMSA, Tenascin C, thrombin, MUC1 were already exploited for radiolabelling. Aptamer-based radiopharmaceuticals were primarily developed for imaging and therapy of cancer diseases, metabolic disorders and others. The aptamers are mainly radiolabeled with technetium-99 m for SPECT (Single photon emission computed tomography), PET (Positron emission tomography) imaging. Very few aptamers were published related to PET imaging, and there is only one study of radiolabeled aptamers for therapy by Bandekar *et al.* [77] Other radiolabeled aptamers have only been tested for preclinical applications or in the course of preclinical assesement.

Molecular nuclear imaging technique is a diagnostic process of non-invasive visualization of any disease *in-vivo* at molecular level with high precision. For nuclear imaging, the probes used for radiolabelling has to be modified accordingly. Aptamers are the most promising candidates with versatile modification capibility, can be easily engineered for various imaging and other diagnostic purposes.

The first radiolabeled aptamer for nuclear imaging was discovered by Charlton et al. A DNA aptamer, NX21909, was selected against human neutrophil elastase, an enzyme which is secreted by neutrophils and macrophages during inflammation to kill pathogens. [78]

Aptamer TTA1, an RNA aptamer targeting the extracellular matrix protein tenascin C (TN-C), was the first radiolabeled aptamer which was used as molecular cancer imaging agent. Aptamer TTA1 was generated by a cross-over SELEX involving the purified recombinant TN-C protein and TN-C-positive U251 glioblastoma cells. [79, 80]

#### **7. Lightup aptasensors for diagnostic applications**

There are a unique group of aptamers (generally RNA aptamers) which can bind specifically with their cognate fluorogen molecules like DFHBI, thiazole orange, thioflavin T etc. [49, 81, 82] The non-fluorescent moelcules (native unbound state) become fluorescent (bound state) after binding to the aptamers and these "light-up" aptamers generate fluorescence signal. In the omni-presence of target molecules (small pre-miRNAs) and malachite green (fluorogen) light up aptasensors 'malaswitch' exhibit fluorescence enhancement. [83] We can engineer the small-molecule specific aptamers (like aptamers for some pesticides, toxins, small metaboltes) in such a way, that combined with light-up aptamers, they can generate a detectable signal. Light up aptasensors are promising alternative biosensor for label free sensitive detection of small molecules. [84]

#### **8. Future perspectives**

With more focus on *in vivo* studies for potential clinical applications, aptamers can be developed in combination with DNA nanostructures, nanomaterial, or microfluidic devices as diagnostic probes or therapeutic agents for cancers, infectious diseases, genetic, metabolic, neurological disorders, lifestyle diseases and several others. The use of aptamers as targeting agents in drug delivery can also be explored. Aptamers might be exploited to develop portable, low-cost and robust diagnostic kit using simple devices for real-time and on-site POC (point-of-care) detection and monitoring, instead of the laborious and time-consuming diagnostic tests currently available only in clinical labs. Regarding therapeutics approach, there is still untapped potential in the combination of the target recognition and strong

**53**

**Author details**

Czech Republic, Prague, Czech Republic

provided the original work is properly cited.

\*Address all correspondence to: arghya.sett@gmail.com

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Arghya Sett

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

management system.

binding property of aptamers with exquisitely designed nanomaterials. It can be used as an effective alternative drug delivery platform. Variety of materials such as liposomes, polymer vesicles and silica nanoparticles, combined with DNA/RNA aptamers, has shown feasibility for use in *in vivo* targeted drug delivery. [85, 86] The integration of diagnostic capability with therapeutic interventions termed, as "Theranostics" is critical to address the challenges of disease heterogeneity and adaptation. Although aptamers have immense potential as theranostic agents, tailor-made modifications, validation of experiments need to be executed before aptamer-based drug delivery can reach clinical trials and eventually the patient

#### *Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

*Theranostics - An Old Concept in New Clothing*

kill pathogens. [78]

cells. [79, 80]

Till date, a plethora of aptamers have been modified or labelled with radioactive molecules. Aptamers against several important biomarkers like PMSA, Tenascin C, thrombin, MUC1 were already exploited for radiolabelling. Aptamer-based radiopharmaceuticals were primarily developed for imaging and therapy of cancer diseases, metabolic disorders and others. The aptamers are mainly radiolabeled with technetium-99 m for SPECT (Single photon emission computed tomography), PET (Positron emission tomography) imaging. Very few aptamers were published related to PET imaging, and there is only one study of radiolabeled aptamers for therapy by Bandekar *et al.* [77] Other radiolabeled aptamers have only been tested

for preclinical applications or in the course of preclinical assesement.

**7. Lightup aptasensors for diagnostic applications**

label free sensitive detection of small molecules. [84]

**8. Future perspectives**

Molecular nuclear imaging technique is a diagnostic process of non-invasive visualization of any disease *in-vivo* at molecular level with high precision. For nuclear imaging, the probes used for radiolabelling has to be modified accordingly. Aptamers are the most promising candidates with versatile modification capibility,

The first radiolabeled aptamer for nuclear imaging was discovered by Charlton et al. A DNA aptamer, NX21909, was selected against human neutrophil elastase, an enzyme which is secreted by neutrophils and macrophages during inflammation to

Aptamer TTA1, an RNA aptamer targeting the extracellular matrix protein tenascin C (TN-C), was the first radiolabeled aptamer which was used as molecular cancer imaging agent. Aptamer TTA1 was generated by a cross-over SELEX involving the purified recombinant TN-C protein and TN-C-positive U251 glioblastoma

There are a unique group of aptamers (generally RNA aptamers) which can bind specifically with their cognate fluorogen molecules like DFHBI, thiazole orange, thioflavin T etc. [49, 81, 82] The non-fluorescent moelcules (native unbound state) become fluorescent (bound state) after binding to the aptamers and these "light-up" aptamers generate fluorescence signal. In the omni-presence of target molecules (small pre-miRNAs) and malachite green (fluorogen) light up aptasensors 'malaswitch' exhibit fluorescence enhancement. [83] We can engineer the small-molecule specific aptamers (like aptamers for some pesticides, toxins, small metaboltes) in such a way, that combined with light-up aptamers, they can generate a detectable signal. Light up aptasensors are promising alternative biosensor for

With more focus on *in vivo* studies for potential clinical applications, aptamers can be developed in combination with DNA nanostructures, nanomaterial, or microfluidic devices as diagnostic probes or therapeutic agents for cancers, infectious diseases, genetic, metabolic, neurological disorders, lifestyle diseases and several others. The use of aptamers as targeting agents in drug delivery can also be explored. Aptamers might be exploited to develop portable, low-cost and robust diagnostic kit using simple devices for real-time and on-site POC (point-of-care) detection and monitoring, instead of the laborious and time-consuming diagnostic tests currently available only in clinical labs. Regarding therapeutics approach, there is still untapped potential in the combination of the target recognition and strong

can be easily engineered for various imaging and other diagnostic purposes.

**52**

binding property of aptamers with exquisitely designed nanomaterials. It can be used as an effective alternative drug delivery platform. Variety of materials such as liposomes, polymer vesicles and silica nanoparticles, combined with DNA/RNA aptamers, has shown feasibility for use in *in vivo* targeted drug delivery. [85, 86] The integration of diagnostic capability with therapeutic interventions termed, as "Theranostics" is critical to address the challenges of disease heterogeneity and adaptation. Although aptamers have immense potential as theranostic agents, tailor-made modifications, validation of experiments need to be executed before aptamer-based drug delivery can reach clinical trials and eventually the patient management system.

### **Author details**

Arghya Sett Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

\*Address all correspondence to: arghya.sett@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Famulok M, Mayer G. Aptamers and SELEX in chemistry & biology. Vol. 21, Chemistry and Biology. 2014.

[2] Nimjee SM, White RR, Becker RC, Sullenger BA. Aptamers as Therapeutics. Annu Rev Pharmacol Toxicol [Internet]. 2017 Jan 6;57:61-79. Available from: https://pubmed.ncbi. nlm.nih.gov/28061688

[3] Bauer M, Strom M, Hammond DS, Shigdar S. Anything You Can Do, I Can Do Better: Can Aptamers Replace Antibodies in Clinical Diagnostic Applications? Molecules. 2019;24(23):4377.

[4] Cai S, Yan J, Xiong H, Liu Y, Peng D, Liu Z. Investigations on the interface of nucleic acid aptamers and binding targets. Analyst [Internet]. 2018;143(22):5317-38. Available from: http://dx.doi.org/10.1039/C8AN01467A

[5] Elskens JP, Elskens JM, Madder A. Chemical Modification of Aptamers for Increased Binding Affinity in Diagnostic Applications: Current Status and Future Prospects. Int J Mol Sci. 2020;21(12):4522.

[6] Breaker RR, Joyce GF. The Expanding View of RNA and DNA Function. Chem Biol [Internet]. 2014;21(9):1059-65. Available from: http://linkinghub.elsevier.com/retrieve/ pii/S1074552114002373

[7] Doudna JA, Cech TR. The chemical repertoire of natural ribozymes. Nature. 2002;418(6894):222-8.

[8] Horiya S, Li X, Kawai G, Saito R, Katoh A, Kobayashi K, et al. RNA LEGO: magnesium-dependent formation of specific RNA assemblies through kissing interactions. Chem Biol. 2003;10(7):645-54.

[9] Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res [Internet]. 2003 Jul 1;31(13):3406-15. Available from: http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC169194/

[10] Wolfe BR, Porubsky NJ, Zadeh JN, Dirks RM, Pierce NA. Constrained multistate sequence design for nucleic acid reaction pathway engineering. J Am Chem Soc. 2017;139(8):3134-44.

[11] Khati M. The Future of Aptamers in Medicine. J Clin Pathol [Internet]. 2010;63(6):480-7. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/20360137

[12] Cibiel A, Dupont DM, Ducongé F. Methods to identify aptamers against cell surface biomarkers. Pharmaceuticals. 2011;4(9):1216-35.

[13] Soper SA, Brown K, Ellington A, Frazier B, Garcia-Manero G, Gau V, et al. Point-of-care biosensor systems for cancer diagnostics/ prognostics. Biosens Bioelectron. 2006;21(10):1932-42.

[14] Brody EN, Willis MC, Smith JD, Jayasena S, Zichi D, Gold L. The use of aptamers in large arrays for molecular diagnostics. Vol. 4, Molecular Diagnosis. 1999. p. 381-8.

[15] Le Basle Y, Chennell P, Tokhadze N, Astier A, Sautou V. Physicochemical stability of monoclonal antibodies: a review. J Pharm Sci. 2020;109(1):169-90.

[16] Bruno JG. Long shelf life of a lyophilized DNA aptamer beacon assay. J Fluoresc. 2017;27(2):439-41.

[17] Kozlowski S, Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv Drug Deliv Rev. 2006;58(5-6):707-22.

**55**

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

> [26] Hofmann H-P, Limmer S, Hornung V, Sprinzl M. Ni2+-binding RNA motifs with an asymmetric purinerich internal loop and a GA base pair.

Rna. 1997;3(11):1289-300.

2009;19(13):3619-22.

2017;183:104-20.

2017;19(8).

pone.0153001

[27] Huizenga DE, Szostak JW. A DNA aptamer that binds adenosine and ATP. Biochemistry. 1995;34(2):656-65.

[28] Miyachi Y, Shimizu N, Ogino C, Fukuda H, Kondo A. Selection of a DNA aptamer that binds 8-OHdG using GMP-agarose. Bioorg Med Chem Lett.

[29] Sett A, Borthakur BB, Sharma JD, Kataki AC, Bora U. DNA aptamer probes for detection of estrogen receptor α positive carcinomas. Transl Res.

[30] Sett A, Borthakur BB, Bora U. Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas. Clin Transl Oncol.

[31] Ahirwar R, Vellarikkal SK, Sett A, Sivasubbu S, Scaria V, Bora U, et al. Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer. PLoS One [Internet]. 2016;11(4):e0153001. Available from: http://journals.plos. org/plosone/article?id=10.1371/journal.

[32] Boese BJ, Breaker RR. In vitro selection and characterization of cellulose-binding DNA aptamers. Nucleic Acids Res. 2007;35(19):6378-88.

[33] Betat H, Vogel S, Struhalla M, Förster H-H, Famulok M, Welzel P, et al. Aptamers that recognize the lipid moiety of the antibiotic moenomycin A. Biol Chem. 2003;384(10-11):1497-500.

[34] Dwivedi HP, Smiley RD, Jaykus L-A. Selection and characterization of DNA aptamers with binding selectivity to

Longobardo I, Condorelli G, Marotta P, Affuso A, et al. A Neutralizing RNA Aptamer against EGFR Causes Selective

[18] Esposito CL, Passaro D,

Apoptotic Cell Death. PLoS One [Internet]. 2011;6(9):e24071. Available from: http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=3167817&t ool=pmcentrez&rendertype=abstract

[19] Šmuc T, Ahn I-Y, Ulrich H. Nucleic acid aptamers as high affinity ligands in biotechnology and biosensorics. J Pharm Biomed Anal.

[20] Majumder P, Gomes KN, Ulrich H. Aptamers: from bench side research towards patented molecules with therapeutic

applications. Expert Opin Ther Pat [Internet]. 2009 Nov 1;19(11):1603- 13. Available from: https://doi. org/10.1517/13543770903313746

[21] Meek KN, Rangel AE, Heemstra JM. Enhancing aptamer function and stability via in vitro selection using modified nucleic acids. Methods. 2016

[22] Kedzierski S, Khoshnejad M, Caltagirone GT. Synthetic antibodies: the emerging field of aptamers. Bioprocess J. 2012;11:46-9.

[23] Kaur H, Bruno JG, Kumar A, Sharma TK. Aptamers in the

[24] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature.

[25] Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to

bacteriophage T4 DNA polymerase. Science. 1990;249(4968):505-10.

1990;346(6287):818.

Therapeutics and Diagnostics Pipelines. Theranostics [Internet]. 2018 Jul 1;8(15):4016-32. Available from: https:// pubmed.ncbi.nlm.nih.gov/30128033

2013;81:210-7.

Aug;106:29-36.

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

[18] Esposito CL, Passaro D, Longobardo I, Condorelli G, Marotta P, Affuso A, et al. A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death. PLoS One [Internet]. 2011;6(9):e24071. Available from: http://www.pubmedcentral.nih. gov/articlerender.fcgi?artid=3167817&t ool=pmcentrez&rendertype=abstract

[19] Šmuc T, Ahn I-Y, Ulrich H. Nucleic acid aptamers as high affinity ligands in biotechnology and biosensorics. J Pharm Biomed Anal. 2013;81:210-7.

[20] Majumder P, Gomes KN, Ulrich H. Aptamers: from bench side research towards patented molecules with therapeutic applications. Expert Opin Ther Pat [Internet]. 2009 Nov 1;19(11):1603- 13. Available from: https://doi. org/10.1517/13543770903313746

[21] Meek KN, Rangel AE, Heemstra JM. Enhancing aptamer function and stability via in vitro selection using modified nucleic acids. Methods. 2016 Aug;106:29-36.

[22] Kedzierski S, Khoshnejad M, Caltagirone GT. Synthetic antibodies: the emerging field of aptamers. Bioprocess J. 2012;11:46-9.

[23] Kaur H, Bruno JG, Kumar A, Sharma TK. Aptamers in the Therapeutics and Diagnostics Pipelines. Theranostics [Internet]. 2018 Jul 1;8(15):4016-32. Available from: https:// pubmed.ncbi.nlm.nih.gov/30128033

[24] Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346(6287):818.

[25] Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249(4968):505-10.

[26] Hofmann H-P, Limmer S, Hornung V, Sprinzl M. Ni2+-binding RNA motifs with an asymmetric purinerich internal loop and a GA base pair. Rna. 1997;3(11):1289-300.

[27] Huizenga DE, Szostak JW. A DNA aptamer that binds adenosine and ATP. Biochemistry. 1995;34(2):656-65.

[28] Miyachi Y, Shimizu N, Ogino C, Fukuda H, Kondo A. Selection of a DNA aptamer that binds 8-OHdG using GMP-agarose. Bioorg Med Chem Lett. 2009;19(13):3619-22.

[29] Sett A, Borthakur BB, Sharma JD, Kataki AC, Bora U. DNA aptamer probes for detection of estrogen receptor α positive carcinomas. Transl Res. 2017;183:104-20.

[30] Sett A, Borthakur BB, Bora U. Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas. Clin Transl Oncol. 2017;19(8).

[31] Ahirwar R, Vellarikkal SK, Sett A, Sivasubbu S, Scaria V, Bora U, et al. Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer. PLoS One [Internet]. 2016;11(4):e0153001. Available from: http://journals.plos. org/plosone/article?id=10.1371/journal. pone.0153001

[32] Boese BJ, Breaker RR. In vitro selection and characterization of cellulose-binding DNA aptamers. Nucleic Acids Res. 2007;35(19):6378-88.

[33] Betat H, Vogel S, Struhalla M, Förster H-H, Famulok M, Welzel P, et al. Aptamers that recognize the lipid moiety of the antibiotic moenomycin A. Biol Chem. 2003;384(10-11):1497-500.

[34] Dwivedi HP, Smiley RD, Jaykus L-A. Selection and characterization of DNA aptamers with binding selectivity to

**54**

*Theranostics - An Old Concept in New Clothing*

[1] Famulok M, Mayer G. Aptamers and SELEX in chemistry & biology. Vol. 21,

prediction. Nucleic Acids Res [Internet]. 2003 Jul 1;31(13):3406-15. Available from: http://www.ncbi.nlm.nih.gov/

[10] Wolfe BR, Porubsky NJ, Zadeh JN, Dirks RM, Pierce NA. Constrained multistate sequence design for nucleic acid reaction pathway engineering. J Am

Chem Soc. 2017;139(8):3134-44.

pubmed/20360137

2011;4(9):1216-35.

for cancer diagnostics/

2006;21(10):1932-42.

stability of monoclonal

2020;109(1):169-90.

2006;58(5-6):707-22.

1999. p. 381-8.

[12] Cibiel A, Dupont DM, Ducongé F. Methods to identify aptamers against cell surface biomarkers. Pharmaceuticals.

[11] Khati M. The Future of Aptamers in Medicine. J Clin Pathol [Internet]. 2010;63(6):480-7. Available from: http://www.ncbi.nlm.nih.gov/

[13] Soper SA, Brown K, Ellington A, Frazier B, Garcia-Manero G, Gau V, et al. Point-of-care biosensor systems

prognostics. Biosens Bioelectron.

[14] Brody EN, Willis MC, Smith JD, Jayasena S, Zichi D, Gold L. The use of aptamers in large arrays for molecular diagnostics. Vol. 4, Molecular Diagnosis.

[15] Le Basle Y, Chennell P, Tokhadze N, Astier A, Sautou V. Physicochemical

antibodies: a review. J Pharm Sci.

[16] Bruno JG. Long shelf life of a lyophilized DNA aptamer beacon assay.

[17] Kozlowski S, Swann P. Current and future issues in the manufacturing and development of monoclonal antibodies. Adv Drug Deliv Rev.

J Fluoresc. 2017;27(2):439-41.

pmc/articles/PMC169194/

[2] Nimjee SM, White RR, Becker RC,

Therapeutics. Annu Rev Pharmacol Toxicol [Internet]. 2017 Jan 6;57:61-79. Available from: https://pubmed.ncbi.

[3] Bauer M, Strom M, Hammond DS, Shigdar S. Anything You Can Do, I Can Do Better: Can Aptamers Replace Antibodies in Clinical Diagnostic

Chemistry and Biology. 2014.

**References**

Sullenger BA. Aptamers as

nlm.nih.gov/28061688

Applications? Molecules.

[4] Cai S, Yan J, Xiong H, Liu Y, Peng D, Liu Z. Investigations on the interface of nucleic acid aptamers and binding targets. Analyst [Internet]. 2018;143(22):5317-38. Available from: http://dx.doi.org/10.1039/C8AN01467A

[5] Elskens JP, Elskens JM, Madder A. Chemical Modification of Aptamers for Increased Binding Affinity in Diagnostic Applications: Current Status and Future Prospects. Int J Mol Sci.

[7] Doudna JA, Cech TR. The chemical repertoire of natural ribozymes. Nature.

Saito R, Katoh A, Kobayashi K, et al. RNA LEGO: magnesium-dependent formation of specific RNA assemblies through kissing interactions. Chem Biol.

[9] Zuker M. Mfold web server for nucleic acid folding and hybridization

2019;24(23):4377.

2020;21(12):4522.

[6] Breaker RR, Joyce GF. The Expanding View of RNA and DNA Function. Chem Biol [Internet]. 2014;21(9):1059-65. Available from: http://linkinghub.elsevier.com/retrieve/

pii/S1074552114002373

2002;418(6894):222-8.

2003;10(7):645-54.

[8] Horiya S, Li X, Kawai G,

Campylobacter jejuni using whole-cell SELEX. Appl Microbiol Biotechnol. 2010;87(6):2323-34.

[35] Wang G, Liu J, Chen K, Xu Y, Liu B, Liao J, et al. Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX. Sci Rep. 2017;7(1):1-10.

[36] Pobanz K, Lupták A. Improving the odds: Influence of starting pools on in vitro selection outcomes. Methods. 2016;106:14-20.

[37] Stoltenburg R, Nikolaus N, Strehlitz B. Capture-SELEX: selection of DNA aptamers for aminoglycoside antibiotics. J Anal Methods Chem. 2012;2012.

[38] Paniel N, Istamboulié G, Triki A, Lozano C, Barthelmebs L, Noguer T. Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor. Talanta. 2017;162:232-40.

[39] Taylor AI, Houlihan G, Holliger P. Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics. Cold Spring Harb Perspect Biol. 2019 Jun;11(6).

[40] Lee J, Schwieter KE, Watkins AM, Kim DS, Yu H, Schwarz KJ, et al. Expanding the limits of the second genetic code with ribozymes. Nat Commun. 2019 Nov;10(1):5097.

[41] Morihiro K, Kasahara Y, Obika S. Biological applications of xeno nucleic acids. Mol Biosyst. 2017 Jan;13(2):235-45.

[42] Rangel AE, Chen Z, Ayele TM, Heemstra JM. In vitro selection of an XNA aptamer capable of smallmolecule recognition. Nucleic Acids Res. 2018;46(16).

[43] Vorobyeva MA, Davydova AS, Vorobjev PE, Venyaminova AG. Key Aspects of Nucleic Acid Library Design for in Vitro Selection. Int J Mol Sci [Internet]. 2018 Feb 5;19(2):470. Available from: https://pubmed.ncbi. nlm.nih.gov/29401748

[44] Jayasena SD. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin Chem. 1999;45(9):1628-50.

[45] Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov [Internet]. 2010;9(7):537- 50. Available from: http://dx.doi. org/10.1038/nrd3141

[46] Xiang D, Shigdar S, Qiao G, Wang T, Kouzani AZ, Zhou S-F, et al. Nucleic Acid Aptamer-Guided Cancer Therapeutics and Diagnostics: the Next Generation of Cancer Medicine. Theranostics [Internet]. 2015 Jan 1;5(1):23-42. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/ PMC4265746/

[47] Cho EJ, Lee J-W, Ellington AD. Applications of aptamers as sensors. Annu Rev Anal Chem (Palo Alto Calif). 2009;2:241-64.

[48] Li S, Xu H, Ding H, Huang Y, Cao X, Yang G, et al. Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX. 2009;(February):327-36.

[49] Autour A, Bouhedda F, Cubi R, Ryckelynck M. Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahighthroughput screening. Methods. 2019;161:46-53.

[50] Barciszewski J, Medgaard M, Koch T, Kurreck J, Erdmann VA. Locked Nucleic Acid Aptamers. In: Nucleic Acid and Peptide Aptamers: Methods and Protocols [Internet]. 2009. p. 165-86. Available from: http://www.springerlink.com/ index/10.1007/978-1-59745-557-2

**57**

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

> [58] Lee J-H, Canny MD, De Erkenez A, Krilleke D, Ng Y-S,

2005;102(52):18902-7.

Shima DT, et al. A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc Natl Acad Sci.

[59] Lipi F, Chen S, Chakravarthy M, Rakesh S, Veedu RN. In vitro evolution

[60] Kimoto M, Matsunaga K, Hirao I. Evolving Aptamers with Unnatural Base Pairs. Curr Protoc Chem Biol.

[61] Ng EWM, Shima DT, Calias P, Cunningham ET, Guyer DR, Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006;5(2):123-32.

[62] Sampson T. Aptamers and SELEX:

[63] Shamah SM, Healy JM, Cload ST. Complex target SELEX. Acc Chem Res.

[64] Lao Y-H, Phua KKL, Leong KW. Aptamer nanomedicine for cancer therapeutics: barriers and potential

[65] Carvalho J, Paiva A, Campello MPC, Paulo A, Mergny J-L, Salgado GF, et al. Aptamer-based targeted Delivery of a G-quadruplex Ligand in Cervical Cancer Cells. Sci Rep. 2019;9(1):1-12.

[66] Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN, Harzstark AL, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal

for translation. ACS Nano.

The technology. World Pat Inf.

of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies. RNA Biol [Internet]. 2016 Dec 1;13(12):1232- 45. Available from: https://doi.org/10.10

80/15476286.2016.1236173

2017;9(4):315-39.

2003;25(2):123-9.

2008;41(1):130-8.

2015;9(3):2235-54.

[51] Veedu RN, Wengel J. Locked nucleic acids: promising nucleic acid analogs for therapeutic applications. Chem Biodivers. 2010;7(3):536-42.

[52] Darfeuille F, Hansen JB, Orum H, Di Primo C, Toulmé J-J. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1. Nucleic Acids Res [Internet]. 2004 Jun 4;32(10):3101-7. Available from: https://www.ncbi.nlm.

nih.gov/pubmed/15181175

2009;17(15):5420-5.

[53] Langkjær N, Pasternak A, Wengel J. UNA (unlocked nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid duplex stability. Bioorg Med Chem.

[54] Wittung P, Nielsen PE, Buchardt O, Egholm M, Norde B. DNA-like double helix formed by peptide nucleic acid. Nature. 1994;368(6471):561-3.

[55] Floege J, Ostendorf T, Janssen U, Burg M, Radeke HH, Vargeese C, et al. Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. Am J

Pathol. 1999;154(1):169-79.

[56] Santosh B, Yadava PK. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics. Biomed Res Int [Internet]. 2014;2014:1-

13. Available from: http://www. pubmedcentral.nih.gov/articlerender. fcgi?artid=4090538&tool=pmcentrez&

[57] Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, et al. 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165) inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem. 1998;273(32):20556-67.

rendertype=abstract

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

*Theranostics - An Old Concept in New Clothing*

Campylobacter jejuni using whole-cell SELEX. Appl Microbiol Biotechnol.

Aspects of Nucleic Acid Library Design for in Vitro Selection. Int J Mol Sci [Internet]. 2018 Feb 5;19(2):470. Available from: https://pubmed.ncbi.

[44] Jayasena SD. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin Chem.

[45] Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov [Internet]. 2010;9(7):537- 50. Available from: http://dx.doi.

[46] Xiang D, Shigdar S, Qiao G, Wang T, Kouzani AZ, Zhou S-F, et al. Nucleic Acid Aptamer-Guided Cancer Therapeutics and Diagnostics: the Next Generation of Cancer Medicine. Theranostics [Internet]. 2015 Jan 1;5(1):23-42. Available from: http:// www.ncbi.nlm.nih.gov/pmc/articles/

[47] Cho EJ, Lee J-W, Ellington AD. Applications of aptamers as sensors. Annu Rev Anal Chem (Palo Alto Calif).

[48] Li S, Xu H, Ding H, Huang Y, Cao X, Yang G, et al. Identification of an aptamer targeting hnRNP A1 by tissue slide-based SELEX.

2009;(February):327-36.

[49] Autour A, Bouhedda F,

Cubi R, Ryckelynck M. Optimization of fluorogenic RNA-based biosensors using droplet-based microfluidic ultrahighthroughput screening. Methods.

[50] Barciszewski J, Medgaard M, Koch T, Kurreck J, Erdmann VA. Locked Nucleic Acid Aptamers. In: Nucleic Acid and Peptide Aptamers: Methods and Protocols [Internet]. 2009. p. 165-86. Available from: http://www.springerlink.com/ index/10.1007/978-1-59745-557-2

nlm.nih.gov/29401748

1999;45(9):1628-50.

org/10.1038/nrd3141

PMC4265746/

2009;2:241-64.

2019;161:46-53.

[35] Wang G, Liu J, Chen K, Xu Y, Liu B, Liao J, et al. Selection and characterization of DNA aptamer against glucagon receptor by cell-SELEX. Sci Rep. 2017;7(1):1-10.

[36] Pobanz K, Lupták A. Improving the odds: Influence of starting pools on in vitro selection outcomes. Methods.

[37] Stoltenburg R, Nikolaus N, Strehlitz B. Capture-SELEX: selection of DNA aptamers for aminoglycoside antibiotics. J Anal Methods Chem.

[38] Paniel N, Istamboulié G, Triki A, Lozano C, Barthelmebs L, Noguer T. Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor. Talanta. 2017;162:232-40.

[39] Taylor AI, Houlihan G, Holliger P. Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics. Cold Spring Harb Perspect Biol. 2019

[40] Lee J, Schwieter KE, Watkins AM, Kim DS, Yu H, Schwarz KJ, et al. Expanding the limits of the second genetic code with ribozymes. Nat Commun. 2019 Nov;10(1):5097.

[41] Morihiro K, Kasahara Y, Obika S. Biological applications of xeno nucleic acids. Mol Biosyst. 2017

[42] Rangel AE, Chen Z, Ayele TM, Heemstra JM. In vitro selection of an XNA aptamer capable of smallmolecule recognition. Nucleic Acids Res.

[43] Vorobyeva MA, Davydova AS, Vorobjev PE, Venyaminova AG. Key

Jan;13(2):235-45.

2018;46(16).

2010;87(6):2323-34.

2016;106:14-20.

2012;2012.

Jun;11(6).

**56**

[51] Veedu RN, Wengel J. Locked nucleic acids: promising nucleic acid analogs for therapeutic applications. Chem Biodivers. 2010;7(3):536-42.

[52] Darfeuille F, Hansen JB, Orum H, Di Primo C, Toulmé J-J. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1. Nucleic Acids Res [Internet]. 2004 Jun 4;32(10):3101-7. Available from: https://www.ncbi.nlm. nih.gov/pubmed/15181175

[53] Langkjær N, Pasternak A, Wengel J. UNA (unlocked nucleic acid): a flexible RNA mimic that allows engineering of nucleic acid duplex stability. Bioorg Med Chem. 2009;17(15):5420-5.

[54] Wittung P, Nielsen PE, Buchardt O, Egholm M, Norde B. DNA-like double helix formed by peptide nucleic acid. Nature. 1994;368(6471):561-3.

[55] Floege J, Ostendorf T, Janssen U, Burg M, Radeke HH, Vargeese C, et al. Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. Am J Pathol. 1999;154(1):169-79.

[56] Santosh B, Yadava PK. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics. Biomed Res Int [Internet]. 2014;2014:1- 13. Available from: http://www. pubmedcentral.nih.gov/articlerender. fcgi?artid=4090538&tool=pmcentrez& rendertype=abstract

[57] Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, et al. 2′-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165) inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain. J Biol Chem. 1998;273(32):20556-67.

[58] Lee J-H, Canny MD, De Erkenez A, Krilleke D, Ng Y-S, Shima DT, et al. A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc Natl Acad Sci. 2005;102(52):18902-7.

[59] Lipi F, Chen S, Chakravarthy M, Rakesh S, Veedu RN. In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies. RNA Biol [Internet]. 2016 Dec 1;13(12):1232- 45. Available from: https://doi.org/10.10 80/15476286.2016.1236173

[60] Kimoto M, Matsunaga K, Hirao I. Evolving Aptamers with Unnatural Base Pairs. Curr Protoc Chem Biol. 2017;9(4):315-39.

[61] Ng EWM, Shima DT, Calias P, Cunningham ET, Guyer DR, Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006;5(2):123-32.

[62] Sampson T. Aptamers and SELEX: The technology. World Pat Inf. 2003;25(2):123-9.

[63] Shamah SM, Healy JM, Cload ST. Complex target SELEX. Acc Chem Res. 2008;41(1):130-8.

[64] Lao Y-H, Phua KKL, Leong KW. Aptamer nanomedicine for cancer therapeutics: barriers and potential for translation. ACS Nano. 2015;9(3):2235-54.

[65] Carvalho J, Paiva A, Campello MPC, Paulo A, Mergny J-L, Salgado GF, et al. Aptamer-based targeted Delivery of a G-quadruplex Ligand in Cervical Cancer Cells. Sci Rep. 2019;9(1):1-12.

[66] Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN, Harzstark AL, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal

cell carcinoma. Invest New Drugs. 2014;32(1):178-87.

[67] Hoellenriegel J, Zboralski D, Maasch C, Rosin NY, Wierda WG, Keating MJ, et al. The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization. Blood. 2014;123(7):1032-9.

[68] Kruschinski MGSC-CMJTDZZR. Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 alone and in combination with bendamustine and rituximab in patients with relapsed chronic lymphocytic leukemia (CLL): results from a phase IIa study [Abstract 1635]. 55th American Society of Hematology Annual Meeting and Exhibition. 2013.

[69] Pestourie C, Tavitian B, Duconge F. Aptamers against extracellular targets for in vivo applications. Biochimie. 2005;87(9-10):921-30.

[70] Zhou J, Rossi JJ. Cell-specific aptamer-mediated targeted drug delivery. Oligonucleotides. 2011;21(1):1-10.

[71] Li N, Nguyen HH, Byrom M, Ellington AD. Inhibition of cell proliferation by an anti-EGFR aptamer. PLoS One. 2011;6(6):e20299.

[72] Hicke BJ, Stephens AW. Escort aptamers: a delivery service for diagnosis and therapy. J Clin Invest. 2000;106(8):923-8.

[73] Gijs M, Aerts A, Impens N, Baatout S, Luxen A. Aptamers as radiopharmaceuticals for nuclear imaging and therapy. Nucl Med Biol. 2016;43(4):253-71.

[74] Inkster JAH, Adam MJ, Storr T, Ruth TJ. Labeling of an antisense oligonucleotide with [18F] FPy5yne. Nucleosides, Nucleotides and Nucleic Acids. 2009;28(11-12):1131-43.

[75] Mercier F, Paris J, Kaisin G, Thonon D, Flagothier J, Teller N, et al. General method for labeling siRNA by click chemistry with fluorine-18 for the purpose of PET imaging. Bioconjug Chem. 2011;22(1):108-14.

[76] Lange CW, VanBrocklin HF, Taylor SE. Photoconjugation of 3-azido-5-nitrobenzyl-[18F] fluoride to an oligonucleotide aptamer. J Label Compd Radiopharm. 2002;45(3):257-68.

[77] Bandekar A, Zhu C, Jindal R, Bruchertseifer F, Morgenstern A, Sofou S. Anti–prostate-specific membrane antigen liposomes loaded with 225Ac for potential targeted antivascular α-particle therapy of cancer. J Nucl Med. 2014;55(1):107-14.

[78] Charlton J, Sennello J, Smith D. In vivo imaging of inflammation using an aptamer inhibitor of human neutrophil elastase. Chem Biol. 1997;4(11):809-16.

[79] Hicke BJ, Marion C, Chang Y-F, Gould T, Lynott CK, Parma D, et al. Tenascin-C aptamers are generated using tumor cells and purified protein. J Biol Chem. 2001;276(52):48644-54.

[80] Hicke BJ, Stephens AW, Gould T, Chang Y-F, Lynott CK, Heil J, et al. Tumor targeting by an aptamer. J Nucl Med. 2006;47(4):668-78.

[81] Ouellet J. RNA Fluorescence with Light-Up Aptamers. Front Chem [Internet]. 2016;4(June):1-12.

[82] Bouhedda F, Autour A, Ryckelynck M. Light-up RNA aptamers and their cognate fluorogens: from their development to their applications. Int J Mol Sci. 2018;19(1):44.

[83] Sett A, Zara L, Dausse E, Toulme J-J. Engineering light-up aptamers for the detection of RNA hairpins through kissing interaction. Anal Chem. 2020 Jun 4;0(ja).

**59**

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

[84] Durand G, Lisi S, Ravelet C, Dausse E, Peyrin E, Toulmé JJ. Riboswitches based on kissing complexes for the detection of small ligands. Angew Chemie - Int Ed.

[85] Germer K, Leonard M, Zhang X. RNA aptamers and their therapeutic and diagnostic applications. Vol. 4, International Journal of Biochemistry and Molecular Biology. 2013. p. 27-40.

[86] Tan W, Wang H, Chen Y, Zhang X, Zhu H, Yang C, et al. Molecular aptamers for drug delivery. Trends Biotechnol. 2011;29(12):634-40.

2014;53(27):6942-5.

*Aptamers: Magic Bullet for Theranostic Applications DOI: http://dx.doi.org/10.5772/intechopen.95403*

[84] Durand G, Lisi S, Ravelet C, Dausse E, Peyrin E, Toulmé JJ. Riboswitches based on kissing complexes for the detection of small ligands. Angew Chemie - Int Ed. 2014;53(27):6942-5.

*Theranostics - An Old Concept in New Clothing*

[75] Mercier F, Paris J, Kaisin G, Thonon D, Flagothier J, Teller N, et al. General method for labeling siRNA by click chemistry with fluorine-18 for the purpose of PET imaging. Bioconjug

[76] Lange CW, VanBrocklin HF,

Radiopharm. 2002;45(3):257-68.

[77] Bandekar A, Zhu C, Jindal R, Bruchertseifer F, Morgenstern A, Sofou S. Anti–prostate-specific membrane antigen liposomes loaded with 225Ac for potential targeted antivascular α-particle therapy of cancer. J Nucl Med. 2014;55(1):107-14.

[78] Charlton J, Sennello J, Smith D. In vivo imaging of inflammation using an aptamer inhibitor of human neutrophil elastase. Chem Biol. 1997;4(11):809-16.

[79] Hicke BJ, Marion C, Chang Y-F, Gould T, Lynott CK, Parma D, et al. Tenascin-C aptamers are generated using tumor cells and purified protein. J Biol Chem. 2001;276(52):48644-54.

[80] Hicke BJ, Stephens AW, Gould T, Chang Y-F, Lynott CK, Heil J, et al. Tumor targeting by an aptamer. J Nucl

[81] Ouellet J. RNA Fluorescence with Light-Up Aptamers. Front Chem [Internet]. 2016;4(June):1-12.

Ryckelynck M. Light-up RNA aptamers and their cognate fluorogens: from their development to their applications. Int J

[83] Sett A, Zara L, Dausse E, Toulme J-J. Engineering light-up aptamers for the detection of RNA hairpins through kissing interaction. Anal Chem. 2020

Med. 2006;47(4):668-78.

[82] Bouhedda F, Autour A,

Mol Sci. 2018;19(1):44.

Jun 4;0(ja).

Taylor SE. Photoconjugation of 3-azido-5-nitrobenzyl-[18F] fluoride to an oligonucleotide aptamer. J Label Compd

Chem. 2011;22(1):108-14.

cell carcinoma. Invest New Drugs.

[67] Hoellenriegel J, Zboralski D, Maasch C, Rosin NY, Wierda WG, Keating MJ, et al. The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization. Blood.

[68] Kruschinski MGSC-CMJTDZZR. Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 alone and in combination with bendamustine and rituximab in patients with relapsed chronic lymphocytic leukemia (CLL): results from a phase IIa study [Abstract 1635]. 55th American Society of Hematology Annual Meeting

[69] Pestourie C, Tavitian B, Duconge F. Aptamers against extracellular targets for in vivo applications. Biochimie.

[70] Zhou J, Rossi JJ. Cell-specific aptamer-mediated targeted drug delivery. Oligonucleotides.

[71] Li N, Nguyen HH, Byrom M, Ellington AD. Inhibition of cell

[72] Hicke BJ, Stephens AW. Escort aptamers: a delivery service for diagnosis and therapy. J Clin Invest.

[73] Gijs M, Aerts A, Impens N, Baatout S, Luxen A. Aptamers as radiopharmaceuticals for nuclear imaging and therapy. Nucl Med Biol.

[74] Inkster JAH, Adam MJ, Storr T, Ruth TJ. Labeling of an antisense oligonucleotide with [18F] FPy5yne. Nucleosides, Nucleotides and Nucleic Acids. 2009;28(11-12):1131-43.

PLoS One. 2011;6(6):e20299.

proliferation by an anti-EGFR aptamer.

2014;32(1):178-87.

2014;123(7):1032-9.

and Exhibition. 2013.

2005;87(9-10):921-30.

2011;21(1):1-10.

2000;106(8):923-8.

2016;43(4):253-71.

**58**

[85] Germer K, Leonard M, Zhang X. RNA aptamers and their therapeutic and diagnostic applications. Vol. 4, International Journal of Biochemistry and Molecular Biology. 2013. p. 27-40.

[86] Tan W, Wang H, Chen Y, Zhang X, Zhu H, Yang C, et al. Molecular aptamers for drug delivery. Trends Biotechnol. 2011;29(12):634-40.

**61**

**Table 1.**

*The main characteristics of EVs [1, 2].*

**Chapter 4**

**Abstract**

targeting modification

**1. Introduction**

Extracellular Vesicles: "Stealth

Transport Aircrafts" for Drugs

conjunction with the application of EVs in the treatment of COVID-19.

**Keywords:** extracellular vesicles, exosomes, drug carrier, drug loading,

**Vesicle Size (nm) Density (g/mL) Origin Markers**

Exosomes 30-150 1.13-1.18 Endosomes Tetraspanins, Alix, TSG101 Microvesicles 50-1000 1.16-1.19 Plasma membrane Intergrins, Selectins, CD40

Extracellular vesicles (EVs) are a collective term for tiny vesicles with a phospholipid bilayer structure that are actively secreted by cells. Almost all known cell types can be secreted. The two main categories of EVs are exosomes and microvesicles (**Table 1**). Exosomes (30-150 nm in diameter) are intraluminal vesicles, formed by the invagination of the multivesicular endosome membrane, and are released into the extracellular space after the multivesicular endosomes fuse with the cell membrane [1]. Microvesicles (50–1,000 nm in diameter) are a group of highly heterogeneous EVs characterized in that their origin and secretion are budding through the plasma membrane [1]. Considering the complexity of identifying its biogenesis, the size of the vesicle is the most widely used parameter for classifying EV types,

Extracellular vesicles (EVs) can deliver many types of drugs with their natural source material transport properties, inherent long-term blood circulation capabilities and excellent biocompatibility, and have great potential in the field of drug carrier. Modification of the content and surface of EVs according to the purpose of treatment has become a research focus to improve the drug load and the targeting of EVs. EVs can maximize the stability of the drugs, prevent immune clearance and achieve accurate delivery. Therefore, EVs can be described as "stealth transport aircrafts" for drugs. This chapter will respectively introduce the application of natural EVs as cell substitutes in cell therapy and engineered EVs as carriers of nucleic acids, proteins, small molecule drugs and therapeutic viral particles in disease treatment. It will also explain the drug loading and modification strategies of EVs, the source and characteristics of EVs. In addition, the commercialization progress of EVs drugs will be mentioned here, and the problems in their applications will be discussed in

*Chunying Liu, Xuejing Lin and Changqing Su*

#### **Chapter 4**

## Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs

*Chunying Liu, Xuejing Lin and Changqing Su*

#### **Abstract**

Extracellular vesicles (EVs) can deliver many types of drugs with their natural source material transport properties, inherent long-term blood circulation capabilities and excellent biocompatibility, and have great potential in the field of drug carrier. Modification of the content and surface of EVs according to the purpose of treatment has become a research focus to improve the drug load and the targeting of EVs. EVs can maximize the stability of the drugs, prevent immune clearance and achieve accurate delivery. Therefore, EVs can be described as "stealth transport aircrafts" for drugs. This chapter will respectively introduce the application of natural EVs as cell substitutes in cell therapy and engineered EVs as carriers of nucleic acids, proteins, small molecule drugs and therapeutic viral particles in disease treatment. It will also explain the drug loading and modification strategies of EVs, the source and characteristics of EVs. In addition, the commercialization progress of EVs drugs will be mentioned here, and the problems in their applications will be discussed in conjunction with the application of EVs in the treatment of COVID-19.

**Keywords:** extracellular vesicles, exosomes, drug carrier, drug loading, targeting modification

#### **1. Introduction**

Extracellular vesicles (EVs) are a collective term for tiny vesicles with a phospholipid bilayer structure that are actively secreted by cells. Almost all known cell types can be secreted. The two main categories of EVs are exosomes and microvesicles (**Table 1**). Exosomes (30-150 nm in diameter) are intraluminal vesicles, formed by the invagination of the multivesicular endosome membrane, and are released into the extracellular space after the multivesicular endosomes fuse with the cell membrane [1]. Microvesicles (50–1,000 nm in diameter) are a group of highly heterogeneous EVs characterized in that their origin and secretion are budding through the plasma membrane [1]. Considering the complexity of identifying its biogenesis, the size of the vesicle is the most widely used parameter for classifying EV types,


#### **Table 1.**

*The main characteristics of EVs [1, 2].*

and on this basis they are described as small EVs or medium and large EVs. In this article, unless otherwise specified, the term "EVs" generally refers to small EVs.

In recent years, people's understanding of the biogenesis, composition, function and mechanism of EVs has continued to deepen [3–5]. Their application in clinical treatment has also attracted more and more attention. One of the most useful properties of EVs is their ability to cross barriers, such as the plasma membrane and blood/brain barrier. This makes them very suitable for delivering therapeutic molecules. With their natural source material transport properties, inherent longterm blood circulation capabilities and excellent biocompatibility, EVs can deliver a variety of chemical drugs, proteins, nucleic acids, gene drugs and other drugs. They have great potential in the field of drug carriers. CD47 is the ligand for signal regulatory protein alpha (SIRPα), and CD47-SIRPα binding initiates the 'don't eat me' signal that inhibits phagocytosis. Therefore, CD47 on EVs prevents them from being engulfed by immune cells [6]. EVs are more efficient than their synthetic analog liposomes. The application of EVs as drug delivery carriers is like putting a "stealth coat" on the drug molecules, which can maximize the stability of the drugs, reduce the immune system's clearance of them, and make "precise delivery" possible. Therefore, EVs can be described as "stealth transport aircrafts" for drugs. EVs therapy has shown great application prospects from oncology to regenerative medicine.

#### **2. Therapeutic application of natural EVs as cell substitutes**

A number of studies have shown that EVs derived from mesenchymal stem cells (MSCs) can be used for stem cell replacement therapy [7–21]. In most cases, it is not clear which component of the unmodified EVs exerts curative effects. The researchers' operations are only the separation and purification of EVs produced by therapeutic cells. The curative effects are based on the biological functions of the donor cells, such as the regulation of the immune environment, the repair of damaged cells and the promotion of angiogenesis.

At present, the most extensive research is the attempt to use stem cell-derived EVs for disease treatment. The main application ranges are to repair and regenerate tissues and organs. Such researches involve central nervous system diseases [7, 8], cardiovascular diseases [9–12] and other organ damage repair and regeneration [13–21].

#### **2.1 EVs derived from stem cells and the treatment of central nervous system diseases**

In the treatment of central nervous system disease, there is a blood-brain barrier, which often results in that drugs can not reach the diseased site and work well. Stem cells have been gradually used in the treatment of central nervous system diseases in recent years. A large number of research results have been obtained [22, 23]. However, there are still potential risks faced by direct stem cell transplantation, such as tumorigenicity, infection, transplant failure, graft versus host disease, hemorrhagic cystitis, and long-term complications [24].

The application of stem cell EVs avoids a variety of potential risks of direct stem cell transplantation. EVs have low immunogenicity and are easy to preserve and transport, showing unique advantages as a "cell-free stem cell therapy technology". Spinal cord injury (SCI) is one of the deadliest diseases. The complex inhibitory microenvironment needs to be fully mitigated. EVs derived from MSCs have the function of microenvironmental regulation. Studies have established innovative implantation strategies using human MSC-derived EVs immobilized in

**63**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

peptide-modified adhesive hydrogels (Exo-pGel) [7]. Exo-pGel plays an important role in nerve recovery and urinary tissue protection by effectively reducing inflammation and oxidation [7]. In addition, small extracellular vesiclesderived from embryonic stem cells (ESC-sEVs) can significantly reduce the time-related aging of hippocampal neural stem cells (H-NSCs) through intravenous injection into vascular dementia (VD) rats. ESC-sEVs can restore the damaged proliferation and neuronal differentiation ability, and reverse cognitive impairment. The application of ESC-sEVs may be a new cell-free treatment tool for VD and other diseases related

**2.2 EVs derived from stem cells and the treatment of cardiovascular diseases**

Stem cells can be induced to differentiate into cardiomyocytes. Early studies believed that the transplanted stem cells can differentiate into heart cells and necrotic cells in the body to repair damaged myocardium and maintain heart function [25]. At present, a large number of preclinical studies have found that EVs derived from transplanted stem cells also have the function of myocardial repair [26, 27]. EVs mainly promote myocardial regeneration by activating cardiac precursor cells, promoting the survival and proliferation of cardiomyocytes, inhibiting their apoptosis, promoting cardiac angiogenesis, reducing infarct size and tissue fibrosis, and regulating inflammation. Extracellular vesicles secreted by cardiovascular precursor cells (hCVPC-EVs) derived from human pluripotent stem cells (hPSCs) play a role in protecting the heart in myocardial infarction by improving cardiomyocyte survival and angiogenesis [9]. Mouse ESC-derived EVs promote angiogenesis, cardiomyocyte survival and proliferation, reduce cardiac fibrosis, and improve cardiac function by carrying miR-294-3p [10]. IPSC-derived EVs inhibit cardiomyocyte apoptosis through miR-21 and miR-210 loaded, and also have a cardioprotective effect [11]. Exosomes produced by immature bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a.

**2.3 EVs derived from stem cells and the damage repair and regeneration of other** 

With the continuous discovery of the repair and regeneration effects of stem cell EVs in brain tissue and cardiovascular tissues and organs, the application of stem cell EVs in the repair and regeneration of other tissues has also made a lot of

MSC-derived EVs reduce radiation-induced lung injury through miRNA-214-3p [13]. Replacing autologous cells with EVs derived from hair follicle papillary cell spheres can promote hair growth [14]. Human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exo) can inhibit pyrolysis and repair muscle ischemic injury by releasing circular RNA circHIPK3 [15]. Hertwig's EVs derived from epithelial root sheath cells promote the regeneration of dentin plasma tissue [16]. Exosomes from neural progenitor cells retain photoreceptor cells during retinal degeneration (RD) by inactivating microglia. This suggests that NPC-exos and its

Aging is the process of cell and tissue dysfunction. Small extracellular vesicles (sEVs) isolated from primary fibroblasts from young human donors can improve certain biomarkers of cellular senescence from elderly and Hutchinson-Gilford progeria donors. Studies have shown that sEVs have GST activity to improve aging-related tissue damage [18]. In obesity diseases, EVs derived from adipocytes, as new adipokines, are related to the body's metabolic homeostasis. EVs released

contents may be the mechanism of stem cell therapy to treat RD [17].

They can reduce the necrotic lesions of atherosclerosis [12].

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

to aging [8].

**organs**

progresses.

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

*Theranostics - An Old Concept in New Clothing*

and on this basis they are described as small EVs or medium and large EVs. In this article, unless otherwise specified, the term "EVs" generally refers to small EVs.

**2. Therapeutic application of natural EVs as cell substitutes**

cells and the promotion of angiogenesis.

hemorrhagic cystitis, and long-term complications [24].

A number of studies have shown that EVs derived from mesenchymal stem cells (MSCs) can be used for stem cell replacement therapy [7–21]. In most cases, it is not clear which component of the unmodified EVs exerts curative effects. The researchers' operations are only the separation and purification of EVs produced by therapeutic cells. The curative effects are based on the biological functions of the donor cells, such as the regulation of the immune environment, the repair of damaged

At present, the most extensive research is the attempt to use stem cell-derived EVs for disease treatment. The main application ranges are to repair and regenerate tissues and organs. Such researches involve central nervous system diseases [7, 8], cardiovascular diseases [9–12] and other organ damage repair and regeneration [13–21].

In the treatment of central nervous system disease, there is a blood-brain barrier, which often results in that drugs can not reach the diseased site and work well. Stem cells have been gradually used in the treatment of central nervous system diseases in recent years. A large number of research results have been obtained [22, 23]. However, there are still potential risks faced by direct stem cell transplantation, such as tumorigenicity, infection, transplant failure, graft versus host disease,

**2.1 EVs derived from stem cells and the treatment of central nervous system** 

The application of stem cell EVs avoids a variety of potential risks of direct stem cell transplantation. EVs have low immunogenicity and are easy to preserve and transport, showing unique advantages as a "cell-free stem cell therapy technology". Spinal cord injury (SCI) is one of the deadliest diseases. The complex inhibitory microenvironment needs to be fully mitigated. EVs derived from MSCs have the function of microenvironmental regulation. Studies have established innovative implantation strategies using human MSC-derived EVs immobilized in

In recent years, people's understanding of the biogenesis, composition, function and mechanism of EVs has continued to deepen [3–5]. Their application in clinical treatment has also attracted more and more attention. One of the most useful properties of EVs is their ability to cross barriers, such as the plasma membrane and blood/brain barrier. This makes them very suitable for delivering therapeutic molecules. With their natural source material transport properties, inherent longterm blood circulation capabilities and excellent biocompatibility, EVs can deliver a variety of chemical drugs, proteins, nucleic acids, gene drugs and other drugs. They have great potential in the field of drug carriers. CD47 is the ligand for signal regulatory protein alpha (SIRPα), and CD47-SIRPα binding initiates the 'don't eat me' signal that inhibits phagocytosis. Therefore, CD47 on EVs prevents them from being engulfed by immune cells [6]. EVs are more efficient than their synthetic analog liposomes. The application of EVs as drug delivery carriers is like putting a "stealth coat" on the drug molecules, which can maximize the stability of the drugs, reduce the immune system's clearance of them, and make "precise delivery" possible. Therefore, EVs can be described as "stealth transport aircrafts" for drugs. EVs therapy has shown great application prospects from oncology to regenerative

**62**

medicine.

**diseases**

peptide-modified adhesive hydrogels (Exo-pGel) [7]. Exo-pGel plays an important role in nerve recovery and urinary tissue protection by effectively reducing inflammation and oxidation [7]. In addition, small extracellular vesiclesderived from embryonic stem cells (ESC-sEVs) can significantly reduce the time-related aging of hippocampal neural stem cells (H-NSCs) through intravenous injection into vascular dementia (VD) rats. ESC-sEVs can restore the damaged proliferation and neuronal differentiation ability, and reverse cognitive impairment. The application of ESC-sEVs may be a new cell-free treatment tool for VD and other diseases related to aging [8].

#### **2.2 EVs derived from stem cells and the treatment of cardiovascular diseases**

Stem cells can be induced to differentiate into cardiomyocytes. Early studies believed that the transplanted stem cells can differentiate into heart cells and necrotic cells in the body to repair damaged myocardium and maintain heart function [25]. At present, a large number of preclinical studies have found that EVs derived from transplanted stem cells also have the function of myocardial repair [26, 27]. EVs mainly promote myocardial regeneration by activating cardiac precursor cells, promoting the survival and proliferation of cardiomyocytes, inhibiting their apoptosis, promoting cardiac angiogenesis, reducing infarct size and tissue fibrosis, and regulating inflammation. Extracellular vesicles secreted by cardiovascular precursor cells (hCVPC-EVs) derived from human pluripotent stem cells (hPSCs) play a role in protecting the heart in myocardial infarction by improving cardiomyocyte survival and angiogenesis [9]. Mouse ESC-derived EVs promote angiogenesis, cardiomyocyte survival and proliferation, reduce cardiac fibrosis, and improve cardiac function by carrying miR-294-3p [10]. IPSC-derived EVs inhibit cardiomyocyte apoptosis through miR-21 and miR-210 loaded, and also have a cardioprotective effect [11]. Exosomes produced by immature bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a. They can reduce the necrotic lesions of atherosclerosis [12].

#### **2.3 EVs derived from stem cells and the damage repair and regeneration of other organs**

With the continuous discovery of the repair and regeneration effects of stem cell EVs in brain tissue and cardiovascular tissues and organs, the application of stem cell EVs in the repair and regeneration of other tissues has also made a lot of progresses.

MSC-derived EVs reduce radiation-induced lung injury through miRNA-214-3p [13]. Replacing autologous cells with EVs derived from hair follicle papillary cell spheres can promote hair growth [14]. Human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exo) can inhibit pyrolysis and repair muscle ischemic injury by releasing circular RNA circHIPK3 [15]. Hertwig's EVs derived from epithelial root sheath cells promote the regeneration of dentin plasma tissue [16]. Exosomes from neural progenitor cells retain photoreceptor cells during retinal degeneration (RD) by inactivating microglia. This suggests that NPC-exos and its contents may be the mechanism of stem cell therapy to treat RD [17].

Aging is the process of cell and tissue dysfunction. Small extracellular vesicles (sEVs) isolated from primary fibroblasts from young human donors can improve certain biomarkers of cellular senescence from elderly and Hutchinson-Gilford progeria donors. Studies have shown that sEVs have GST activity to improve aging-related tissue damage [18]. In obesity diseases, EVs derived from adipocytes, as new adipokines, are related to the body's metabolic homeostasis. EVs released

from brown adipose tissue or adipose stem cells can help control the remodeling of white adipose tissue, making it brown and maintaining metabolic homeostasis. EVs have been considered as new regulators of diseases such as insulin resistance, diabetes and non-alcoholic fatty liver. The results provide new treatment strategies for obesity and metabolic diseases [19].

In addition, some reports suggest that some EVs derived from mesenchymal stem cells contain some tumor suppressor molecules. For example, it has been reported that miR-206 in exosomes derived from bone marrow mesenchymal stem cells could inhibit the progression of osteosarcoma by targeting TRA2B [20]. The exosomes derived from human umbilical cord mesenchymal stem cells deliver miRNA-375 to delay the progression of esophageal squamous cell carcinoma [21]. However, although EVs contain these small RNAs that have been reported to exert anti-cancer effects, they also contain a large number of growth factors and proangiogenesis factors. When these substances are transported to tumor cells by EVs, can EVs derived from MSCs still exert a tumor suppressor effect? This needs more research to prove.

At present, cell replacement therapy based on the characteristics of donor cells has been studied earlier and more frequently in the field of EVs. There is also a clearer understanding of the components that play a major role. With the continuous increase of clinical needs, people began to try to modify the surfaces and contents of EVs to adapt to more disease treatments.

#### **3. Application of engineered EVs as carriers of nucleic acid drugs in disease treatment**

Although natural EVs have been used for cell replacement therapy based on their source and achieved good results, their therapeutic range is far from meeting the current treatment needs. One of the most important therapeutic areas is the treatment of malignant tumors. The secretion ability of EVs in malignant tumor itself is enhanced and contributes to tumor progression. Considering that MSCderived EVs generally contain high levels of growth factors and pro-angiogenic factors, most natural EVs are not suitable for tumor therapy, except that EVs derived from antigen-presenting cells can be used as tumor vaccines to activate anti-tumor immune responses [28]. Based on the biological characteristics of EVs, it has become the focus of researchers and biopharmaceutical companies to transform EVs as carriers of multiple drugs.

Most diseases have characteristic down-regulation of small RNA expression. Small RNA is the main content of extracellular vesicles, the most abundant and the most easily carried component. Therefore, EVs can be used to carry and deliver small RNA and other gene therapy systems. This section will discuss the progress of engineered EVs to deliver nucleic acid drugs and the strategies of drug loading and targeting.

#### **3.1 Research progress of engineered EVs to deliver nucleic acid drugs**

There are three main problems in the development of nucleic acid drugs: the instability of nucleic acid molecules in the body, potential side effects and difficulties in drug delivery systems. The most important one is the development of delivery systems. Because a good drug delivery system can improve drug stability and target cell absorption efficiency, and can reduce its side effects. At present, the commonly used delivery vehicles in the field of nucleic acid drugs are mainly adeno-associated virus (AAV) and liposomal nanoparticles (LNPs). A small number of companies also use lentivirus (LV) and exosomes as delivery vehicles.

**65**

**Figure 1.**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

the therapeutic effect of engineered EVs [30–33].

The packaging capacity of AAV is small (≤5kb). AAV will be used more than once in patients for therapeutic purposes and the second use will cause the body to produce a strong immune response. The safety of LNPs is relatively high, and the carrier capacity and delivery efficiency can meet the current demand for drug carriers. However, the organ selectivity of LNPs is still relatively limited. The main delivery area is concentrated in the liver, and the delivery efficiency to other tissues

EVs are now candidate carriers for nucleic acid drugs by virtue of their advan-

In addition, researchers are also committed to modifying the surfaces of EVs to improve their targeting. Many results show that this strategy can indeed improve

The researchers combined ligand-coupled superparamagnetic nanoparticles with specific membrane proteins of blood exosomes to achieve the separation, purification and tumor targeting of exosomes [30]. The chemotherapy drug doxorubicin (Dox) and the cholesterol-modified single-stranded miRNA-21 inhibitor (chol-miR21i) were co-loaded onto the exosomes. Then the cationic endolysin peptide was absorbed on the negatively charged membrane surface of exosomes to promote the cytoplasmic release of the packaged cargo (**Figure 1**). The research results showed that these effectively released drugs and RNA simultaneously interfered with nuclear DNA activity and down-regulated the expression of oncogenes, thereby significantly inhibiting tumor growth and reducing side effects [30].

Chimeric antigen receptors (CAR) are cell surface receptors that recognize specific proteins (antigens). Tumor cells express their specific antigens. Modification of EVs surfaces with CAR targeting tumor antigens enables EVs to target tumors for

*Schematic representation of engineered blood exosomes for effective gene/chemo combined antitumor therapy [30].*

tages. The red blood cell extracellular vesicles (RBCEVs) have a large loading capacity (≤11kb), can be loaded with many types (including DNA, mRNA, antisense oligonucleotides, siRNA and other nucleic acid types), and contain very little nucleic acid. The advantages make them high-quality natural blank nucleic acid carriers. RBCEVs can be delivered to many different organs and tissues. In mouse experiments, the delivery effects of lung, liver, kidney, bone tissue, immune cells, etc. are all obvious [29]. Moreover, the raw materials used to produce RBCEVs are mainly blood from type O blood donors. This means large quantities of raw materials are readily available, and costs are controllable. Carmine Therapeutics focuses on the research and development of nucleic acid delivery technology using RBCEVs

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

and organs is relatively low.

as carriers.

*Theranostics - An Old Concept in New Clothing*

for obesity and metabolic diseases [19].

contents of EVs to adapt to more disease treatments.

research to prove.

**disease treatment**

EVs as carriers of multiple drugs.

from brown adipose tissue or adipose stem cells can help control the remodeling of white adipose tissue, making it brown and maintaining metabolic homeostasis. EVs have been considered as new regulators of diseases such as insulin resistance, diabetes and non-alcoholic fatty liver. The results provide new treatment strategies

In addition, some reports suggest that some EVs derived from mesenchymal stem cells contain some tumor suppressor molecules. For example, it has been reported that miR-206 in exosomes derived from bone marrow mesenchymal stem cells could inhibit the progression of osteosarcoma by targeting TRA2B [20]. The exosomes derived from human umbilical cord mesenchymal stem cells deliver miRNA-375 to delay the progression of esophageal squamous cell carcinoma [21]. However, although EVs contain these small RNAs that have been reported to exert anti-cancer effects, they also contain a large number of growth factors and proangiogenesis factors. When these substances are transported to tumor cells by EVs, can EVs derived from MSCs still exert a tumor suppressor effect? This needs more

At present, cell replacement therapy based on the characteristics of donor cells has been studied earlier and more frequently in the field of EVs. There is also a clearer understanding of the components that play a major role. With the continuous increase of clinical needs, people began to try to modify the surfaces and

**3. Application of engineered EVs as carriers of nucleic acid drugs in** 

Although natural EVs have been used for cell replacement therapy based on their source and achieved good results, their therapeutic range is far from meeting the current treatment needs. One of the most important therapeutic areas is the treatment of malignant tumors. The secretion ability of EVs in malignant tumor itself is enhanced and contributes to tumor progression. Considering that MSCderived EVs generally contain high levels of growth factors and pro-angiogenic factors, most natural EVs are not suitable for tumor therapy, except that EVs derived from antigen-presenting cells can be used as tumor vaccines to activate anti-tumor immune responses [28]. Based on the biological characteristics of EVs, it has become the focus of researchers and biopharmaceutical companies to transform

Most diseases have characteristic down-regulation of small RNA expression. Small RNA is the main content of extracellular vesicles, the most abundant and the most easily carried component. Therefore, EVs can be used to carry and deliver small RNA and other gene therapy systems. This section will discuss the progress of engineered EVs to deliver nucleic acid drugs and the strategies of drug loading and targeting.

There are three main problems in the development of nucleic acid drugs: the instability of nucleic acid molecules in the body, potential side effects and difficulties in drug delivery systems. The most important one is the development of delivery systems. Because a good drug delivery system can improve drug stability and target cell absorption efficiency, and can reduce its side effects. At present, the commonly used delivery vehicles in the field of nucleic acid drugs are mainly adeno-associated virus (AAV) and liposomal nanoparticles (LNPs). A small number of companies also use lentivirus (LV) and exosomes as delivery vehicles.

**3.1 Research progress of engineered EVs to deliver nucleic acid drugs**

**64**

The packaging capacity of AAV is small (≤5kb). AAV will be used more than once in patients for therapeutic purposes and the second use will cause the body to produce a strong immune response. The safety of LNPs is relatively high, and the carrier capacity and delivery efficiency can meet the current demand for drug carriers. However, the organ selectivity of LNPs is still relatively limited. The main delivery area is concentrated in the liver, and the delivery efficiency to other tissues and organs is relatively low.

EVs are now candidate carriers for nucleic acid drugs by virtue of their advantages. The red blood cell extracellular vesicles (RBCEVs) have a large loading capacity (≤11kb), can be loaded with many types (including DNA, mRNA, antisense oligonucleotides, siRNA and other nucleic acid types), and contain very little nucleic acid. The advantages make them high-quality natural blank nucleic acid carriers. RBCEVs can be delivered to many different organs and tissues. In mouse experiments, the delivery effects of lung, liver, kidney, bone tissue, immune cells, etc. are all obvious [29]. Moreover, the raw materials used to produce RBCEVs are mainly blood from type O blood donors. This means large quantities of raw materials are readily available, and costs are controllable. Carmine Therapeutics focuses on the research and development of nucleic acid delivery technology using RBCEVs as carriers.

In addition, researchers are also committed to modifying the surfaces of EVs to improve their targeting. Many results show that this strategy can indeed improve the therapeutic effect of engineered EVs [30–33].

The researchers combined ligand-coupled superparamagnetic nanoparticles with specific membrane proteins of blood exosomes to achieve the separation, purification and tumor targeting of exosomes [30]. The chemotherapy drug doxorubicin (Dox) and the cholesterol-modified single-stranded miRNA-21 inhibitor (chol-miR21i) were co-loaded onto the exosomes. Then the cationic endolysin peptide was absorbed on the negatively charged membrane surface of exosomes to promote the cytoplasmic release of the packaged cargo (**Figure 1**). The research results showed that these effectively released drugs and RNA simultaneously interfered with nuclear DNA activity and down-regulated the expression of oncogenes, thereby significantly inhibiting tumor growth and reducing side effects [30].

Chimeric antigen receptors (CAR) are cell surface receptors that recognize specific proteins (antigens). Tumor cells express their specific antigens. Modification of EVs surfaces with CAR targeting tumor antigens enables EVs to target tumors for

*Schematic representation of engineered blood exosomes for effective gene/chemo combined antitumor therapy [30].*

drug delivery. Modified EVs with CAR can serve as a biosafety delivery platform for the CRISPR/Cas9 system to improve their tumor selectivity. Compared with unmodified EVs, CAR-EVs accumulate rapidly in tumors and effectively release the CRISPR/Cas9 system targeting MYC oncogenes in vitro and in vivo [31].

Rabies virus glycoprotein (RVG) is neurogenic. At present, it has become the most active guide molecule for brain targeted drugs. Lysosomal-associated membrane glycoprotein 2b (Lamp2b) is the membrane surface protein of EVs. RVG fused with Lamp2b is located on the surface of the EV to achieve brain targeting. Engineered Lamp2b-RVG-circSCMH1-extracellular vesicles (Lamp2b-RVGcircSCMH1-EVs) can selectively deliver circSCMH1 to the brain. The treatment can improve the functional recovery of mice and monkeys after stroke [32].

In addition, EVs without modification for targeting have also shown certain curative effects. The miR-214 inhibitor was transfected into HEK293T cells. Their exosomes Exo-anti-214 can reverse the resistance of gastric cancer to DDP [33]. HEK293T cells were transfected with HGF siRNA and their exosomes were harvested. In vivo and in vitro experiments have shown that exosomes loaded with HGF siRNA can inhibit the proliferation and migration of cancer cells and vascular cells [33].

#### **3.2 Methods of loading nucleic acid drugs into engineered EVs**

Methods of loading nucleic acids into EVs include: chemical reagent transfection, electroporation transfection, transfection of donor cells, protein and characteristic sequence targeting methods. The application scope and advantages and disadvantages of different methods are shown in **Table 2**.

The use of proteins that can bind to specific RNA sequences (**Figure 2**) or the conservative sequences of Exosome-enriched RNAs (eRNAs) to achieve active packaging is a promising direction. The researchers used the specificity of protein binding to the RNA sequence to develop EXOtic devices for mRNA delivery [38]. Archaeal ribosomal protein L7Ae specifically binds to the C/Dbox RNA structure [40–42]. Based on this, L7Ae was conjugated to the C-terminus of CD63. C/D box


**67**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

was inserted into the 3′-untranslated region (3′-UTR) of the reporter gene. Therefore, the mRNA encoding the reporter protein could be well incorporated into exosomes via the interaction between L7Ae and the C/D box in the 3′-UTR. Exosomes containing the RNA packaging device (CD63-L7Ae), targeting module (RVG-Lamp2b to target CHRNA7), cytosolic delivery helper (Cx43 S368A) and mRNA (nluc-C/Dbox) were efficiently produced from exosome producer cells by the exosome production booster. The engineered exosomes were delivered to target cells and the mRNA was delivered into the target cell cytosol with the help of the cytosolic delivery helper. Finally, protein encoded in the mRNA was expressed in the target cells [38] (**Figure 2**). In the future, researchers need to obtain more specific RNA sequence binding proteins and conserved sequences of eRNAs through

*EXOtic devices for mRNA delivery. A schematic illustration of the EXOtic devices [38].*

**4. Application of engineered EVs as protein transporters in disease** 

**4.1 Research progress of engineered EVs as protein transporters for disease** 

Compared with the previous small molecule compound drugs, protein drugs have the characteristics of high activity, strong specificity, low toxicity, clear biological functions, and are beneficial to clinical application. However, protein drugs are unstable in the internal and external environments, and may undergo a variety of complex chemical degradation and physical changes, such as aggregation, precipitation, racemization, hydrolysis, and deamidation. Protein drugs have short half-life, high clearance rate, large molecular weight, poor permeability, susceptibility to the

The lack of protein and malfunction are important causes of many diseases. For example, the occurrence of malignant tumors is related to the lack of certain tumor suppressor factors and malfunctions. Therefore, increasing the corresponding protein level is one of the ways to treat diseases. Considering the risk of genome changes, researchers aim to deliver therapeutic protein molecules to the lesion through effective drug delivery vehicles. This section will introduce the use of EVs to transport protein molecules for the prevention and treatment of tumors, immune diseases, cardiovascular diseases, atherosclerosis, myocardial infarction and other diseases.

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

bioinformatics analysis.

**treatment**

**Figure 2.**

**treatment**

#### **Table 2.**

*Methods of loading nucleic acid drugs into engineered EVs.*

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

**Figure 2.**

*Theranostics - An Old Concept in New Clothing*

drug delivery. Modified EVs with CAR can serve as a biosafety delivery platform for the CRISPR/Cas9 system to improve their tumor selectivity. Compared with unmodified EVs, CAR-EVs accumulate rapidly in tumors and effectively release the

Rabies virus glycoprotein (RVG) is neurogenic. At present, it has become the most active guide molecule for brain targeted drugs. Lysosomal-associated membrane glycoprotein 2b (Lamp2b) is the membrane surface protein of EVs. RVG fused with Lamp2b is located on the surface of the EV to achieve brain targeting. Engineered Lamp2b-RVG-circSCMH1-extracellular vesicles (Lamp2b-RVG-

circSCMH1-EVs) can selectively deliver circSCMH1 to the brain. The treatment can

In addition, EVs without modification for targeting have also shown certain curative effects. The miR-214 inhibitor was transfected into HEK293T cells. Their exosomes Exo-anti-214 can reverse the resistance of gastric cancer to DDP [33]. HEK293T cells were transfected with HGF siRNA and their exosomes were harvested. In vivo and in vitro experiments have shown that exosomes loaded with HGF siRNA can inhibit the proliferation and migration of cancer cells and vascular

Methods of loading nucleic acids into EVs include: chemical reagent transfection, electroporation transfection, transfection of donor cells, protein and characteristic sequence targeting methods. The application scope and advantages and

The use of proteins that can bind to specific RNA sequences (**Figure 2**) or the conservative sequences of Exosome-enriched RNAs (eRNAs) to achieve active packaging is a promising direction. The researchers used the specificity of protein binding to the RNA sequence to develop EXOtic devices for mRNA delivery [38]. Archaeal ribosomal protein L7Ae specifically binds to the C/Dbox RNA structure [40–42]. Based on this, L7Ae was conjugated to the C-terminus of CD63. C/D box

**Methods Application scope Merit and demerit References**

Broad-spectrum. Easy to operate, but EVs should

mRNA and miRNA. High specificity of loading, but

be purified before and after

Easy to operate, but EVs should be purified before and after

Purify EVs after transfection, but the effect of the transfected molecule on the donor cell should be taken into account

the therapeutic molecules will be modified. Whether this will affect the efficacy remains to be [34]

[35]

[33, 36, 37]

[38, 39]

transfection.

transfection.

(e.g. biotoxicity).

determined.

CRISPR/Cas9 system targeting MYC oncogenes in vitro and in vivo [31].

improve the functional recovery of mice and monkeys after stroke [32].

**3.2 Methods of loading nucleic acid drugs into engineered EVs**

disadvantages of different methods are shown in **Table 2**.

The most commonly used method, but not for miRNA, shRNA, mRNA containing chemical modification.

*Methods of loading nucleic acid drugs into engineered EVs.*

Broad spectrum, but not for biotoxic molecules.

**66**

**Table 2.**

cells [33].

Chemical reagent transfection

Electroporation transfection

Transfection of donor cells

Protein and characteristic sequence targeting

*EXOtic devices for mRNA delivery. A schematic illustration of the EXOtic devices [38].*

was inserted into the 3′-untranslated region (3′-UTR) of the reporter gene. Therefore, the mRNA encoding the reporter protein could be well incorporated into exosomes via the interaction between L7Ae and the C/D box in the 3′-UTR. Exosomes containing the RNA packaging device (CD63-L7Ae), targeting module (RVG-Lamp2b to target CHRNA7), cytosolic delivery helper (Cx43 S368A) and mRNA (nluc-C/Dbox) were efficiently produced from exosome producer cells by the exosome production booster. The engineered exosomes were delivered to target cells and the mRNA was delivered into the target cell cytosol with the help of the cytosolic delivery helper. Finally, protein encoded in the mRNA was expressed in the target cells [38] (**Figure 2**). In the future, researchers need to obtain more specific RNA sequence binding proteins and conserved sequences of eRNAs through bioinformatics analysis.

#### **4. Application of engineered EVs as protein transporters in disease treatment**

The lack of protein and malfunction are important causes of many diseases. For example, the occurrence of malignant tumors is related to the lack of certain tumor suppressor factors and malfunctions. Therefore, increasing the corresponding protein level is one of the ways to treat diseases. Considering the risk of genome changes, researchers aim to deliver therapeutic protein molecules to the lesion through effective drug delivery vehicles. This section will introduce the use of EVs to transport protein molecules for the prevention and treatment of tumors, immune diseases, cardiovascular diseases, atherosclerosis, myocardial infarction and other diseases.

#### **4.1 Research progress of engineered EVs as protein transporters for disease treatment**

Compared with the previous small molecule compound drugs, protein drugs have the characteristics of high activity, strong specificity, low toxicity, clear biological functions, and are beneficial to clinical application. However, protein drugs are unstable in the internal and external environments, and may undergo a variety of complex chemical degradation and physical changes, such as aggregation, precipitation, racemization, hydrolysis, and deamidation. Protein drugs have short half-life, high clearance rate, large molecular weight, poor permeability, susceptibility to the

destruction of enzymes, bacteria and body fluids in the receptor, and low bioavailability of non-injection administration. These problems greatly limit the use of protein drugs. Although researchers have improved the stability and absorption efficiency of protein drugs through methods such as PEG modification, microsphere sustained release, and liposome embedding, they still look forward to the emergence of better drug carriers. The application of EVs has brought dawn to this field.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. Delivery of TRAIL through EVs can efficiently induce cancer cell apoptosis. When combined with dinaciclib, they inhibit the growth of drug-resistant tumors [43]. Immunosuppressive drugs must be taken after organ transplantation, but long-term use of these drugs increases the risk of infection and other serious diseases. Using bioengineering methods, researchers prepared exosome-like nanovesicles (NV) displaying the dual target cargo of PD-L1/CTLA-4. These NVs enhanced the PD-L1/PD-1 and CTLA-4/CD80 immunosuppressive pathways and could be used as prospective immunosuppressive agents in organ transplantation [44]. Using extracellular nanovesicles to package CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping can avoid off-target mutagenesis and immunogenicity. And this method can achieve effective genome editing in a variety of cell types that are difficult to transfect, including human induced pluripotent stem cells (iPS), neurons and myoblasts [45]. Catalase could be loaded into exosomes by incubating at room temperature, saponins penetrating the membrane, repeated freezing and thawing and mechanical extrusion for the treatment of Parkinson's disease (PD) [46].

Surface modification of EVs carrying protein drugs can greatly improve their targeting. In the study of stroke, nerve growth factor (NGF) exerts various neuroprotective functions after ischemia. NGF was loaded into EVs with RVG peptide modification on the surface. Through systemic administration, NGF was effectively delivered to the ischemic cortex. The delivered NGF reduced inflammation by remodeling microglia polarization, promoted cell survival, and increased the number of neuroblast marker doublecortin-positive cells. The results of the study indicated the potential therapeutic effect of NGF@Exo (RVG) on stroke [47]. In addition, integrin αVβ5 exhibits tropism for the liver while integrin α6Vβ4 and integrin α6β1 target lung [48, 49]. The iRGD specifically recognizes αV integrins on the surface of tumor cells [50]. RVG and c(RGDyK) peptides target brain tissue [51]. Klotho protein has the property of binding to circulating endothelial progenitor cells (EPCs) [52]. And chimeric antigen receptor (CAR) targeting specific tumor antigens and so on. These guiding molecules are utilized either by fusion with EVs membrane surface proteins (such as Lamp2b, VSVG, CD63, and other transmembrane proteins, etc.), or by chemical cross-linking on the surface of EVs to achieve the EVs targeting modification. Liu et al. summarized the surface modification strategies to improve the targeting of EVs (**Figure 3**) [53]. In addition, EVs derived from antigen-presenting cells with tumor antigens can be used as tumor vaccines to activate anti-tumor immune responses.

#### **4.2 Methods of loading protein drugs into EVs**

How to load protein drugs into EVs? There are currently the following strategies:

**69**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

natural packaging process. Subsequent separation and purification of EVs in the cell culture supernatant is sufficient [54]. Although this method seems simple and easy to implement, cytotoxicity, mixed interactions and impaired biological responses will provide great obstacles to the production of EVs. And the loading efficiency of the target protein is relatively low. Therefore, researchers have carried out various attempts to specifically load target proteins into EVs. For example, the fusion of therapeutic proteins with the constituent proteins of EVs and the specific modifica-

The therapeutic proteins are fused with the constituent proteins of EVs. Then they will be distributed into EVs mediated by the constituent proteins. This method can improve the specificity of protein loading into EVs. The fused constituent proteins of EVs that have been tried include: CD63, Nef [55], vesicular stomatitis virus glycoprotein (VSVG) [56], apolipoprotein E (ApoE) [57], lysosome-associated

In addition, based on the idea of fusion proteins, researchers have developed a conditional loading method called "exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs)" [59]. The principle is to couple the exosomal membrane protein CD9 with CIBN, and CRY 2 with the therapeutic protein. After light excitation, CIBN and CRY2 interact, and the therapeutic protein can be loaded into EVs through "photoreversible protein-protein interaction" [59]. All in all, the fusion expression of therapeutic proteins with the constituent proteins of EVs can indeed increase the enrichment level of therapeutic proteins in EVs. However, whether the fusion protein affects the uptake and function of the therapeutic protein by the recipient cells needs to be verified. Therefore, exploring the fusion of peptides that can play a sorting role with therapeutic proteins and minimize the impact on protein functions will become one of the research hotspots

*4.2.2 Fusion of therapeutic protein with the constituent proteins of EVs*

membrane glycoprotein 2 (LAMP2B) [58], etc.

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

tion of therapeutic proteins.

*Design strategies for therapeutic exosome targeting [53].*

**Figure 3.**

in the field of engineered EVs.

#### *4.2.1 Expression of therapeutic protein in donor cells*

Transfect donor cells with plasmids carrying the gene of interest. The cell will synthesize the target protein. These proteins are then secreted into EVs through a

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

*Theranostics - An Old Concept in New Clothing*

treatment of Parkinson's disease (PD) [46].

activate anti-tumor immune responses.

**4.2 Methods of loading protein drugs into EVs**

*4.2.1 Expression of therapeutic protein in donor cells*

destruction of enzymes, bacteria and body fluids in the receptor, and low bioavailability of non-injection administration. These problems greatly limit the use of protein drugs. Although researchers have improved the stability and absorption efficiency of protein drugs through methods such as PEG modification, microsphere sustained release, and liposome embedding, they still look forward to the emergence

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. Delivery of TRAIL through EVs can efficiently induce cancer cell apoptosis. When combined with dinaciclib, they inhibit the growth of drug-resistant tumors [43]. Immunosuppressive drugs must be taken after organ transplantation, but long-term use of these drugs increases the risk of infection and other serious diseases. Using bioengineering methods, researchers prepared exosome-like nanovesicles (NV) displaying the dual target cargo of PD-L1/CTLA-4. These NVs enhanced the PD-L1/PD-1 and CTLA-4/CD80 immunosuppressive pathways and could be used as prospective immunosuppressive agents in organ transplantation [44]. Using extracellular nanovesicles to package CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping can avoid off-target mutagenesis and immunogenicity. And this method can achieve effective genome editing in a variety of cell types that are difficult to transfect, including human induced pluripotent stem cells (iPS), neurons and myoblasts [45]. Catalase could be loaded into exosomes by incubating at room temperature, saponins penetrating the membrane, repeated freezing and thawing and mechanical extrusion for the

Surface modification of EVs carrying protein drugs can greatly improve their targeting. In the study of stroke, nerve growth factor (NGF) exerts various neuroprotective functions after ischemia. NGF was loaded into EVs with RVG peptide modification on the surface. Through systemic administration, NGF was effectively delivered to the ischemic cortex. The delivered NGF reduced inflammation by remodeling microglia polarization, promoted cell survival, and increased the number of neuroblast marker doublecortin-positive cells. The results of the study indicated the potential therapeutic effect of NGF@Exo (RVG) on stroke [47]. In addition, integrin αVβ5 exhibits tropism for the liver while integrin α6Vβ4 and integrin α6β1 target lung [48, 49]. The iRGD specifically recognizes αV integrins on the surface of tumor cells [50]. RVG and c(RGDyK) peptides target brain tissue [51]. Klotho protein has the property of binding to circulating endothelial progenitor cells (EPCs) [52]. And chimeric antigen receptor (CAR) targeting specific tumor antigens and so on. These guiding molecules are utilized either by fusion with EVs membrane surface proteins (such as Lamp2b, VSVG, CD63, and other transmembrane proteins, etc.), or by chemical cross-linking on the surface of EVs to achieve the EVs targeting modification. Liu et al. summarized the surface modification strategies to improve the targeting of EVs (**Figure 3**) [53]. In addition, EVs derived from antigen-presenting cells with tumor antigens can be used as tumor vaccines to

How to load protein drugs into EVs? There are currently the following strategies:

Transfect donor cells with plasmids carrying the gene of interest. The cell will synthesize the target protein. These proteins are then secreted into EVs through a

of better drug carriers. The application of EVs has brought dawn to this field.

**68**

**Figure 3.** *Design strategies for therapeutic exosome targeting [53].*

natural packaging process. Subsequent separation and purification of EVs in the cell culture supernatant is sufficient [54]. Although this method seems simple and easy to implement, cytotoxicity, mixed interactions and impaired biological responses will provide great obstacles to the production of EVs. And the loading efficiency of the target protein is relatively low. Therefore, researchers have carried out various attempts to specifically load target proteins into EVs. For example, the fusion of therapeutic proteins with the constituent proteins of EVs and the specific modification of therapeutic proteins.

#### *4.2.2 Fusion of therapeutic protein with the constituent proteins of EVs*

The therapeutic proteins are fused with the constituent proteins of EVs. Then they will be distributed into EVs mediated by the constituent proteins. This method can improve the specificity of protein loading into EVs. The fused constituent proteins of EVs that have been tried include: CD63, Nef [55], vesicular stomatitis virus glycoprotein (VSVG) [56], apolipoprotein E (ApoE) [57], lysosome-associated membrane glycoprotein 2 (LAMP2B) [58], etc.

In addition, based on the idea of fusion proteins, researchers have developed a conditional loading method called "exosomes for protein loading via optically reversible protein-protein interaction (EXPLORs)" [59]. The principle is to couple the exosomal membrane protein CD9 with CIBN, and CRY 2 with the therapeutic protein. After light excitation, CIBN and CRY2 interact, and the therapeutic protein can be loaded into EVs through "photoreversible protein-protein interaction" [59].

All in all, the fusion expression of therapeutic proteins with the constituent proteins of EVs can indeed increase the enrichment level of therapeutic proteins in EVs. However, whether the fusion protein affects the uptake and function of the therapeutic protein by the recipient cells needs to be verified. Therefore, exploring the fusion of peptides that can play a sorting role with therapeutic proteins and minimize the impact on protein functions will become one of the research hotspots in the field of engineered EVs.

#### *4.2.3 Specific modification of therapeutic protein*

Currently, known protein modifications that can target therapeutic proteins into EVs mainly include two types. One is ubiquitination modification. The fusion of ubiquitin to the C-terminus of therapeutic protein can make the concentration of the fused therapeutic protein in EVs increased by nearly 10 times [60]. The other is to fuse the N-terminus of the therapeutic protein with a palmitoylated or myristoylated peptide, which can further increase the therapeutic protein in EVs [61]. However, it is still unknown whether the modification of proteins, especially ubiquitination, will cause the degradation of the therapeutic protein by the proteasome and affect its function in the recipient cell.

#### *4.2.4 Combined with mechanical methods to produce small vesicles containing therapeutic proteins*

Expression of therapeutic protein in donor cells, combined with mechanical methods that pass through different pores, can produce small vesicles containing the therapeutic proteins [46, 62]. In addition, there are methods such as incubation at room temperature, permeabilization with saponin, freeze-thaw cycles and sonication, [46]. There are two main problems with engineered EVs obtained by mechanical methods. One is that the technical requirements for the separation and purification of EVs are relatively high. The second is the maintenance of the integrity and biological activity of EVs. The composition of EVs actively produced by cells is different from the composition of mechanically produced EVs. The difference may affect the efficacy of engineered EVs. In the future application research of EVs, these two problems need to be solved and proved urgently.

So, what are the possible development directions for the existing cytotoxicity and the interaction of biological functions? The expression of tumor suppressor protein molecules may cause cytotoxicity to donor cells, which is not conducive to the production of EVs. If an inducible expression system is established, the coding DNA containing the inducible promoter is introduced into the donor cell to make the donor cell produce EVs containing the coding DNA, which will avoid cytotoxicity to the donor cell. Then prepare EVs containing small molecules that induce DNA expression. The two types of EVs can be used in combination to express tumor suppressor molecules in target cells. It can play a therapeutic role without affecting the production efficiency of EVs. The dual targeting of the two EVs will greatly reduce the impact of engineered EVs on non-targeted tissues. Because single-component EVs are randomly engulfed by cells and will not affect the cells. This may become one of the follow-up development directions in this field.

#### **5. Application of engineered EVs as carriers of small compounds in disease treatment**

Chemotherapeutics and traditional Chinese medicine ingredients with anticancer effects are often used in the clinical treatment of a variety of malignant tumors. However, their toxic, side effects and short half-life limit their efficacy. The packaging and transportation with EVs will improve the targeting of chemotherapeutic drugs, increase the uptake efficiency of tumor cells, promote drug stability, reduce the use concentration, and reduce toxic side effects on other organs and normal tissues [63].

The hydrophobic drug curcumin could be packaged into exosomes by direct mixing for tumor treatment [64]. Paclitaxel (PTX) was loaded into EVs secreted by

**71**

research [70].

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

obstruction in 25% of patients, almost no adverse reactions [67].

macrophages by different methods such as room temperature incubation, electroporation and sonication. Studies have found that ultrasound treatment increases the load of EVs on drug molecules and the sustained release [65]. Small compounds can also be naturally secreted into EVs by incubating with donor cells. Incubation with paclitaxel make mesenchymal stromal cells produce microvesicles containing paclitaxel [66]. Injecting methotrexate-containing plasma membrane microvesicles (MTX-TMP) from apoptotic human tumor cells into the bile duct lumen of extrahepatic CCA patients could mobilize and activate neutrophils, and relieve the bile duct

At present, small molecule drugs are often loaded into EVs by passive loading methods, such as direct mixing, incubation, ultrasonic treatment, vortexing, saponin permeation, repeated freezing and thawing, and mechanical extrusion. The disadvantages of these methods have always existed, that is, the loss and quality reduction of EVs caused by multiple purifications. In addition, long-term in vitro processing and the physical and chemical properties of drug molecules will also affect the biological activity and stability of EVs. Therefore, before EVs can be widely used in treatment, the storage methods and stability factors of EVs are also

**6. Application of engineered EVs as virus carriers in gene therapy**

Why are EVs a "stealth cap" for drugs? Because we know viruses to use them exactly like this. In nature, viruses "hijack" EVs to secrete and infect other cells. This method helps to provide a "cover" for the virus to prevent the virus from being cleared by the immune system or neutralized by antibodies, such as the infection

In gene therapy, currently widely used adeno-associated virus (AAV), oncolytic adenovirus (OAV) and lentivirus (LV) mediated gene therapy can cause the body's immune response. After the same kind of AAV is used once, the body will produce a strong immune response, making the second injection ineffective. If EVs encapsulate viral particles to mediate their delivery, perhaps the therapeutic effect will

Studies have shown that AAV isolated from conditioned media could bind to exosomes (exo-AAV) [68]. Compared with conventional AAV, exo-AAV was more resistant to neutralizing antibodies. After systemic injection in mice, compared with conventional AAV, exo-AAV delivered genes to the brain more efficiently at low vector doses. Importantly, no cytotoxicity was found in exo-AAV transduced cells. This may make exo-AAV widely used as a neuroscience research tool [68]. Compared with non-targeted modified EV-AAV, the expression of brain-targeting peptides on the surfaces of EVs can significantly enhance transduction [69].

In gene therapy of ophthalmic diseases, transferring genes to the retina is challenging. Because it requires a carrier system to overcome physical and biochemical barriers to enter and spread throughout the retinal tissue. After the exo-AAV was injected into the vitreous cavity (IVT), it was found that the expression of exo-AAV was better than the traditional AAV. Exo-AAV exhibited a deeper penetration in the retina, effectively reaching the inner core and outer plexus, and to a lesser extent the outer nuclear layer. Exo-AAV is a reliable mouse retina gene delivery tool. Its simplicity of production and isolation makes it widely used in basic eye

Due to the low efficiency of gene delivery to the inner ear sensory hair cells. AAV is not so advanced in the field of gene therapy for hearing impairment. Studies have shown that Exo-AAV1-GFP is more effective than traditional AAV1-GFP, whether

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

worthy of research.

be better.

process of HAV, HBV and HCV.

#### *Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

*Theranostics - An Old Concept in New Clothing*

*4.2.3 Specific modification of therapeutic protein*

some and affect its function in the recipient cell.

*therapeutic proteins*

Currently, known protein modifications that can target therapeutic proteins into EVs mainly include two types. One is ubiquitination modification. The fusion of ubiquitin to the C-terminus of therapeutic protein can make the concentration of the fused therapeutic protein in EVs increased by nearly 10 times [60]. The other is to fuse the N-terminus of the therapeutic protein with a palmitoylated or myristoylated peptide, which can further increase the therapeutic protein in EVs [61]. However, it is still unknown whether the modification of proteins, especially ubiquitination, will cause the degradation of the therapeutic protein by the protea-

*4.2.4 Combined with mechanical methods to produce small vesicles containing* 

EVs, these two problems need to be solved and proved urgently.

one of the follow-up development directions in this field.

**disease treatment**

other organs and normal tissues [63].

Expression of therapeutic protein in donor cells, combined with mechanical methods that pass through different pores, can produce small vesicles containing the therapeutic proteins [46, 62]. In addition, there are methods such as incubation at room temperature, permeabilization with saponin, freeze-thaw cycles and sonication, [46]. There are two main problems with engineered EVs obtained by mechanical methods. One is that the technical requirements for the separation and purification of EVs are relatively high. The second is the maintenance of the integrity and biological activity of EVs. The composition of EVs actively produced by cells is different from the composition of mechanically produced EVs. The difference may affect the efficacy of engineered EVs. In the future application research of

So, what are the possible development directions for the existing cytotoxicity and the interaction of biological functions? The expression of tumor suppressor protein molecules may cause cytotoxicity to donor cells, which is not conducive to the production of EVs. If an inducible expression system is established, the coding DNA containing the inducible promoter is introduced into the donor cell to make the donor cell produce EVs containing the coding DNA, which will avoid cytotoxicity to the donor cell. Then prepare EVs containing small molecules that induce DNA expression. The two types of EVs can be used in combination to express tumor suppressor molecules in target cells. It can play a therapeutic role without affecting the production efficiency of EVs. The dual targeting of the two EVs will greatly reduce the impact of engineered EVs on non-targeted tissues. Because single-component EVs are randomly engulfed by cells and will not affect the cells. This may become

**5. Application of engineered EVs as carriers of small compounds in** 

Chemotherapeutics and traditional Chinese medicine ingredients with anticancer effects are often used in the clinical treatment of a variety of malignant tumors. However, their toxic, side effects and short half-life limit their efficacy. The packaging and transportation with EVs will improve the targeting of chemotherapeutic drugs, increase the uptake efficiency of tumor cells, promote drug stability, reduce the use concentration, and reduce toxic side effects on

The hydrophobic drug curcumin could be packaged into exosomes by direct mixing for tumor treatment [64]. Paclitaxel (PTX) was loaded into EVs secreted by

**70**

macrophages by different methods such as room temperature incubation, electroporation and sonication. Studies have found that ultrasound treatment increases the load of EVs on drug molecules and the sustained release [65]. Small compounds can also be naturally secreted into EVs by incubating with donor cells. Incubation with paclitaxel make mesenchymal stromal cells produce microvesicles containing paclitaxel [66]. Injecting methotrexate-containing plasma membrane microvesicles (MTX-TMP) from apoptotic human tumor cells into the bile duct lumen of extrahepatic CCA patients could mobilize and activate neutrophils, and relieve the bile duct obstruction in 25% of patients, almost no adverse reactions [67].

At present, small molecule drugs are often loaded into EVs by passive loading methods, such as direct mixing, incubation, ultrasonic treatment, vortexing, saponin permeation, repeated freezing and thawing, and mechanical extrusion. The disadvantages of these methods have always existed, that is, the loss and quality reduction of EVs caused by multiple purifications. In addition, long-term in vitro processing and the physical and chemical properties of drug molecules will also affect the biological activity and stability of EVs. Therefore, before EVs can be widely used in treatment, the storage methods and stability factors of EVs are also worthy of research.

#### **6. Application of engineered EVs as virus carriers in gene therapy**

Why are EVs a "stealth cap" for drugs? Because we know viruses to use them exactly like this. In nature, viruses "hijack" EVs to secrete and infect other cells. This method helps to provide a "cover" for the virus to prevent the virus from being cleared by the immune system or neutralized by antibodies, such as the infection process of HAV, HBV and HCV.

In gene therapy, currently widely used adeno-associated virus (AAV), oncolytic adenovirus (OAV) and lentivirus (LV) mediated gene therapy can cause the body's immune response. After the same kind of AAV is used once, the body will produce a strong immune response, making the second injection ineffective. If EVs encapsulate viral particles to mediate their delivery, perhaps the therapeutic effect will be better.

Studies have shown that AAV isolated from conditioned media could bind to exosomes (exo-AAV) [68]. Compared with conventional AAV, exo-AAV was more resistant to neutralizing antibodies. After systemic injection in mice, compared with conventional AAV, exo-AAV delivered genes to the brain more efficiently at low vector doses. Importantly, no cytotoxicity was found in exo-AAV transduced cells. This may make exo-AAV widely used as a neuroscience research tool [68]. Compared with non-targeted modified EV-AAV, the expression of brain-targeting peptides on the surfaces of EVs can significantly enhance transduction [69].

In gene therapy of ophthalmic diseases, transferring genes to the retina is challenging. Because it requires a carrier system to overcome physical and biochemical barriers to enter and spread throughout the retinal tissue. After the exo-AAV was injected into the vitreous cavity (IVT), it was found that the expression of exo-AAV was better than the traditional AAV. Exo-AAV exhibited a deeper penetration in the retina, effectively reaching the inner core and outer plexus, and to a lesser extent the outer nuclear layer. Exo-AAV is a reliable mouse retina gene delivery tool. Its simplicity of production and isolation makes it widely used in basic eye research [70].

Due to the low efficiency of gene delivery to the inner ear sensory hair cells. AAV is not so advanced in the field of gene therapy for hearing impairment. Studies have shown that Exo-AAV1-GFP is more effective than traditional AAV1-GFP, whether

injected in mouse cochlear explants in vitro or directly injected into the cochlea in vivo. Exo-AAV was not toxic in the body. Exo-AAV1 gene therapy partially rescued the hearing in a mouse model of hereditary deafness. It was a powerful hair cell gene delivery system that could be used for gene therapy of deafness [71].

Oncolytic viruses show unique anti-cancer mechanisms in cancer treatment. Chemotherapeutic drugs combined with oncolytic viruses showed stronger cytotoxicity and oncolytic effects. Someone has studied the systemic delivery of oncolytic adenovirus and paclitaxel encapsulated by EVs. The results have shown that this combination therapy enhanced anticancer effects in lung cancer models both in vitro and in vivo. EVs play a key role in the effective transmission of oncolytic viruses and chemotherapeutic drugs [72].

#### **7. Sources of EVs that can be used for drug delivery**

EVs currently used for therapeutic research are mainly derived from the following sources: mesenchymal stem cells (MSCs), dendritic cells (DCs), tumor cells, red blood cells, macrophages and plants. EVs from different sources have different biological characteristics. Materials should be selected according to the purpose of treatment. The characteristics, advantages and disadvantages of EVs from different sources will be described below.

#### **7.1 Mesenchymal stem cells**

The MSCs involved in the study of EVs include adipose-derived MSCs, bone marrow MSCs, progenitor cells from different tissues, and so on. MSCs can be extracted from the patient's bone marrow, fat, or other tissues. EVs derived from MSCs are very attractive. Because they have anti-inflammatory, anti-apoptotic and anti-microbial capability, and promote angiogenesis and the repair and regeneration of damaged tissues. As mentioned above, EVs derived from MSCs have been widely used in the treatment of central nervous system diseases, cardiovascular diseases, bone and cartilage damage repair and regeneration, wound repair, and other organ damage repair and regeneration [7–21].

#### **7.2 Dendritic cells**

One potential source of therapeutic EVs is immature dendritic cells (imDCs). EVs secreted by imDCs lack surface markers such as CD40, CD86, MHC-I and MHC-II. Therefore, they have low immunogenicity. They can be isolated from CD34+ cells from the patient's peripheral blood. It is one of the preferred sources of therapeutic EVs.

#### **7.3 Tumor cells**

The use of EVs derived from tumor cells to deliver drugs and vaccines for immunotherapy is very promising. Tumor EVs can deliver antigens to dendritic cells, thereby inducing T cell-mediated immune responses to tumor cells [73]. As tumorderived EVs specifically express Tetraspanins, they can target different tissues. This makes it possible to use tumor-derived EVs for tumor-targeting and selective drug delivery [74]. However, tumor-derived EVs also have many potential risks. Due to the presence of Tetraspanins, Urokinase plasminogen activator, Cathepsin D, Vimentin and other molecules derived from the surface of tumor cells [75, 76], they may promote tumor proliferation and metastasis, and Immunosuppressive effect [77–79].

**73**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

Blood EVs mainly secreted by reticulocytes (RTC) are a potential source of safe and sufficient EVs. Because they integrate various membrane proteins including Transferrin (Tf) receptors, but they do not have any immune and cancer stimulating activity [30]. Red blood cell EVs (RBCEV) also have the following advantages: large load; low self-nucleic acid content (red blood cells without nucleus and mitochondria); they can be delivered to a variety of different organs and tissues; large quantities of raw materials and easily available (the raw materials for producing RBCEVs are mainly O-type Blood of blood donors). Using blood EVs as carriers can efficiently target tumors to co-deliver chemotherapeutics and nucleic acid drugs. Significant tumor growth inhibitory effects were observed in tumor-bearing mice.

Macrophages are an important immune cell in the antigen-presenting cell family. EVs derived from immune cells can mimic immune cells to target tumor cells. Macrophage EVs can transfer miRNAs or proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, in the study of tumor treatment of EVs, in addition to using the targeting properties of macrophages-derived EVs, the influence of their contents must also be considered. It has been reported that the contents of EVs derived from macrophages can be removed. Then the EVs were used to carry chemotherapeutic

Based on reliable sources and safety, fruits and plants have been used as alternative sources for the isolation of EVs for clinical use [81]. Plant-derived EVs have similar structural characteristics to animal cell-derived EVs. EVs from different plant sources have the physiological functions of the plant from which they are derived. For example, lemon-derived EVs have certain anti-cancer effects. Some researchers have tried to isolate lemon-derived EVs (LDEVs) for the treatment of gastric cancer. LDEVs caused s-phase arrest of gastric cancer cell cycle and induced cell apoptosis. LDEVs could be retained in the organs of the gastrointestinal tract and had strong anti-tumor activity against gastric cancer [82]. The isolated plant EVs can also be used after being engineered. Some researchers isolated EVs from grapefruit, modified the EVs in a targeted manner, and then loaded the anti-tumor drugs doxorubicin and curcumin. These modified EVs could target inflammatory

Plant-derived EVs have a wide range of sources, are safe and non-toxic, have low immunogenicity, low cost, and are edible. They have great clinical application

So far, no EVs drugs have entered the clinic. Codiak BioSciences, a leading company in the development of engineered EVs as a new type of biopharmaceutical, uses its proprietary engEx platform to engineer EVs with different characteristics, load them

drugs to achieve targeted therapy of triple-negative breast cancer [80].

tumors and have anti-inflammatory effects in mouse models [83].

**8. Commercialization progress and potential problems of EVs**

potential as edible chemotherapeutic drug carriers.

**8.1 Progress in the commercialization of EVs**

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

There were no obvious side effects [30].

**7.4 Red blood cells**

**7.5 Macrophages**

**7.6 Plant-derived EVs**

#### **7.4 Red blood cells**

*Theranostics - An Old Concept in New Clothing*

viruses and chemotherapeutic drugs [72].

from different sources will be described below.

other organ damage repair and regeneration [7–21].

**7.1 Mesenchymal stem cells**

**7.2 Dendritic cells**

therapeutic EVs.

**7.3 Tumor cells**

**7. Sources of EVs that can be used for drug delivery**

injected in mouse cochlear explants in vitro or directly injected into the cochlea in vivo. Exo-AAV was not toxic in the body. Exo-AAV1 gene therapy partially rescued the hearing in a mouse model of hereditary deafness. It was a powerful hair cell gene delivery system that could be used for gene therapy of deafness [71].

Oncolytic viruses show unique anti-cancer mechanisms in cancer treatment. Chemotherapeutic drugs combined with oncolytic viruses showed stronger cytotoxicity and oncolytic effects. Someone has studied the systemic delivery of oncolytic adenovirus and paclitaxel encapsulated by EVs. The results have shown that this combination therapy enhanced anticancer effects in lung cancer models both in vitro and in vivo. EVs play a key role in the effective transmission of oncolytic

EVs currently used for therapeutic research are mainly derived from the following sources: mesenchymal stem cells (MSCs), dendritic cells (DCs), tumor cells, red blood cells, macrophages and plants. EVs from different sources have different biological characteristics. Materials should be selected according to the purpose of treatment. The characteristics, advantages and disadvantages of EVs

The MSCs involved in the study of EVs include adipose-derived MSCs, bone marrow MSCs, progenitor cells from different tissues, and so on. MSCs can be extracted from the patient's bone marrow, fat, or other tissues. EVs derived from MSCs are very attractive. Because they have anti-inflammatory, anti-apoptotic and anti-microbial capability, and promote angiogenesis and the repair and regeneration of damaged tissues. As mentioned above, EVs derived from MSCs have been widely used in the treatment of central nervous system diseases, cardiovascular diseases, bone and cartilage damage repair and regeneration, wound repair, and

One potential source of therapeutic EVs is immature dendritic cells (imDCs). EVs secreted by imDCs lack surface markers such as CD40, CD86, MHC-I and MHC-II. Therefore, they have low immunogenicity. They can be isolated from CD34+ cells from the patient's peripheral blood. It is one of the preferred sources of

The use of EVs derived from tumor cells to deliver drugs and vaccines for immu-

notherapy is very promising. Tumor EVs can deliver antigens to dendritic cells, thereby inducing T cell-mediated immune responses to tumor cells [73]. As tumorderived EVs specifically express Tetraspanins, they can target different tissues. This makes it possible to use tumor-derived EVs for tumor-targeting and selective drug delivery [74]. However, tumor-derived EVs also have many potential risks. Due to the presence of Tetraspanins, Urokinase plasminogen activator, Cathepsin D, Vimentin and other molecules derived from the surface of tumor cells [75, 76], they may promote tumor proliferation and metastasis, and Immunosuppressive effect [77–79].

**72**

Blood EVs mainly secreted by reticulocytes (RTC) are a potential source of safe and sufficient EVs. Because they integrate various membrane proteins including Transferrin (Tf) receptors, but they do not have any immune and cancer stimulating activity [30]. Red blood cell EVs (RBCEV) also have the following advantages: large load; low self-nucleic acid content (red blood cells without nucleus and mitochondria); they can be delivered to a variety of different organs and tissues; large quantities of raw materials and easily available (the raw materials for producing RBCEVs are mainly O-type Blood of blood donors). Using blood EVs as carriers can efficiently target tumors to co-deliver chemotherapeutics and nucleic acid drugs. Significant tumor growth inhibitory effects were observed in tumor-bearing mice. There were no obvious side effects [30].

#### **7.5 Macrophages**

Macrophages are an important immune cell in the antigen-presenting cell family. EVs derived from immune cells can mimic immune cells to target tumor cells. Macrophage EVs can transfer miRNAs or proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, in the study of tumor treatment of EVs, in addition to using the targeting properties of macrophages-derived EVs, the influence of their contents must also be considered. It has been reported that the contents of EVs derived from macrophages can be removed. Then the EVs were used to carry chemotherapeutic drugs to achieve targeted therapy of triple-negative breast cancer [80].

#### **7.6 Plant-derived EVs**

Based on reliable sources and safety, fruits and plants have been used as alternative sources for the isolation of EVs for clinical use [81]. Plant-derived EVs have similar structural characteristics to animal cell-derived EVs. EVs from different plant sources have the physiological functions of the plant from which they are derived. For example, lemon-derived EVs have certain anti-cancer effects. Some researchers have tried to isolate lemon-derived EVs (LDEVs) for the treatment of gastric cancer. LDEVs caused s-phase arrest of gastric cancer cell cycle and induced cell apoptosis. LDEVs could be retained in the organs of the gastrointestinal tract and had strong anti-tumor activity against gastric cancer [82]. The isolated plant EVs can also be used after being engineered. Some researchers isolated EVs from grapefruit, modified the EVs in a targeted manner, and then loaded the anti-tumor drugs doxorubicin and curcumin. These modified EVs could target inflammatory tumors and have anti-inflammatory effects in mouse models [83].

Plant-derived EVs have a wide range of sources, are safe and non-toxic, have low immunogenicity, low cost, and are edible. They have great clinical application potential as edible chemotherapeutic drug carriers.

### **8. Commercialization progress and potential problems of EVs**

#### **8.1 Progress in the commercialization of EVs**

So far, no EVs drugs have entered the clinic. Codiak BioSciences, a leading company in the development of engineered EVs as a new type of biopharmaceutical, uses its proprietary engEx platform to engineer EVs with different characteristics, load them

with various types of therapeutic molecules and change their orientation, so that they can reach specific cellular targets. Recently, Evox Therapeutics Ltd. and Eli Lilly and Co. reached a cooperation agreement to apply its exosome technology to the system to deliver RNA interference and antisense oligonucleotide drugs to the central nervous system, treating five unspecified Neurological diseases. Carmine Therapeutics is also a gene therapy company based on EVs, established in 2019. Carmine's REGENT technology platform focuses on using red blood cell extracellular vesicles (RBCEV) as drug delivery vehicles. Mantra Bio also joined the emerging group of exosome drug development companies. With the deepening of research, more and more companies will join the field of EVs treatment.

#### **8.2 EVs treatment and COVID-19**

The Severe Acute Respiratory Syndrome (which first appeared in December 2019) related to the new coronavirus (COVID-19) has rapidly developed into a pandemic, and the morbidity and mortality rates are increasing worldwide. COVID-19 respiratory tract infection is characterized by an imbalanced immune response, leading to an increased possibility of severe respiratory disease and multiple organ disease.

Because EVs derived from MSCs have anti-inflammatory, anti-apoptotic and anti-microbial capability, promote angiogenesis and the repair and regeneration of damaged tissues. In related lung disease models, including acute lung injury and sepsis, systemic administration of MSC-EVs preparations can modulate immune responses. In a mouse model of pneumonia induced by Escherichia coli, it was found that MSC-EVs administration could enhance the phagocytosis of bacteria. In the pig model, MSC-EVs could reduce influenza virus-induced acute lung injury by inhibiting influenza virus replication. In other disease models, the disease alleviation effect of MSC-EVs on the inflammatory immune response has also been observed. It is speculated that they may also have anti-COVID-19 efficacy. In cell therapy research for COVID-19, some registered clinical trials have turned their targets to EVs in the conditioned medium of MSCs. MSC-EVs can be administered intravenously (ChiCTR2000030484) or by inhalation (NCT04276987, ChiCTR2000030261).

However, before using MSC-EVs for COVID-19 patients, many other issues should be considered, such as the huge heterogeneity of MSC-EVs composition and source. In fact, comparing MSC-EVs harvested from the conditioned medium of bone marrow MSCs derived from different donors, there are significant differences in cytokine content and different therapeutic effects. In addition to immune regulation, MSC-EVs can also control other biological processes and may cause unpredictable side effects. For example, increasing the risk of thrombosis.

In short, in order to reduce the risk of potential life-threatening side effects, International Society for Extracellular Vesicles (ISEV) and International Society for Cell and Gene Therapy (ISCT) strongly require that the clinical data from reasonable clinical trial should be carefully weighed. The EV preparations with good characteristics and produced under strict GMP conditions and appropriate regulatory supervision could be used. Any application of EVs should be carefully evaluated [84].

#### **8.3 Potential problems in the industrialization of EVs**

The potential application of EVs in new diagnostic and therapeutic strategies has attracted increasing attention. However, due to the inherent complex biogenesis of EVs and their huge heterogeneity in size, composition and source, the research of

**75**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

variety of diseases will bring more and greater surprises.

This work was supported by the National Key R&D Program of China (2018YFA0900900), and the National Natural Science Foundation of China

EVs still faces huge challenges. It is necessary to establish a standardized method to solve the heterogeneity of EVs and the analysis of pre-processing and analysis of sources of variability in the study of EVs. The quality standards, extraction specifications and especially the stability of preparation conditions for therapeutic EVs

In addition, the diversity and uncertainty of EVs content are also issues that need to be considered in the application. Before metastasis, malignant tumor cells use EVs to modify the microenvironment of the organ targeted by cancer metastasis, making it a suitable "soil" for tumor cell growth. The contents of EVs secreted by most tumor cells play a role in promoting tumor metastasis and progression. As mentioned earlier in this article, macrophage EVs can transfer miRNAs and proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, if EVs from such sources are used as drug carriers, it is particularly important to first remove the adverse effects of their

As an important medium of intercellular communication, EVs play an important physiological function and are also involved in the occurrence and development of a variety of diseases. In recent years, there have been numerous studies on the treatment of related diseases with EVs from different cell sources, and EV has shown its unique advantages in drug transportation. EVs are similar to natural liposomes, which can enhance the function of EVs to treat specific diseases through targeting modification and delivery of functional active substances and other technical modifications according to the characteristics of different diseases. EVs with improved function have shown obvious advantages in the treatment of tumors and difficult diseases of central nervous system. However, the clinical application of EVs technology is still in its infancy, and the challenges it faces are accompanied by the possibility of numerous new discoveries and new technologies. We expect that with the continuous in-depth research, EVs as a new drug carrier in the treatment of a

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

also need to be clarified.

contents.

**9. Conclusion**

**Acknowledgements**

(81773251 and 81702735).

#### *Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

EVs still faces huge challenges. It is necessary to establish a standardized method to solve the heterogeneity of EVs and the analysis of pre-processing and analysis of sources of variability in the study of EVs. The quality standards, extraction specifications and especially the stability of preparation conditions for therapeutic EVs also need to be clarified.

In addition, the diversity and uncertainty of EVs content are also issues that need to be considered in the application. Before metastasis, malignant tumor cells use EVs to modify the microenvironment of the organ targeted by cancer metastasis, making it a suitable "soil" for tumor cell growth. The contents of EVs secreted by most tumor cells play a role in promoting tumor metastasis and progression. As mentioned earlier in this article, macrophage EVs can transfer miRNAs and proteins to tumor cells, mediate tumor cell resistance to chemotherapy, promote cell invasion and other regulatory effects. Therefore, if EVs from such sources are used as drug carriers, it is particularly important to first remove the adverse effects of their contents.

#### **9. Conclusion**

*Theranostics - An Old Concept in New Clothing*

will join the field of EVs treatment.

**8.2 EVs treatment and COVID-19**

disease.

ChiCTR2000030261).

with various types of therapeutic molecules and change their orientation, so that they can reach specific cellular targets. Recently, Evox Therapeutics Ltd. and Eli Lilly and Co. reached a cooperation agreement to apply its exosome technology to the system to deliver RNA interference and antisense oligonucleotide drugs to the central nervous system, treating five unspecified Neurological diseases. Carmine Therapeutics is also a gene therapy company based on EVs, established in 2019. Carmine's REGENT technology platform focuses on using red blood cell extracellular vesicles (RBCEV) as drug delivery vehicles. Mantra Bio also joined the emerging group of exosome drug development companies. With the deepening of research, more and more companies

The Severe Acute Respiratory Syndrome (which first appeared in December 2019) related to the new coronavirus (COVID-19) has rapidly developed into a pandemic, and the morbidity and mortality rates are increasing worldwide. COVID-19 respiratory tract infection is characterized by an imbalanced immune response, leading to an increased possibility of severe respiratory disease and multiple organ

Because EVs derived from MSCs have anti-inflammatory, anti-apoptotic and anti-microbial capability, promote angiogenesis and the repair and regeneration of damaged tissues. In related lung disease models, including acute lung injury and sepsis, systemic administration of MSC-EVs preparations can modulate immune responses. In a mouse model of pneumonia induced by Escherichia coli, it was found that MSC-EVs administration could enhance the phagocytosis of bacteria. In the pig model, MSC-EVs could reduce influenza virus-induced acute lung injury by inhibiting influenza virus replication. In other disease models, the disease alleviation effect of MSC-EVs on the inflammatory immune response has also been observed. It is speculated that they may also have anti-COVID-19 efficacy. In cell therapy research for COVID-19, some registered clinical trials have turned their targets to EVs in the conditioned medium of MSCs. MSC-EVs can be administered intravenously (ChiCTR2000030484) or by inhalation (NCT04276987,

However, before using MSC-EVs for COVID-19 patients, many other issues should be considered, such as the huge heterogeneity of MSC-EVs composition and source. In fact, comparing MSC-EVs harvested from the conditioned medium of bone marrow MSCs derived from different donors, there are significant differences in cytokine content and different therapeutic effects. In addition to immune regulation, MSC-EVs can also control other biological processes and may cause unpredict-

In short, in order to reduce the risk of potential life-threatening side effects, International Society for Extracellular Vesicles (ISEV) and International Society for Cell and Gene Therapy (ISCT) strongly require that the clinical data from reasonable clinical trial should be carefully weighed. The EV preparations with good characteristics and produced under strict GMP conditions and appropriate regulatory supervision could be used. Any application of EVs should be carefully

The potential application of EVs in new diagnostic and therapeutic strategies has attracted increasing attention. However, due to the inherent complex biogenesis of EVs and their huge heterogeneity in size, composition and source, the research of

able side effects. For example, increasing the risk of thrombosis.

**8.3 Potential problems in the industrialization of EVs**

**74**

evaluated [84].

As an important medium of intercellular communication, EVs play an important physiological function and are also involved in the occurrence and development of a variety of diseases. In recent years, there have been numerous studies on the treatment of related diseases with EVs from different cell sources, and EV has shown its unique advantages in drug transportation. EVs are similar to natural liposomes, which can enhance the function of EVs to treat specific diseases through targeting modification and delivery of functional active substances and other technical modifications according to the characteristics of different diseases. EVs with improved function have shown obvious advantages in the treatment of tumors and difficult diseases of central nervous system. However, the clinical application of EVs technology is still in its infancy, and the challenges it faces are accompanied by the possibility of numerous new discoveries and new technologies. We expect that with the continuous in-depth research, EVs as a new drug carrier in the treatment of a variety of diseases will bring more and greater surprises.

#### **Acknowledgements**

This work was supported by the National Key R&D Program of China (2018YFA0900900), and the National Natural Science Foundation of China (81773251 and 81702735).

*Theranostics - An Old Concept in New Clothing*

### **Author details**

Chunying Liu, Xuejing Lin and Changqing Su\* Department of Molecular Oncology, National Center for Liver Cancer, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China

\*Address all correspondence to: suchangqing@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**77**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

NSCs by activating lysosomes to improve cognitive dysfunction in vascular dementia. Adv Sci (Weinh). 2020;7:1903330. DOI: 10.1002/

[9] Wu Q, Wang J, Tan WLW, Jiang Y, Wang S, Li Q, et al. Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis. Cell Death Dis. 2020;11:354. DOI: 10.1038/

[10] Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, et al. Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circulation research. 2015;117:52-64. DOI: 10.1161/ CIRCRESAHA.117.305990

[11] Wang Y, Zhang L, Li Y, Chen L, Wang X, Guo W, et al. Exosomes/ microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium. International journal of cardiology. 2015;192:61-9. DOI: 10.1016/j.

[12] Bouchareychas L, Duong P, Covarrubias S, Alsop E, Phu TA, Chung A, et al. Macrophage exosomes resolve atherosclerosis by regulating hematopoiesis and inflammation via microRNA cargo. Cell Rep. 2020;32:107881. DOI: 10.1016/j.

[13] Lei X, He N, Zhu L, Zhou M, Zhang K, Wang C, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate radiation-induced lung injury via miRNA-214-3p. Antioxid Redox Signal. 2020. DOI: 10.1089/ars.2019.7965

advs.201903330

s41419-020-2508-y

ijcard.2015.05.020

celrep.2020.107881

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

[1] Gurunathan S, Kang MH, Jeyaraj M, Qasim M, Kim JH. Review of the isolation, characterization, biological function, and multifarious therapeutic

approaches of exosomes. Cells. 2019;8:307. DOI: 10.3390/cells8040307

[2] Shao H, Im H, Castro CM, Breakefield X, Weissleder R, Lee H. New technologies for analysis of extracellular vesicles. Chem Rev. 2018;118:1917-50. DOI: 10.1021/acs.

[3] Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569-

[4] Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255-89. DOI: 10.1146/

annurev-cellbio-101512-122326

[5] van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213-28. DOI: 10.1038/

[6] Kamerkar S, LeBleu VS, Sugimoto H,

Yang S, Ruivo CF, Melo SA, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017;546: 498-503. DOI: 10.1038/nature22341

[7] Li L, Zhang Y, Mu J, Chen J,

[8] Hu G, Xia Y, Zhang J, Chen Y, Yuan J, Niu X, et al. ESC-sEVs rejuvenate senescent hippocampal

acs.nanolett.0c00929

Zhang C, Cao H, et al. Transplantation of human mesenchymal stem cellderived exosomes immobilized in an adhesive hydrogel for effective treatment of spinal cord injury. Nano Lett. 2020;20:4298-305. DOI: 10.1021/

chemrev.7b00534

nrm.2017.125

79. DOI: 10.1038/nri855

**References**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

#### **References**

*Theranostics - An Old Concept in New Clothing*

**76**

**Author details**

Shanghai, China

Chunying Liu, Xuejing Lin and Changqing Su\*

provided the original work is properly cited.

\*Address all correspondence to: suchangqing@gmail.com

Department of Molecular Oncology, National Center for Liver Cancer, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Gurunathan S, Kang MH, Jeyaraj M, Qasim M, Kim JH. Review of the isolation, characterization, biological function, and multifarious therapeutic approaches of exosomes. Cells. 2019;8:307. DOI: 10.3390/cells8040307

[2] Shao H, Im H, Castro CM, Breakefield X, Weissleder R, Lee H. New technologies for analysis of extracellular vesicles. Chem Rev. 2018;118:1917-50. DOI: 10.1021/acs. chemrev.7b00534

[3] Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2:569- 79. DOI: 10.1038/nri855

[4] Colombo M, Raposo G, Théry C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255-89. DOI: 10.1146/ annurev-cellbio-101512-122326

[5] van Niel G, D'Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213-28. DOI: 10.1038/ nrm.2017.125

[6] Kamerkar S, LeBleu VS, Sugimoto H, Yang S, Ruivo CF, Melo SA, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017;546: 498-503. DOI: 10.1038/nature22341

[7] Li L, Zhang Y, Mu J, Chen J, Zhang C, Cao H, et al. Transplantation of human mesenchymal stem cellderived exosomes immobilized in an adhesive hydrogel for effective treatment of spinal cord injury. Nano Lett. 2020;20:4298-305. DOI: 10.1021/ acs.nanolett.0c00929

[8] Hu G, Xia Y, Zhang J, Chen Y, Yuan J, Niu X, et al. ESC-sEVs rejuvenate senescent hippocampal NSCs by activating lysosomes to improve cognitive dysfunction in vascular dementia. Adv Sci (Weinh). 2020;7:1903330. DOI: 10.1002/ advs.201903330

[9] Wu Q, Wang J, Tan WLW, Jiang Y, Wang S, Li Q, et al. Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis. Cell Death Dis. 2020;11:354. DOI: 10.1038/ s41419-020-2508-y

[10] Khan M, Nickoloff E, Abramova T, Johnson J, Verma SK, Krishnamurthy P, et al. Embryonic stem cell-derived exosomes promote endogenous repair mechanisms and enhance cardiac function following myocardial infarction. Circulation research. 2015;117:52-64. DOI: 10.1161/ CIRCRESAHA.117.305990

[11] Wang Y, Zhang L, Li Y, Chen L, Wang X, Guo W, et al. Exosomes/ microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium. International journal of cardiology. 2015;192:61-9. DOI: 10.1016/j. ijcard.2015.05.020

[12] Bouchareychas L, Duong P, Covarrubias S, Alsop E, Phu TA, Chung A, et al. Macrophage exosomes resolve atherosclerosis by regulating hematopoiesis and inflammation via microRNA cargo. Cell Rep. 2020;32:107881. DOI: 10.1016/j. celrep.2020.107881

[13] Lei X, He N, Zhu L, Zhou M, Zhang K, Wang C, et al. Mesenchymal stem cell-derived extracellular vesicles attenuate radiation-induced lung injury via miRNA-214-3p. Antioxid Redox Signal. 2020. DOI: 10.1089/ars.2019.7965 [14] Hu S, Li Z, Lutz H, Huang K, Su T, Cores J, et al. Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling. Sci Adv. 2020;6:eaba1685. DOI: 10.1126/sciadv.aba1685

[15] Yan B, Zhang Y, Liang C, Liu B, Ding F, Wang Y, et al. Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway. Theranostics. 2020;10:6728-42. DOI: 10.7150/thno.42259

[16] Zhang S, Yang Y, Jia S, Chen H, Duan Y, Li X, et al. Exosome-like vesicles derived from Hertwig's epithelial root sheath cells promote the regeneration of dentin-pulp tissue. Theranostics. 2020;10:5914-31. DOI: 10.7150/thno.43156

[17] Bian B, Zhao C, He X, Gong Y, Ren C, Ge L, et al. Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia. J Extracell Vesicles. 2020;9:1748931. DOI: 10.1080/20013078.2020.1748931

[18] Fafián-Labora JA, Rodríguez-Navarro JA, O'Loghlen A. Small extracellular vesicles have GST activity and ameliorate senescence-related tissue damage. Cell Metab. 2020;32:71-86 e5. DOI: 10.1016/j.cmet.2020.06.004

[19] Li CJ, Fang QH, Liu ML, Lin JN. Current understanding of the role of Adipose-derived Extracellular Vesicles in Metabolic Homeostasis and Diseases: Communication from the distance between cells/tissues. Theranostics. 2020;10:7422-35. DOI: 10.7150/thno.42167

[20] Zhang H, Wang J, Ren T, Huang Y, Liang X, Yu Y, et al. Bone marrow mesenchymal stem cellderived exosomal miR-206 inhibits osteosarcoma progression by targeting TRA2B. Cancer Lett. 2020;490:54-65. DOI: 10.1016/j.canlet.2020.07.008

[21] He Z, Li W, Zheng T, Liu D, Zhao S. Human umbilical cord mesenchymal stem cells-derived exosomes deliver microRNA-375 to downregulate ENAH and thus retard esophageal squamous cell carcinoma progression. J Exp Clin Cancer Res. 2020;39:140. DOI: 10.1186/ s13046-020-01631-w

[22] Gazdic M, Volarevic V, Harrell CR, Fellabaum C, Jovicic N, Arsenijevic N, et al. Stem cells therapy for spinal cord injury. Int J Mol Sci. 2018 Mar 30;19:1039. DOI: 10.3390/ijms19041039

[23] González C, Bonilla S, Flores AI, Cano E, Liste I. An update on human stem cell-based therapy in Parkinson's disease. Curr Stem Cell Res Ther. 2016;11:561-8. DOI: 10.2174/1574888x10 666150531172612

[24] Tang W. Challenges and advances in stem cell therapy. Biosci Trends. 2019;13:286. DOI: 10.5582/ bst.2019.01241

[25] Isomi M, Sadahiro T, Ieda M. Progress and challenge of cardiac regeneration to treat heart failure. J Cardiol. 2019;73:97-101. DOI: 10.1016/j. jjcc.2018.10.002

[26] Adamiak M, Cheng G, Bobis-Wozowicz S, Zhao L, Kedracka-Krok S, Samanta A, et al. Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs. Circ Res. 2018;122:296-309. DOI: 10.1161/ CIRCRESAHA.117.311769

[27] Dougherty JA, Kumar N, Noor M, Angelos MG, Khan M, Chen CA. Extracellular vesicles released by human induced-pluripotent stem cell-derived cardiomyocytes promote angiogenesis. Front Physiol. 2018;9:1794. DOI: 10.3389/fphys.2018.01794

[28] Kato T, Fahrmann JF, Hanash SM, Vykoukal J. Extracellular vesicles mediate B cell immune response and

**79**

s12248-016-0015-y

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29:341-5. DOI: 10.1038/

[36] Luo Q, Guo D, Liu G, Chen G, Hang M, Jin M. Exosomes from miR-126-overexpressing Adscs are therapeutic in relieving acute myocardial ischaemic injury. Cell Physiol Biochem. 2017;44:2105-16. DOI:

[37] Zhang H, Wang Y, Bai M, Wang J, Zhu K, Liu R, et al. Exosomes serve as nanoparticles to suppress tumor growth and angiogenesis in gastric cancer by delivering hepatocyte growth factor siRNA. Cancer Sci. 2017;109:629-41.

nbt.1807

10.1159/000485949

DOI: 10.1111/cas.13488

[38] Kojima R, Bojar D, Rizzi G, Hamri GC, El-Baba MD, Saxena P, et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. Nat Commun. 2018;9:1305. DOI: 10.1038/s41467-018-03733-8

[39] Batagov AO, Kuznetsov VA, Kurochkin IV. Identification of nucleotide patterns enriched in secreted RNAs as putative cis-acting elements targeting them to exosome nano-vesicles. BMC Genomics. 2011;12 Suppl 3(Suppl 3):S18. DOI: 10.1186/1471-2164-12-S3-S18

[40] Ausländer S, Ausländer D,

[41] Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T. Synthetic human

cell fate regulation by proteindriven RNA switches. Nat Commun. 2011;2:160. DOI: 10.1038/ncomms1157

[42] Saito H, Kobayashi T, Hara T, Fujita Y, Hayashi K, Furushima R, et al. Synthetic translational regulation by

DOI: 10.1038/nature11149

Müller M, Wieland M, Fussenegger M. Programmable single-cell mammalian biocomputers. Nature. 2012;487:123-7.

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

are a potential target for cancer therapy. Cells. 2020;9:1518. DOI: 10.3390/

[29] Usman WM, Pham TC, Kwok YY, Vu LT, Ma V, Peng B, et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat Commun. 2018;9:2359. DOI: 10.1038/

[30] Zhan Q, Yi K, Qi H, Li S, Li X, Wang Q, et al. Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy. Theranostics. 2020;10:7889-905. DOI:

[31] Xu Q, Zhang Z, Zhao L, Qin Y, Cai H, Geng Z, et al. Tropism-facilitated delivery of CRISPR/Cas9 system with chimeric antigen receptor-extracellular vesicles against B-cell malignancies. J Control Release. 2020;326:455-67. DOI:

[32] Yang L, Han B, Zhang Z, Wang S, Bai Y, Zhang Y, et al. Extracellular vesicle-mediated delivery of circular RNA SCMH1 promotes functional recovery in rodent and nonhuman primate ischemic stroke models. Circulation. 2020;142:556-74. DOI: 10.1161/CIRCULATIONAHA.120.045765

[33] Wang X, Zhang H, Bai M, Ning T, Ge S, Deng T, et al. Exosomes serve as nanoparticles to deliver anti-miR-214 to reverse chemoresistance to cisplatin in gastric cancer. Mol Ther. 2018;26:774-83. DOI: 10.1016/j.ymthe.2018.01.001

[34] Yang T, Fogarty B, LaForge B, Aziz S, Pham T, Lai L, et al. Delivery of small interfering RNA to inhibit vascular endothelial growth factor in zebrafish using natural brain endothelia cell-secreted exosome nanovesicles for the treatment of brain cancer. AAPS J. 2016;19:475-86. DOI: 10.1208/

[35] Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of

10.1016/j.jconrel.2020.07.033

cells9061518

s41467-018-04791-8

10.7150/thno.45028

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

are a potential target for cancer therapy. Cells. 2020;9:1518. DOI: 10.3390/ cells9061518

*Theranostics - An Old Concept in New Clothing*

[21] He Z, Li W, Zheng T, Liu D, Zhao S. Human umbilical cord mesenchymal stem cells-derived exosomes deliver microRNA-375 to downregulate ENAH and thus retard esophageal squamous cell carcinoma progression. J Exp Clin Cancer Res. 2020;39:140. DOI: 10.1186/

[22] Gazdic M, Volarevic V, Harrell CR, Fellabaum C, Jovicic N, Arsenijevic N, et al. Stem cells therapy for spinal cord injury. Int J Mol Sci. 2018 Mar 30;19:1039. DOI: 10.3390/ijms19041039

[23] González C, Bonilla S, Flores AI, Cano E, Liste I. An update on human stem cell-based therapy in Parkinson's disease. Curr Stem Cell Res Ther. 2016;11:561-8. DOI: 10.2174/1574888x10

s13046-020-01631-w

666150531172612

bst.2019.01241

jjcc.2018.10.002

[24] Tang W. Challenges and

advances in stem cell therapy. Biosci Trends. 2019;13:286. DOI: 10.5582/

[25] Isomi M, Sadahiro T, Ieda M. Progress and challenge of cardiac regeneration to treat heart failure. J Cardiol. 2019;73:97-101. DOI: 10.1016/j.

[26] Adamiak M, Cheng G, Bobis-Wozowicz S, Zhao L, Kedracka-Krok S, Samanta A, et al. Induced pluripotent stem cell (iPSC)-derived extracellular vesicles are safer and more effective for cardiac repair than iPSCs. Circ Res. 2018;122:296-309. DOI: 10.1161/

[27] Dougherty JA, Kumar N, Noor M, Angelos MG, Khan M, Chen CA. Extracellular vesicles released by human induced-pluripotent stem cell-derived cardiomyocytes promote angiogenesis. Front Physiol. 2018;9:1794. DOI: 10.3389/fphys.2018.01794

[28] Kato T, Fahrmann JF, Hanash SM, Vykoukal J. Extracellular vesicles mediate B cell immune response and

CIRCRESAHA.117.311769

[14] Hu S, Li Z, Lutz H, Huang K, Su T, Cores J, et al. Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling. Sci Adv. 2020;6:eaba1685.

DOI: 10.1126/sciadv.aba1685

10.7150/thno.42259

10.7150/thno.43156

[15] Yan B, Zhang Y, Liang C, Liu B, Ding F, Wang Y, et al. Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway. Theranostics. 2020;10:6728-42. DOI:

[16] Zhang S, Yang Y, Jia S, Chen H, Duan Y, Li X, et al. Exosome-like vesicles derived from Hertwig's epithelial root sheath cells promote the regeneration of dentin-pulp tissue. Theranostics. 2020;10:5914-31. DOI:

[17] Bian B, Zhao C, He X, Gong Y, Ren C, Ge L, et al. Exosomes derived from neural progenitor cells preserve photoreceptors during retinal

[18] Fafián-Labora JA, Rodríguez-Navarro JA, O'Loghlen A. Small extracellular vesicles have GST activity and ameliorate senescence-related tissue damage. Cell Metab. 2020;32:71-86 e5. DOI: 10.1016/j.cmet.2020.06.004

[19] Li CJ, Fang QH, Liu ML, Lin JN. Current understanding of the role of Adipose-derived Extracellular Vesicles in Metabolic Homeostasis and Diseases: Communication from the distance between cells/tissues. Theranostics. 2020;10:7422-35. DOI: 10.7150/thno.42167

[20] Zhang H, Wang J, Ren T, Huang Y, Liang X, Yu Y, et al. Bone marrow mesenchymal stem cellderived exosomal miR-206 inhibits osteosarcoma progression by targeting TRA2B. Cancer Lett. 2020;490:54-65. DOI: 10.1016/j.canlet.2020.07.008

degeneration by inactivating microglia. J Extracell Vesicles. 2020;9:1748931. DOI: 10.1080/20013078.2020.1748931

**78**

[29] Usman WM, Pham TC, Kwok YY, Vu LT, Ma V, Peng B, et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat Commun. 2018;9:2359. DOI: 10.1038/ s41467-018-04791-8

[30] Zhan Q, Yi K, Qi H, Li S, Li X, Wang Q, et al. Engineering blood exosomes for tumor-targeting efficient gene/chemo combination therapy. Theranostics. 2020;10:7889-905. DOI: 10.7150/thno.45028

[31] Xu Q, Zhang Z, Zhao L, Qin Y, Cai H, Geng Z, et al. Tropism-facilitated delivery of CRISPR/Cas9 system with chimeric antigen receptor-extracellular vesicles against B-cell malignancies. J Control Release. 2020;326:455-67. DOI: 10.1016/j.jconrel.2020.07.033

[32] Yang L, Han B, Zhang Z, Wang S, Bai Y, Zhang Y, et al. Extracellular vesicle-mediated delivery of circular RNA SCMH1 promotes functional recovery in rodent and nonhuman primate ischemic stroke models. Circulation. 2020;142:556-74. DOI: 10.1161/CIRCULATIONAHA.120.045765

[33] Wang X, Zhang H, Bai M, Ning T, Ge S, Deng T, et al. Exosomes serve as nanoparticles to deliver anti-miR-214 to reverse chemoresistance to cisplatin in gastric cancer. Mol Ther. 2018;26:774-83. DOI: 10.1016/j.ymthe.2018.01.001

[34] Yang T, Fogarty B, LaForge B, Aziz S, Pham T, Lai L, et al. Delivery of small interfering RNA to inhibit vascular endothelial growth factor in zebrafish using natural brain endothelia cell-secreted exosome nanovesicles for the treatment of brain cancer. AAPS J. 2016;19:475-86. DOI: 10.1208/ s12248-016-0015-y

[35] Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29:341-5. DOI: 10.1038/ nbt.1807

[36] Luo Q, Guo D, Liu G, Chen G, Hang M, Jin M. Exosomes from miR-126-overexpressing Adscs are therapeutic in relieving acute myocardial ischaemic injury. Cell Physiol Biochem. 2017;44:2105-16. DOI: 10.1159/000485949

[37] Zhang H, Wang Y, Bai M, Wang J, Zhu K, Liu R, et al. Exosomes serve as nanoparticles to suppress tumor growth and angiogenesis in gastric cancer by delivering hepatocyte growth factor siRNA. Cancer Sci. 2017;109:629-41. DOI: 10.1111/cas.13488

[38] Kojima R, Bojar D, Rizzi G, Hamri GC, El-Baba MD, Saxena P, et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. Nat Commun. 2018;9:1305. DOI: 10.1038/s41467-018-03733-8

[39] Batagov AO, Kuznetsov VA, Kurochkin IV. Identification of nucleotide patterns enriched in secreted RNAs as putative cis-acting elements targeting them to exosome nano-vesicles. BMC Genomics. 2011;12 Suppl 3(Suppl 3):S18. DOI: 10.1186/1471-2164-12-S3-S18

[40] Ausländer S, Ausländer D, Müller M, Wieland M, Fussenegger M. Programmable single-cell mammalian biocomputers. Nature. 2012;487:123-7. DOI: 10.1038/nature11149

[41] Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T. Synthetic human cell fate regulation by proteindriven RNA switches. Nat Commun. 2011;2:160. DOI: 10.1038/ncomms1157

[42] Saito H, Kobayashi T, Hara T, Fujita Y, Hayashi K, Furushima R, et al. Synthetic translational regulation by

an L7Ae-kink-turn RNP switch. Nat Chem Biol. 2010;6:71-8. DOI: 10.1038/ nchembio.273

[43] Ke C, Hou H, Li J, Su K, Huang C, Lin Y, et al. Extracellular vesicle delivery of TRAIL eradicates resistant tumor growth in combination with CDK inhibition by Dinaciclib. Cancers (Basel). 2020;12:1157. DOI: 10.3390/ cancers12051157

[44] Xu Z, Tsai HI, Xiao Y, Wu Y, Su D, Yang M, et al. Engineering programmed death ligand-1/cytotoxic T-lymphocyteassociated antigen-4 dual-targeting nanovesicles for immunosuppressive therapy in transplantation. ACS Nano. 2020;14:7959-69. DOI: 10.1021/ acsnano.9b09065

[45] Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, et al. Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat Commun. 2020;11:1334. DOI: 10.1038/ s41467-020-14957-y

[46] Haney MJ, Klyachko NL, Zhao Y, Gupta R, Plotnikova EG, He Z, et al. Exosomes as drug delivery vehicles for Parkinson's disease therapy. J Control Release. 2015;207:18-30. DOI: 10.1016/j. jconrel.2015.03.033

[47] Yang J, Wu S, Hou L, Zhu D, Yin S, Yang G, et al. Therapeutic effects of simultaneous delivery of nerve growth factor mRNA and protein via exosomes on cerebral ischemia. Mol Ther Nucleic Acids. 2020;21:512-22. DOI: 10.1016/j. omtn.2020.06.013

[48] Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527:329-35. DOI: 10.1038/nature15756

[49] Rak J. Cancer: Organ-seeking vesicles. Nature. 2015;527:312-4. DOI: 10.1038/nature15642

[50] Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials. 2014;35:2383-90. DOI: 10.1016/j. biomaterials.2013.11.083

[51] Yang J, Zhang X, Chen X, Wang L, Yang G. Exosome mediated delivery of miR-124 promotes neurogenesis after ischemia. Mol Ther Nucleic Acids. 2017;7:278-87. DOI: 10.1016/j. omtn.2017.04.010

[52] Chen W, Yang M, Bai J, Li X, Kong X, Gao Y, et al. Exosome-modified tissue engineered blood vessel for endothelial progenitor cell capture and targeted siRNA delivery. Macromol Biosci. 2018;18. DOI: 10.1002/mabi.201700242

[53] Liu C, Su C. Design strategies and application progress of therapeutic exosomes. Theranostics. 2019;9:1015-28. DOI: 10.7150/thno.30853

[54] Hall J, Prabhakar S, Balaj L, Lai CP, Cerione RA, Breakefield XO. Delivery of therapeutic proteins via extracellular vesicles: Review and potential treatments for Parkinson's disease, glioma, and schwannoma. Cell Mol Neurobiol. 2016;36:417-27. DOI: 10.1007/s10571-015-0309-0

[55] Di Bonito P, Chiozzini C, Arenaccio C, Anticoli S, Manfredi F, Olivetta E, et al. Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation. Int J Nanomedicine. 2017;12:4579-91. DOI: 10.2147/IJN.S131309

[56] Meyer C, Losacco J, Stickney Z, Li L, Marriott G, Lu B. Pseudotyping exosomes for enhanced protein delivery in mammalian cells. Int J Nanomedicine. 2017;12:3153-70. DOI: 10.2147/IJN. S133430

[57] Zheng P, Luo Q, Wang W, Li J, Wang T, Wang P, et al. Tumor-associated

**81**

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs*

[65] Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et al. Development of exosomeencapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 2015;12:655-64. DOI: 10.1016/j.

[66] Pascucci L, Coccè V, Bonomi A, Ami D, Ceccarelli P, Ciusani E, et al. Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery. J Control Release. 2014;192:262-70. DOI: 10.1016/j.

[67] Gao Y, Zhang H, Zhou N, Xu P, Wang J, Jin X, et al. Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma. Nat Biomed Eng. 2020;4:743-53. DOI:

10.1038/s41551-020-0583-0

[69] György B, Fitzpatrick Z,

[70] Wassmer SJ, Carvalho LS, György B, Vandenberghe LH,

Crommentuijn MH, Mu D, Maguire CA. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo. Biomaterials. 2014;35:7598-609. DOI: 10.1016/j.biomaterials.2014.05.032

Maguire CA. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection. Sci Rep. 2017;7:45329. DOI:

[71] György B, Sage C, Indzhykulian AA,

Scheffer DI, Brisson AR, Tan S, et al. Rescue of hearing by gene delivery to inner-ear hair cells using

10.1038/gt.2016.11

10.1038/srep45329

[68] Hudry E, Martin C, Gandhi S, György B, Scheffer DI, Mu D, et al. Exosome-associated AAV vector as a robust and convenient neuroscience tool. Gene Ther. 2016;23:380-92. DOI:

nano.2015.10.012

jconrel.2014.07.042

*DOI: http://dx.doi.org/10.5772/intechopen.94502*

macrophages-derived exosomes promote the migration of gastric cancer cells by transfer of functional Apolipoprotein E.

Cell Death Dis. 2018;9:434. DOI: 10.1038/s41419-018-0465-5

[58] Simpson RJ, Jensen SS, Lim JW. Proteomic profiling of exosomes: current perspectives. Proteomics. 2008;8:4083-99. DOI: 10.1002/

[59] Yim N, Ryu SW, Choi K, Lee KR,

engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module. Nat Commun. 2016;7:12277. DOI: 10.1038/ncomms12277

[60] Cheng Y, Schorey JS. Targeting soluble proteins to exosomes using a ubiquitin tag. Biotechnol Bioeng. 2015;113:1315-24. DOI: 10.1002/

[61] Shen B, Wu N, Yang JM, Gould SJ.

[62] Wan Y, Wang L, Zhu C, Zheng Q, Wang G, Tong J, et al. Aptamerconjugated extracellular nanovesicles for targeted drug delivery. Cancer Res. 2018;78:798-808. DOI: 10.1158/0008-

[63] Zhang D, Qin X, Wu T, Qiao Q, Song Q, Zhang Z. Extracellular vesicles based self-grown gold nanopopcorn for combinatorial chemo-photothermal therapy. Biomaterials. 2019;197:220-8. DOI: 10.1016/j.biomaterials.2019.01.024

[64] Zarovni N, Corrado A, Guazzi P, Zocco D, Lari E, Radano G, et al. Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods. 2015;87:46-58. DOI: 10.1016/j.ymeth.2015.05.028

5472.CAN-17-2880

Protein targeting to exosomes/ microvesicles by plasma membrane anchors. J Biol Chem. 2011;286:14383- 95. DOI: 10.1074/jbc.M110.208660

Lee S, Choi H, et al. Exosome

pmic.200800109

bit.25884

*Extracellular Vesicles: "Stealth Transport Aircrafts" for Drugs DOI: http://dx.doi.org/10.5772/intechopen.94502*

macrophages-derived exosomes promote the migration of gastric cancer cells by transfer of functional Apolipoprotein E. Cell Death Dis. 2018;9:434. DOI: 10.1038/s41419-018-0465-5

*Theranostics - An Old Concept in New Clothing*

[50] Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, et al. A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials. 2014;35:2383-90. DOI: 10.1016/j.

[51] Yang J, Zhang X, Chen X, Wang L, Yang G. Exosome mediated delivery of miR-124 promotes neurogenesis after ischemia. Mol Ther Nucleic Acids. 2017;7:278-87. DOI: 10.1016/j.

[52] Chen W, Yang M, Bai J, Li X, Kong X, Gao Y, et al. Exosome-modified tissue engineered blood vessel for endothelial progenitor cell capture and targeted siRNA delivery. Macromol Biosci. 2018;18. DOI: 10.1002/mabi.201700242

[53] Liu C, Su C. Design strategies and application progress of therapeutic exosomes. Theranostics. 2019;9:1015-28.

DOI: 10.7150/thno.30853

[54] Hall J, Prabhakar S, Balaj L, Lai CP, Cerione RA, Breakefield XO. Delivery of therapeutic proteins via extracellular vesicles: Review and potential treatments for Parkinson's disease, glioma, and schwannoma. Cell Mol Neurobiol. 2016;36:417-27. DOI:

10.1007/s10571-015-0309-0

[55] Di Bonito P, Chiozzini C, Arenaccio C, Anticoli S, Manfredi F, Olivetta E, et al. Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation. Int J Nanomedicine. 2017;12:4579-91. DOI:

[56] Meyer C, Losacco J, Stickney Z, Li L, Marriott G, Lu B. Pseudotyping exosomes for enhanced protein delivery in mammalian cells. Int J Nanomedicine. 2017;12:3153-70. DOI: 10.2147/IJN.

[57] Zheng P, Luo Q, Wang W, Li J, Wang T, Wang P, et al. Tumor-associated

10.2147/IJN.S131309

S133430

biomaterials.2013.11.083

omtn.2017.04.010

an L7Ae-kink-turn RNP switch. Nat Chem Biol. 2010;6:71-8. DOI: 10.1038/

[43] Ke C, Hou H, Li J, Su K, Huang C, Lin Y, et al. Extracellular vesicle delivery of TRAIL eradicates resistant tumor growth in combination with CDK inhibition by Dinaciclib. Cancers (Basel). 2020;12:1157. DOI: 10.3390/

[44] Xu Z, Tsai HI, Xiao Y, Wu Y, Su D, Yang M, et al. Engineering programmed death ligand-1/cytotoxic T-lymphocyteassociated antigen-4 dual-targeting nanovesicles for immunosuppressive therapy in transplantation. ACS Nano. 2020;14:7959-69. DOI: 10.1021/

[45] Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, et al. Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat Commun. 2020;11:1334. DOI: 10.1038/

[46] Haney MJ, Klyachko NL, Zhao Y, Gupta R, Plotnikova EG, He Z, et al. Exosomes as drug delivery vehicles for Parkinson's disease therapy. J Control Release. 2015;207:18-30. DOI: 10.1016/j.

[47] Yang J, Wu S, Hou L, Zhu D, Yin S, Yang G, et al. Therapeutic effects of simultaneous delivery of nerve growth factor mRNA and protein via exosomes on cerebral ischemia. Mol Ther Nucleic Acids. 2020;21:512-22. DOI: 10.1016/j.

nchembio.273

cancers12051157

acsnano.9b09065

s41467-020-14957-y

jconrel.2015.03.033

omtn.2020.06.013

[48] Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527:329-35.

DOI: 10.1038/nature15756

10.1038/nature15642

[49] Rak J. Cancer: Organ-seeking vesicles. Nature. 2015;527:312-4. DOI:

**80**

[58] Simpson RJ, Jensen SS, Lim JW. Proteomic profiling of exosomes: current perspectives. Proteomics. 2008;8:4083-99. DOI: 10.1002/ pmic.200800109

[59] Yim N, Ryu SW, Choi K, Lee KR, Lee S, Choi H, et al. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module. Nat Commun. 2016;7:12277. DOI: 10.1038/ncomms12277

[60] Cheng Y, Schorey JS. Targeting soluble proteins to exosomes using a ubiquitin tag. Biotechnol Bioeng. 2015;113:1315-24. DOI: 10.1002/ bit.25884

[61] Shen B, Wu N, Yang JM, Gould SJ. Protein targeting to exosomes/ microvesicles by plasma membrane anchors. J Biol Chem. 2011;286:14383- 95. DOI: 10.1074/jbc.M110.208660

[62] Wan Y, Wang L, Zhu C, Zheng Q, Wang G, Tong J, et al. Aptamerconjugated extracellular nanovesicles for targeted drug delivery. Cancer Res. 2018;78:798-808. DOI: 10.1158/0008- 5472.CAN-17-2880

[63] Zhang D, Qin X, Wu T, Qiao Q, Song Q, Zhang Z. Extracellular vesicles based self-grown gold nanopopcorn for combinatorial chemo-photothermal therapy. Biomaterials. 2019;197:220-8. DOI: 10.1016/j.biomaterials.2019.01.024

[64] Zarovni N, Corrado A, Guazzi P, Zocco D, Lari E, Radano G, et al. Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches. Methods. 2015;87:46-58. DOI: 10.1016/j.ymeth.2015.05.028

[65] Kim MS, Haney MJ, Zhao Y, Mahajan V, Deygen I, Klyachko NL, et al. Development of exosomeencapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 2015;12:655-64. DOI: 10.1016/j. nano.2015.10.012

[66] Pascucci L, Coccè V, Bonomi A, Ami D, Ceccarelli P, Ciusani E, et al. Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery. J Control Release. 2014;192:262-70. DOI: 10.1016/j. jconrel.2014.07.042

[67] Gao Y, Zhang H, Zhou N, Xu P, Wang J, Jin X, et al. Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma. Nat Biomed Eng. 2020;4:743-53. DOI: 10.1038/s41551-020-0583-0

[68] Hudry E, Martin C, Gandhi S, György B, Scheffer DI, Mu D, et al. Exosome-associated AAV vector as a robust and convenient neuroscience tool. Gene Ther. 2016;23:380-92. DOI: 10.1038/gt.2016.11

[69] György B, Fitzpatrick Z, Crommentuijn MH, Mu D, Maguire CA. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo. Biomaterials. 2014;35:7598-609. DOI: 10.1016/j.biomaterials.2014.05.032

[70] Wassmer SJ, Carvalho LS, György B, Vandenberghe LH, Maguire CA. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection. Sci Rep. 2017;7:45329. DOI: 10.1038/srep45329

[71] György B, Sage C, Indzhykulian AA, Scheffer DI, Brisson AR, Tan S, et al. Rescue of hearing by gene delivery to inner-ear hair cells using

exosome-associated AAV. Mol Ther. 2017;25:379-91. DOI: 10.1016/j. ymthe.2016.12.010

[72] Garofalo M, Saari H, Somersalo P, Crescenti D, Kuryk L, Aksela L, et al. Antitumor effect of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment. J Control Release. 2018;283:223-34. DOI: 10.1016/j.jconrel.2018.05.015

[73] Wolfers J, Lozier A, Raposo G, Regnault A, Théry C, Masurier C, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med. 2001;7:297-303. DOI: 10.1038/85438

[74] Rana S, Yue S, Stadel D, Zöller M. Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection. Int J Biochem Cell Biol. 2012;44:1574-84. DOI: 10.1016/j. biocel.2012.06.018

[75] Zomer A, Maynard C, Verweij FJ, Kamermans A, Schäfer R, Beerling E, et al. In vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior. Cell. 2015;161:1046-57. DOI: 10.1016/j. cell.2015.04.042

[76] Harris DA, Patel SH, Gucek M, Hendrix A, Westbroek W, Taraska JW. Exosomes released from breast cancer carcinomas stimulate cell movement. PLoS One. 2015;10:e0117495. DOI: 10.1371/journal.pone.0117495

[77] Tomihari M, Chung JS, Akiyoshi H, Cruz PD, Jr., Ariizumi K. DC-HIL/ glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells. Cancer Res. 2010;70:5778-87. DOI: 10.1158/0008-5472.CAN-09-2538

[78] Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL. Fas ligand-positive membranous vesicles isolated from sera of patients with oral

cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res. 2005;11:1010-20.

[79] Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin. 2017;38:754-63. DOI: 10.1038/ aps.2017.12

[80] Li S, Wu Y, Ding F, Yang J, Li J, Gao X, et al. Engineering macrophagederived exosomes for targeted chemotherapy of triple-negative breast cancer. Nanoscale. 2020;12:10854-62. DOI: 10.1039/d0nr00523a

[81] Ju S, Mu J, Dokland T, Zhuang X, Wang Q, Jiang H, et al. Grape exosomelike nanoparticles induce intestinal stem cells and protect mice from DSSinduced colitis. Mol Ther. 2013;21:1345- 57. DOI: 10.1038/mt.2013.64

Section 3

Theranostics with

Nanomaterials

**83**

[82] Yang M, Liu X, Luo Q, Xu L, Chen F. An efficient method to isolate lemon derived extracellular vesicles for gastric cancer therapy. J Nanobiotechnology. 2020;18:100. DOI: 10.1186/s12951-020-00656-9

[83] Wang Q, Ren Y, Mu J, Egilmez NK, Zhuang X, Deng Z, et al. Grapefruitderived nanovectors use an activated leukocyte trafficking pathway to deliver therapeutic agents to inflammatory tumor sites. Cancer Res. 2015;75:2520-9. DOI: 10.1158/0008-5472.CAN-14-3095

[84] Börger V, Weiss DJ, Anderson JD, Borràs FE, Bussolati B, Carter DRF, et al. International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19. Cytotherapy. 2020;22:482-5. DOI: 10.1016/j. jcyt.2020.05.002

Section 3

## Theranostics with Nanomaterials

*Theranostics - An Old Concept in New Clothing*

cancer induce apoptosis of activated T lymphocytes. Clin Cancer Res.

[79] Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin. 2017;38:754-63. DOI: 10.1038/

[80] Li S, Wu Y, Ding F, Yang J, Li J, Gao X, et al. Engineering macrophage-

chemotherapy of triple-negative breast cancer. Nanoscale. 2020;12:10854-62.

[81] Ju S, Mu J, Dokland T, Zhuang X, Wang Q, Jiang H, et al. Grape exosomelike nanoparticles induce intestinal stem cells and protect mice from DSSinduced colitis. Mol Ther. 2013;21:1345-

derived exosomes for targeted

DOI: 10.1039/d0nr00523a

57. DOI: 10.1038/mt.2013.64

10.1186/s12951-020-00656-9

[83] Wang Q, Ren Y, Mu J, Egilmez NK, Zhuang X, Deng Z, et al. Grapefruitderived nanovectors use an activated leukocyte trafficking pathway to deliver therapeutic agents to inflammatory tumor sites. Cancer Res. 2015;75:2520-9. DOI: 10.1158/0008-5472.CAN-14-3095

[84] Börger V, Weiss DJ, Anderson JD, Borràs FE, Bussolati B, Carter DRF, et al. International Society for

Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19. Cytotherapy. 2020;22:482-5. DOI: 10.1016/j.

jcyt.2020.05.002

[82] Yang M, Liu X, Luo Q, Xu L, Chen F. An efficient method to isolate lemon derived extracellular vesicles for gastric cancer therapy. J Nanobiotechnology. 2020;18:100. DOI:

2005;11:1010-20.

aps.2017.12

exosome-associated AAV. Mol Ther. 2017;25:379-91. DOI: 10.1016/j.

[72] Garofalo M, Saari H, Somersalo P, Crescenti D, Kuryk L, Aksela L, et al. Antitumor effect of oncolytic virus and paclitaxel encapsulated in extracellular vesicles for lung cancer treatment. J Control Release. 2018;283:223-34. DOI:

10.1016/j.jconrel.2018.05.015

DOI: 10.1038/85438

biocel.2012.06.018

cell.2015.04.042

[73] Wolfers J, Lozier A, Raposo G, Regnault A, Théry C, Masurier C, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med. 2001;7:297-303.

[74] Rana S, Yue S, Stadel D, Zöller M. Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection. Int J Biochem Cell Biol. 2012;44:1574-84. DOI: 10.1016/j.

[75] Zomer A, Maynard C, Verweij FJ, Kamermans A, Schäfer R, Beerling E,

phenocopying of metastatic behavior. Cell. 2015;161:1046-57. DOI: 10.1016/j.

[76] Harris DA, Patel SH, Gucek M, Hendrix A, Westbroek W, Taraska JW. Exosomes released from breast cancer carcinomas stimulate cell movement. PLoS One. 2015;10:e0117495. DOI: 10.1371/journal.pone.0117495

[77] Tomihari M, Chung JS, Akiyoshi H, Cruz PD, Jr., Ariizumi K. DC-HIL/ glycoprotein Nmb promotes growth of melanoma in mice by inhibiting the activation of tumor-reactive T cells. Cancer Res. 2010;70:5778-87. DOI: 10.1158/0008-5472.CAN-09-2538

[78] Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL. Fas ligand-positive membranous vesicles isolated from sera of patients with oral

et al. In vivo imaging reveals extracellular vesicle-mediated

ymthe.2016.12.010

**82**

**Chapter 5**

**Abstract**

bioremediation

**1. Introduction**

**85**

Graphene-Based Nanosystems:

*Marlene Lúcio, Eduarda Fernandes, Hugo Gonçalves,*

Theranostics and Bioremediation

Since its revolutionary discovery in 2004, graphene— a two-dimensional (2D) nanomaterial consisting of single-layer carbon atoms packed in a honeycomb lattice — was thoroughly discussed for a broad variety of applications including quantum physics, nanoelectronics, energy efficiency, and catalysis. Graphene and graphenebased nanomaterials (GBNs) have also captivated the interest of researchers for innovative biomedical applications since the first publication on the use of graphene as a nanocarrier for the delivery of anticancer drugs in 2008. Today, GBNs have evolved into hybrid combinations of graphene and other elements (e.g., drugs or other bioactive compounds, polymers, lipids, and nanoparticles). In the context of developing theranostic (therapeutic + diagnostic) tools, which combine multiple therapies with imaging strategies to track the distribution of therapeutic agents in the body, the multipurpose character of the GBNs hybrid systems has been further explored. Because each therapy and imaging strategy has inherent advantages and disadvantages, a mixture of complementary strategies is interesting as it will result in a synergistic theranostic effect. The flexibility of GBNs cannot be limited to their biomedical applications and, these nanosystems emerge as a viable choice for an indirect effect on health by their future use as environmental cleaners. Indeed, GBNs can be used in bioremediation approaches alone or combined with other techniques such as phytoremediation. In summary, without ignoring the difficulties that GBNs still present before being deemed translatable to clinical and environmental applications, the purpose of this chapter is to provide an overview of the remarkable potential of GBNs on health by presenting examples of their versatility

**Keywords:** Graphene-based nanomaterials, graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, cancer theranostics, green synthesis,

Since its first serendipitous but groundbreaking discovery by Geim and Novoselov in 2004 [1], followed by the 2010 Nobel Prize in Physics, graphene has

Versatile Nanotools for

*Sofia Machado, Andreia C. Gomes*

*and Maria Elisabete C.D. Real Oliveira*

as nanotools for theranostics and bioremediation.

**Chapter 5**

## Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation

*Marlene Lúcio, Eduarda Fernandes, Hugo Gonçalves, Sofia Machado, Andreia C. Gomes and Maria Elisabete C.D. Real Oliveira*

#### **Abstract**

Since its revolutionary discovery in 2004, graphene— a two-dimensional (2D) nanomaterial consisting of single-layer carbon atoms packed in a honeycomb lattice — was thoroughly discussed for a broad variety of applications including quantum physics, nanoelectronics, energy efficiency, and catalysis. Graphene and graphenebased nanomaterials (GBNs) have also captivated the interest of researchers for innovative biomedical applications since the first publication on the use of graphene as a nanocarrier for the delivery of anticancer drugs in 2008. Today, GBNs have evolved into hybrid combinations of graphene and other elements (e.g., drugs or other bioactive compounds, polymers, lipids, and nanoparticles). In the context of developing theranostic (therapeutic + diagnostic) tools, which combine multiple therapies with imaging strategies to track the distribution of therapeutic agents in the body, the multipurpose character of the GBNs hybrid systems has been further explored. Because each therapy and imaging strategy has inherent advantages and disadvantages, a mixture of complementary strategies is interesting as it will result in a synergistic theranostic effect. The flexibility of GBNs cannot be limited to their biomedical applications and, these nanosystems emerge as a viable choice for an indirect effect on health by their future use as environmental cleaners. Indeed, GBNs can be used in bioremediation approaches alone or combined with other techniques such as phytoremediation. In summary, without ignoring the difficulties that GBNs still present before being deemed translatable to clinical and environmental applications, the purpose of this chapter is to provide an overview of the remarkable potential of GBNs on health by presenting examples of their versatility as nanotools for theranostics and bioremediation.

**Keywords:** Graphene-based nanomaterials, graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, cancer theranostics, green synthesis, bioremediation

#### **1. Introduction**

Since its first serendipitous but groundbreaking discovery by Geim and Novoselov in 2004 [1], followed by the 2010 Nobel Prize in Physics, graphene has drawn tremendous interest from scientists from every direction to exploit many of its special features. Indeed, graphene and graphene-based nanomaterials (GBNs) have distinctive mechanical, electronic, optical, and chemical properties [2–4]. Graphene and GBNs can therefore be found in numerous applications in the areas of electronics, physics, and material science [5–7]. In recent times, considering an emerging opinion on the eco-friendly characteristics of graphene and its derivatives, researchers have agreed to use these nanomaterials in other fields of science, for example in medical [8–13] and environmental applications in bioremediation [14–21].

GBNs are graphene-like structures that can be obtained from graphene or graphite as the starting material, but that possess sp<sup>2</sup> and sp<sup>3</sup> hybridized carbon atoms and differ from one another in terms of surface chemistry, number of defects and lateral dimensions (**Table 1**). GBNs include graphene derivatives, such as graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQDs). GO is a highly oxidized form of graphene that contains oxygen functional groups (e.g., epoxide –O–; carboxyl –COOH; hydroxyl –OH) either in the plane or at the edges. rGO is a reduced form of GO where most of its functional oxygen groups have been removed. As a result of oxygen removal processes, rGO has more in-plane defects than GO and graphene. On the other hand, due to oxidation processes, GO has more defects than pristine graphene. GQDs consist of one or more layers (up to ten layers) of graphene or rGOs with a lateral size below 30 nm.

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Many fascinating properties of graphene, including strong thermal and electrical conductivity, large surface area and excellent mechanical properties, have been discovered since 2004 (**Table 1**). Further data on the properties of graphene can be found elsewhere [37, 38]. Herein, we focused on the properties of graphene and GBNs that are most significant for their biomedical and environmental applications and emphasized how these exceptional properties are connected to the special 2D

Because of the 2D carbon atomic honeycomb arrangement, each carbon atom is covalently bound to three neighbouring atoms inside a graphene layer. The tight C-C covalent bonds are responsible for graphene's extraordinary structural rigidity and a single defect-free graphene sheet is thus approximately 200 times mechanically stronger than steel. This explains the outstanding mechanical parameters of graphene: Young's modulus of 1 TPa, Poisson's ratio of 0.149 GPa and fracture

The mechanical properties of GO and rGO are significantly affected compared to graphene and depend on the surface groups and defects left over from oxidation or other treatment processes. However, the rigidity of these GBNs is still particularly high. Graphene's extraordinary structural rigidity and the still excellent mechanical properties of GBNs mean that these nanomaterials can potentially be used in medical devices, hydrogels, biodegradable films, electrospun fibres and other tissue engineering scaffolds to fill or strengthen the structures of these materials [39].

Graphene is a monoatomic layer of sp<sup>2</sup> hybridized carbon atoms arranged as a honeycomb lattice. The π–π bonds below and above the carbon atomic plane impart exceptional thermal and electrical conductivity to graphene. In fact, a carbon atom normally has four electrons for bonding, but in graphene every atom allocates a single unbound electron that walks freely through the crystal lattice and leads to excellent electrical and thermal conductivity [28]. Defect-free graphene has therefore been reported to have a thermal conductivity between 4500 and 5200 W/mK [28]. Additionally, graphene exhibits an ultra-high electron mobility (25 <sup>10</sup><sup>4</sup> cm2

<sup>V</sup>s) and an electrical conductivity of 10<sup>4</sup> S/cm [29] (**Table 1**).

/

carbon atomic honeycomb structure of graphene and its derivatives.

**3. Properties of graphene and GBNs**

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

**3.1 Mechanical properties**

strength of 130 GPa [27] (**Table 1**).

**3.2 Thermal and electrical properties**

**87**

One of the most interesting applications of GBNs in the medicine field is its use as theranostic tools, i.e. taking advantage of its properties to provide a combination strategy for both therapy and diagnosis [8]. Multiple combinations of different therapeutic and diagnostic strategies are currently being used to achieve a therapeutic effect with GBNs. Since each strategy has inherent advantages and limitations, a combination of complementary strategies can result in a synergistic theranostic effect [8]. Of all diseases, the synergistic theranostic effects of GBNs can be more significant in cancer. In fact, despite all the resources expended in clinical advances, cancer remains the world's leading cause of death, with a confirmed mortality rate of 8.8 million by 2015. In addition, the World Health Organization (WHO) and International Agency for Research on Cancer (IARC) expect all cases of cancer to rise to 21.2 million by 2030 [22, 23]. With conventional approaches to cancer treatment, such as chemotherapy and radiation, tumor-initiating cells also designated as cancer stem cells (CSCs), are hard to eradicate [24]. The survival of residual CSCs is therefore believed to drive the onset of tumor recurrence, distant metastasis, and drug resistance, which is a major clinical problem for effective cancer treatment [24]. Therefore, new cancer therapy approaches such as GBNs are urgently necessary to address this clinical need [8, 24].

Another field that requires investment in research is the use of GBNs in bioremediation. Air, water, and soil pollution is a worldwide challenge for the environment and human society [17, 18, 25]. The removal from the environment of multiple pollutants, including inorganic and organic compounds, is a growing concern [17, 18, 25]. The most harmful and hazardous pollutants that have been the focus of the GBNs' bioremediation research will be discussed in this chapter and listed according to the following classes: volatile organic compounds, inorganic metals, organic dyes, polycyclic aromatic hydrocarbons, pharmaceuticals, pesticides. In water sources and the atmosphere, these chemical pollutants also have the property of degrading and producing carcinogenic and mutagenic compounds [20]. In addition, microbial drug resistance can also be caused by bioaccumulation of contaminants such as pharmaceutical drugs, pesticides and their by-products in water bodies [20]. Therefore, pollution damages ecosystems but also affects human health, and the large number of pollutants emitted annually by industries and households have had a major impact on the environment and human existence.

This chapter presents an overview of the properties of graphene and GBNs and their synthesis by classical and green methods. In addition, the use of GBNs will be described either in medicine (as theranostic tools) or in bioremediation (as adsorbents and photocatalysts) and these different aspects will be presented as part of their versatile beneficial use when applied to human health.

#### **2. Graphene and GBNs**

Graphene is a single layer of sp2 hybridized carbon atoms bound together in a planar 2D honeycomb structure.

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

GBNs are graphene-like structures that can be obtained from graphene or graphite as the starting material, but that possess sp<sup>2</sup> and sp<sup>3</sup> hybridized carbon atoms and differ from one another in terms of surface chemistry, number of defects and lateral dimensions (**Table 1**). GBNs include graphene derivatives, such as graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQDs). GO is a highly oxidized form of graphene that contains oxygen functional groups (e.g., epoxide –O–; carboxyl –COOH; hydroxyl –OH) either in the plane or at the edges. rGO is a reduced form of GO where most of its functional oxygen groups have been removed. As a result of oxygen removal processes, rGO has more in-plane defects than GO and graphene. On the other hand, due to oxidation processes, GO has more defects than pristine graphene. GQDs consist of one or more layers (up to ten layers) of graphene or rGOs with a lateral size below 30 nm.

#### **3. Properties of graphene and GBNs**

Many fascinating properties of graphene, including strong thermal and electrical conductivity, large surface area and excellent mechanical properties, have been discovered since 2004 (**Table 1**). Further data on the properties of graphene can be found elsewhere [37, 38]. Herein, we focused on the properties of graphene and GBNs that are most significant for their biomedical and environmental applications and emphasized how these exceptional properties are connected to the special 2D carbon atomic honeycomb structure of graphene and its derivatives.

#### **3.1 Mechanical properties**

drawn tremendous interest from scientists from every direction to exploit many of its special features. Indeed, graphene and graphene-based nanomaterials (GBNs) have distinctive mechanical, electronic, optical, and chemical properties [2–4]. Graphene and GBNs can therefore be found in numerous applications in the areas of electronics, physics, and material science [5–7]. In recent times, considering an emerging opinion on the eco-friendly characteristics of graphene and its derivatives, researchers have agreed to use these nanomaterials in other fields of science, for example in medical [8–13] and environmental applications in bioremediation

One of the most interesting applications of GBNs in the medicine field is its use as theranostic tools, i.e. taking advantage of its properties to provide a combination strategy for both therapy and diagnosis [8]. Multiple combinations of different therapeutic and diagnostic strategies are currently being used to achieve a therapeutic effect with GBNs. Since each strategy has inherent advantages and limitations, a combination of complementary strategies can result in a synergistic

theranostic effect [8]. Of all diseases, the synergistic theranostic effects of GBNs can be more significant in cancer. In fact, despite all the resources expended in clinical advances, cancer remains the world's leading cause of death, with a confirmed mortality rate of 8.8 million by 2015. In addition, the World Health Organization (WHO) and International Agency for Research on Cancer (IARC) expect all cases of cancer to rise to 21.2 million by 2030 [22, 23]. With conventional approaches to cancer treatment, such as chemotherapy and radiation, tumor-initiating cells also designated as cancer stem cells (CSCs), are hard to eradicate [24]. The survival of residual CSCs is therefore believed to drive the onset of tumor recurrence, distant metastasis, and drug resistance, which is a major clinical problem for effective cancer treatment [24]. Therefore, new cancer therapy approaches such as GBNs are

Another field that requires investment in research is the use of GBNs in bioremediation. Air, water, and soil pollution is a worldwide challenge for the environment and human society [17, 18, 25]. The removal from the environment of multiple pollutants, including inorganic and organic compounds, is a growing concern [17, 18, 25]. The most harmful and hazardous pollutants that have been the focus of the GBNs' bioremediation research will be discussed in this chapter and listed according to the following classes: volatile organic compounds, inorganic metals, organic dyes, polycyclic aromatic hydrocarbons, pharmaceuticals, pesticides. In water sources and the atmosphere, these chemical pollutants also have the property of degrading and producing carcinogenic and mutagenic compounds [20]. In addition, microbial drug resistance can also be caused by bioaccumulation of contaminants such as pharmaceutical drugs, pesticides and their by-products in water bodies [20]. Therefore, pollution damages ecosystems but also affects human health, and the large number of pollutants emitted annually by industries and households have had a major impact on the environment and human existence. This chapter presents an overview of the properties of graphene and GBNs and their synthesis by classical and green methods. In addition, the use of GBNs will be described either in medicine (as theranostic tools) or in bioremediation (as adsorbents and photocatalysts) and these different aspects will be presented as part of

Graphene is a single layer of sp2 hybridized carbon atoms bound together in a

urgently necessary to address this clinical need [8, 24].

their versatile beneficial use when applied to human health.

**2. Graphene and GBNs**

**86**

planar 2D honeycomb structure.

[14–21].

*Theranostics - An Old Concept in New Clothing*

Because of the 2D carbon atomic honeycomb arrangement, each carbon atom is covalently bound to three neighbouring atoms inside a graphene layer. The tight C-C covalent bonds are responsible for graphene's extraordinary structural rigidity and a single defect-free graphene sheet is thus approximately 200 times mechanically stronger than steel. This explains the outstanding mechanical parameters of graphene: Young's modulus of 1 TPa, Poisson's ratio of 0.149 GPa and fracture strength of 130 GPa [27] (**Table 1**).

The mechanical properties of GO and rGO are significantly affected compared to graphene and depend on the surface groups and defects left over from oxidation or other treatment processes. However, the rigidity of these GBNs is still particularly high. Graphene's extraordinary structural rigidity and the still excellent mechanical properties of GBNs mean that these nanomaterials can potentially be used in medical devices, hydrogels, biodegradable films, electrospun fibres and other tissue engineering scaffolds to fill or strengthen the structures of these materials [39].

#### **3.2 Thermal and electrical properties**

Graphene is a monoatomic layer of sp<sup>2</sup> hybridized carbon atoms arranged as a honeycomb lattice. The π–π bonds below and above the carbon atomic plane impart exceptional thermal and electrical conductivity to graphene. In fact, a carbon atom normally has four electrons for bonding, but in graphene every atom allocates a single unbound electron that walks freely through the crystal lattice and leads to excellent electrical and thermal conductivity [28]. Defect-free graphene has therefore been reported to have a thermal conductivity between 4500 and 5200 W/mK [28]. Additionally, graphene exhibits an ultra-high electron mobility (25 <sup>10</sup><sup>4</sup> cm2 / <sup>V</sup>s) and an electrical conductivity of 10<sup>4</sup> S/cm [29] (**Table 1**).


Defects caused by GO and rGO manufacturing lead to disruption of graphene sp2 bonding orbitals and the addition of abundant surface groups that impede electron and heat flow, thereby reducing electronic and thermal conductivity of these GBNs [10, 40]. However, the electrical conductivity can be greatly improved upon GO reduction and conversion into rGO, although it is always smaller than that of graphene, as even after reduction, the rGO contains residual sp3 bonded carbon to oxygen, which interferes with the electron movement through the rest of the sp2 clusters [10, 40, 41]. As a result of its superior electrical conductivity and thermal properties, graphene is the nanomaterial of choice for electronic applications, but also for biomedical applications for cell potential assessment and as a substrate for biosen-

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

The first special physicochemical characteristics of graphene are its high surface area combined with the sp<sup>2</sup> network (**Table 1**). These two characteristics confer great reactivity to graphene. The graphene planar and electron networks can engage

additions, and reactions to carbine insertion. Moreover, the sp2 network enables π-π stacking interactions with aromatic structures existent in therapeutic agents, or biomolecules [26]. Finally, pristine graphene has a water contact angle of 95–100° [46] indicative of a hydrophobic nature, which means that therapeutic agents may also establish hydrophobic interactions with graphene via van der Waals interactions. The problem with the extreme hydrophobicity of graphene is the difficulty of dispersing it in aqueous media requiring the use of surfactants or other stabilizing

GO preserves unmodified areas of graphene, which are hydrophobic and capable

functionalization. However, it can be said that GO has a higher loading potential as it has additional epoxide and hydroxyl groups (**Table 1**) capable of forming hydrogen bonds and weak interactions with other groups of the therapeutic agents [47].

functionalized groups that are ionized at certain pH values (e.g. carboxyl groups are negatively charged at pH values greater than ≈4.5) [48]. The presence of ionizable groups and negative charges enhances the reactivity of GO, as additional electrostatic interactions can be established with therapeutic agents. Moreover, charged groups also reduce the water contact angle of GO to 30.7°, improving aqueous solubility and consequently improving colloidal stability [10, 40, 48]. In contrast, rGO (**Table 1**) contains higher number of defects that occurred during GO oxygen removal making it less hydrophobic than graphene (but more hydrophobic than

In conclusion, the physicochemical attributes of graphene and rGO make these materials suitable for the loading and delivery of hydrophobic or aromatic bearing therapeutic agents, but their hydrophobic nature creates problems of colloidal stability. In the context of the loading and delivery of therapeutic agents, GO is the GBN that reunites the best physicochemical features: large surface area; capacity of establishing π–π interactions, hydrogen bonds, hydrophobic interactions and electrostatic interactions; amphiphilic nature and colloidal stability [8, 10, 40].

In terms of electronic transitions, pristine graphene is considered to have a zero-band gap, i.e., no distance between the valence band and the conduction band

in various electrophilic replacement reactions such as click reactions, cyclo-

of establishing π–π interactions adequate for drug loading and non-covalent

In addition, GO has an amphiphilic nature, since it possesses other oxygen

sors and conductive cell culture devices [42–45].

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

agents to avoid agglomeration in biological fluids [10].

**3.3 Physicochemical properties**

GO) and less reactive than GO [41].

**3.4 Optical properties**

**89**

*Abbreviations and symbols: GBNs – Graphene based nanomaterials; GO – Graphene oxide; rGO – Reduced graphene oxide; GQDs – Graphene quantum dots; n/a – not available; NIR –near infrared;* E *– Young's modulus;* FS *– Fracture strength;* κ *– Thermal conductivity;* σ *– Electrical conductivity.*

#### **Table 1.**

*Summary of the properties of the family of graphene nanomaterials.*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

Defects caused by GO and rGO manufacturing lead to disruption of graphene sp2 bonding orbitals and the addition of abundant surface groups that impede electron and heat flow, thereby reducing electronic and thermal conductivity of these GBNs [10, 40]. However, the electrical conductivity can be greatly improved upon GO reduction and conversion into rGO, although it is always smaller than that of graphene, as even after reduction, the rGO contains residual sp3 bonded carbon to oxygen, which interferes with the electron movement through the rest of the sp2 clusters [10, 40, 41].

As a result of its superior electrical conductivity and thermal properties, graphene is the nanomaterial of choice for electronic applications, but also for biomedical applications for cell potential assessment and as a substrate for biosensors and conductive cell culture devices [42–45].

#### **3.3 Physicochemical properties**

**Graphene or GBNs**

**Structure and physicochemical properties**

*Theranostics - An Old Concept in New Clothing*

Graphene • Monoatomic layer of sp<sup>2</sup>

GO • Sp3 and sp2 domains

groups • Amphiphilic

rGO • Sp3 and larger sp2

GO)

GQDs • Small sp<sup>3</sup> and sp2

**Table 1.**

**88**

arranged as a honeycomb lattice • Hydrophobic • Establishes π-π stacking and hydrophobic interactions [26]

hybridized carbon atoms

with oxygen functional

• Establishes π-π stacking, H bonds, electrostatic and hydrophobic interactions [31]

domains than GO with less hydrophilic groups • Hydrophobic (less than graphene and more than

• Establishes π-π stacking, and hydrophobic interactions [31]

domains with oxygen functional groups • Amphiphilic

• Establishes π-π stacking, H bonds, electrostatic and hydrophobic interactions [31]

*strength;* κ *– Thermal conductivity;* σ *– Electrical conductivity.*

*Summary of the properties of the family of graphene nanomaterials.*

**Mechanical Properties**

• E = 1000 GPa • FS = 130 GPa [27]

• E = 220 GPa • FS = 120 GPa [32, 33]

• E = 250 GPa • FS n/a [32]

*Abbreviations and symbols: GBNs – Graphene based nanomaterials; GO – Graphene oxide; rGO – Reduced graphene oxide; GQDs – Graphene quantum dots; n/a – not available; NIR –near infrared;* E *– Young's modulus;* FS *– Fracture*

**Electrical and thermal Properties**

• σ = 10<sup>4</sup> S/cm • κ = 5000 W/ mK [28, 29]

• σ = 10<sup>1</sup> S/cm • κ = 0.5–1 W/ mK [29, 34]

• <sup>σ</sup> = 2 <sup>10</sup><sup>2</sup> S/cm • κ = 3–51 W/mK [34, 35]

• n/a • n/a • Intrinsic

**Optical properties**

• 97.7% optical transmittance • NIR absorption [30]

• Intrinsic

range

photoluminescence with UV excitation and tuneable emission in UV–Vis

• NIR absorption [31]

• 60–90% optical transmittance • Strong

[31] • Enhanced NIR absorption (6 times higher than GO)

[36]

range

photoluminescence quenching effect

photoluminescence with UV excitation and tuneable emission in UV–Vis

• NIR absorption [31]

The first special physicochemical characteristics of graphene are its high surface area combined with the sp<sup>2</sup> network (**Table 1**). These two characteristics confer great reactivity to graphene. The graphene planar and electron networks can engage in various electrophilic replacement reactions such as click reactions, cycloadditions, and reactions to carbine insertion. Moreover, the sp2 network enables π-π stacking interactions with aromatic structures existent in therapeutic agents, or biomolecules [26]. Finally, pristine graphene has a water contact angle of 95–100° [46] indicative of a hydrophobic nature, which means that therapeutic agents may also establish hydrophobic interactions with graphene via van der Waals interactions. The problem with the extreme hydrophobicity of graphene is the difficulty of dispersing it in aqueous media requiring the use of surfactants or other stabilizing agents to avoid agglomeration in biological fluids [10].

GO preserves unmodified areas of graphene, which are hydrophobic and capable of establishing π–π interactions adequate for drug loading and non-covalent functionalization. However, it can be said that GO has a higher loading potential as it has additional epoxide and hydroxyl groups (**Table 1**) capable of forming hydrogen bonds and weak interactions with other groups of the therapeutic agents [47]. In addition, GO has an amphiphilic nature, since it possesses other oxygen functionalized groups that are ionized at certain pH values (e.g. carboxyl groups are negatively charged at pH values greater than ≈4.5) [48]. The presence of ionizable groups and negative charges enhances the reactivity of GO, as additional electrostatic interactions can be established with therapeutic agents. Moreover, charged groups also reduce the water contact angle of GO to 30.7°, improving aqueous solubility and consequently improving colloidal stability [10, 40, 48]. In contrast, rGO (**Table 1**) contains higher number of defects that occurred during GO oxygen removal making it less hydrophobic than graphene (but more hydrophobic than GO) and less reactive than GO [41].

In conclusion, the physicochemical attributes of graphene and rGO make these materials suitable for the loading and delivery of hydrophobic or aromatic bearing therapeutic agents, but their hydrophobic nature creates problems of colloidal stability. In the context of the loading and delivery of therapeutic agents, GO is the GBN that reunites the best physicochemical features: large surface area; capacity of establishing π–π interactions, hydrogen bonds, hydrophobic interactions and electrostatic interactions; amphiphilic nature and colloidal stability [8, 10, 40].

#### **3.4 Optical properties**

In terms of electronic transitions, pristine graphene is considered to have a zero-band gap, i.e., no distance between the valence band and the conduction band [49–51]. This property makes graphene an outstanding electron conductive material, but a material that is unable to reach electronic excited states capable of optical excitation and visible emission. Pristine graphene is also a low-absorption nonphotoluminescent material with a 97.7% light transmittance of the total incident light across a wide range of wavelengths [30]. Defect-free or unmodified graphene is therefore not completely suitable for biomedical imaging as its light absorption and optical image contrast are poor. In addition, only when the size of the graphene is reduced to a nanoscale (e.g., in the case of GBNs) can photoluminescence be caused by an increase in the bandgap. In this regard, GO and GQDs are more interesting for biomedical imaging applications due to their intrinsic photoluminescence [49–51]. The bandgap changes that occur during GO reduction decreases rGO photoluminescence capacities.

*3.4.3 Edges*

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

**4. Synthesis of GBNs**

**4.1 Classical methods**

*4.1.1 Top-down methods*

ical, or physical [55].

**91**

Depending on the chemical structure of the GBNs edges different emission can be obtained: carbene-like edges have a zig-zag conformation that reduces the band gap energy resulting in a red-shift emission, whereas carbyne like armchair conformation increases the band gap energy resulting in a blue-shift emission [49–51]. Other important optical property that has been exploited for biosensing is the GBNs ability to act as efficient fluorescent quenchers for a variety of fluorophores

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Finally, a fundamental optical property is the capacity of graphene and its GBNs

Despite the enormous increase in the number of literature studies on graphene synthesis, the large-scale commercial development of graphene is still difficult to achieve [35]. Indeed, the development of cost-effective, highly reliable and scalable synthesis processes with high product yields and quality is a major challenge [35, 53]. In this chapter we will briefly present the methods to synthesize GBNs

The classical methods (**Figure 1**) used in the synthesis of GBNs can be classified

One of the most famous mechanical methods is the exfoliation of graphene from graphite firstly described by Geim and Novoselov [1]. This method is remarkably simple and consists of repeatedly gluing a graphite flake with adhesive tape and sticking it and peeling a dozen times [1, 56]. This process can cut a 1 μm thick graphite flake into a single-layer, thin graphene sample that is afterwards transferred to a clean substrate (Si/SiO2) by gently pressing the tape. Post-heat treatment

Chemical methods range from oxidation processes to other nano-cutting strategies using electrochemical or hydrothermal/solvothermal special oxidation. Oxidation may be handled by a one-or two-step method. The first step uses oxidizing agents (e.g., nitric acid, sulphuric acid, potassium chlorate, potassium permanganate) to oxidize graphite-based materials using the Hummer method or a modified version of the Hummer method [35, 54]. Graphite oxidation breaks the sp2 hybridized carbon sheets into a graphite sp2 domain surrounded by sp3 domains and several defects. Oxidized graphite is a stacked structure similar to graphite, but with a wider spacing between graphite sheets and several oxygen functional groups

Top-down methods of GBNs'synthesis start with graphite or other carbon sources such as carbon nanotubes, fullerenes or larger graphene sheets that are cut into smaller monoatomic carbon pieces. These methods may be mechanical, chem-

derivatives to have strong absorption in the NIR range, which means that these nanomaterials are capable of converting photons into heat by NIR irradiation, making them powerful agents for photothermal therapy [52]. In this matter, the reduction of GO to rGO in order to partially restore the aromatic, conjugate character of the graphene sheets increases the absorption of NIR by >6-fold [36].

through nonradiative electronic fluorophore-to-GBN energy transfer.

categorizing these methods in classical and green methods.

into two categories: top-down and bottom-up [35, 54].

may be used to remove residues of glue from the tape [37].

The origin of GBNs photoluminescence is still widely discussed and not completely elucidated, but three mechanisms have been proposed to explain this property [49–51]:

#### *3.4.1 Quantum confinement effect*

In the GBNs structure, the photoluminescent properties are determined by the confinement effect of the π and π\* electronic levels sites of the sp<sup>2</sup> clusters determined by the bandgap of σ and σ\* states of the sp3 matrix. Upon excitation, an electron from the valence band is promoted to the conduction band leaving a hole behind after absorbing a photon with higher energy than the band-gap energy [50]. This causes the formation of an exciton (a state of excited electron, also referred to as electron–hole pair). When the exciton returns to a lower level this results in the emission of fluorescence [50].

The natural separation distance between the positive charge (hole) and negative charge (electron) in the exciton is designated as the Bohr radius. If the size of the nanomaterial is smaller than the Bohr radius, there will be an electron confinement effect. Excitons have an infinite Bohr radius in graphene. Thus, GBNs, being graphene fragments of any size, will have a quantum confinement effect and, consequently, a photoluminescent effect [50]. GBNs also have a size-dependent photoluminescence as the space between the energy levels (bandgap) can be tuned to the lateral size of the nanomaterial. Smaller sizes have larger band gaps and emit at lower wavelengths, while larger ones have smaller band gaps and emit at higher wavelengths [50].

#### *3.4.2 Surface state*

Changing the surface state by the presence of impurities, defects or surface functionalization causes the formation of trap states, i.e., the exciton can be trapped under these conditions leading to a lower-energy radiative emission resulting in a red-shift emission [49–51]. This is what happens, for example, in oxidative graphite exfoliation processes to obtain GO, a process that induces the functionalization of the surface with oxygen functional groups, reducing the band gap energy and therefore causing fluorescence emission at higher wavelengths. This strategy can be used to enhance fluorescent emission in the near infrared (NIR) region known as 'biological window' where the autofluorescence from haemoglobin and biological tissue is negligible and therefore the signal-to-noise ratio can be improved [49–51].

Another proposed mechanism to change emission properties and produce a more emissive material is the creation of conjugated π domain upon a careful choice of the surface functionalization [49–51].

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

#### *3.4.3 Edges*

[49–51]. This property makes graphene an outstanding electron conductive material, but a material that is unable to reach electronic excited states capable of optical excitation and visible emission. Pristine graphene is also a low-absorption nonphotoluminescent material with a 97.7% light transmittance of the total incident light across a wide range of wavelengths [30]. Defect-free or unmodified graphene is therefore not completely suitable for biomedical imaging as its light absorption and optical image contrast are poor. In addition, only when the size of the graphene is reduced to a nanoscale (e.g., in the case of GBNs) can photoluminescence be caused by an increase in the bandgap. In this regard, GO and GQDs are more interesting for biomedical imaging applications due to their intrinsic photoluminescence [49–51]. The bandgap changes that occur during GO reduction decreases

The origin of GBNs photoluminescence is still widely discussed and not completely elucidated, but three mechanisms have been proposed to explain this

In the GBNs structure, the photoluminescent properties are determined by the confinement effect of the π and π\* electronic levels sites of the sp<sup>2</sup> clusters determined by the bandgap of σ and σ\* states of the sp3 matrix. Upon excitation, an electron from the valence band is promoted to the conduction band leaving a hole behind after absorbing a photon with higher energy than the band-gap energy [50]. This causes the formation of an exciton (a state of excited electron, also referred to as electron–hole pair). When the exciton returns to a lower level this results in the

The natural separation distance between the positive charge (hole) and negative charge (electron) in the exciton is designated as the Bohr radius. If the size of the nanomaterial is smaller than the Bohr radius, there will be an electron confinement effect. Excitons have an infinite Bohr radius in graphene. Thus, GBNs, being graphene fragments of any size, will have a quantum confinement effect and, consequently, a photoluminescent effect [50]. GBNs also have a size-dependent photoluminescence as the space between the energy levels (bandgap) can be tuned to the lateral size of the nanomaterial. Smaller sizes have larger band gaps and emit at lower wavelengths, while larger ones have smaller band gaps and emit at higher

Changing the surface state by the presence of impurities, defects or surface functionalization causes the formation of trap states, i.e., the exciton can be trapped under these conditions leading to a lower-energy radiative emission resulting in a red-shift emission [49–51]. This is what happens, for example, in oxidative graphite exfoliation processes to obtain GO, a process that induces the functionalization of the surface with oxygen functional groups, reducing the band gap energy and therefore causing fluorescence emission at higher wavelengths. This strategy can be used to enhance fluorescent emission in the near infrared (NIR) region known as 'biological window' where the autofluorescence from haemoglobin and biological tissue is negligible and therefore the signal-to-noise ratio can be improved [49–51]. Another proposed mechanism to change emission properties and produce a more emissive material is the creation of conjugated π domain upon a careful choice

rGO photoluminescence capacities.

*Theranostics - An Old Concept in New Clothing*

*3.4.1 Quantum confinement effect*

emission of fluorescence [50].

wavelengths [50].

*3.4.2 Surface state*

**90**

of the surface functionalization [49–51].

property [49–51]:

Depending on the chemical structure of the GBNs edges different emission can be obtained: carbene-like edges have a zig-zag conformation that reduces the band gap energy resulting in a red-shift emission, whereas carbyne like armchair conformation increases the band gap energy resulting in a blue-shift emission [49–51].

Other important optical property that has been exploited for biosensing is the GBNs ability to act as efficient fluorescent quenchers for a variety of fluorophores through nonradiative electronic fluorophore-to-GBN energy transfer.

Finally, a fundamental optical property is the capacity of graphene and its GBNs derivatives to have strong absorption in the NIR range, which means that these nanomaterials are capable of converting photons into heat by NIR irradiation, making them powerful agents for photothermal therapy [52]. In this matter, the reduction of GO to rGO in order to partially restore the aromatic, conjugate character of the graphene sheets increases the absorption of NIR by >6-fold [36].

#### **4. Synthesis of GBNs**

Despite the enormous increase in the number of literature studies on graphene synthesis, the large-scale commercial development of graphene is still difficult to achieve [35]. Indeed, the development of cost-effective, highly reliable and scalable synthesis processes with high product yields and quality is a major challenge [35, 53]. In this chapter we will briefly present the methods to synthesize GBNs categorizing these methods in classical and green methods.

#### **4.1 Classical methods**

The classical methods (**Figure 1**) used in the synthesis of GBNs can be classified into two categories: top-down and bottom-up [35, 54].

#### *4.1.1 Top-down methods*

Top-down methods of GBNs'synthesis start with graphite or other carbon sources such as carbon nanotubes, fullerenes or larger graphene sheets that are cut into smaller monoatomic carbon pieces. These methods may be mechanical, chemical, or physical [55].

One of the most famous mechanical methods is the exfoliation of graphene from graphite firstly described by Geim and Novoselov [1]. This method is remarkably simple and consists of repeatedly gluing a graphite flake with adhesive tape and sticking it and peeling a dozen times [1, 56]. This process can cut a 1 μm thick graphite flake into a single-layer, thin graphene sample that is afterwards transferred to a clean substrate (Si/SiO2) by gently pressing the tape. Post-heat treatment may be used to remove residues of glue from the tape [37].

Chemical methods range from oxidation processes to other nano-cutting strategies using electrochemical or hydrothermal/solvothermal special oxidation. Oxidation may be handled by a one-or two-step method. The first step uses oxidizing agents (e.g., nitric acid, sulphuric acid, potassium chlorate, potassium permanganate) to oxidize graphite-based materials using the Hummer method or a modified version of the Hummer method [35, 54]. Graphite oxidation breaks the sp2 hybridized carbon sheets into a graphite sp2 domain surrounded by sp3 domains and several defects. Oxidized graphite is a stacked structure similar to graphite, but with a wider spacing between graphite sheets and several oxygen functional groups

is the most widely used as it enables low-cost, large-scale production of high-quality materials [37, 55]. The main disadvantages of this method are the high toxicity of the chemical reaction by-product and the need for a fine choice of precursors. CVD production of graphene sheets occurs mainly in two stages [37, 55]. In the first step, the precursor (carbon containing gas) is injected into the reaction chamber. The chamber is subjected to high temperatures and the gas is pyrolyzed inside the chamber to obtain dissociated carbon atoms. This stage must occur on the surface of the substrate to avoid precipitation of carbon clusters during the gaseous phase [37, 55]. The second stage occurs due to the precursor's pyrolysis and corresponds to the deposition of a single atomic layer on the substrate. After the deposition and diffusion of the desired material on the substrate, the by-products dissociate from

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Bottom-up methods of synthesis are considered time-consuming and face challenges, therefore focusing on top-down methods that generate GO and rGO are more popular, particularly for the use of GBNs in theranostic applications [54].

As a new category of carbon materials, GBNs have attracted considerable attention due to their tunable photoluminescent properties, low toxicity, strong biocompatibility and excellent photostability [8]. However, despite their general use, standard GBNs'synthetic methods are generally expensive [57], complex and require toxic reagents [58]. The biocompatibility of the carbon content of GBNs may therefore be compromised by the toxicity associated with their classic production methods. Alternatively to classical approaches, GBNs' green approach synthesis, for example, by substituting chemical reducing agents for natural products, is a promising and fascinating field where the resulting material and synthetic processes are biocompatible and can be more safely integrated into living systems for bioapplications [59]. It is therefore important to invest in more sustainable, environ-

Nowadays, various green methods have been reported with interesting applications to produce GBNs alone or conjugated with other substances that enhance their bioactive effect. For example, most of the chemical methods used to date to produce graphene include harsh oxidizers and organic solvents, all of which are environmentally hazardous [60]. Alternatively to chemical methods, green graphene synthesis can be performed by electrochemical exfoliation of graphite into graphene sheets using a molten salt mixture. The molten salts are environmentally friendly and allow the interaction of alkali ions with graphite, which enables the formation of graphene nanosheets and flakes. In addition, this process reduces the number of defects in the graphene structure compared to classic chemical-rich processes [60]. GO's synthesis using classical methods is also very harmful to the environment. For example, in the Hummers method, approximately 1000 times more water than graphite must be used to remove excess oxidants after oxidation reactions, resulting in a large amount of wastewater containing mixed acids and heavy metal ions typically detected on GO sheets [61]. In addition, these methods are all timeconsuming, and take a few to hundreds of hours of oxidation. The oxidation time can be shortened to around 1 h simply by using stronger oxidizing mixtures that contribute to further contamination [61]. An alternative green approach to these classical methods is the electrolytic oxidation of graphite water. The GO obtained shows similar chemical composition, structure, and properties to those accomplished by the classical Hummers method and enables ultra-fast oxidation of the graphene lattice within a few seconds, which is more than 100 times faster than

the substrate and are pumped out of the chamber [37, 55].

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

mentally friendly, and biocompatible techniques.

currently available methods [61].

**93**

**4.2 Green methods**

#### **Figure 1.**

*Classical methods for the synthesis of Graphene-based Nanomaterials (GBNs). Abbreviations: CVD – Carbon Vapor Deposition; GO – Graphene Oxide; rGO – reduced Graphene Oxide; UV – Ultraviolet light.*

[35, 54]. In the second step, the oxidized graphite is exfoliated in GO sheets or in smaller parts such as GQDs using mechanical forces in aqueous solutions (sonication and centrifugation) [35, 54]. After obtaining GO sheets, it is possible to remove some of its oxygen functional groups by converting GO to rGO. This can be accomplished by thermal and UV treatment of GO or by chemical reduction using hydrazine, ascorbic acid, sodium borohydride, or hydroquinone [35, 54]. Electrochemical techniques include using chemical agents to assist in the growth of carbon electrodes. Carbon electrodes are broken up by electrochemical cutting, allowing for GBNs to be produced. The applied electric field draws the carbon particles from electrodes through graphite layer intercalation and radical reaction [55]. On the hydrothermal/ solvothermal oxidations defect-based carbon materials as GO and carbon nanotubes are cut under high temperature and pressure due to the action of strong alkaline medium. Some special photo-Fenton reactions may also break up GO to form GQDs. Among the physical methods of synthesis, arc discharge, laser ablation or reactive ion etching (RIE) nanolithography are the most widely used. RIE is one of the most efficient for controlling the size and chemical surface of GQDs and is also favorable for the study of some photoluminescent mechanisms [55].

#### *4.1.2 Bottom-up methods*

Bottom-up methods are based on the use of simple carbon molecules to build more complex structures such as graphene. These methods include the epitaxial growth of graphene layers on metal carbides by sublimation or by chemical vapor deposition (CVD) directly on metal surfaces [37]. It also includes organic synthesisbased methods in which intramolecular oxidative reactions using polycyclic aromatic hydrocarbons (PAHs) are widely used. Among the bottom-up methods, CVD *Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

is the most widely used as it enables low-cost, large-scale production of high-quality materials [37, 55]. The main disadvantages of this method are the high toxicity of the chemical reaction by-product and the need for a fine choice of precursors. CVD production of graphene sheets occurs mainly in two stages [37, 55]. In the first step, the precursor (carbon containing gas) is injected into the reaction chamber. The chamber is subjected to high temperatures and the gas is pyrolyzed inside the chamber to obtain dissociated carbon atoms. This stage must occur on the surface of the substrate to avoid precipitation of carbon clusters during the gaseous phase [37, 55]. The second stage occurs due to the precursor's pyrolysis and corresponds to the deposition of a single atomic layer on the substrate. After the deposition and diffusion of the desired material on the substrate, the by-products dissociate from the substrate and are pumped out of the chamber [37, 55].

Bottom-up methods of synthesis are considered time-consuming and face challenges, therefore focusing on top-down methods that generate GO and rGO are more popular, particularly for the use of GBNs in theranostic applications [54].

#### **4.2 Green methods**

[35, 54]. In the second step, the oxidized graphite is exfoliated in GO sheets or in smaller parts such as GQDs using mechanical forces in aqueous solutions (sonication and centrifugation) [35, 54]. After obtaining GO sheets, it is possible to remove some of its oxygen functional groups by converting GO to rGO. This can be accomplished by thermal and UV treatment of GO or by chemical reduction using hydrazine, ascorbic acid, sodium borohydride, or hydroquinone [35, 54]. Electrochemical techniques include using chemical agents to assist in the growth of carbon electrodes. Carbon electrodes are broken up by electrochemical cutting, allowing for GBNs to be produced. The applied electric field draws the carbon particles from electrodes through graphite layer intercalation and radical reaction [55]. On the hydrothermal/ solvothermal oxidations defect-based carbon materials as GO and carbon nanotubes are cut under high temperature and pressure due to the action of strong alkaline medium. Some special photo-Fenton reactions may also break up GO to form GQDs. Among the physical methods of synthesis, arc discharge, laser ablation or reactive ion etching (RIE) nanolithography are the most widely used. RIE is one of the most efficient for controlling the size and chemical surface of GQDs and is also favorable

*Classical methods for the synthesis of Graphene-based Nanomaterials (GBNs). Abbreviations: CVD – Carbon Vapor Deposition; GO – Graphene Oxide; rGO – reduced Graphene Oxide; UV – Ultraviolet light.*

Bottom-up methods are based on the use of simple carbon molecules to build more complex structures such as graphene. These methods include the epitaxial growth of graphene layers on metal carbides by sublimation or by chemical vapor deposition (CVD) directly on metal surfaces [37]. It also includes organic synthesisbased methods in which intramolecular oxidative reactions using polycyclic aromatic hydrocarbons (PAHs) are widely used. Among the bottom-up methods, CVD

for the study of some photoluminescent mechanisms [55].

*4.1.2 Bottom-up methods*

**92**

**Figure 1.**

*Theranostics - An Old Concept in New Clothing*

As a new category of carbon materials, GBNs have attracted considerable attention due to their tunable photoluminescent properties, low toxicity, strong biocompatibility and excellent photostability [8]. However, despite their general use, standard GBNs'synthetic methods are generally expensive [57], complex and require toxic reagents [58]. The biocompatibility of the carbon content of GBNs may therefore be compromised by the toxicity associated with their classic production methods. Alternatively to classical approaches, GBNs' green approach synthesis, for example, by substituting chemical reducing agents for natural products, is a promising and fascinating field where the resulting material and synthetic processes are biocompatible and can be more safely integrated into living systems for bioapplications [59]. It is therefore important to invest in more sustainable, environmentally friendly, and biocompatible techniques.

Nowadays, various green methods have been reported with interesting applications to produce GBNs alone or conjugated with other substances that enhance their bioactive effect. For example, most of the chemical methods used to date to produce graphene include harsh oxidizers and organic solvents, all of which are environmentally hazardous [60]. Alternatively to chemical methods, green graphene synthesis can be performed by electrochemical exfoliation of graphite into graphene sheets using a molten salt mixture. The molten salts are environmentally friendly and allow the interaction of alkali ions with graphite, which enables the formation of graphene nanosheets and flakes. In addition, this process reduces the number of defects in the graphene structure compared to classic chemical-rich processes [60].

GO's synthesis using classical methods is also very harmful to the environment. For example, in the Hummers method, approximately 1000 times more water than graphite must be used to remove excess oxidants after oxidation reactions, resulting in a large amount of wastewater containing mixed acids and heavy metal ions typically detected on GO sheets [61]. In addition, these methods are all timeconsuming, and take a few to hundreds of hours of oxidation. The oxidation time can be shortened to around 1 h simply by using stronger oxidizing mixtures that contribute to further contamination [61]. An alternative green approach to these classical methods is the electrolytic oxidation of graphite water. The GO obtained shows similar chemical composition, structure, and properties to those accomplished by the classical Hummers method and enables ultra-fast oxidation of the graphene lattice within a few seconds, which is more than 100 times faster than currently available methods [61].

The classical synthesis of rGO poses the same problems. The most used reducing agents, such as hydrazine, dimethylhydrazine and sodium borohydride, are highly toxic and remaining trace amounts of these toxic agents can have harmful effects, especially for bio-related applications [62]. In addition, the handling of hazardous waste produced by GO's reduction reaction to the production of rGO may dramatically increase costs on an industrial scale. Efforts have been made over the past few years to counteract toxicity issues by using natural reducing agents [62]. For example, plant extracts (aqueous leaf extracts of *Colocasia esculenta*, *Mesua ferralinn*, *Citrus sinensis*, tea polyphenol [62–65]), microorganisms (bacteria and baker's yeast [62, 66]), amino acids [67], bio-antioxidants (melatonin) [68], non-harmful acids (hydriodic acids, trifluoracetic acid), glucose and glucosamine [62, 69] are used as green reducing agents. Although the degree of reduction of GO by these strategies is typically lower than that of the hydrazine–based method, the excellent biocompatibility of the obtained rGO sheets may enhance their ability to be used in biological and biomedical fields [62].

Preparative methods of GQDs, which are typically manufactured with strong acids or organic solvents, often face severe challenges, and post-treatment with complicated methods remain necessary. Thus, raw materials made from natural renewable resources should be identified, as well as separation and post-treatment procedures that can be performed without complicated processes and without heavy/polluting waste generation [70]. For example, GQDs were synthesized using cotton cellulose, where cellulose and water were part of the reaction mechanism, in the absence of all other dangerous and chemical materials [71]. In another study, GQDs were synthesized by an organic solvent-free methodology using only deionized water and glucose as a precursor [72]. Microwave-assisted synthesis is another technique that has been reported to be able to produce, for example, carbon quantum dots in one-step using roasted chickpeas as carbon source [73] and also aqueous soluble GQDs using cow milk [73]. In another study, GQDs were produced by a simple, eco-friendly and single-pot hydrothermal reaction, with starch as a precursor [74]. In addition, a simple and high-yielding hydrothermal method has been reported to produce GQDs from glucose [75]. GQDs have also been produced using coal tar pitch, a by-product of the coking industries, oxidized with hydrogen peroxide under mild conditions [76]. Finally, also using hydrogen peroxide under mild conditions it is possible to produce GQDs by a greener hydrothermal synthesis using GO as precursor and without involving any harsh reagents [77].

diagnostic strategies. The therapeutic and diagnostic strategies of GBNs will be presented together with some examples of their use in the following subsections.

*Theranostic strategies of Graphene-based Nanomaterials. Abbreviations: CT—Computed Tomography; IR-TI—Infrared Thermal Imaging; MHT—Magnetic Hyperthermia Therapy; MRI—Magnetic Resonance Imaging; PAI—Photoacoustic Imaging; PAT—Photoacoustic Therapy; PDT—Photodynamic Therapy; PET— Positron Emission Tomography; PL—Photoluminescence; PTT—Photothermal Therapy; SERS—Super Enhanced Raman Spectroscopy; SPECT—Single Photon Emission Computed Tomography; USI—Ultrasound*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

The GBNs' intrinsic properties have paved the way for the advancement of

Chemotherapy implies the use of anticancer drugs which, by several mechanisms (i.e., interfering with angiogenesis and cell division), may result in cellular damage/stress and may lead to cell death if apoptosis is triggered. The chemotherapeutic arsenal is widely known as it is the basis of classical cancer therapy [8]. Furthermore, by incorporating these drugs into nanocarriers like GBNs, the toxic effects of anticancer drugs in healthy cells that are not affected by cancer can be reduced [8]. Indeed, GBNs (especially GO) have a high drug loading ratio of hydrophilic and lipophilic anticancer drugs, due to the combination of a large surface area and the presence of delocalized π electrons, as well as chemical polar groups [8]. The diverse range of potential chemical interactions between anticancer drugs and GBNs has conferred to these nanocarriers an important role in chemotherapy, as drug loading ratios can exceed 200 wt%, which is unusually high compared to other nanocarriers [78]. For example, the commercial liposomal formulations Caelyx® and Doxil® containing the anticancer drug doxorubicin have a drug load of 16 wt %, while the majority of GBNs' formulations can meet the drug loading values from 55 wt % to 133 wt % [79–85]. GBNs loaded with another anticancer drug, paclitaxel, also achieved a remarkably effective drug loading of 90 wt % compared to commercial formulations containing this drug: Taxol® and

Abraxane® with a drug loading of 1 wt % and 11 wt %, respectively [86].

Gene therapy requires the incorporation of genes, gene segments or oligonucleotides in nanocarriers that provide protection against enzyme-induced degradation and/or inactivation of the genetic material [8, 87]. When used in cancer, the mechanism of action of this therapeutic strategy is based on: (i) deactivation of oncogenes; (ii) substitution of non-functioning tumor suppressor genes; (iii) inducing cell death or repair of normal cell function; (iv) defense of normal cells from

**5.1 Therapeutic strategies**

**Figure 2.**

*Imaging.*

**95**

*5.1.1 Chemotherapy and gene therapy*

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

approaches to chemotherapy and gene therapy.

In conclusion, green synthesis of GBNs is an essential area of research that should be promoted within the scientific community once it presents many advantages: (a) it is inexpensive and renewable precursors are easily obtained; (b) it is environmentally-friendly, once no hazardous reagents are needed; (c) it involves simple methods, usually in one-step or one-pot; (d) normally avoids any complicated post-processes [71]; (e) originates products with great biocompatibility [72].

#### **5. Applications of GBNs in therapy and diagnostics (theranostics)**

As described earlier, the interesting properties of GBNs have placed these nanomaterials as ideal for creation of theranostic strategies particularly used in the therapy and therapy monitorization (diagnostic) of cancer [8]. Current treatments involve several variations of different strategies that can be used for therapeutic and diagnostic ends. Because each strategy has various inherent advantages and different mechanisms of actuation, a mixture of complementary strategies may result in a synergistic effect. **Figure 2** presents a schematization of the main therapeutic and

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

**Figure 2.**

The classical synthesis of rGO poses the same problems. The most used reducing agents, such as hydrazine, dimethylhydrazine and sodium borohydride, are highly toxic and remaining trace amounts of these toxic agents can have harmful effects, especially for bio-related applications [62]. In addition, the handling of hazardous waste produced by GO's reduction reaction to the production of rGO may dramatically increase costs on an industrial scale. Efforts have been made over the past few years to counteract toxicity issues by using natural reducing agents [62]. For example, plant extracts (aqueous leaf extracts of *Colocasia esculenta*, *Mesua ferralinn*, *Citrus sinensis*, tea polyphenol [62–65]), microorganisms (bacteria and baker's yeast [62, 66]), amino acids [67], bio-antioxidants (melatonin) [68], non-harmful acids (hydriodic acids, trifluoracetic acid), glucose and glucosamine [62, 69] are used as green reducing agents. Although the degree of reduction of GO by these strategies is typically lower than that of the hydrazine–based method, the excellent biocompatibility of the obtained rGO sheets may enhance their ability to be used in biological

Preparative methods of GQDs, which are typically manufactured with strong acids or organic solvents, often face severe challenges, and post-treatment with complicated methods remain necessary. Thus, raw materials made from natural renewable resources should be identified, as well as separation and post-treatment procedures that can be performed without complicated processes and without heavy/polluting waste generation [70]. For example, GQDs were synthesized using cotton cellulose, where cellulose and water were part of the reaction mechanism, in the absence of all other dangerous and chemical materials [71]. In another study, GQDs were synthesized by an organic solvent-free methodology using only deionized water and glucose as a precursor [72]. Microwave-assisted synthesis is another technique that has been reported to be able to produce, for example, carbon quantum dots in one-step using roasted chickpeas as carbon source [73] and also aqueous soluble GQDs using cow milk [73]. In another study, GQDs were produced by a simple, eco-friendly and single-pot hydrothermal reaction, with starch as a precursor [74]. In addition, a simple and high-yielding hydrothermal method has been reported to produce GQDs from glucose [75]. GQDs have also been produced using coal tar pitch, a by-product of the coking industries, oxidized with hydrogen peroxide under mild conditions [76]. Finally, also using hydrogen peroxide under mild conditions it is possible to produce GQDs by a greener hydrothermal synthesis using GO as precursor and without involving any harsh

In conclusion, green synthesis of GBNs is an essential area of research that should be promoted within the scientific community once it presents many advantages: (a) it is inexpensive and renewable precursors are easily obtained; (b) it is environmentally-friendly, once no hazardous reagents are needed; (c) it involves simple methods, usually in one-step or one-pot; (d) normally avoids any complicated post-processes [71]; (e) originates products with great biocompatibility [72].

**5. Applications of GBNs in therapy and diagnostics (theranostics)**

As described earlier, the interesting properties of GBNs have placed these nanomaterials as ideal for creation of theranostic strategies particularly used in the therapy and therapy monitorization (diagnostic) of cancer [8]. Current treatments involve several variations of different strategies that can be used for therapeutic and diagnostic ends. Because each strategy has various inherent advantages and different mechanisms of actuation, a mixture of complementary strategies may result in a synergistic effect. **Figure 2** presents a schematization of the main therapeutic and

and biomedical fields [62].

*Theranostics - An Old Concept in New Clothing*

reagents [77].

**94**

*Theranostic strategies of Graphene-based Nanomaterials. Abbreviations: CT—Computed Tomography; IR-TI—Infrared Thermal Imaging; MHT—Magnetic Hyperthermia Therapy; MRI—Magnetic Resonance Imaging; PAI—Photoacoustic Imaging; PAT—Photoacoustic Therapy; PDT—Photodynamic Therapy; PET— Positron Emission Tomography; PL—Photoluminescence; PTT—Photothermal Therapy; SERS—Super Enhanced Raman Spectroscopy; SPECT—Single Photon Emission Computed Tomography; USI—Ultrasound Imaging.*

diagnostic strategies. The therapeutic and diagnostic strategies of GBNs will be presented together with some examples of their use in the following subsections.

#### **5.1 Therapeutic strategies**

#### *5.1.1 Chemotherapy and gene therapy*

The GBNs' intrinsic properties have paved the way for the advancement of approaches to chemotherapy and gene therapy.

Chemotherapy implies the use of anticancer drugs which, by several mechanisms (i.e., interfering with angiogenesis and cell division), may result in cellular damage/stress and may lead to cell death if apoptosis is triggered. The chemotherapeutic arsenal is widely known as it is the basis of classical cancer therapy [8]. Furthermore, by incorporating these drugs into nanocarriers like GBNs, the toxic effects of anticancer drugs in healthy cells that are not affected by cancer can be reduced [8]. Indeed, GBNs (especially GO) have a high drug loading ratio of hydrophilic and lipophilic anticancer drugs, due to the combination of a large surface area and the presence of delocalized π electrons, as well as chemical polar groups [8]. The diverse range of potential chemical interactions between anticancer drugs and GBNs has conferred to these nanocarriers an important role in chemotherapy, as drug loading ratios can exceed 200 wt%, which is unusually high compared to other nanocarriers [78]. For example, the commercial liposomal formulations Caelyx® and Doxil® containing the anticancer drug doxorubicin have a drug load of 16 wt %, while the majority of GBNs' formulations can meet the drug loading values from 55 wt % to 133 wt % [79–85]. GBNs loaded with another anticancer drug, paclitaxel, also achieved a remarkably effective drug loading of 90 wt % compared to commercial formulations containing this drug: Taxol® and Abraxane® with a drug loading of 1 wt % and 11 wt %, respectively [86].

Gene therapy requires the incorporation of genes, gene segments or oligonucleotides in nanocarriers that provide protection against enzyme-induced degradation and/or inactivation of the genetic material [8, 87]. When used in cancer, the mechanism of action of this therapeutic strategy is based on: (i) deactivation of oncogenes; (ii) substitution of non-functioning tumor suppressor genes; (iii) inducing cell death or repair of normal cell function; (iv) defense of normal cells from

drug-induced toxicity or activation of immune cells for the destruction of cancer cells [8, 87]. The same favorable properties of GBNs for chemotherapy are valid for explaining their use in gene therapy. Indeed, GBNs have shown the ability to efficiently condense genetic material by π-π stacking interactions, avoiding endonuclease's degradation of nucleic acids [40, 88, 89].

**5.2 Diagnostic strategies**

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

GBNs possess attractive optical features applied to the monitoring of therapeutic efficacy. As a result, GBNs act as dye-free labeling to follow the delivery of therapeutic nanosystems to cells. Due to the quantum confinement effect that exists when the sizes of GBNs are smaller than their exciton Bohr radii, the nano-sized material has non-blinking photoluminescence (PL) and photostability [8]. GBNs therefore emit low-energy fluorescence when excited by high-energy light (usually UV or visible light) and GBNs' fluorescence intensity remains strong under confocal laser lighting. GQDs are among the most used GBNs for their PL [88, 89, 99, 115,

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Upon conjugation of GBNs with upconversion luminescence nanoparticles (UCNPs), such as: NaYF4:Yb3+, Er3+ or NaYF4: Yb3+, Tm3+ an anti-Stoke emission occurs when two or more low-energy photons from NIR light are absorbed to generate higher energy emissions in the visible region. The conjugation with UCNPs confers to GBNs an even more fascinating PL property, as in this case excitation with NIR light produces emission at lower wavelengths. The advantages of this upconversion PL are due to the use of NIR light excitation, which reduces

autofluorescence of biological tissues and increases penetration depths, thus reduc-

The fluorescence quenching capability demonstrated by GBNs resulting from fluorescence resonance energy transfer (FRET) or non-radiative dipole–dipole interactions between fluorescence species and GBNs is also important. The fluorescence quenching effect is used as an external diagnostic feature that enables the release of GBNs' cargo to be identified [8]. Indeed, when GBNs interact with fluorescent cargo (drugs or other active substances) they reduce their fluorescence

emission, but when the cargo is released, the fluorescence emission is reset

Infrared thermal imaging (IR-TI) is a diagnostic strategy based on thermal changes due to radiation absorption. Light absorbed and not lost by emission results in heat that can be registered as an image [8]. As a result, the GBNs photothermal conversion properties used in PTT can also be used as a therapy-guiding strategy under an IR-TI non-labelling technique. The use of the NIR laser to trigger a PTT effect can be detected by means of a visible thermal field signal, which is especially important because of its non-invasive nature and because it provides real-time images [8]. Provided that PTT is one of the treatment modalities most commonly used by GBN-producing researchers for biomedical applications, IR-TI is also widely used, as both strategies (PTT and IR-TI) are often used together

Raman scattering-based spectroscopy can be used as a diagnostic technique to obtain morphological and chemical information from accessible tissue surfaces, e.g., skin, gastrointestinal tract, or intraoperatively. This imaging technique combines the surface imaging of the tissues with the Raman spectra provided by its molecular components [8]. When visible or NIR light interacts with the surface material it originates inelastic scattering of photons (Raman scattering) that display a shift in

ing photo-damage of healthy tissues [8, 95, 101, 121, 124].

[92–99, 105–108, 110, 112, 113, 119, 120, 124, 131–133].

*5.2.3 Raman spectroscopy and surface enhanced Raman spectroscopy*

*5.2.1 Photoluminescence*

125–129].

[79, 130].

**97**

*5.2.2 Infrared thermal imaging*

#### *5.1.2 Hyperthermia*

Hyperthermia is a therapeutic strategy that causes the temperature rise to kill cancer cells. Mild hyperthermia (temperature rise to 43–50°C) induces increased membrane permeability, defective membrane transport, metabolic signaling disturbance leading to cell apoptosis. Extreme hyperthermia (temperature rise >50°C) causes necrotic cell death due to cell membrane disruption and protein denaturation [8].

The optical and thermal properties of GBNs make these nanosystems desirable for their use in the hyperthermia treatment of cancer cells. GBNs have a wide absorption in the NIR region (700–1100 nm) and can convert it into thermal energy causing local hyperthermia. At the same time, the hyperthermia effect can reduce GBNs' oxygen functional groups causing the release of gas. The formation and collapse of gas bubbles contributes to the development of a microcavitation environment often responsible for the death of cancer cells. Hyperthermia therapy strategy, based on the conversion of absorbed NIR to thermal energy, is known as photothermal therapy (PTT) [8, 90, 91]. Over the last 6 years, PTT has been the therapeutic strategy most explored by researchers working with GBNs [83, 92–97]. This is primarily because this strategy has the advantage of not needing cell internalization of GBNs while maintaining a deep penetration of biological tissues [8]. The efficacy of PTT to destroy cancer cells has also been improved by conjugation of GBNs with other narrow-bandgap materials [84, 85, 98–112]. Moreover, while GO has been the perfect GBN for chemotherapy and gene therapy, rGO is the preferred nanomaterial for PTT because it has an NIR absorption 6 times higher than GO [8, 113].

Another strategy to increase the death of cancer cells by hyperthermia is to combine magnetic hyperthermia (MHT) with PTT through conjugation of GBNs with magnetic nanoparticles (MNPs) [82, 113]. MNPs exposed to an external alternating magnetic field can convert magnetic energy into thermal energy by Néel or Brownian relaxation mechanisms. When the application of the magnetic field is faster than the relaxation time of the MNPs, the delay in magnetic moment relaxation induces MHT [8].

#### *5.1.3 Photodynamic therapy*

Recently, GBNs have also been applied to photodynamic therapy (PDT) strategy used to kill cancer cells [8, 52, 114]. This strategy requires a photosensitizer (PS) agent to be loaded into the GBNs by π-π stacking and/or hydrophobic interactions. Upon photon absorption, the PS agent will be excited to a singlet state after which it decays into a low-energy excited triplet state through intersystem crossing. Then, in the excited triplet state, PS transfers an electron to: (i) different molecules producing reactive oxygen species (ROS): *O*• <sup>2</sup> , *H*2*O*2, *HO*• or (ii) oxygen originating <sup>1</sup> *O*2. ROS interact with cellular components of cancer cells (lipids, proteins, nucleic acids) causing oxidative stress and ultimately cell death [8, 52, 114].

PDT is commonly used in conjunction with PTT to benefit from the synergistic influence of both therapeutic strategies [101, 108, 115–124].

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

#### **5.2 Diagnostic strategies**

#### *5.2.1 Photoluminescence*

drug-induced toxicity or activation of immune cells for the destruction of cancer cells [8, 87]. The same favorable properties of GBNs for chemotherapy are valid for explaining their use in gene therapy. Indeed, GBNs have shown the ability to efficiently condense genetic material by π-π stacking interactions, avoiding

Hyperthermia is a therapeutic strategy that causes the temperature rise to kill cancer cells. Mild hyperthermia (temperature rise to 43–50°C) induces increased membrane permeability, defective membrane transport, metabolic signaling disturbance leading to cell apoptosis. Extreme hyperthermia (temperature rise >50°C) causes necrotic cell death due to cell membrane disruption and protein

The optical and thermal properties of GBNs make these nanosystems desirable

for their use in the hyperthermia treatment of cancer cells. GBNs have a wide absorption in the NIR region (700–1100 nm) and can convert it into thermal energy causing local hyperthermia. At the same time, the hyperthermia effect can reduce GBNs' oxygen functional groups causing the release of gas. The formation and collapse of gas bubbles contributes to the development of a microcavitation environment often responsible for the death of cancer cells. Hyperthermia therapy strategy, based on the conversion of absorbed NIR to thermal energy, is known as photothermal therapy (PTT) [8, 90, 91]. Over the last 6 years, PTT has been the therapeutic strategy most explored by researchers working with GBNs [83, 92–97]. This is primarily because this strategy has the advantage of not needing cell internalization of GBNs while maintaining a deep penetration of biological tissues [8]. The efficacy of PTT to destroy cancer cells has also been improved by conjugation of GBNs with other narrow-bandgap materials [84, 85, 98–112]. Moreover, while GO has been the perfect GBN for chemotherapy and gene therapy, rGO is the preferred nanomaterial for PTT because it has an NIR absorption 6 times higher

Another strategy to increase the death of cancer cells by hyperthermia is to combine magnetic hyperthermia (MHT) with PTT through conjugation of GBNs with magnetic nanoparticles (MNPs) [82, 113]. MNPs exposed to an external alternating magnetic field can convert magnetic energy into thermal energy by Néel or Brownian relaxation mechanisms. When the application of the magnetic field is faster than the relaxation time of the MNPs, the delay in magnetic moment relaxa-

Recently, GBNs have also been applied to photodynamic therapy (PDT) strategy used to kill cancer cells [8, 52, 114]. This strategy requires a photosensitizer (PS) agent to be loaded into the GBNs by π-π stacking and/or hydrophobic interactions. Upon photon absorption, the PS agent will be excited to a singlet state after which it decays into a low-energy excited triplet state through intersystem crossing. Then, in the excited triplet state, PS transfers an electron to: (i) different molecules produc-

<sup>2</sup> , *H*2*O*2, *HO*•

PDT is commonly used in conjunction with PTT to benefit from the synergistic

ROS interact with cellular components of cancer cells (lipids, proteins, nucleic

acids) causing oxidative stress and ultimately cell death [8, 52, 114].

influence of both therapeutic strategies [101, 108, 115–124].

or (ii) oxygen originating <sup>1</sup>

*O*2.

endonuclease's degradation of nucleic acids [40, 88, 89].

*Theranostics - An Old Concept in New Clothing*

*5.1.2 Hyperthermia*

denaturation [8].

than GO [8, 113].

tion induces MHT [8].

**96**

*5.1.3 Photodynamic therapy*

ing reactive oxygen species (ROS): *O*•

GBNs possess attractive optical features applied to the monitoring of therapeutic efficacy. As a result, GBNs act as dye-free labeling to follow the delivery of therapeutic nanosystems to cells. Due to the quantum confinement effect that exists when the sizes of GBNs are smaller than their exciton Bohr radii, the nano-sized material has non-blinking photoluminescence (PL) and photostability [8]. GBNs therefore emit low-energy fluorescence when excited by high-energy light (usually UV or visible light) and GBNs' fluorescence intensity remains strong under confocal laser lighting. GQDs are among the most used GBNs for their PL [88, 89, 99, 115, 125–129].

Upon conjugation of GBNs with upconversion luminescence nanoparticles (UCNPs), such as: NaYF4:Yb3+, Er3+ or NaYF4: Yb3+, Tm3+ an anti-Stoke emission occurs when two or more low-energy photons from NIR light are absorbed to generate higher energy emissions in the visible region. The conjugation with UCNPs confers to GBNs an even more fascinating PL property, as in this case excitation with NIR light produces emission at lower wavelengths. The advantages of this upconversion PL are due to the use of NIR light excitation, which reduces autofluorescence of biological tissues and increases penetration depths, thus reducing photo-damage of healthy tissues [8, 95, 101, 121, 124].

The fluorescence quenching capability demonstrated by GBNs resulting from fluorescence resonance energy transfer (FRET) or non-radiative dipole–dipole interactions between fluorescence species and GBNs is also important. The fluorescence quenching effect is used as an external diagnostic feature that enables the release of GBNs' cargo to be identified [8]. Indeed, when GBNs interact with fluorescent cargo (drugs or other active substances) they reduce their fluorescence emission, but when the cargo is released, the fluorescence emission is reset [79, 130].

#### *5.2.2 Infrared thermal imaging*

Infrared thermal imaging (IR-TI) is a diagnostic strategy based on thermal changes due to radiation absorption. Light absorbed and not lost by emission results in heat that can be registered as an image [8]. As a result, the GBNs photothermal conversion properties used in PTT can also be used as a therapy-guiding strategy under an IR-TI non-labelling technique. The use of the NIR laser to trigger a PTT effect can be detected by means of a visible thermal field signal, which is especially important because of its non-invasive nature and because it provides real-time images [8]. Provided that PTT is one of the treatment modalities most commonly used by GBN-producing researchers for biomedical applications, IR-TI is also widely used, as both strategies (PTT and IR-TI) are often used together [92–99, 105–108, 110, 112, 113, 119, 120, 124, 131–133].

#### *5.2.3 Raman spectroscopy and surface enhanced Raman spectroscopy*

Raman scattering-based spectroscopy can be used as a diagnostic technique to obtain morphological and chemical information from accessible tissue surfaces, e.g., skin, gastrointestinal tract, or intraoperatively. This imaging technique combines the surface imaging of the tissues with the Raman spectra provided by its molecular components [8]. When visible or NIR light interacts with the surface material it originates inelastic scattering of photons (Raman scattering) that display a shift in

frequency. The energy shift provides information on the vibrational modes in the system. GBNs usually demonstrate the required Raman scattering intensity, exhibiting the standard D, G and 2-D band characteristics of the vibrational modes in the range 1000–3000 cm<sup>1</sup> . As a result, the delivery of GBNs used as cancer therapeutic tools can be followed by Raman imaging of the tissues [8].

*5.2.7 Magnetic resonance imaging*

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

better image [113].

animal health [17–20, 25, 135].

bioremediation efficiency.

*6.1.1 Adsorption*

characteristics [25]:

reusing;

adsorption.

**99**

**6. Application of GBNs in bioremediation**

Magnetic resonance imaging (MRI) consists of the application of radiofrequency

GBNs offer a holistic approach to health. In fact, in the previous sections, we described the great potential of GBNs in human health due to their role in therapy and diagnosis. Moreover, GBNs or their functionalized derivatives are cutting-edge materials used in bioremediation, and their remarkable properties can be used to mitigate environmental contaminants, as well as to improve human, plant, and

The following sections describe the properties of GBNs that favor their use in bioremediation and the major pollutants on which GBNs have demonstrated their

Graphene oxidation to GO and rGO reinforces its properties and improves its hydrophilic nature, thereby enhancing its ability to associate with contaminants either physically or chemically. This association can be processed by adsorption of contaminants on the surface of GBNs or by the oxidation breakdown of contaminants, by photocatalysis or other advanced oxidation processes (AOPs) [18].

One of the most widely used processes for bioremediation is chemical and physical adsorption. The adsorption capacity of materials depends on several

i. good mechanical strength for handling and possibly regenerating and

ii. strong wettability to ensure use in the adsorption of water pollutants;

iv. large surface area and different functional groups to promote chemical

As previously described GBNs obey to all these requirements and hold great potential as adsorbent materials. GBNs have a large surface area and excellent

iii. high porosity in favor of physical adsorption;

**6.1 GBNs properties and processes involved in bioremediation**

pulses and is derived from the interaction between the water protons and the magnetic field applied. The resulting image is produced by the pattern of absorption and emission of the electromagnetic wave [8]. In order to increase the visibility of anatomical structures, contrast agents (MRI probes) are used to reduce the relaxation times of water protons inside body tissues [8]. The unusual wide surface area and high loading capacity of the GBNs have also proven to be very advantageous for carrying MRI probes [81–83, 113, 124, 130]. In addition, the high molecular weight of GBNs can reduce the rotational motion of the water proton, increase the relaxation time and increase the *in vivo* half-life of the MRI contrast agent, resulting in a

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Raman imaging is an even more effective diagnostic strategy when GBNs are associated with gold and silver nanoparticles. In this case, the Raman signals of GBNs are significantly improved by the surface enhanced Raman scattering (SERS). Indeed, SERS occurs when molecules are adsorbed or located near a metallic nanostructure, i.e., the improvement of the Raman scattering occurs due to the resonant interaction of light with the surface plasmons that are excited at the surface of the metallic nanostructure. Using this strategy, SERS can be used to combine microscope cell imaging with Raman spectroscopy, mapping the presence of GBNs in the tumor tissue of the cell [8, 103].

#### *5.2.4 Ultrasound Imaging*

Using the electrical properties of GBNs, it is possible to image these nanosystems in their journey through the body using ultrasound imaging (USI) strategies [96, 102]. USI is therefore based on the conversion of electrical signals to ultrasound waves that penetrate the body and biological tissues. Some of these ultrasound waves are reflected and transformed by a transducer into electrical signals that are handled and displayed as an image [8].

#### *5.2.5 Photoacoustic imaging*

Photoacoustic Imaging (PAI) is another diagnostic strategy that benefits from the NIR absorption capacity of GBNs and enables monitoring their distribution in body tissues. When tissues are irradiated with NIR short laser pulses, locally dispersed GBNs absorb energy and generate heat that leads to thermoelastic expansion followed by contraction and consequent emission of mechanical pressure waves at ultrasonic frequencies [8]. Periodic sound waves produced can be sensed by ultrasonic transducers creating an image by mapping the original absorbed energy distribution [8]. Among the GBNs, rGO has gained interest as a PAI contrast agent due to its higher NIR absorbance properties [85, 96, 125, 134]. In spite of the improved PAI properties of rGO, GO nanomaterials compensate for their lower NIR absorption with higher loading capacity. In some studies, thus, GO nanomaterials were loaded with other narrow-band gap materials as a solution to increase NIR absorption and thus also attained PAI diagnostic modality [80, 100, 105, 110].

#### *5.2.6 Tomography*

Tomography is a nuclear medicine imaging technique where a cyclotron is used to produce short or ultra-short-lived radionuclides that decay with the emission of: (i) positron, in the case of Positron Emission Tomography (PET); (ii) γ rays in the case of Single Photon Emission Computed Tomography (SPECT); and multiple X-rays in the case of Computed Tomography (CT). All these techniques rely on differential levels of the radiation attenuation within the body to create threedimensional, high-contrast anatomical images that allow for delineation between various structures [8].

The physicochemical properties of GBNs promote the loading of these nanocarriers with radionuclides that enable tomography imaging of tissues [85, 102, 124, 132].

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

#### *5.2.7 Magnetic resonance imaging*

frequency. The energy shift provides information on the vibrational modes in the system. GBNs usually demonstrate the required Raman scattering intensity, exhibiting the standard D, G and 2-D band characteristics of the vibrational modes

Raman imaging is an even more effective diagnostic strategy when GBNs are associated with gold and silver nanoparticles. In this case, the Raman signals of GBNs are significantly improved by the surface enhanced Raman scattering (SERS). Indeed, SERS occurs when molecules are adsorbed or located near a metallic nanostructure, i.e., the improvement of the Raman scattering occurs due to the resonant interaction of light with the surface plasmons that are excited at the surface of the metallic nanostructure. Using this strategy, SERS can be used to combine microscope cell imaging with Raman spectroscopy, mapping the presence of GBNs in the

Using the electrical properties of GBNs, it is possible to image these nanosystems

Photoacoustic Imaging (PAI) is another diagnostic strategy that benefits from the NIR absorption capacity of GBNs and enables monitoring their distribution in body tissues. When tissues are irradiated with NIR short laser pulses, locally dispersed GBNs absorb energy and generate heat that leads to thermoelastic expansion followed by contraction and consequent emission of mechanical pressure waves at ultrasonic frequencies [8]. Periodic sound waves produced can be sensed by ultrasonic transducers creating an image by mapping the original absorbed energy distribution [8]. Among the GBNs, rGO has gained interest as a PAI contrast agent due to its higher NIR absorbance properties [85, 96, 125, 134]. In spite of the improved PAI properties of rGO, GO nanomaterials compensate for their lower NIR absorption with higher loading capacity. In some studies, thus, GO nanomaterials were loaded with other narrow-band gap materials as a solution to increase NIR absorp-

tion and thus also attained PAI diagnostic modality [80, 100, 105, 110].

Tomography is a nuclear medicine imaging technique where a cyclotron is used to produce short or ultra-short-lived radionuclides that decay with the emission of: (i) positron, in the case of Positron Emission Tomography (PET); (ii) γ rays in the case of Single Photon Emission Computed Tomography (SPECT); and multiple X-rays in the case of Computed Tomography (CT). All these techniques rely on differential levels of the radiation attenuation within the body to create threedimensional, high-contrast anatomical images that allow for delineation between

The physicochemical properties of GBNs promote the loading of these nanocarriers with radionuclides that enable tomography imaging of tissues

in their journey through the body using ultrasound imaging (USI) strategies [96, 102]. USI is therefore based on the conversion of electrical signals to ultrasound waves that penetrate the body and biological tissues. Some of these ultrasound waves are reflected and transformed by a transducer into electrical signals that are

therapeutic tools can be followed by Raman imaging of the tissues [8].

. As a result, the delivery of GBNs used as cancer

in the range 1000–3000 cm<sup>1</sup>

*Theranostics - An Old Concept in New Clothing*

tumor tissue of the cell [8, 103].

handled and displayed as an image [8].

*5.2.4 Ultrasound Imaging*

*5.2.5 Photoacoustic imaging*

*5.2.6 Tomography*

various structures [8].

[85, 102, 124, 132].

**98**

Magnetic resonance imaging (MRI) consists of the application of radiofrequency pulses and is derived from the interaction between the water protons and the magnetic field applied. The resulting image is produced by the pattern of absorption and emission of the electromagnetic wave [8]. In order to increase the visibility of anatomical structures, contrast agents (MRI probes) are used to reduce the relaxation times of water protons inside body tissues [8]. The unusual wide surface area and high loading capacity of the GBNs have also proven to be very advantageous for carrying MRI probes [81–83, 113, 124, 130]. In addition, the high molecular weight of GBNs can reduce the rotational motion of the water proton, increase the relaxation time and increase the *in vivo* half-life of the MRI contrast agent, resulting in a better image [113].

#### **6. Application of GBNs in bioremediation**

GBNs offer a holistic approach to health. In fact, in the previous sections, we described the great potential of GBNs in human health due to their role in therapy and diagnosis. Moreover, GBNs or their functionalized derivatives are cutting-edge materials used in bioremediation, and their remarkable properties can be used to mitigate environmental contaminants, as well as to improve human, plant, and animal health [17–20, 25, 135].

The following sections describe the properties of GBNs that favor their use in bioremediation and the major pollutants on which GBNs have demonstrated their bioremediation efficiency.

#### **6.1 GBNs properties and processes involved in bioremediation**

Graphene oxidation to GO and rGO reinforces its properties and improves its hydrophilic nature, thereby enhancing its ability to associate with contaminants either physically or chemically. This association can be processed by adsorption of contaminants on the surface of GBNs or by the oxidation breakdown of contaminants, by photocatalysis or other advanced oxidation processes (AOPs) [18].

#### *6.1.1 Adsorption*

One of the most widely used processes for bioremediation is chemical and physical adsorption. The adsorption capacity of materials depends on several characteristics [25]:


As previously described GBNs obey to all these requirements and hold great potential as adsorbent materials. GBNs have a large surface area and excellent

mechanical properties. GBNs also have favorable wettability and different functional surface and edge groups (in these aspects GO has more adsorbent properties than rGO) [18]. As far as porosity is concerned, highly porous GBNs have recently been developed by chemical activation of GO precursors with KOH [25]. Other GBN derivatives functionalized with metal/oxide composites or magnetic nanoparticles may also improve the adsorption capacity of GBNs or demonstrate advantages in the magnetic separation of contaminants adsorbed and re-use of adsorbents by adsorption–desorption cycles [20, 25, 135]. GBNs can also be functionalized with chelating compounds like ethylenediamine tetraacetic acid (EDTA) which favors adsorption of metal ions [19]. However, while the functionalization of GBNs may improve the adsorption capacity of some specific contaminant, it may also limit its use for a more generic type of adsorbate [20].

*6.1.2 Oxidation and photocatalysis*

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

**6.2 Atmospheric pollutants**

*6.2.1 Gas pollutants*

GO-presenting oxygen functional groups also lead to redox reactions and make different contaminants environmentally friendly and degradable [18]. This removes the problem of waste management that exists in the case of adsorption. Radicalbased oxidation processes, also referred to as AOPs, specifically turn organic entities into environmentally compatible harmless entities, including various minerals, less toxic fragments of carbon-based contaminants, and neutral entities such as water and carbon dioxide [18]. Photocatalysis is also an AOP that is effective in the degradation of various organic pollutants by GBNs and their composites. GBNs with a zero-band gap are capable of absorbing light over a wide spectrum. This allows the electrons to be excited from the valence band to the conduction band, forming holes in the valence band. Both electrons and holes are involved in redox reactions that produce many radicals (e.g., hydroxyl radicals) [18]. These radicals serve as potent oxidizers across the surface of GBNs and are responsible for photocatalysis of organic contaminants enabling the destruction of dyes and other organic matter from wastewater [18]. It is also helpful to reduce the band gap of GBNs by loading them with materials such as titanium (TiO2) and zinc oxide (ZnO) to allow efficient

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Gas pollutants have risen over decades due to industrial developments and have

GO and other GBNs as well as their modified forms are good adsorbents for the reactive removal of these toxic gases. Most of the research, however, concentrated on NH3 adsorption using GBNs and composites modified by metal oxide. Owing to the presence of diverse active defect sites, such as the hydroxyl and epoxy functional groups and their neighboring carbon atoms, NH3 adsorption on GO is usually

A wide number of volatile organic compounds (VOCs) are responsible for the growth of cancer in people all over the world, according to the WHO [20]. Formaldehyde which comes from paint and decorating materials, is one of VOCs and the major indoor air pollutant responsible for the sick building syndrome [25].

become one of the most significant issues in the modern world. Because of its widespread combustion from vehicles, forest fires and manufacturing processes, CO2 is the air pollutant of most concern [20]. The ability to block infrared irradiation in the stratospheric layer exacerbates the greenhouse effect and, thus, global warming [20]. Chlorofluorocarbon (CFC), a gas used in freezers, refrigerators, and air-conditioners, is another chemical specie which causes serious damage to the atmosphere. CFC has the property of interacting with ozone causing damage to the ozone layer, responsible for filtering UV irradiation from sunlight [20]. Inorganic gaseous pollutants such as SO2, NO2, NH3 and H2S are also implicated in the phenomenon of acid rain [20, 25]. Monuments and buildings damage, flora degradation, a reduction in soil pH, pollution of the bodies of water and human diseases are the environmental effects of acid rain in large cities; however, it is very difficult to measure them economically [20]. In addition, possible health hazards such as respiratory irritation and damage to the central nervous system have been associ-

use of solar irradiation during photocatalytic decomposition [17].

ated with long-term exposure to these contaminants [20].

greater than that on other GBNs [25].

*6.2.2 Volatile organic compounds*

**101**

With regard to the chemical versatility of GBNs, this material is certainly advantageous in comparison with other adsorbents [15, 19, 20]. For example, GO has oxygen-functionalized groups (e.g., COOH) which are deprotonated at a broad range of pH values (≈ pH > 4.5) and therefore negatively charged groups establish electrostatic interactions with cationic pollutants [19]. Oxygen-functionalized groups also enable hydrogen bonding with adsorbate compounds. These interactions may be established between hydrogen with a partial positive charge and an electronegative atom such as chlorine, fluorine, or oxygen [20]. Hydrogen bonding can therefore be formed between hydrogen atoms present in the functional moieties of GBNs and partially negatively charged atoms of the adsorbate molecule [20]. Hydrophobic interaction is driven by the entropic effect that occurs when ordered water molecules are banned from nonpolar carbon surface of GBNs. Hydrophobic interaction is also another significant contribution to the adsorption of hydrophobic/amphiphilic contaminants to GBNs [19]. Finally, GBNs have the possibility to establish π-π interactions with aromatic rings from contaminants, which may be the only interaction established or may be strengthened by simultaneous electrostatic interactions in cases where aromatic contaminants are also charged [19, 20].

**Figure 3** illustrates the possible adsorption mechanisms of the different pollutant compounds on GBN adsorbents.

**Figure 3.** *Common chemical interactions established between Graphene-based Nanomaterials and pollutants.*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

#### *6.1.2 Oxidation and photocatalysis*

mechanical properties. GBNs also have favorable wettability and different functional surface and edge groups (in these aspects GO has more adsorbent properties than rGO) [18]. As far as porosity is concerned, highly porous GBNs have recently been developed by chemical activation of GO precursors with KOH [25]. Other GBN derivatives functionalized with metal/oxide composites or magnetic nanoparticles may also improve the adsorption capacity of GBNs or demonstrate advantages in the magnetic separation of contaminants adsorbed and re-use of adsorbents by adsorption–desorption cycles [20, 25, 135]. GBNs can also be functionalized with chelating compounds like ethylenediamine tetraacetic acid (EDTA) which favors adsorption of metal ions [19]. However, while the functionalization of GBNs may improve the adsorption capacity of some specific contami-

nant, it may also limit its use for a more generic type of adsorbate [20].

pollutant compounds on GBN adsorbents.

*Theranostics - An Old Concept in New Clothing*

**Figure 3.**

**100**

With regard to the chemical versatility of GBNs, this material is certainly advantageous in comparison with other adsorbents [15, 19, 20]. For example, GO has oxygen-functionalized groups (e.g., COOH) which are deprotonated at a broad range of pH values (≈ pH > 4.5) and therefore negatively charged groups establish electrostatic interactions with cationic pollutants [19]. Oxygen-functionalized groups also enable hydrogen bonding with adsorbate compounds. These interactions may be established between hydrogen with a partial positive charge and an electronegative atom such as chlorine, fluorine, or oxygen [20]. Hydrogen bonding can therefore be formed between hydrogen atoms present in the functional moieties of GBNs and partially negatively charged atoms of the adsorbate molecule [20]. Hydrophobic interaction is driven by the entropic effect that occurs when ordered water molecules are banned from nonpolar carbon surface of GBNs. Hydrophobic interaction is also another significant contribution to the adsorption of hydrophobic/amphiphilic contaminants to GBNs [19]. Finally, GBNs have the possibility to establish π-π interactions with aromatic rings from contaminants, which may be the only interaction established or may be strengthened by simultaneous electrostatic interactions in cases where aromatic contaminants are also charged [19, 20]. **Figure 3** illustrates the possible adsorption mechanisms of the different

*Common chemical interactions established between Graphene-based Nanomaterials and pollutants.*

GO-presenting oxygen functional groups also lead to redox reactions and make different contaminants environmentally friendly and degradable [18]. This removes the problem of waste management that exists in the case of adsorption. Radicalbased oxidation processes, also referred to as AOPs, specifically turn organic entities into environmentally compatible harmless entities, including various minerals, less toxic fragments of carbon-based contaminants, and neutral entities such as water and carbon dioxide [18]. Photocatalysis is also an AOP that is effective in the degradation of various organic pollutants by GBNs and their composites. GBNs with a zero-band gap are capable of absorbing light over a wide spectrum. This allows the electrons to be excited from the valence band to the conduction band, forming holes in the valence band. Both electrons and holes are involved in redox reactions that produce many radicals (e.g., hydroxyl radicals) [18]. These radicals serve as potent oxidizers across the surface of GBNs and are responsible for photocatalysis of organic contaminants enabling the destruction of dyes and other organic matter from wastewater [18]. It is also helpful to reduce the band gap of GBNs by loading them with materials such as titanium (TiO2) and zinc oxide (ZnO) to allow efficient use of solar irradiation during photocatalytic decomposition [17].

#### **6.2 Atmospheric pollutants**

#### *6.2.1 Gas pollutants*

Gas pollutants have risen over decades due to industrial developments and have become one of the most significant issues in the modern world. Because of its widespread combustion from vehicles, forest fires and manufacturing processes, CO2 is the air pollutant of most concern [20]. The ability to block infrared irradiation in the stratospheric layer exacerbates the greenhouse effect and, thus, global warming [20]. Chlorofluorocarbon (CFC), a gas used in freezers, refrigerators, and air-conditioners, is another chemical specie which causes serious damage to the atmosphere. CFC has the property of interacting with ozone causing damage to the ozone layer, responsible for filtering UV irradiation from sunlight [20]. Inorganic gaseous pollutants such as SO2, NO2, NH3 and H2S are also implicated in the phenomenon of acid rain [20, 25]. Monuments and buildings damage, flora degradation, a reduction in soil pH, pollution of the bodies of water and human diseases are the environmental effects of acid rain in large cities; however, it is very difficult to measure them economically [20]. In addition, possible health hazards such as respiratory irritation and damage to the central nervous system have been associated with long-term exposure to these contaminants [20].

GO and other GBNs as well as their modified forms are good adsorbents for the reactive removal of these toxic gases. Most of the research, however, concentrated on NH3 adsorption using GBNs and composites modified by metal oxide. Owing to the presence of diverse active defect sites, such as the hydroxyl and epoxy functional groups and their neighboring carbon atoms, NH3 adsorption on GO is usually greater than that on other GBNs [25].

#### *6.2.2 Volatile organic compounds*

A wide number of volatile organic compounds (VOCs) are responsible for the growth of cancer in people all over the world, according to the WHO [20]. Formaldehyde which comes from paint and decorating materials, is one of VOCs and the major indoor air pollutant responsible for the sick building syndrome [25].

In order to reduce harm to the environment and human health caused by VOCs, GBNs, in particular GO, have recently been employed in several studies for VOC removal by adsorption and photocatalytic decomposition [20, 25].

*6.3.2 Inorganic anions*

amounts of NO3

*pharmaceuticals*

central nervous system [18].

nervous system [20].

**103**

Some inorganic anions, F, NO3

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

and PO4

, SO4

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

amounts in water, and they may also cause water pollution and should be removed, although they are less harmful than heavy metal ions [25]. The presence of large

water enriched with nutrients that induce excessive growth of algae). Due to the negative anion charge, GO is not so successful for inorganic anion adsorption [25]. GBNs and functionalized GBNs, however, have been identified as efficient in inorganic anion adsorption. For example, the surface exchange between F in solution and hydroxyl ions promotes the adsorption of these anions to GBNs [25, 137].

*6.3.3 Organic pollutants: dyes, polycyclic aromatic hydrocarbons, pesticides, and*

Dyes are a class of organic compounds commonly found as water contaminants that are released from a wide variety of sources, such as printing, textiles, dyeing, paper production, tanning, and painting industries. Most dyes are durable and difficult to biodegrade and have complex molecular structures. By altering the color of water, the presence of dyes in water causes disturbance of the photosynthetic phase of aquatic plants, thus suppressing sunshine, creating an imbalance in the entire aquatic environment [18, 25]. In addition, certain dyes are detrimental to human beings. Most dyes are dissolved in water and are either cationic or anionic. By establishing electrostatic interactions, GO exhibits high adsorption of cationic dyes, but between GO anionic groups and anionic dyes, there is strong electrostatic repulsion. However, because of additional π-π stacking interactions, GBNs and composites can still be excellent adsorbents for cationic and anionic dyes [18, 25]. Another class of organic pollutants composed of repeated aromatic ring structures is polycyclic aromatic hydrocarbons (PAHs) [18]. They are non-charged and non-polar molecules produced from different methods, such as petroleum products burning, incomplete biomass combustion, coal mining [18], etc. PAHs have adverse effects on human health and are believed to cause cancer of the skin, blood, bladder, liver and cardiovascular diseases [18]. Owing to insufficient waste management, leakage and accidents, monocyclic aromatic hydrocarbons such as toluene, xylene, benzene are also largely excreted from industry causing damage to the human

Many pesticides are organic aromatic compounds still commonly used in agriculture, dairy, and insect control. In addition, pesticides have also been used in domestic gardening and veterinary practice by common citizens. Therefore, the systematic usage of pesticides is of concern owing to their neurotoxicity, carcinogenic potential and involvement in other pathologies [20, 138]. Moreover, the toxicity of organophosphorus pesticides lies in the fact that these compounds are inhibitors of acetylcholinesterase enzymes, which contribute to dysfunction of the

Pharmaceutical drugs are also organic contaminants that have harmful effects on the environment and human health. Even at low concentrations, these chemicals are very difficult to remove and between 30 and 90% remain undegradable and are

Numerous investigations have demonstrated potential in the use of GO and other GBNs for the adsorption of PAHs, phenolic compounds, pesticides, and pharmaceutical drugs [20, 21, 25, 39, 135, 138]. In general, there are five potential interactions, including hydrophobic effects, π-π stacking, hydrogen bonds, and covalent and electrostatic interactions, which are assumed to be responsible for the

excreted as active compounds in the environment [20, 135].

<sup>2</sup>, ClO4

, PO4

<sup>3</sup>, in water, for instance, can induce eutrophication (i.e.,

<sup>3</sup>, are still found in large

#### **6.3 Water pollutants**

One of the long-lasting concerns in the past few decades has been aquatic contamination due to industrial activities. Groundwater, surface water and wastewater systems contain many pollutants [14, 17, 20, 25]. Anions and heavy metal cations as well as organic compounds are significant contaminants (e.g., dye from textile factories, pesticides, and pharmaceuticals) [14, 17, 20, 21, 25]. Aqueous pollutants arising from waste oil from numerous oil leakage incidents and eventually from biological contaminants may also be identified [17, 20].

#### *6.3.1 Inorganic metals and metalloid cations*

Owing to their high toxicity to plants, animals and human beings, heavy metals are the most substantial contaminants in water. The most prevalent heavy metals in contaminated waters are Hg, Pb, Ag, Cu, Cd, Cr, Zn, Ni, Co and Mn [21, 25]. Most metal ions are found in cationic forms, but certain metals are present in anions such as Cr (VI) within CrO4 <sup>2</sup>, Cr2O7 <sup>2</sup> [21, 25]. The most important metalloid ion with high toxicity is arsenic present in the form of As (V) in H2AsO4 and HAsO4<sup>2</sup> [135]. Arsenic is frequently present in soils and rocks in the form of minerals that are mobilized into groundwater by natural weathering, geochemical reactions, biological activity, volcanic emissions and industrial activities [135]. The high degree of exposure to arsenic by water is a calamity for developing countries. More than 100 million people from densely populated countries, including Bangladesh, China, India, Pakistan, Taiwan, and Mexico, and more than 70% of people from Asian continents, live at risk of arsenic-contaminated ground water and are drinking potable water contaminated with excessive levels of arsenic [25]. Huge amounts of adverse problems are caused by exposure to elevated concentrations of arsenic from drinking water and are commonly associated with skin lesions and hyperkeratosis as adverse effects, whereas long-term exposure leads to cancers of the skin, kidneys, liver and prostate. In addition, arsenic also affects nervous and cardiovascular system functions [25, 135].

Adsorption is probably the most efficient way to eliminate aquatic heavy metal ions, because bioprocessing and chemical reactions like photocatalysis are unable to destroy the metal ions. Due to the numerous functional groups on the surface, GO is a potential adsorbent for metal ion complexation by both electrostatic and coordination methods (e.g., upon GO functionalization with EDTA) [25]. Arsenic removal has become imperative, but most treatment processes are expensive, except for adsorption, which is affordable, convenient and easy to handle. For water treatment, GO and its composite-based membranes, thin films, paper-like materials, and solid composite materials have gained notoriety and have shown efficient and high potential for arsenic removal [135, 136]. The numerous oxygen functional groups are responsible for both higher adsorption and desorption potential of GO. With a change in solvent pH, arsenic desorption from the GO surface contributes to GO regeneration, which can be used to repeat adsorption–desorption processes, thus increasing adsorption efficiency and reducing costs [135]. The adsorption capability, selectivity, thermal and chemical stability of GO can be enhanced by surface modifications. Moreover, conjugation of GO with magnetic nanoparticles also facilitates the magneto-responsive separation of depleted adsorbents from water [135].

#### *6.3.2 Inorganic anions*

In order to reduce harm to the environment and human health caused by VOCs, GBNs, in particular GO, have recently been employed in several studies for VOC

One of the long-lasting concerns in the past few decades has been aquatic contamination due to industrial activities. Groundwater, surface water and wastewater systems contain many pollutants [14, 17, 20, 25]. Anions and heavy metal cations as well as organic compounds are significant contaminants (e.g., dye from textile factories, pesticides, and pharmaceuticals) [14, 17, 20, 21, 25]. Aqueous pollutants arising from waste oil from numerous oil leakage incidents and eventu-

Owing to their high toxicity to plants, animals and human beings, heavy metals are the most substantial contaminants in water. The most prevalent heavy metals in contaminated waters are Hg, Pb, Ag, Cu, Cd, Cr, Zn, Ni, Co and Mn [21, 25]. Most metal ions are found in cationic forms, but certain metals are present in anions such

[135]. Arsenic is frequently present in soils and rocks in the form of minerals that are mobilized into groundwater by natural weathering, geochemical reactions, biological activity, volcanic emissions and industrial activities [135]. The high degree of exposure to arsenic by water is a calamity for developing countries. More than 100 million people from densely populated countries, including Bangladesh, China, India, Pakistan, Taiwan, and Mexico, and more than 70% of people from Asian continents, live at risk of arsenic-contaminated ground water and are drinking potable water contaminated with excessive levels of arsenic [25]. Huge amounts of adverse problems are caused by exposure to elevated concentrations of arsenic from drinking water and are commonly associated with skin lesions and hyperkeratosis as adverse effects, whereas long-term exposure leads to cancers of the skin, kidneys, liver and prostate. In addition, arsenic also affects nervous and cardiovascular

Adsorption is probably the most efficient way to eliminate aquatic heavy metal ions, because bioprocessing and chemical reactions like photocatalysis are unable to destroy the metal ions. Due to the numerous functional groups on the surface, GO is a potential adsorbent for metal ion complexation by both electrostatic and coordination methods (e.g., upon GO functionalization with EDTA) [25]. Arsenic removal has become imperative, but most treatment processes are expensive, except for adsorption, which is affordable, convenient and easy to handle. For water treatment, GO and its composite-based membranes, thin films, paper-like materials, and solid composite materials have gained notoriety and have shown efficient and high potential for arsenic removal [135, 136]. The numerous oxygen functional groups are responsible for both higher adsorption and desorption potential of GO. With a change in solvent pH, arsenic desorption from the GO surface contributes to GO regeneration, which can be used to repeat adsorption–desorption processes, thus increasing adsorption efficiency and reducing costs [135]. The adsorption capability, selectivity, thermal and chemical stability of GO can be enhanced by surface modifications. Moreover, conjugation of GO with magnetic nanoparticles also facilitates the magneto-responsive separation of depleted adsorbents from water [135].

<sup>2</sup> [21, 25]. The most important metalloid ion with

and HAsO4<sup>2</sup>

removal by adsorption and photocatalytic decomposition [20, 25].

ally from biological contaminants may also be identified [17, 20].

<sup>2</sup>, Cr2O7

high toxicity is arsenic present in the form of As (V) in H2AsO4

*6.3.1 Inorganic metals and metalloid cations*

*Theranostics - An Old Concept in New Clothing*

**6.3 Water pollutants**

as Cr (VI) within CrO4

system functions [25, 135].

**102**

Some inorganic anions, F, NO3 , SO4 <sup>2</sup>, ClO4 , PO4 <sup>3</sup>, are still found in large amounts in water, and they may also cause water pollution and should be removed, although they are less harmful than heavy metal ions [25]. The presence of large amounts of NO3 and PO4 <sup>3</sup>, in water, for instance, can induce eutrophication (i.e., water enriched with nutrients that induce excessive growth of algae). Due to the negative anion charge, GO is not so successful for inorganic anion adsorption [25]. GBNs and functionalized GBNs, however, have been identified as efficient in inorganic anion adsorption. For example, the surface exchange between F in solution and hydroxyl ions promotes the adsorption of these anions to GBNs [25, 137].

#### *6.3.3 Organic pollutants: dyes, polycyclic aromatic hydrocarbons, pesticides, and pharmaceuticals*

Dyes are a class of organic compounds commonly found as water contaminants that are released from a wide variety of sources, such as printing, textiles, dyeing, paper production, tanning, and painting industries. Most dyes are durable and difficult to biodegrade and have complex molecular structures. By altering the color of water, the presence of dyes in water causes disturbance of the photosynthetic phase of aquatic plants, thus suppressing sunshine, creating an imbalance in the entire aquatic environment [18, 25]. In addition, certain dyes are detrimental to human beings. Most dyes are dissolved in water and are either cationic or anionic. By establishing electrostatic interactions, GO exhibits high adsorption of cationic dyes, but between GO anionic groups and anionic dyes, there is strong electrostatic repulsion. However, because of additional π-π stacking interactions, GBNs and composites can still be excellent adsorbents for cationic and anionic dyes [18, 25].

Another class of organic pollutants composed of repeated aromatic ring structures is polycyclic aromatic hydrocarbons (PAHs) [18]. They are non-charged and non-polar molecules produced from different methods, such as petroleum products burning, incomplete biomass combustion, coal mining [18], etc. PAHs have adverse effects on human health and are believed to cause cancer of the skin, blood, bladder, liver and cardiovascular diseases [18]. Owing to insufficient waste management, leakage and accidents, monocyclic aromatic hydrocarbons such as toluene, xylene, benzene are also largely excreted from industry causing damage to the human central nervous system [18].

Many pesticides are organic aromatic compounds still commonly used in agriculture, dairy, and insect control. In addition, pesticides have also been used in domestic gardening and veterinary practice by common citizens. Therefore, the systematic usage of pesticides is of concern owing to their neurotoxicity, carcinogenic potential and involvement in other pathologies [20, 138]. Moreover, the toxicity of organophosphorus pesticides lies in the fact that these compounds are inhibitors of acetylcholinesterase enzymes, which contribute to dysfunction of the nervous system [20].

Pharmaceutical drugs are also organic contaminants that have harmful effects on the environment and human health. Even at low concentrations, these chemicals are very difficult to remove and between 30 and 90% remain undegradable and are excreted as active compounds in the environment [20, 135].

Numerous investigations have demonstrated potential in the use of GO and other GBNs for the adsorption of PAHs, phenolic compounds, pesticides, and pharmaceutical drugs [20, 21, 25, 39, 135, 138]. In general, there are five potential interactions, including hydrophobic effects, π-π stacking, hydrogen bonds, and covalent and electrostatic interactions, which are assumed to be responsible for the

adsorption of organic compounds on the GBNs'surface [15, 19, 20, 25] (**Figure 3**). In the case of GO and other GBNs, the majority of investigations have shown that ππ association plays an important role in the adsorption of aromatic organic contaminants [25]. In comparison, the latest major methods used to treat these pollutants are AOPs and the chemical-microbial depletion [20].

standard and reproducible methods that can be scaled up to reduce production costs while maintaining a minimal presence of residual contaminants. In addition, although many studies have shown that GBN's adsorbents have been recycled, these studies are still scarce and more innovative research work needs to be explored in the future to achieve convenient separation and regeneration of GBN's adsorbents.

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

This work was supported by Fundação para a Ciência e Tecnologia (FCT) in the framework of the Strategic Funding Funding [UID/FIS/04650/2019], and by the project CONCERT [POCI-01-0145-FEDER-032651 and PTDC/NAN-MAT/326512017], co-financed by the European Regional Development Fund (ERDF), through COM-PETE 2020, under Portugal 2020, and FCT I.P. M Lúcio thanks FCT and ERDF for doctoral position [CTTI-150/18-CF (1)] in the ambit of the project CONCERT. Eduarda Fernandes acknowledges FCT for PhD grant (SFRH/BD/147938/2019).

**Acknowledgements**

**Abbreviations**

AOPs Advanced oxidation processes

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

CVD Chemical vapor deposition CT Computed Tomography

EDTA Ethylenediamine tetraacetic acid FRET Fluorescence resonance energy transfer

GBNs Graphene-based nanomaterials GQDs Graphene quantum dots

IARC International Agency for Research on Cancer

rGO Reduced graphene oxide

IR-TI Infrared Thermal Imaging MHT Magnetic Hyperthermia Therapy

PAHs Polycyclic aromatic hydrocarbons

PET Positron Emission Tomography

SERS Super Enhanced Raman Spectroscopy

SPECT Single Photon Emission Computed Tomography UCNPs Upconversion luminescence nanoparticles

MNPs Magnetic nanoparticles MRI Magnetic Resonance Imaging

PAI Photoacoustic Imaging PAT Photoacoustic Therapy PDT Photodynamic Therapy

PL Photoluminescence PS Photosensitizer PTT Photothermal Therapy RIE Reactive ion etching ROS Reactive oxygen species

USI Ultrasound Imaging

**105**

VOCs Volatile organic compounds WHO World Health Organization

CFC Chlorofluorocarbon CSCs Cancer stem cells

GO Graphene oxide

NIR Near infrared

#### *6.3.4 Oil and its derivatives*

The significant rise in the discovery of crude oil and the increase in the production of petroleum derivatives have caused negative and long-term destruction of various habitats [20]. One of the most important pollution issues happening very frequently in the ocean or seashore is oil leakage from reservoirs, ships, or oil drilling facilities. In order to minimize the harmful impact on marine ecology, the adsorption of leaking oils from polluted seawater has been an important area of study [17]. Latest experiments have successfully investigated the adsorption of oil emulsions on GBNs, demonstrating excellent adsorption capacities. Extremely porous GBNs (sponges, hydrogels and xerogels) are recently developed as cuttingedge oil adsorbents; many of them are conjugated with magnetic metallic nanospheres and typically have high recyclability [17, 20].

#### *6.3.5 Biological contaminants*

A significant process for public health safety is also the disinfection of the water supply and indoor air to remove common harmful pathogens, like bacteria (e.g., *E. coli*, *F. Solani*), and viruses (e.g., EV71 and H9N2 virus). In these cases, the use of GBNs together with UVC light is also effective for decontamination by photocatalysis [17].

#### **7. Conclusions and prospects**

In this chapter, the current progress on the use of various GBNs in the treatment of cancer and bioremediation has been reviewed. The extraordinary properties of GBNs have also been described with special focus in those that favor the biomedical applications of this material, i.e., the large surface area, the large number of unsaturated π-bonds, the mechanical strength, the NIR absorption properties, the PL capacity, etc. The versatility of GBNs is indicated as a feature that can be explored in the most diverse biomedical fields. In this sense, the use of GBNs in cancer theranostic strategies has been discussed. Successful research studies using GBNs for the loading of anticancer drugs or nucleic acids in synergistic chemotherapy, gene therapy and photothermal/photodynamic therapy have been revised in the field of cancer therapy. GBNs have also been described as imaging diagnostic tools used to track the path of therapeutic delivery in target tissues. Finally, the application of GBNs for photocatalysis and adsorption was described as a means of environmental decontamination, i.e., bioremediation.

It is clear from all the revised research that GBNs have a great future in biomedical applications, either as therapeutic tools or as bioremediation strategies, where specifically GO can be considered one of the most advanced and promising adsorbents. However, despite successful attempts to use GBNs in the biomedical field, there are still several challenges that need to be overcome prior to their widespread commercial or clinical use. First, green methods must be used to develop environmentally sustainable approaches to the production of GBNs. Some attempts at green synthesis have been made, but they are still far from proposing

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

standard and reproducible methods that can be scaled up to reduce production costs while maintaining a minimal presence of residual contaminants. In addition, although many studies have shown that GBN's adsorbents have been recycled, these studies are still scarce and more innovative research work needs to be explored in the future to achieve convenient separation and regeneration of GBN's adsorbents.

#### **Acknowledgements**

adsorption of organic compounds on the GBNs'surface [15, 19, 20, 25] (**Figure 3**). In the case of GO and other GBNs, the majority of investigations have shown that ππ association plays an important role in the adsorption of aromatic organic contaminants [25]. In comparison, the latest major methods used to treat these pollutants

The significant rise in the discovery of crude oil and the increase in the production of petroleum derivatives have caused negative and long-term destruction of various habitats [20]. One of the most important pollution issues happening very frequently in the ocean or seashore is oil leakage from reservoirs, ships, or oil drilling facilities. In order to minimize the harmful impact on marine ecology, the adsorption of leaking oils from polluted seawater has been an important area of study [17]. Latest experiments have successfully investigated the adsorption of oil emulsions on GBNs, demonstrating excellent adsorption capacities. Extremely porous GBNs (sponges, hydrogels and xerogels) are recently developed as cutting-

A significant process for public health safety is also the disinfection of the water supply and indoor air to remove common harmful pathogens, like bacteria (e.g., *E. coli*, *F. Solani*), and viruses (e.g., EV71 and H9N2 virus). In these cases, the use of

In this chapter, the current progress on the use of various GBNs in the treatment of cancer and bioremediation has been reviewed. The extraordinary properties of GBNs have also been described with special focus in those that favor the biomedical applications of this material, i.e., the large surface area, the large number of unsaturated π-bonds, the mechanical strength, the NIR absorption properties, the PL capacity, etc. The versatility of GBNs is indicated as a feature that can be explored in the most diverse biomedical fields. In this sense, the use of GBNs in cancer theranostic strategies has been discussed. Successful research studies using GBNs for the loading of anticancer drugs or nucleic acids in synergistic chemotherapy, gene therapy and photothermal/photodynamic therapy have been revised in the field of cancer therapy. GBNs have also been described as imaging diagnostic tools

edge oil adsorbents; many of them are conjugated with magnetic metallic

GBNs together with UVC light is also effective for decontamination by

used to track the path of therapeutic delivery in target tissues. Finally, the

environmental decontamination, i.e., bioremediation.

application of GBNs for photocatalysis and adsorption was described as a means of

It is clear from all the revised research that GBNs have a great future in biomedical applications, either as therapeutic tools or as bioremediation strategies, where specifically GO can be considered one of the most advanced and promising adsorbents. However, despite successful attempts to use GBNs in the biomedical field, there are still several challenges that need to be overcome prior to their widespread commercial or clinical use. First, green methods must be used to develop environmentally sustainable approaches to the production of GBNs. Some attempts at green synthesis have been made, but they are still far from proposing

nanospheres and typically have high recyclability [17, 20].

are AOPs and the chemical-microbial depletion [20].

*Theranostics - An Old Concept in New Clothing*

*6.3.4 Oil and its derivatives*

*6.3.5 Biological contaminants*

**7. Conclusions and prospects**

photocatalysis [17].

**104**

This work was supported by Fundação para a Ciência e Tecnologia (FCT) in the framework of the Strategic Funding Funding [UID/FIS/04650/2019], and by the project CONCERT [POCI-01-0145-FEDER-032651 and PTDC/NAN-MAT/326512017], co-financed by the European Regional Development Fund (ERDF), through COM-PETE 2020, under Portugal 2020, and FCT I.P. M Lúcio thanks FCT and ERDF for doctoral position [CTTI-150/18-CF (1)] in the ambit of the project CONCERT. Eduarda Fernandes acknowledges FCT for PhD grant (SFRH/BD/147938/2019).

#### **Abbreviations**


*Theranostics - An Old Concept in New Clothing*

#### **Author details**

Marlene Lúcio1,2\*, Eduarda Fernandes1 , Hugo Gonçalves<sup>3</sup> , Sofia Machado2 , Andreia C. Gomes2,4 and Maria Elisabete C.D. Real Oliveira<sup>1</sup>

1 Departamento de Física da Universidade do Minho, CF-UM-UP, Centro de Física das Universidades do Minho e Porto, Braga, Portugal

**References**

81.109

[1] Novoselov KS, Geim AK, Morozov SV, Jiang D, Zhang Y,

[2] Castro Neto AH, Guinea F,

Dubonos SV, Grigorieva IV, Firsov AA. Electric Field Effect in Atomically Thin Carbon Films. Science. 2004;306(5696): 666-669. DOI:10.1126/science.1102896

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

technology-and-its-applications-forelectronic-devices: IntechOpen; 2015.

[8] Viseu T, Lopes CM, Fernandes E, Oliveira MECDR, Lúcio M. A Systematic Review and Critical Analysis of the Role of Graphene-Based Nanomaterials in Cancer Theranostics. Pharmaceutics. 2018;10(4):282. DOI:10.3390/ pharmaceutics10040282

[9] Reina G, González-Domínguez JM, Criado A, Vázquez E, Bianco A,

Prato M. Promises, facts and challenges for graphene in biomedical applications. Chemical Society Reviews. 2017;46(15): 4400-4416. DOI:10.1039/C7CS00363C

[10] Goenka S, Sant V, Sant S. Graphenebased nanomaterials for drug delivery and tissue engineering. Journal of Controlled Release. 2014;173:75-88. DOI:

[11] Kim MG, Park JY, Shon Y, Shim G, Oh YK. Pharmaceutical applications of graphene-based nanosheets. Curr Pharm Biotechnol. 2014;14(12): 1016-1026. DOI: 10.2174/ 1389201015666140113113222

[12] Yang K, Feng L, Shi X, Liu Z. Nanographene in biomedicine: theranostic applications. Chem Soc Rev. 2013;42(2): 530-547. DOI:10.1039/c2cs35342c

[13] Chen Y, Tan C, Zhang H, Wang L. Two-dimensional graphene analogues for biomedical applications. Chem Soc Rev. 2015;44(9):2681-2701. DOI:

[14] Kommu A, Singh JK. A review on graphene-based materials for removal of toxic pollutants from wastewater. Soft Materials. 2020:1-26. DOI:10.1080/

[15] Kim S, Park CM, Jang M, Son A, Her N, Yu M, Snyder S, Kim D-H,

10.1039/c4cs00300d

1539445x.2020.1739710

10.1016/j.jconrel.2013.10.017

DOI:10.5772/61316

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Peres NMR, Novoselov KS, Geim AK. The electronic properties of graphene. Reviews of Modern Physics. 2009;81(1): 109-162. DOI:10.1103/RevModPhys.

[3] Wang F, Shi S. Optical Properties of Graphene. In: Avouris P, Low T, Heinz TF, editors. 2D Materials: Properties and Devices. Cambridge: Cambridge University Press; 2017. p. 38-51. DOI:10.1017/9781316681619.004

[4] Allen MJ, Tung VC, Kaner RB. Honeycomb Carbon: A Review of Graphene. Chemical Reviews. 2010;110 (1):132-145. DOI:10.1021/cr900070d

[5] Randviir EP, Brownson DAC, Banks CE. A decade of graphene research: production, applications and outlook. Materials Today. 2014;17(9):

[6] Aïssa B, Memon NK, Ali A,

[7] Sood AK, Lund I, Puri YR, Efstathiadis H, Haldar P, Dhar NK,

Lewis J, Dubey M, Zakar E,

Khraisheh MK. Recent Progress in the Growth and Applications of Graphene as a Smart Material: A Review. Frontiers in Materials. 2015;2(58). DOI:10.3389/

Wijewarnasuriya P, Polla DL, Fritze M. Review of Graphene Technology and Its Applications for Electronic Devices. In: Ebrahimi F, editor. Graphene - New Trends and Developments. Available from: https://www.intechopen.com/ books/graphene-new-trends-anddevelopments/review-of-graphene-

426-432. DOI:10.1016/j. mattod.2014.06.001

fmats.2015.00058

**107**

2 Departamento de Biologia, CBMA, Centro de Biologia Molecular e Ambiental, Universidade do Minho, Braga, Portugal

3 Paralab, SA, Valbom, Portugal

4 IB-S, Institute of Science and Innovation for Bio-Sustainability, Portugal

\*Address all correspondence to: mlucio@fisica.uminho.pt

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

#### **References**

[1] Novoselov KS, Geim AK, Morozov SV, Jiang D, Zhang Y, Dubonos SV, Grigorieva IV, Firsov AA. Electric Field Effect in Atomically Thin Carbon Films. Science. 2004;306(5696): 666-669. DOI:10.1126/science.1102896

[2] Castro Neto AH, Guinea F, Peres NMR, Novoselov KS, Geim AK. The electronic properties of graphene. Reviews of Modern Physics. 2009;81(1): 109-162. DOI:10.1103/RevModPhys. 81.109

[3] Wang F, Shi S. Optical Properties of Graphene. In: Avouris P, Low T, Heinz TF, editors. 2D Materials: Properties and Devices. Cambridge: Cambridge University Press; 2017. p. 38-51. DOI:10.1017/9781316681619.004

[4] Allen MJ, Tung VC, Kaner RB. Honeycomb Carbon: A Review of Graphene. Chemical Reviews. 2010;110 (1):132-145. DOI:10.1021/cr900070d

[5] Randviir EP, Brownson DAC, Banks CE. A decade of graphene research: production, applications and outlook. Materials Today. 2014;17(9): 426-432. DOI:10.1016/j. mattod.2014.06.001

[6] Aïssa B, Memon NK, Ali A, Khraisheh MK. Recent Progress in the Growth and Applications of Graphene as a Smart Material: A Review. Frontiers in Materials. 2015;2(58). DOI:10.3389/ fmats.2015.00058

[7] Sood AK, Lund I, Puri YR, Efstathiadis H, Haldar P, Dhar NK, Lewis J, Dubey M, Zakar E, Wijewarnasuriya P, Polla DL, Fritze M. Review of Graphene Technology and Its Applications for Electronic Devices. In: Ebrahimi F, editor. Graphene - New Trends and Developments. Available from: https://www.intechopen.com/ books/graphene-new-trends-anddevelopments/review-of-graphenetechnology-and-its-applications-forelectronic-devices: IntechOpen; 2015. DOI:10.5772/61316

[8] Viseu T, Lopes CM, Fernandes E, Oliveira MECDR, Lúcio M. A Systematic Review and Critical Analysis of the Role of Graphene-Based Nanomaterials in Cancer Theranostics. Pharmaceutics. 2018;10(4):282. DOI:10.3390/ pharmaceutics10040282

[9] Reina G, González-Domínguez JM, Criado A, Vázquez E, Bianco A, Prato M. Promises, facts and challenges for graphene in biomedical applications. Chemical Society Reviews. 2017;46(15): 4400-4416. DOI:10.1039/C7CS00363C

[10] Goenka S, Sant V, Sant S. Graphenebased nanomaterials for drug delivery and tissue engineering. Journal of Controlled Release. 2014;173:75-88. DOI: 10.1016/j.jconrel.2013.10.017

[11] Kim MG, Park JY, Shon Y, Shim G, Oh YK. Pharmaceutical applications of graphene-based nanosheets. Curr Pharm Biotechnol. 2014;14(12): 1016-1026. DOI: 10.2174/ 1389201015666140113113222

[12] Yang K, Feng L, Shi X, Liu Z. Nanographene in biomedicine: theranostic applications. Chem Soc Rev. 2013;42(2): 530-547. DOI:10.1039/c2cs35342c

[13] Chen Y, Tan C, Zhang H, Wang L. Two-dimensional graphene analogues for biomedical applications. Chem Soc Rev. 2015;44(9):2681-2701. DOI: 10.1039/c4cs00300d

[14] Kommu A, Singh JK. A review on graphene-based materials for removal of toxic pollutants from wastewater. Soft Materials. 2020:1-26. DOI:10.1080/ 1539445x.2020.1739710

[15] Kim S, Park CM, Jang M, Son A, Her N, Yu M, Snyder S, Kim D-H,

**Author details**

Marlene Lúcio1,2\*, Eduarda Fernandes1

*Theranostics - An Old Concept in New Clothing*

Universidade do Minho, Braga, Portugal

provided the original work is properly cited.

**106**

3 Paralab, SA, Valbom, Portugal

Andreia C. Gomes2,4 and Maria Elisabete C.D. Real Oliveira<sup>1</sup>

das Universidades do Minho e Porto, Braga, Portugal

\*Address all correspondence to: mlucio@fisica.uminho.pt

, Hugo Gonçalves<sup>3</sup>

1 Departamento de Física da Universidade do Minho, CF-UM-UP, Centro de Física

2 Departamento de Biologia, CBMA, Centro de Biologia Molecular e Ambiental,

4 IB-S, Institute of Science and Innovation for Bio-Sustainability, Portugal

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Sofia Machado2

,

Yoon Y. Aqueous removal of inorganic and organic contaminants by graphenebased nanoadsorbents: A review. Chemosphere. 2018;212:1104-1124. DOI: 10.1016/j.chemosphere.2018.09.033

[16] Lü K, Zhao G, Wang X. A brief review of graphene-based material synthesis and its application in environmental pollution management. Chinese Science Bulletin. 2012;57(11): 1223-1234. DOI:10.1007/s11434-012- 4986-5

[17] Tsang CHA, Kwok HYH, Cheng Z, Leung DYC. The applications of graphene-based materials in pollutant control and disinfection. Progress in Solid State Chemistry. 2017;45-46:1-8. DOI: 10.1016/j.progsolidstchem.2017.02.001

[18] Thakur K, Kandasubramanian B. Graphene and Graphene Oxide-Based Composites for Removal of Organic Pollutants: A Review. Journal of Chemical & Engineering Data. 2019;64(3):833-867. DOI:10.1021/acs.jced.8b01057

[19] Xu J, Lv H, Yang S-T, Luo J. Preparation of graphene adsorbents and their applications in water purification. Reviews in Inorganic Chemistry. 2013; 33(2-3):139-160. DOI:10.1515/revic-2013-0007

[20] Fraga TJM, Carvalho MN, Ghislandi MG, Motta Sobrinho MA. Functionalized Graphene-Based Materials as Innovative Adsorbents of Organic Pollutants: A Concise Overview. Brazilian Journal of Chemical Engineering. 2019;36(1):1-31. DOI: 10.1590/0104-6632.20190361s20180283

[21] Pérez-Ramírez EF, Luz-Asunción M, Martínez-Hernández AL, Velasco-Santos C. Graphene Materials to Remove Organic Pollutants and Heavy Metals from Water: Photocatalysis and Adsorption. In: Cao W, editor. Semiconductor Photocatalysis - Materials, Mechanisms and Applications. Available from:

https://www.intechopen.com/books/ semiconductor-photocatalysis-materialsmechanisms-and-applications/graphenematerials-to-remove-organic-pollutantsand-heavy-metals-from-waterphotocatalysis-and-adsor: Intechopen; 2016. DOI:10.5772/62777

2008;8(3):902-907. DOI:10.1021/

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

Casalongue H, Vinh D, Dai H. Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. Journal of the American Chemical Society. 2011;133 (17):6825-6831. DOI:10.1021/ja2010175

[37] Soldano C, Mahmood A, Dujardin E. Production, properties and potential of

graphene. Carbon. 2010;48(8): 2127-2150. DOI:10.1016/j. carbon.2010.01.058

[38] Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, Ruoff RS. Graphene and Graphene Oxide: Synthesis,

10.1002/adma.201001068

07622-w

Properties, and Applications. Advanced Materials. 2010;22(35):3906-3924. DOI:

[39] Zhao C, Pei S, Ma J, Song Z, Xia H, Song X, Qi H, Yang Y. Influence of graphene oxide nanosheets on the cotransport of cu-tetracycline multipollutants in saturated porous media. Environ Sci Pollut Res Int. 2020;27(10): 10846-10856. DOI:10.1007/s11356-020-

[40] Zhao H, Ding R, Zhao X, Li Y, Qu L, Pei H, Yildirimer L, Wu Z, Zhang W. Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering. Drug Discovery Today. 2017;22(9):1302-1317. DOI: 10.1016/j.drudis.2017.04.002

[41] Bagri A, Mattevi C, Acik M, Chabal YJ, Chhowalla M, Shenoy VB. Structural evolution during the reduction of chemically derived graphene oxide. Nature Chemistry. 2010;2(7):581-587.

[42] Cohen-Karni T, Qing Q, Li Q, Fang Y, Lieber CM. Graphene and Nanowire Transistors for Cellular Interfaces and Electrical Recording. Nano Letters. 2010;10(3):1098-1102.

DOI:10.1038/nchem.686

DOI:10.1021/nl1002608

[43] Cohen-Karni T, Langer R,

Kohane DS. The Smartest Materials: The

[29] Gao W, Alemany LB, Ci L, Ajayan PM. New insights into the structure and reduction of graphite oxide. Nature Chemistry. 2009;1(5): 403-408. DOI:10.1038/nchem.281

1308-1308. DOI:10.1126/

10.1016/j.cbi.2016.11.019

10.1021/nl801384y

[31] Gulzar A, Yang P, He F, Xu J, Yang D, Xu L, Jan MO. Bioapplications of graphene constructed functional nanomaterials. Chemico-Biological Interactions. 2017;262:69-89. DOI:

[32] Gómez-Navarro C, Burghard M, Kern K. Elastic Properties of Chemically Derived Single Graphene Sheets. Nano Letters. 2008;8(7):2045-2049. DOI:

[33] Suk JW, Piner RD, An J, Ruoff RS. Mechanical Properties of Monolayer Graphene Oxide. ACS Nano. 2010;4 (11):6557-6564. DOI:10.1021/nn101781v

[34] Renteria JD, Ramirez S, Malekpour H,

Conductivity of Free-Standing Reduced Graphene Oxide Films Annealed at High Temperature. Advanced Functional Materials. 2015;25(29):4664-4672. DOI:

Alonso B, Centeno A, Zurutuza A, Cocemasov AI, Nika DL, Balandin AA.

Strongly Anisotropic Thermal

10.1002/adfm.201501429

[35] Syama S, Mohanan PV. Comprehensive Application of Graphene: Emphasis on Biomedical Concerns. Nano-Micro Letters. 2019;11 (1):6. DOI:10.1007/s40820-019-0237-5

[36] Robinson JT, Tabakman SM, Liang Y, Wang H, Sanchez

**109**

science.1156965

[30] Nair RR, Blake P, Grigorenko AN, Novoselov KS, Booth TJ, Stauber T, Peres NMR, Geim AK. Fine Structure Constant Defines Visual Transparency of Graphene. Science. 2008;320(5881):

nl0731872

[22] WHO. GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012. Lyon, France: International Agency for Research on Cancer, 2012.

[23] WHO. Cancer prevention and control in the context of an integrated approach Report by the Secretariat of WHO, 2016.

[24] Fiorillo M, Verre AF, Iliut M, Peiris-Pagés M, Ozsvari B, Gandara R, Cappello AR, Sotgia F, Vijayaraghavan A, Lisanti MP. Graphene oxide selectively targets cancer stem cells, across multiple tumor types: implications for non-toxic cancer treatment, via "differentiation-based nano-therapy". Oncotarget. 2015;6(6): 3553-3562. DOI:10.18632/ oncotarget.3348

[25] Wang S, Sun H, Ang HM, Tadé MO. Adsorptive remediation of environmental pollutants using novel graphene-based nanomaterials. Chemical Engineering Journal. 2013;226: 336-347. DOI:10.1016/j.cej.2013.04.070

[26] Loh KP, Bao Q, Ang PK, Yang J. The chemistry of graphene. Journal of Materials Chemistry. 2010;20(12): 2277-2289. DOI:10.1039/B920539J

[27] Lee C, Wei X, Kysar JW, Hone J. Measurement of the Elastic Properties and Intrinsic Strength of Monolayer Graphene. Science. 2008;321(5887): 385-388. DOI:10.1126/science.1157996

[28] Balandin AA, Ghosh S, Bao W, Calizo I, Teweldebrhan D, Miao F, Lau CN. Superior Thermal Conductivity of Single-Layer Graphene. Nano Letters. *Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

2008;8(3):902-907. DOI:10.1021/ nl0731872

Yoon Y. Aqueous removal of inorganic and organic contaminants by graphenebased nanoadsorbents: A review.

*Theranostics - An Old Concept in New Clothing*

https://www.intechopen.com/books/ semiconductor-photocatalysis-materialsmechanisms-and-applications/graphenematerials-to-remove-organic-pollutants-

[22] WHO. GLOBOCAN 2012: Estimated

and-heavy-metals-from-waterphotocatalysis-and-adsor: Intechopen;

Cancer Incidence, Mortality and Prevalence Worldwide in 2012. Lyon, France: International Agency for Research on Cancer, 2012.

[23] WHO. Cancer prevention and control in the context of an integrated approach Report by the Secretariat of

Pagés M, Ozsvari B, Gandara R,

Vijayaraghavan A, Lisanti MP. Graphene oxide selectively targets cancer stem cells, across multiple tumor types: implications for non-toxic cancer treatment, via "differentiation-based nano-therapy". Oncotarget. 2015;6(6):

Cappello AR, Sotgia F,

3553-3562. DOI:10.18632/

Adsorptive remediation of

oncotarget.3348

[24] Fiorillo M, Verre AF, Iliut M, Peiris-

[25] Wang S, Sun H, Ang HM, Tadé MO.

Chemical Engineering Journal. 2013;226: 336-347. DOI:10.1016/j.cej.2013.04.070

[26] Loh KP, Bao Q, Ang PK, Yang J. The chemistry of graphene. Journal of Materials Chemistry. 2010;20(12): 2277-2289. DOI:10.1039/B920539J

[27] Lee C, Wei X, Kysar JW, Hone J. Measurement of the Elastic Properties and Intrinsic Strength of Monolayer Graphene. Science. 2008;321(5887): 385-388. DOI:10.1126/science.1157996

[28] Balandin AA, Ghosh S, Bao W, Calizo I, Teweldebrhan D, Miao F, Lau CN. Superior Thermal Conductivity of Single-Layer Graphene. Nano Letters.

environmental pollutants using novel graphene-based nanomaterials.

2016. DOI:10.5772/62777

WHO, 2016.

Chemosphere. 2018;212:1104-1124. DOI: 10.1016/j.chemosphere.2018.09.033

[16] Lü K, Zhao G, Wang X. A brief review of graphene-based material synthesis and its application in

environmental pollution management. Chinese Science Bulletin. 2012;57(11): 1223-1234. DOI:10.1007/s11434-012-

[17] Tsang CHA, Kwok HYH, Cheng Z, Leung DYC. The applications of graphene-based materials in pollutant control and disinfection. Progress in Solid State Chemistry. 2017;45-46:1-8. DOI: 10.1016/j.progsolidstchem.2017.02.001

[18] Thakur K, Kandasubramanian B. Graphene and Graphene Oxide-Based Composites for Removal of Organic Pollutants: A Review. Journal of Chemical & Engineering Data. 2019;64(3):833-867.

DOI:10.1021/acs.jced.8b01057

2013-0007

[19] Xu J, Lv H, Yang S-T, Luo J.

[20] Fraga TJM, Carvalho MN, Ghislandi MG, Motta Sobrinho MA. Functionalized Graphene-Based Materials as Innovative Adsorbents of

Organic Pollutants: A Concise

[21] Pérez-Ramírez EF, Luz-

Heavy Metals from Water:

**108**

Photocatalysis and Adsorption. In: Cao W, editor. Semiconductor

Photocatalysis - Materials, Mechanisms and Applications. Available from:

Overview. Brazilian Journal of Chemical Engineering. 2019;36(1):1-31. DOI: 10.1590/0104-6632.20190361s20180283

Asunción M, Martínez-Hernández AL, Velasco-Santos C. Graphene Materials to Remove Organic Pollutants and

Preparation of graphene adsorbents and their applications in water purification. Reviews in Inorganic Chemistry. 2013; 33(2-3):139-160. DOI:10.1515/revic-

4986-5

[29] Gao W, Alemany LB, Ci L, Ajayan PM. New insights into the structure and reduction of graphite oxide. Nature Chemistry. 2009;1(5): 403-408. DOI:10.1038/nchem.281

[30] Nair RR, Blake P, Grigorenko AN, Novoselov KS, Booth TJ, Stauber T, Peres NMR, Geim AK. Fine Structure Constant Defines Visual Transparency of Graphene. Science. 2008;320(5881): 1308-1308. DOI:10.1126/ science.1156965

[31] Gulzar A, Yang P, He F, Xu J, Yang D, Xu L, Jan MO. Bioapplications of graphene constructed functional nanomaterials. Chemico-Biological Interactions. 2017;262:69-89. DOI: 10.1016/j.cbi.2016.11.019

[32] Gómez-Navarro C, Burghard M, Kern K. Elastic Properties of Chemically Derived Single Graphene Sheets. Nano Letters. 2008;8(7):2045-2049. DOI: 10.1021/nl801384y

[33] Suk JW, Piner RD, An J, Ruoff RS. Mechanical Properties of Monolayer Graphene Oxide. ACS Nano. 2010;4 (11):6557-6564. DOI:10.1021/nn101781v

[34] Renteria JD, Ramirez S, Malekpour H, Alonso B, Centeno A, Zurutuza A, Cocemasov AI, Nika DL, Balandin AA. Strongly Anisotropic Thermal Conductivity of Free-Standing Reduced Graphene Oxide Films Annealed at High Temperature. Advanced Functional Materials. 2015;25(29):4664-4672. DOI: 10.1002/adfm.201501429

[35] Syama S, Mohanan PV. Comprehensive Application of Graphene: Emphasis on Biomedical Concerns. Nano-Micro Letters. 2019;11 (1):6. DOI:10.1007/s40820-019-0237-5

[36] Robinson JT, Tabakman SM, Liang Y, Wang H, Sanchez

Casalongue H, Vinh D, Dai H. Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. Journal of the American Chemical Society. 2011;133 (17):6825-6831. DOI:10.1021/ja2010175

[37] Soldano C, Mahmood A, Dujardin E. Production, properties and potential of graphene. Carbon. 2010;48(8): 2127-2150. DOI:10.1016/j. carbon.2010.01.058

[38] Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, Ruoff RS. Graphene and Graphene Oxide: Synthesis, Properties, and Applications. Advanced Materials. 2010;22(35):3906-3924. DOI: 10.1002/adma.201001068

[39] Zhao C, Pei S, Ma J, Song Z, Xia H, Song X, Qi H, Yang Y. Influence of graphene oxide nanosheets on the cotransport of cu-tetracycline multipollutants in saturated porous media. Environ Sci Pollut Res Int. 2020;27(10): 10846-10856. DOI:10.1007/s11356-020- 07622-w

[40] Zhao H, Ding R, Zhao X, Li Y, Qu L, Pei H, Yildirimer L, Wu Z, Zhang W. Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering. Drug Discovery Today. 2017;22(9):1302-1317. DOI: 10.1016/j.drudis.2017.04.002

[41] Bagri A, Mattevi C, Acik M, Chabal YJ, Chhowalla M, Shenoy VB. Structural evolution during the reduction of chemically derived graphene oxide. Nature Chemistry. 2010;2(7):581-587. DOI:10.1038/nchem.686

[42] Cohen-Karni T, Qing Q, Li Q, Fang Y, Lieber CM. Graphene and Nanowire Transistors for Cellular Interfaces and Electrical Recording. Nano Letters. 2010;10(3):1098-1102. DOI:10.1021/nl1002608

[43] Cohen-Karni T, Langer R, Kohane DS. The Smartest Materials: The Future of Nanoelectronics in Medicine. ACS Nano. 2012;6(8):6541-6545. DOI: 10.1021/nn302915s

[44] Nguyen P, Berry V. Graphene Interfaced with Biological Cells: Opportunities and Challenges. The Journal of Physical Chemistry Letters. 2012;3(8):1024-1029. DOI:10.1021/ jz300033g

[45] Artiles MS, Rout CS, Fisher TS. Graphene-based hybrid materials and devices for biosensing. Advanced Drug Delivery Reviews. 2011;63(14): 1352-1360. DOI:10.1016/j. addr.2011.07.005

[46] Taherian F, Marcon V, van der Vegt NFA, Leroy F. What Is the Contact Angle of Water on Graphene? Langmuir. 2013;29(5):1457-1465. DOI: 10.1021/la304645w

[47] Kim J, Cote LJ, Kim F, Yuan W, Shull KR, Huang J. Graphene Oxide Sheets at Interfaces. Journal of the American Chemical Society. 2010;132 (23):8180-8186. DOI:10.1021/ja102777p

[48] Park S, An J, Jung I, Piner RD, An SJ, Li X, Velamakanni A, Ruoff RS. Colloidal Suspensions of Highly Reduced Graphene Oxide in a Wide Variety of Organic Solvents. Nano Letters. 2009;9(4):1593-1597. DOI: 10.1021/nl803798y

[49] Hong G, Diao S, Antaris AL, Dai H. Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy. Chemical Reviews. 2015;115(19): 10816-10906. DOI:10.1021/acs. chemrev.5b00008

[50] Zhu S, Song Y, Zhao X, Shao J, Zhang J, Yang B. The photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): current state and future perspective. Nano Research. 2015;8(2):355-381. DOI: 10.1007/s12274-014-0644-3

[51] Demchenko AP, Dekaliuk MO. Novel fluorescent carbonic nanomaterials for sensing and imaging. Methods and Applications in Fluorescence. 2013;1(4). DOI:10.1088/ 2050-6120/1/4/042001

nanocomposite. Ultrason Sonochem. 2017;35(Pt A):397-404. DOI:10.1016/j.

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

[66] Salas EC, Sun Z, Lüttge A, Tour JM. Reduction of Graphene Oxide via Bacterial Respiration. ACS Nano. 2010;4 (8):4852-4856. DOI:10.1021/nn101081t

[67] Gao J, Liu F, Liu Y, Ma N, Wang Z, Zhang X. Environment-Friendly Method To Produce Graphene That Employs Vitamin C and Amino Acid. Chemistry of Materials. 2010;22(7): 2213-2218. DOI:10.1021/cm902635j

[68] Esfandiar A, Akhavan O, Irajizad A. Melatonin as a powerful bio-antioxidant for reduction of graphene oxide. Journal of Materials Chemistry. 2011;21(29): 10907-10914. DOI:10.1039/C1JM10151J

[69] Akhavan O, Ghaderi E, Aghayee S, Fereydooni Y, Talebi A. The use of a glucose-reduced graphene oxide suspension for photothermal cancer therapy. Journal of Materials Chemistry. 2012;22(27):13773-13781. DOI:10.1039/

[70] Iravani S, Varma RS. Green synthesis, biomedical and

18(3):703-727. DOI:10.1007/ s10311-020-00984-0

biotechnological applications of carbon and graphene quantum dots. A review. Environmental Chemistry Letters. 2020;

[71] Chen W, Shen J, Lv G, Li D, Hu Y, Zhou C, Liu X, Dai Z. Green Synthesis of Graphene Quantum Dots from Cotton Cellulose. ChemistrySelect. 2019;4(10):2898-2902. DOI:10.1002/

[72] de Menezes FD, Dos Reis SRR, Pinto SR, Portilho FL, do Vale

Chaves EMF, Helal-Neto E, da Silva de Barros AO, Alencar LMR, de Menezes AS, Dos Santos CC, Saraiva-Souza A, Perini JA, Machado DE, Felzenswalb I, Araujo-Lima CF, Sukhanova A, Nabiev I, Santos-Oliveira R. Graphene quantum dots unraveling: Green synthesis, characterization, radiolabeling with 99mTc, in vivo behavior and

mutagenicity. Mater Sci Eng C Mater Biol

C2JM31396K

slct.201803512

[59] Gurunathan S, Han JW, Kim JH. Green chemistry approach for the synthesis of biocompatible graphene. Int J Nanomedicine. 2013;8:2719-2732. DOI:

[60] Gürünlü B, Taşdelen Yücedağ Ç, Bayramoğlu MR. Green Synthesis of Graphene from Graphite in Molten Salt Medium. Journal of Nanomaterials. 2020;2020:7029601. DOI:10.1155/2020/

[61] Pei S, Wei Q, Huang K, Cheng H-M, Ren W. Green synthesis of graphene oxide by seconds timescale water electrolytic oxidation. Nature

Communications. 2018;9(1):145. DOI:

[62] Kar S, Saha S, Dutta S, Rana D,

International Journal of Research and Scientific Innovation (IJRSI). 2018;V

[63] Thakur S, Karak N. Green reduction

of graphene oxide by aqueous phytoextracts. Carbon. 2012;50(14):

[64] Wang Y, Shi Z, Yin J. Facile Synthesis of Soluble Graphene via a Green Reduction of Graphene Oxide in Tea Solution and Its Biocomposites. ACS Applied Materials & Interfaces. 2011;3(4):1127-1133. DOI:10.1021/

[65] Akhavan O, Kalaee M, Alavi ZS, Ghiasi SMA, Esfandiar A. Increasing the

antioxidant activity of green tea polyphenols in the presence of iron for the reduction of graphene oxide. Carbon. 2012;50(8):3015-3025. DOI: 10.1016/j.carbon.2012.02.087

5331-5339. DOI:10.1016/j. carbon.2012.07.023

10.1038/s41467-017-02479-z

Sadhukhan S, Ghosh TK. A Comprehensive Review over Green Synthesis of Graphene.

ultsonch.2016.10.018

10.2147/IJN.S45174

7029601

(VII):1-12.

am1012613

**111**

[52] Zhang B, Wang Y, Liu J, Zhai G. Recent developments of phototherapy based on graphene family nanomaterials. Current Medicinal Chemistry. 2017;24(3):268-291. DOI: 10.2174/0929867323666161019141817

[53] (SCENIHR) SCoEaNIHR. Position Statement on emerging and newly identified health risks to be drawn to the attention of the European Commission 2014. DOI:10.2875/996348 EW-AS-16-001-EN-N

[54] Smith AT, LaChance AM, Zeng S, Liu B, Sun L. Synthesis, properties, and applications of graphene oxide/reduced graphene oxide and their nanocomposites. Nano Materials Science. 2019;1(1):31-47. DOI:10.1016/j. nanoms.2019.02.004

[55] Wu Y, Wang S, Komvopoulos K. A review of graphene synthesis by indirect and direct deposition methods. Journal of Materials Research. 2020;35(1):76-89. DOI:10.1557/jmr.2019.377

[56] Gonçalves H, Belsley M, Moura C, Stauber T, Schellenberg P. Enhancing visibility of graphene on arbitrary substrates by microdroplet condensation. Applied Physics Letters. 2010;97(23):231905. DOI:10.1063/ 1.3527081

[57] Kumar R, Kumar VB, Gedanken A. Sonochemical synthesis of carbon dots, mechanism, effect of parameters, and catalytic, energy, biomedical and tissue engineering applications. Ultrason Sonochem. 2020;64:105009. DOI: 10.1016/j.ultsonch.2020.105009

[58] Acar Bozkurt P. Sonochemical green synthesis of Ag/graphene

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

nanocomposite. Ultrason Sonochem. 2017;35(Pt A):397-404. DOI:10.1016/j. ultsonch.2016.10.018

Future of Nanoelectronics in Medicine. ACS Nano. 2012;6(8):6541-6545. DOI:

*Theranostics - An Old Concept in New Clothing*

[51] Demchenko AP, Dekaliuk MO.

nanomaterials for sensing and imaging.

Fluorescence. 2013;1(4). DOI:10.1088/

[52] Zhang B, Wang Y, Liu J, Zhai G. Recent developments of phototherapy

nanomaterials. Current Medicinal Chemistry. 2017;24(3):268-291. DOI: 10.2174/0929867323666161019141817

[53] (SCENIHR) SCoEaNIHR. Position Statement on emerging and newly identified health risks to be drawn to the

Commission 2014. DOI:10.2875/996348

[54] Smith AT, LaChance AM, Zeng S, Liu B, Sun L. Synthesis, properties, and applications of graphene oxide/reduced

[55] Wu Y, Wang S, Komvopoulos K. A review of graphene synthesis by indirect and direct deposition methods. Journal of Materials Research. 2020;35(1):76-89.

[56] Gonçalves H, Belsley M, Moura C, Stauber T, Schellenberg P. Enhancing visibility of graphene on arbitrary substrates by microdroplet

condensation. Applied Physics Letters. 2010;97(23):231905. DOI:10.1063/

[57] Kumar R, Kumar VB, Gedanken A. Sonochemical synthesis of carbon dots, mechanism, effect of parameters, and catalytic, energy, biomedical and tissue engineering applications. Ultrason Sonochem. 2020;64:105009. DOI: 10.1016/j.ultsonch.2020.105009

[58] Acar Bozkurt P. Sonochemical green

synthesis of Ag/graphene

Novel fluorescent carbonic

Methods and Applications in

2050-6120/1/4/042001

based on graphene family

attention of the European

graphene oxide and their

DOI:10.1557/jmr.2019.377

1.3527081

nanoms.2019.02.004

nanocomposites. Nano Materials Science. 2019;1(1):31-47. DOI:10.1016/j.

EW-AS-16-001-EN-N

[44] Nguyen P, Berry V. Graphene Interfaced with Biological Cells: Opportunities and Challenges. The Journal of Physical Chemistry Letters. 2012;3(8):1024-1029. DOI:10.1021/

[45] Artiles MS, Rout CS, Fisher TS. Graphene-based hybrid materials and devices for biosensing. Advanced Drug

[46] Taherian F, Marcon V, van der Vegt NFA, Leroy F. What Is the Contact

Langmuir. 2013;29(5):1457-1465. DOI:

[47] Kim J, Cote LJ, Kim F, Yuan W, Shull KR, Huang J. Graphene Oxide Sheets at Interfaces. Journal of the American Chemical Society. 2010;132 (23):8180-8186. DOI:10.1021/ja102777p

[48] Park S, An J, Jung I, Piner RD, An SJ, Li X, Velamakanni A, Ruoff RS. Colloidal Suspensions of Highly Reduced Graphene Oxide in a Wide Variety of Organic Solvents. Nano Letters. 2009;9(4):1593-1597. DOI:

[49] Hong G, Diao S, Antaris AL, Dai H. Carbon Nanomaterials for Biological Imaging and Nanomedicinal Therapy. Chemical Reviews. 2015;115(19): 10816-10906. DOI:10.1021/acs.

[50] Zhu S, Song Y, Zhao X, Shao J,

photoluminescence mechanism in carbon dots (graphene quantum dots, carbon nanodots, and polymer dots): current state and future perspective. Nano Research. 2015;8(2):355-381. DOI:

10.1007/s12274-014-0644-3

Angle of Water on Graphene?

Delivery Reviews. 2011;63(14): 1352-1360. DOI:10.1016/j.

10.1021/nn302915s

jz300033g

addr.2011.07.005

10.1021/la304645w

10.1021/nl803798y

chemrev.5b00008

Zhang J, Yang B. The

**110**

[59] Gurunathan S, Han JW, Kim JH. Green chemistry approach for the synthesis of biocompatible graphene. Int J Nanomedicine. 2013;8:2719-2732. DOI: 10.2147/IJN.S45174

[60] Gürünlü B, Taşdelen Yücedağ Ç, Bayramoğlu MR. Green Synthesis of Graphene from Graphite in Molten Salt Medium. Journal of Nanomaterials. 2020;2020:7029601. DOI:10.1155/2020/ 7029601

[61] Pei S, Wei Q, Huang K, Cheng H-M, Ren W. Green synthesis of graphene oxide by seconds timescale water electrolytic oxidation. Nature Communications. 2018;9(1):145. DOI: 10.1038/s41467-017-02479-z

[62] Kar S, Saha S, Dutta S, Rana D, Sadhukhan S, Ghosh TK. A Comprehensive Review over Green Synthesis of Graphene. International Journal of Research and Scientific Innovation (IJRSI). 2018;V (VII):1-12.

[63] Thakur S, Karak N. Green reduction of graphene oxide by aqueous phytoextracts. Carbon. 2012;50(14): 5331-5339. DOI:10.1016/j. carbon.2012.07.023

[64] Wang Y, Shi Z, Yin J. Facile Synthesis of Soluble Graphene via a Green Reduction of Graphene Oxide in Tea Solution and Its Biocomposites. ACS Applied Materials & Interfaces. 2011;3(4):1127-1133. DOI:10.1021/ am1012613

[65] Akhavan O, Kalaee M, Alavi ZS, Ghiasi SMA, Esfandiar A. Increasing the antioxidant activity of green tea polyphenols in the presence of iron for the reduction of graphene oxide. Carbon. 2012;50(8):3015-3025. DOI: 10.1016/j.carbon.2012.02.087

[66] Salas EC, Sun Z, Lüttge A, Tour JM. Reduction of Graphene Oxide via Bacterial Respiration. ACS Nano. 2010;4 (8):4852-4856. DOI:10.1021/nn101081t

[67] Gao J, Liu F, Liu Y, Ma N, Wang Z, Zhang X. Environment-Friendly Method To Produce Graphene That Employs Vitamin C and Amino Acid. Chemistry of Materials. 2010;22(7): 2213-2218. DOI:10.1021/cm902635j

[68] Esfandiar A, Akhavan O, Irajizad A. Melatonin as a powerful bio-antioxidant for reduction of graphene oxide. Journal of Materials Chemistry. 2011;21(29): 10907-10914. DOI:10.1039/C1JM10151J

[69] Akhavan O, Ghaderi E, Aghayee S, Fereydooni Y, Talebi A. The use of a glucose-reduced graphene oxide suspension for photothermal cancer therapy. Journal of Materials Chemistry. 2012;22(27):13773-13781. DOI:10.1039/ C2JM31396K

[70] Iravani S, Varma RS. Green synthesis, biomedical and biotechnological applications of carbon and graphene quantum dots. A review. Environmental Chemistry Letters. 2020; 18(3):703-727. DOI:10.1007/ s10311-020-00984-0

[71] Chen W, Shen J, Lv G, Li D, Hu Y, Zhou C, Liu X, Dai Z. Green Synthesis of Graphene Quantum Dots from Cotton Cellulose. ChemistrySelect. 2019;4(10):2898-2902. DOI:10.1002/ slct.201803512

[72] de Menezes FD, Dos Reis SRR, Pinto SR, Portilho FL, do Vale Chaves EMF, Helal-Neto E, da Silva de Barros AO, Alencar LMR, de Menezes AS, Dos Santos CC, Saraiva-Souza A, Perini JA, Machado DE, Felzenswalb I, Araujo-Lima CF, Sukhanova A, Nabiev I, Santos-Oliveira R. Graphene quantum dots unraveling: Green synthesis, characterization, radiolabeling with 99mTc, in vivo behavior and mutagenicity. Mater Sci Eng C Mater Biol

Appl. 2019;102:405-414. DOI:10.1016/j. msec.2019.04.058

[73] Basoglu A, Ocak U, Gumrukcuoglu A. Synthesis of Microwave-Assisted Fluorescence Carbon Quantum Dots Using Roasted-Chickpeas and its Applications for Sensitive and Selective Detection of Fe (3+) Ions. J Fluoresc. 2020;30(3): 515-526. DOI:10.1007/s10895-019- 02428-7

[74] Chen W, Li D, Tian L, Xiang W, Wang T, Hu W, Hu Y, Chen S, Chen J, Dai Z. Synthesis of graphene quantum dots from natural polymer starch for cell imaging. Green Chemistry. 2018;20(19): 4438-4442. DOI:10.1039/C8GC02106F

[75] Gu J, Hu MJ, Guo QQ, Ding ZF, Sun XL, Yang J. High-yield synthesis of graphene quantum dots with strong green photoluminescence. RSC Advances. 2014;4(91):50141-50144. DOI:10.1039/C4RA10011E

[76] Liu Q, Zhang J, He H, Huang G, Xing B, Jia J, Zhang C. Green Preparation of High Yield Fluorescent Graphene Quantum Dots from Coal-Tar-Pitch by Mild Oxidation. Nanomaterials. 2018;8(10):844. DOI: 10.3390/nano8100844

[77] Halder A, Godoy-Gallardo M, Ashley J, Feng X, Zhou T, Hosta-Rigau L, Sun Y. One-Pot Green Synthesis of Biocompatible Graphene Quantum Dots and Their Cell Uptake Studies. ACS Applied Bio Materials. 2018;1(2): 452-461. DOI:10.1021/acsabm.8b00170

[78] Augustine S, Singh J, Srivastava M, Sharma M, Das A, Malhotra BD. Recent advances in carbon based nanosystems for cancer theranostics. Biomater Sci. 2017;5(5):901-952. DOI:10.1016/j. msec.2017.02.121

[79] Ding H, Zhang F, Zhao C, Lv Y, Ma G, Wei W, Tian Z. Beyond a Carrier: Graphene Quantum Dots as a Probe for

Programmatically Monitoring Anti-Cancer Drug Delivery, Release, and Response. ACS Applied Materials and Interfaces. 2017;9(33):27396-27401. DOI:10.1021/acsami.7b08824

of Hybrid Reduced Graphene Oxide-Loaded Ultrasmall Gold Nanorod Vesicles for Cancer Therapy. ACS Nano. 2015;9(9):9199-9209. DOI:10.1021/

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

[91] Zhu H, Cheng P, Chen P, Pu K. Recent progress in the development of near-infrared organic photothermal and photodynamic nanotherapeutics. Biomaterials Science. 2018;6(4): 746-765. DOI:10.1039/c7bm01210a

[92] Shim G, Kim D, Kim J, Suh MS, Kim YK, Oh YK. Bacteriomimetic polyγ-glutamic acid surface coating for hemocompatibility and safety of nanomaterials. Nanotoxicology. 2017;11

(6):762-770. DOI:10.1080/ 17435390.2017.1353155

resveratrol loaded

DOI:10.1039/c7tb01600j

[94] Khatun Z, Nurunnabi M,

responsive controlled release of doxorubicin. Nanoscale. 2015;7(24): 10680-10689. DOI:10.1039/c5nr01075f

[95] Li P, Yan Y, Chen B, Zhang P, Wang S, Zhou J, Fan H, Wang Y, Huang X. Lanthanide-doped

10.1039/c7bm01113j

oxide for combined in vivo photoacoustic imaging and

upconversion nanoparticles complexed with nano-oxide graphene used for upconversion fluorescence imaging and photothermal therapy. Biomaterials Science. 2018;6(4):877-884. DOI:

[96] Sheng Z, Song L, Zheng J, Hu D, He M, Zheng M, Gao G, Gong P, Zhang P, Ma Y, Cai L. Protein-assisted fabrication of nano-reduced graphene

photothermal therapy. Biomaterials.

Nafiujjaman M, Reeck GR, Khan HA, Cho KJ, Lee YK. A hyaluronic acid nanogel for photo-chemo theranostics of lung cancer with simultaneous light-

[93] Hai L, He D, He X, Wang K, Yang X, Liu J, Cheng H, Huang X, Shangguan J. Facile fabrication of a

phospholipid@reduced graphene oxide nanoassembly for targeted and nearinfrared laser-triggered chemo/ photothermal synergistic therapy of cancer in vivo. Journal of Materials Chemistry B. 2017;5(29):5783-5792.

[86] Zhang C, Lu T, Tao J, Wan G, Zhao H. Co-delivery of paclitaxel and indocyanine green by PEGylated graphene oxide: a potential integrated nanoplatform for tumor theranostics. Rsc Advances. 2016;6(19):15460-15468.

acsnano.5b03804

DOI:10.1039/c5ra25518j

[87] Oliveira ACN, Fernandes J, Gonçalves A, Gomes AC, Oliveira MECDR. Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO

2019;20(1):29-50. DOI:10.2174/ 1389450119666180703145410

[88] Dong H, Dai W, Ju H, Lu H, Wang S, Xu L, Zhou S-F, Zhang Y, Zhang X. Multifunctional Poly(Llactide)-Polyethylene Glycol-Grafted

Graphene Quantum Dots for

Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved

Therapeutics. Acs Applied Materials & Interfaces. 2015;7(20):11015-11023. DOI:10.1021/acsami.5b02803

[89] Wang X, Sun X, He H, Yang H, Lao J, Song Y, Xia Y, Xu H, Zhang X, Huang F. A two-component active targeting theranostic agent based on graphene quantum dots.

Journal of Materials Chemistry B. 2015;

3(17):3583-3590. DOI:10.1039/

Li Y. Current Approaches of Photothermal Therapy in Treating

Nanotherapeutics. Theranostics. 2016;6(6):762-772. DOI:10.7150/

Cancer Metastasis with

[90] Zou L, Wang H, He B, Zeng L, Tan T, Cao H, He X, Zhang Z, Guo S,

c5tb00211g

thno.14988

**113**

Liposomal System. Current drug targets.

[80] Nie L, Huang P, Li W, Yan X, Jin A, Wang Z, Tang Y, Wang S, Zhang X, Niu G, Chen X. Early-stage imaging of nanocarrier-enhanced chemotherapy response in living subjects by scalable photoacoustic microscopy. ACS Nano. 2014;8(12):12141-12150. DOI:10.1021/ nn505989e

[81] Chen H, Liu F, Lei Z, Ma L, Wang Z. Fe2O3@Au core@shell nanoparticlegraphene nanocomposites as theranostic agents for bioimaging and chemophotothermal synergistic therapy. Rsc Advances. 2015;5(103):84980-84987. DOI:10.1039/c5ra17143a

[82] Chang X, Zhang Y, Xu P, Zhang M, Wu H, Yang S. Graphene oxide/ MnWO4 nanocomposite for magnetic resonance/photoacoustic dual-model imaging and tumor photothermochemotherapy. Carbon. 2018;138: 397-409. DOI:10.1016/j. carbon.2018.07.058

[83] Shi J, Wang B, Chen Z, Liu W, Pan J, Hou L, Zhang Z. A Multi-Functional Tumor Theranostic Nanoplatform for MRI Guided Photothermal-Chemotherapy. Pharm Res. 2016;33(6):1472-1485. DOI: 10.1007/s11095-016-1891-7

[84] Shi J, Wang L, Zhang J, Ma R, Gao J, Liu Y, Zhang C, Zhang Z. A tumortargeting near-infrared laser-triggered drug delivery system based on GO@Ag nanoparticles for chemo-photothermal therapy and X-ray imaging. Biomaterials. 2014;35(22):5847-5861. DOI:10.1016/j.biomaterials.2014.03.042

[85] Song J, Yang X, Jacobson O, Lin L, Huang P, Niu G, Ma Q, Chen X. Sequential Drug Release and Enhanced Photothermal and Photoacoustic Effect *Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

of Hybrid Reduced Graphene Oxide-Loaded Ultrasmall Gold Nanorod Vesicles for Cancer Therapy. ACS Nano. 2015;9(9):9199-9209. DOI:10.1021/ acsnano.5b03804

Appl. 2019;102:405-414. DOI:10.1016/j.

*Theranostics - An Old Concept in New Clothing*

Programmatically Monitoring Anti-Cancer Drug Delivery, Release, and Response. ACS Applied Materials and Interfaces. 2017;9(33):27396-27401. DOI:10.1021/acsami.7b08824

[80] Nie L, Huang P, Li W, Yan X, Jin A, Wang Z, Tang Y, Wang S, Zhang X, Niu G, Chen X. Early-stage imaging of nanocarrier-enhanced chemotherapy response in living subjects by scalable photoacoustic microscopy. ACS Nano. 2014;8(12):12141-12150. DOI:10.1021/

[81] Chen H, Liu F, Lei Z, Ma L, Wang Z. Fe2O3@Au core@shell nanoparticlegraphene nanocomposites as theranostic agents for bioimaging and chemophotothermal synergistic therapy. Rsc Advances. 2015;5(103):84980-84987.

[82] Chang X, Zhang Y, Xu P, Zhang M, Wu H, Yang S. Graphene oxide/ MnWO4 nanocomposite for magnetic resonance/photoacoustic dual-model imaging and tumor photothermochemotherapy. Carbon. 2018;138:

nn505989e

DOI:10.1039/c5ra17143a

397-409. DOI:10.1016/j. carbon.2018.07.058

[83] Shi J, Wang B, Chen Z, Liu W, Pan J, Hou L, Zhang Z. A Multi-Functional Tumor Theranostic Nanoplatform for MRI Guided Photothermal-Chemotherapy. Pharm Res. 2016;33(6):1472-1485. DOI: 10.1007/s11095-016-1891-7

[84] Shi J, Wang L, Zhang J, Ma R, Gao J, Liu Y, Zhang C, Zhang Z. A tumortargeting near-infrared laser-triggered drug delivery system based on GO@Ag nanoparticles for chemo-photothermal

Biomaterials. 2014;35(22):5847-5861. DOI:10.1016/j.biomaterials.2014.03.042

[85] Song J, Yang X, Jacobson O, Lin L, Huang P, Niu G, Ma Q, Chen X. Sequential Drug Release and Enhanced Photothermal and Photoacoustic Effect

therapy and X-ray imaging.

[74] Chen W, Li D, Tian L, Xiang W, Wang T, Hu W, Hu Y, Chen S, Chen J, Dai Z. Synthesis of graphene quantum dots from natural polymer starch for cell imaging. Green Chemistry. 2018;20(19): 4438-4442. DOI:10.1039/C8GC02106F

[75] Gu J, Hu MJ, Guo QQ, Ding ZF, Sun XL, Yang J. High-yield synthesis of graphene quantum dots with strong green photoluminescence. RSC Advances. 2014;4(91):50141-50144.

[76] Liu Q, Zhang J, He H, Huang G, Xing B, Jia J, Zhang C. Green

Preparation of High Yield Fluorescent Graphene Quantum Dots from Coal-

Nanomaterials. 2018;8(10):844. DOI:

[78] Augustine S, Singh J, Srivastava M, Sharma M, Das A, Malhotra BD. Recent advances in carbon based nanosystems for cancer theranostics. Biomater Sci. 2017;5(5):901-952. DOI:10.1016/j.

[79] Ding H, Zhang F, Zhao C, Lv Y, Ma G, Wei W, Tian Z. Beyond a Carrier: Graphene Quantum Dots as a Probe for

[77] Halder A, Godoy-Gallardo M, Ashley J, Feng X, Zhou T, Hosta-Rigau L, Sun Y. One-Pot Green Synthesis of Biocompatible Graphene Quantum Dots and Their Cell Uptake Studies. ACS Applied Bio Materials. 2018;1(2): 452-461. DOI:10.1021/acsabm.8b00170

DOI:10.1039/C4RA10011E

Tar-Pitch by Mild Oxidation.

10.3390/nano8100844

msec.2017.02.121

**112**

msec.2019.04.058

02428-7

[73] Basoglu A, Ocak U, Gumrukcuoglu A. Synthesis of Microwave-Assisted Fluorescence Carbon Quantum Dots Using Roasted-Chickpeas and its Applications for Sensitive and Selective Detection of Fe (3+) Ions. J Fluoresc. 2020;30(3): 515-526. DOI:10.1007/s10895-019-

[86] Zhang C, Lu T, Tao J, Wan G, Zhao H. Co-delivery of paclitaxel and indocyanine green by PEGylated graphene oxide: a potential integrated nanoplatform for tumor theranostics. Rsc Advances. 2016;6(19):15460-15468. DOI:10.1039/c5ra25518j

[87] Oliveira ACN, Fernandes J, Gonçalves A, Gomes AC, Oliveira MECDR. Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO Liposomal System. Current drug targets. 2019;20(1):29-50. DOI:10.2174/ 1389450119666180703145410

[88] Dong H, Dai W, Ju H, Lu H, Wang S, Xu L, Zhou S-F, Zhang Y, Zhang X. Multifunctional Poly(Llactide)-Polyethylene Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics. Acs Applied Materials & Interfaces. 2015;7(20):11015-11023. DOI:10.1021/acsami.5b02803

[89] Wang X, Sun X, He H, Yang H, Lao J, Song Y, Xia Y, Xu H, Zhang X, Huang F. A two-component active targeting theranostic agent based on graphene quantum dots. Journal of Materials Chemistry B. 2015; 3(17):3583-3590. DOI:10.1039/ c5tb00211g

[90] Zou L, Wang H, He B, Zeng L, Tan T, Cao H, He X, Zhang Z, Guo S, Li Y. Current Approaches of Photothermal Therapy in Treating Cancer Metastasis with Nanotherapeutics. Theranostics. 2016;6(6):762-772. DOI:10.7150/ thno.14988

[91] Zhu H, Cheng P, Chen P, Pu K. Recent progress in the development of near-infrared organic photothermal and photodynamic nanotherapeutics. Biomaterials Science. 2018;6(4): 746-765. DOI:10.1039/c7bm01210a

[92] Shim G, Kim D, Kim J, Suh MS, Kim YK, Oh YK. Bacteriomimetic polyγ-glutamic acid surface coating for hemocompatibility and safety of nanomaterials. Nanotoxicology. 2017;11 (6):762-770. DOI:10.1080/ 17435390.2017.1353155

[93] Hai L, He D, He X, Wang K, Yang X, Liu J, Cheng H, Huang X, Shangguan J. Facile fabrication of a resveratrol loaded phospholipid@reduced graphene oxide nanoassembly for targeted and nearinfrared laser-triggered chemo/ photothermal synergistic therapy of cancer in vivo. Journal of Materials Chemistry B. 2017;5(29):5783-5792. DOI:10.1039/c7tb01600j

[94] Khatun Z, Nurunnabi M, Nafiujjaman M, Reeck GR, Khan HA, Cho KJ, Lee YK. A hyaluronic acid nanogel for photo-chemo theranostics of lung cancer with simultaneous lightresponsive controlled release of doxorubicin. Nanoscale. 2015;7(24): 10680-10689. DOI:10.1039/c5nr01075f

[95] Li P, Yan Y, Chen B, Zhang P, Wang S, Zhou J, Fan H, Wang Y, Huang X. Lanthanide-doped upconversion nanoparticles complexed with nano-oxide graphene used for upconversion fluorescence imaging and photothermal therapy. Biomaterials Science. 2018;6(4):877-884. DOI: 10.1039/c7bm01113j

[96] Sheng Z, Song L, Zheng J, Hu D, He M, Zheng M, Gao G, Gong P, Zhang P, Ma Y, Cai L. Protein-assisted fabrication of nano-reduced graphene oxide for combined in vivo photoacoustic imaging and photothermal therapy. Biomaterials.

2013;34(21):5236-5243. DOI:10.1016/j. biomaterials.2013.03.090

[97] Chen L, Zhong X, Yi X, Huang M, Ning P, Liu T, Ge C, Chai Z, Liu Z, Yang K. Radionuclide I-131 labeled reduced graphene oxide for nuclear imaging guided combined radio- and photothermal therapy of cancer. Biomaterials. 2015;66:21-28. DOI: 10.1016/j.biomaterials.2015.06.043

[98] Lin L-S, Yang X, Niu G, Song J, Yang H-H, Chen X. Dual-enhanced photothermal conversion properties of reduced graphene oxide-coated gold superparticles for light-triggered acoustic and thermal theranostics. Nanoscale. 2016;8(4):2116-2122. DOI: 10.1039/c5nr07552a

[99] Li S, Zhou S, Li Y, Li X, Zhu J, Fan L, Yang S. Exceptionally High Payload of the IR780 Iodide on Folic Acid-Functionalized Graphene Quantum Dots for Targeted Photothermal Therapy. ACS Applied Materials and Interfaces. 2017;9(27): 22332-22341. DOI:10.1021/ acsami.7b07267

[100] Qin H, Zhou T, Yang S, Xing D. Fluorescence quenching nanoprobes dedicated to in vivo photoacoustic imaging and high-efficient tumor therapy in deep-seated tissue. Small. 2015;11(22):2675-2686. DOI:10.1002/ smll.201403395

[101] Wang Y, Wang H, Liu D, Song S, Wang X, Zhang H. Graphene oxide covalently grafted upconversion nanoparticles for combined NIR mediated imaging and photothermal/ photodynamic cancer therapy. Biomaterials. 2013;34(31):7715-7724. DOI:10.1016/j.biomaterials.2013.06.045

[102] Jin Y, Wang J, Ke H, Wang S, Dai Z. Graphene oxide modified PLA microcapsules containing gold nanoparticles for ultrasonic/CT bimodal imaging guided photothermal tumor therapy. Biomaterials. 2013;34(20):

4794-4802. DOI:10.1016/j. biomaterials.2013.03.027

[103] Ma X, Qu Q, Zhao Y, Luo Z, Zhao Y, Ng KW, Zhao Y. Graphene oxide wrapped gold nanoparticles for intracellular Raman imaging and drug delivery. Journal of Materials Chemistry B. 2013;1(47):6495-6500. DOI:10.1039/C3TB21385D

Biomaterialia. 2017;53:631-642. DOI:

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

[115] Cao Y, Dong H, Yang Z, Zhong X, Chen Y, Dai W, Zhang X. Aptamer-Conjugated Graphene Quantum Dots/ Porphyrin Derivative Theranostic Agent

MicroRNA Detection and Fluorescence-Guided Photothermal/Photodynamic Synergetic Therapy. ACS applied materials & interfaces. 2017;9(1): 159-166. DOI:10.1021/acsami.6b13150

[116] Viraka Nellore BP, Pramanik A, Chavva SR, Sinha SS, Robinson C, Fan Z, Kanchanapally R, Grennell J, Weaver I, Hamme AT, Ray PC.

Aptamer-conjugated theranostic hybrid graphene oxide with highly selective biosensing and combined therapy capability. Faraday Discuss. 2014;175: 257-271. DOI:10.1039/c4fd00074a

[117] Battogtokh G, Ko YT. Graphene oxide-incorporated pH-responsive folate-albumin-photosensitizer nanocomplex as image-guided dual therapeutics. Journal of controlled release : official journal of the Controlled Release Society. 2016;234:10-20. DOI:

10.1016/j.jconrel.2016.05.007

10.1039/c2cc36297j

[118] Cho Y, Kim H, Choi Y. A graphene oxide-photosensitizer complex as an enzyme-activatable theranostic agent. Chemical communications (Cambridge, England). 2013;49(12):1202-1204. DOI:

[119] Luo S, Yang Z, Tan X, Wang Y, Zeng Y, Wang Y, Li C, Li R, Shi C. Multifunctional Photosensitizer Grafted

Polyethylenimine Dual-Functionalized Nanographene Oxide for Cancer-Targeted Near-Infrared Imaging and Synergistic Phototherapy. ACS Applied Materials and Interfaces. 2016;8(27):

[120] Kalluru P, Vankayala R, Chiang C-S, Hwang KC. Nano-graphene oxidemediated In vivo fluorescence imaging and bimodal photodynamic and

on Polyethylene Glycol and

17176-17186. DOI:10.1021/

acsami.6b05383

for Intracellular Cancer-Related

[109] Nergiz SZ, Gandra N, Tadepalli S, Singamaneni S. Multifunctional Hybrid Nanopatches of Graphene Oxide and Gold Nanostars for Ultraefficient Photothermal Cancer Therapy. ACS Applied Materials & Interfaces. 2014;6(18):16395-16402.

[110] Rong P, Wu J, Liu Z, Ma X, Yu L,

Fluorescence dye loaded nano-graphene

[111] Wang Y-W, Fu Y-Y, Peng Q, Guo S-S, Liu G, Li J, Yang H-H, Chen G-N. Dye-enhanced graphene oxide for

[112] Yu J, Lin YH, Yang L, Huang CC, Chen L, Wang WC, Chen GW, Yan J, Sawettanun S, Lin CH. Improved Anticancer Photothermal Therapy Using the Bystander Effect Enhanced by Antiarrhythmic Peptide Conjugated Dopamine-Modified Reduced Graphene Oxide Nanocomposite. Adv Healthc Mater. 2017;6(2). DOI:10.1002/

[113] Huang G, Zhu X, Li H, Wang L, Chi X, Chen J, Wang X, Chen Z, Gao J. Facile integration of multiple magnetite

combining efficient MRI and thermal therapy. Nanoscale. 2015;7(6): 2667-2675. DOI:10.1039/c4nr06616b

[114] Tabish TA, Zhang S, Winyard PG. Developing the next generation of graphene-based platforms for cancer therapeutics: The potential role of reactive oxygen species. Redox Biology.

nanoparticles for theranostics

2018;15:34-40. DOI:10.1016/j.

redox.2017.11.018

**115**

10.1016/j.actbio.2017.01.078

DOI:10.1021/am504795d

C5RA24752G

Zhou K, Zeng W, Wang W.

photothermal therapy and photoacoustic imaging. Journal of Materials Chemistry B. 2013;1(42): 5762-5767. DOI:10.1039/C3TB20986E

adhm.201600804

for multimodal imaging guided photothermal therapy. RSC Advances. 2016;6(3):1894-1901. DOI:10.1039/

[104] Chen Y-W, Liu T-Y, Chen P-J, Chang P-H, Chen S-Y. A High-Sensitivity and Low-Power Theranostic Nanosystem for Cell SERS Imaging and Selectively Photothermal Therapy Using Anti-EGFR-Conjugated Reduced Graphene Oxide/Mesoporous Silica/ AuNPs Nanosheets. Small. 2016;12(11): 1458-1468. DOI:10.1002/smll.201502917

[105] Gao S, Zhang L, Wang G, Yang K, Chen M, Tian R, Ma Q, Zhu L. Hybrid graphene/Au activatable theranostic agent for multimodalities imaging guided enhanced photothermal therapy. Biomaterials. 2016;79:36-45. DOI: 10.1016/j.biomaterials.2015.11.041

[106] Miao W, Shim G, Kim G, Lee S, Lee HJ, Kim YB, Byun Y, Oh YK. Imageguided synergistic photothermal therapy using photoresponsive imaging agent-loaded graphene-based nanosheets. Journal of controlled release : official journal of the Controlled Release Society. 2015;211:28-36. DOI:10.1016/j. jconrel.2015.05.280

[107] Zheng A, Zhang D, Wu M, Yang H, Liu X, Liu J. Multifunctional human serum albumin-modified reduced graphene oxide for targeted photothermal therapy of hepatocellular carcinoma. Rsc Advances. 2016;6(14): 11167-11175. DOI:10.1039/c5ra24785c

[108] Wu C, Li D,Wang L, Guan X, Tian Y, Yang H, Li S, Liu Y. Single wavelength light-mediated, synergistic bimodal cancer photoablation and amplified photothermal performance by graphene/gold nanostar/ photosensitizer theranostics. Acta

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

Biomaterialia. 2017;53:631-642. DOI: 10.1016/j.actbio.2017.01.078

2013;34(21):5236-5243. DOI:10.1016/j.

*Theranostics - An Old Concept in New Clothing*

4794-4802. DOI:10.1016/j. biomaterials.2013.03.027

[103] Ma X, Qu Q, Zhao Y, Luo Z, Zhao Y, Ng KW, Zhao Y. Graphene oxide wrapped gold nanoparticles for intracellular Raman imaging and drug

[104] Chen Y-W, Liu T-Y, Chen P-J, Chang P-H, Chen S-Y. A High-

Anti-EGFR-Conjugated Reduced Graphene Oxide/Mesoporous Silica/ AuNPs Nanosheets. Small. 2016;12(11): 1458-1468. DOI:10.1002/smll.201502917

Sensitivity and Low-Power Theranostic Nanosystem for Cell SERS Imaging and Selectively Photothermal Therapy Using

[105] Gao S, Zhang L, Wang G, Yang K, Chen M, Tian R, Ma Q, Zhu L. Hybrid graphene/Au activatable theranostic agent for multimodalities imaging guided enhanced photothermal therapy. Biomaterials. 2016;79:36-45. DOI: 10.1016/j.biomaterials.2015.11.041

[106] Miao W, Shim G, Kim G, Lee S, Lee HJ, Kim YB, Byun Y, Oh YK. Image-

nanosheets. Journal of controlled release : official journal of the Controlled Release Society. 2015;211:28-36. DOI:10.1016/j.

[107] Zheng A, Zhang D, Wu M, Yang H, Liu X, Liu J. Multifunctional human serum albumin-modified reduced graphene oxide for targeted

photothermal therapy of hepatocellular carcinoma. Rsc Advances. 2016;6(14): 11167-11175. DOI:10.1039/c5ra24785c

[108] Wu C, Li D,Wang L, Guan X, Tian Y, Yang H, Li S, Liu Y. Single wavelength light-mediated, synergistic bimodal cancer photoablation and amplified photothermal performance by graphene/gold nanostar/ photosensitizer theranostics. Acta

guided synergistic photothermal therapy using photoresponsive imaging

agent-loaded graphene-based

jconrel.2015.05.280

delivery. Journal of Materials Chemistry B. 2013;1(47):6495-6500.

DOI:10.1039/C3TB21385D

[97] Chen L, Zhong X, Yi X, Huang M, Ning P, Liu T, Ge C, Chai Z, Liu Z, Yang K. Radionuclide I-131 labeled reduced graphene oxide for nuclear imaging guided combined radio- and photothermal therapy of cancer. Biomaterials. 2015;66:21-28. DOI: 10.1016/j.biomaterials.2015.06.043

[98] Lin L-S, Yang X, Niu G, Song J, Yang H-H, Chen X. Dual-enhanced photothermal conversion properties of reduced graphene oxide-coated gold superparticles for light-triggered acoustic and thermal theranostics. Nanoscale. 2016;8(4):2116-2122. DOI:

[99] Li S, Zhou S, Li Y, Li X, Zhu J, Fan L, Yang S. Exceptionally High Payload of the IR780 Iodide on Folic Acid-Functionalized Graphene Quantum Dots for Targeted Photothermal Therapy. ACS Applied Materials and Interfaces. 2017;9(27):

[100] Qin H, Zhou T, Yang S, Xing D. Fluorescence quenching nanoprobes dedicated to in vivo photoacoustic imaging and high-efficient tumor therapy in deep-seated tissue. Small. 2015;11(22):2675-2686. DOI:10.1002/

[101] Wang Y, Wang H, Liu D, Song S, Wang X, Zhang H. Graphene oxide covalently grafted upconversion nanoparticles for combined NIR mediated imaging and photothermal/ photodynamic cancer therapy. Biomaterials. 2013;34(31):7715-7724. DOI:10.1016/j.biomaterials.2013.06.045

[102] Jin Y, Wang J, Ke H, Wang S, Dai Z. Graphene oxide modified PLA microcapsules containing gold

nanoparticles for ultrasonic/CT bimodal imaging guided photothermal tumor therapy. Biomaterials. 2013;34(20):

10.1039/c5nr07552a

22332-22341. DOI:10.1021/

acsami.7b07267

smll.201403395

**114**

biomaterials.2013.03.090

[109] Nergiz SZ, Gandra N, Tadepalli S, Singamaneni S. Multifunctional Hybrid Nanopatches of Graphene Oxide and Gold Nanostars for Ultraefficient Photothermal Cancer Therapy. ACS Applied Materials & Interfaces. 2014;6(18):16395-16402. DOI:10.1021/am504795d

[110] Rong P, Wu J, Liu Z, Ma X, Yu L, Zhou K, Zeng W, Wang W. Fluorescence dye loaded nano-graphene for multimodal imaging guided photothermal therapy. RSC Advances. 2016;6(3):1894-1901. DOI:10.1039/ C5RA24752G

[111] Wang Y-W, Fu Y-Y, Peng Q, Guo S-S, Liu G, Li J, Yang H-H, Chen G-N. Dye-enhanced graphene oxide for photothermal therapy and photoacoustic imaging. Journal of Materials Chemistry B. 2013;1(42): 5762-5767. DOI:10.1039/C3TB20986E

[112] Yu J, Lin YH, Yang L, Huang CC, Chen L, Wang WC, Chen GW, Yan J, Sawettanun S, Lin CH. Improved Anticancer Photothermal Therapy Using the Bystander Effect Enhanced by Antiarrhythmic Peptide Conjugated Dopamine-Modified Reduced Graphene Oxide Nanocomposite. Adv Healthc Mater. 2017;6(2). DOI:10.1002/ adhm.201600804

[113] Huang G, Zhu X, Li H, Wang L, Chi X, Chen J, Wang X, Chen Z, Gao J. Facile integration of multiple magnetite nanoparticles for theranostics combining efficient MRI and thermal therapy. Nanoscale. 2015;7(6): 2667-2675. DOI:10.1039/c4nr06616b

[114] Tabish TA, Zhang S, Winyard PG. Developing the next generation of graphene-based platforms for cancer therapeutics: The potential role of reactive oxygen species. Redox Biology. 2018;15:34-40. DOI:10.1016/j. redox.2017.11.018

[115] Cao Y, Dong H, Yang Z, Zhong X, Chen Y, Dai W, Zhang X. Aptamer-Conjugated Graphene Quantum Dots/ Porphyrin Derivative Theranostic Agent for Intracellular Cancer-Related MicroRNA Detection and Fluorescence-Guided Photothermal/Photodynamic Synergetic Therapy. ACS applied materials & interfaces. 2017;9(1): 159-166. DOI:10.1021/acsami.6b13150

[116] Viraka Nellore BP, Pramanik A, Chavva SR, Sinha SS, Robinson C, Fan Z, Kanchanapally R, Grennell J, Weaver I, Hamme AT, Ray PC. Aptamer-conjugated theranostic hybrid graphene oxide with highly selective biosensing and combined therapy capability. Faraday Discuss. 2014;175: 257-271. DOI:10.1039/c4fd00074a

[117] Battogtokh G, Ko YT. Graphene oxide-incorporated pH-responsive folate-albumin-photosensitizer nanocomplex as image-guided dual therapeutics. Journal of controlled release : official journal of the Controlled Release Society. 2016;234:10-20. DOI: 10.1016/j.jconrel.2016.05.007

[118] Cho Y, Kim H, Choi Y. A graphene oxide-photosensitizer complex as an enzyme-activatable theranostic agent. Chemical communications (Cambridge, England). 2013;49(12):1202-1204. DOI: 10.1039/c2cc36297j

[119] Luo S, Yang Z, Tan X, Wang Y, Zeng Y, Wang Y, Li C, Li R, Shi C. Multifunctional Photosensitizer Grafted on Polyethylene Glycol and Polyethylenimine Dual-Functionalized Nanographene Oxide for Cancer-Targeted Near-Infrared Imaging and Synergistic Phototherapy. ACS Applied Materials and Interfaces. 2016;8(27): 17176-17186. DOI:10.1021/ acsami.6b05383

[120] Kalluru P, Vankayala R, Chiang C-S, Hwang KC. Nano-graphene oxidemediated In vivo fluorescence imaging and bimodal photodynamic and

photothermal destruction of tumors. Biomaterials. 2016;95:1-10. DOI: 10.1016/j.biomaterials.2016.04.006

[121] Gulzar A, Xu J, Yang D, Xu L, He F, Gai S, Yang P. Nano-graphene oxide-UCNP-Ce6 covalently constructed nanocomposites for NIR-mediated bioimaging and PTT/PDT combinatorial therapy. Dalton transactions (Cambridge, England : 2003). 2018;47(11):3931-3939. DOI:10.1039/c7dt04141a

[122] Yan X, Hu H, Lin J, Jin AJ, Niu G, Zhang S, Huang P, Shen B, Chen X. Optical and photoacoustic dualmodality imaging guided synergistic photodynamic/photothermal therapies. Nanoscale. 2015;7(6):2520-2526. DOI: 10.1039/c4nr06868h

[123] Taratula O, Patel M, Schumann C, Naleway MA, Pang AJ, He H, Taratula O. Phthalocyanine-loaded graphene nanoplatform for imagingguided combinatorial phototherapy. Int J Nanomedicine. 2015;10:2347-2362. DOI:10.2147/ijn.s81097

[124] Bi H, He F, Dai Y, Xu J, Dong Y, Yang D, Gai S, Li L, Li C, Yang P. Quad-Model Imaging-Guided High-Efficiency Phototherapy Based on Upconversion Nanoparticles and ZnFe2O4 Integrated Graphene Oxide. Inorganic Chemistry. 2018;57(16):9988-9998. DOI:10.1021/ acs.inorgchem.8b01159

[125] Ko NR, Nafiujjaman M, Lee JS, Lim HN, Lee YK, Kwon IK. Graphene quantum dot-based theranostic agents for active targeting of breast cancer. Rsc Advances. 2017;7(19):11420-11427. DOI: 10.1039/c6ra25949a

[126] Thakur M, Mewada A, Pandey S, Bhori M, Singh K, Sharon M, Sharon M. Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system. Materials science & engineering C, Materials for biological applications. 2016;67:468-477. DOI: 10.1016/j.msec.2016.05.007

[127] Zhou L, Zhou L, Ge X, Zhou J,Wei S, Shen J. Multicolor imaging and the anticancer effect of a bifunctional silica nanosystem based on the complex of graphene quantum dots and hypocrellin A. Chemical communications (Cambridge, England). 2015;51(2): 421-424. DOI:10.1039/c4cc06968d

[133] Song S, He S, Tao Y, Wang L, Han F, Chen H, Zhang Z. Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance Dual-Modal Imaging and Photothermal Therapy of Tumor. ACS Applied Materials & Interfaces. 2017;9

*DOI: http://dx.doi.org/10.5772/intechopen.96337*

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation*

(11):9484-9495. DOI:10.1021/

10.1016/j.nano.2015.12.197

138-163. DOI:10.1016/j. psep.2018.07.020

s41598-020-58852-4

jcis.2011.07.032

319-75484-0\_13

**117**

[134] Zhang J, Hu D, Cui H, Gao G, Sheng Z, Cai L. Indocyanine green-loaded polydopamine coated reduced graphene oxide for theranostic applications. Nanomedicine-Nanotechnology Biology and Medicine. 2016;12(2):516-516. DOI:

[135] Siddiqui SI, Chaudhry SA. A review on graphene oxide and its composites preparation and their use for the removal of As3+and As5+ from water under the effect of various parameters: Application of isotherm, kinetic and thermodynamics. Process Safety and Environmental Protection. 2018;119:

[136] Baragano D, Forjan R, Welte L, Gallego JLR. Nanoremediation of As and metals polluted soils by means of graphene oxide nanoparticles. Sci Rep. 2020;10(1):1896. DOI:10.1038/

[137] Li Y, Zhang P, Du Q, Peng X, Liu T, Wang Z, Xia Y, Zhang W, Wang K, Zhu H, Wu D. Adsorption of fluoride from aqueous solution by graphene. J Colloid Interface Sci. 2011;

363(1):348-354. DOI:10.1016/j.

p. 309-327. DOI:10.1007/978-3-

[138] Paramasivan T, Sivarajasekar N, Muthusaravanan S, Subashini R, Prakashmaran J, Sivamani S, Ajmal Koya P. Graphene Family Materials for the Removal of Pesticides from Water. A New Generation Material Graphene: Applications in Water Technology2019.

acsami.7b00490

[128] Nurunnabi M, Khatun Z, Reeck GR, Lee DY, Lee Y-k. Photoluminescent Graphene Nanoparticles for Cancer Phototherapy and Imaging. ACS Applied Materials & Interfaces. 2014;6(15):12413-12421. DOI:10.1021/am504071z

[129] Ge J, Lan M, Zhou B, Liu W, Guo L, Wang H, Jia Q, Niu G, Huang X, Zhou H, Meng X, Wang P, Lee C-S, Zhang W, Han X. A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation. Nature Communications. 2014;5(1):4596. DOI: 10.1038/ncomms5596

[130] Su X, Chan C, Shi J, Tsang MK, Pan Y, Cheng C, Gerile O, Yang M. A graphene quantum dot@Fe(3)O(4) @SiO(2) based nanoprobe for drug delivery sensing and dual-modal fluorescence and MRI imaging in cancer cells. Biosens Bioelectron. 2017;92: 489-495. DOI:10.1016/j. bios.2016.10.076

[131] Su Y-L, Yu T-W, Chiang W-H, Chiu H-C, Chang C-H, Chiang C-S, Hu S-H. Hierarchically Targeted and Penetrated Delivery of Drugs to Tumors by Size-Changeable Graphene Quantum Dot Nanoaircrafts for Photolytic Therapy. Advanced Functional Materials. 2017;27 (23). DOI:10.1002/adfm.201700056

[132] Zhang Y, Zhang H, Wang Y, Wu H, Zeng B, Zhang Y, Tian Q, Yang S. Hydrophilic graphene oxide/ bismuth selenide nanocomposites for CT imaging, photoacoustic imaging, and photothermal therapy. Journal of Materials Chemistry B. 2017;5(9): 1846-1855. DOI:10.1039/c6tb02137a

*Graphene-Based Nanosystems: Versatile Nanotools for Theranostics and Bioremediation DOI: http://dx.doi.org/10.5772/intechopen.96337*

[133] Song S, He S, Tao Y, Wang L, Han F, Chen H, Zhang Z. Indocyanine Green Loaded Magnetic Carbon Nanoparticles for Near Infrared Fluorescence/Magnetic Resonance Dual-Modal Imaging and Photothermal Therapy of Tumor. ACS Applied Materials & Interfaces. 2017;9 (11):9484-9495. DOI:10.1021/ acsami.7b00490

photothermal destruction of tumors. Biomaterials. 2016;95:1-10. DOI: 10.1016/j.biomaterials.2016.04.006

*Theranostics - An Old Concept in New Clothing*

[127] Zhou L, Zhou L, Ge X, Zhou J,Wei S, Shen J. Multicolor imaging and the anticancer effect of a bifunctional silica nanosystem based on the complex of graphene quantum dots and hypocrellin

A. Chemical communications (Cambridge, England). 2015;51(2): 421-424. DOI:10.1039/c4cc06968d

[128] Nurunnabi M, Khatun Z, Reeck GR, Lee DY, Lee Y-k. Photoluminescent Graphene

DOI:10.1021/am504071z

10.1038/ncomms5596

489-495. DOI:10.1016/j. bios.2016.10.076

Nanoparticles for Cancer Phototherapy and Imaging. ACS Applied Materials & Interfaces. 2014;6(15):12413-12421.

[129] Ge J, Lan M, Zhou B, Liu W, Guo L, Wang H, Jia Q, Niu G, Huang X, Zhou H, Meng X, Wang P, Lee C-S, Zhang W, Han X. A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation. Nature Communications. 2014;5(1):4596. DOI:

[130] Su X, Chan C, Shi J, Tsang MK, Pan Y, Cheng C, Gerile O, Yang M. A graphene quantum dot@Fe(3)O(4) @SiO(2) based nanoprobe for drug delivery sensing and dual-modal

fluorescence and MRI imaging in cancer cells. Biosens Bioelectron. 2017;92:

[131] Su Y-L, Yu T-W, Chiang W-H, Chiu H-C, Chang C-H, Chiang C-S, Hu S-H. Hierarchically Targeted and Penetrated Delivery of Drugs to Tumors by Size-Changeable Graphene Quantum Dot Nanoaircrafts for Photolytic Therapy. Advanced Functional Materials. 2017;27 (23). DOI:10.1002/adfm.201700056

[132] Zhang Y, Zhang H, Wang Y, Wu H, Zeng B, Zhang Y, Tian Q, Yang S. Hydrophilic graphene oxide/ bismuth selenide nanocomposites for CT imaging, photoacoustic imaging, and photothermal therapy. Journal of Materials Chemistry B. 2017;5(9): 1846-1855. DOI:10.1039/c6tb02137a

[121] Gulzar A, Xu J, Yang D, Xu L, He F, Gai S, Yang P. Nano-graphene oxide-UCNP-Ce6 covalently constructed nanocomposites for NIR-mediated bioimaging and PTT/PDT combinatorial therapy. Dalton transactions (Cambridge, England : 2003). 2018;47(11):3931-3939.

[122] Yan X, Hu H, Lin J, Jin AJ, Niu G, Zhang S, Huang P, Shen B, Chen X. Optical and photoacoustic dualmodality imaging guided synergistic photodynamic/photothermal therapies. Nanoscale. 2015;7(6):2520-2526. DOI:

[123] Taratula O, Patel M, Schumann C,

[124] Bi H, He F, Dai Y, Xu J, Dong Y, Yang D, Gai S, Li L, Li C, Yang P. Quad-Model Imaging-Guided High-Efficiency Phototherapy Based on Upconversion Nanoparticles and ZnFe2O4 Integrated Graphene Oxide. Inorganic Chemistry. 2018;57(16):9988-9998. DOI:10.1021/

[125] Ko NR, Nafiujjaman M, Lee JS, Lim HN, Lee YK, Kwon IK. Graphene quantum dot-based theranostic agents for active targeting of breast cancer. Rsc Advances. 2017;7(19):11420-11427. DOI:

[126] Thakur M, Mewada A, Pandey S, Bhori M, Singh K, Sharon M, Sharon M.

Milk-derived multi-fluorescent graphene quantum dot-based cancer theranostic system. Materials science & engineering C, Materials for biological applications. 2016;67:468-477. DOI:

10.1016/j.msec.2016.05.007

**116**

Naleway MA, Pang AJ, He H, Taratula O. Phthalocyanine-loaded graphene nanoplatform for imagingguided combinatorial phototherapy. Int J Nanomedicine. 2015;10:2347-2362.

DOI:10.1039/c7dt04141a

10.1039/c4nr06868h

DOI:10.2147/ijn.s81097

acs.inorgchem.8b01159

10.1039/c6ra25949a

[134] Zhang J, Hu D, Cui H, Gao G, Sheng Z, Cai L. Indocyanine green-loaded polydopamine coated reduced graphene oxide for theranostic applications. Nanomedicine-Nanotechnology Biology and Medicine. 2016;12(2):516-516. DOI: 10.1016/j.nano.2015.12.197

[135] Siddiqui SI, Chaudhry SA. A review on graphene oxide and its composites preparation and their use for the removal of As3+and As5+ from water under the effect of various parameters: Application of isotherm, kinetic and thermodynamics. Process Safety and Environmental Protection. 2018;119: 138-163. DOI:10.1016/j. psep.2018.07.020

[136] Baragano D, Forjan R, Welte L, Gallego JLR. Nanoremediation of As and metals polluted soils by means of graphene oxide nanoparticles. Sci Rep. 2020;10(1):1896. DOI:10.1038/ s41598-020-58852-4

[137] Li Y, Zhang P, Du Q, Peng X, Liu T, Wang Z, Xia Y, Zhang W, Wang K, Zhu H, Wu D. Adsorption of fluoride from aqueous solution by graphene. J Colloid Interface Sci. 2011; 363(1):348-354. DOI:10.1016/j. jcis.2011.07.032

[138] Paramasivan T, Sivarajasekar N, Muthusaravanan S, Subashini R, Prakashmaran J, Sivamani S, Ajmal Koya P. Graphene Family Materials for the Removal of Pesticides from Water. A New Generation Material Graphene: Applications in Water Technology2019. p. 309-327. DOI:10.1007/978-3- 319-75484-0\_13

**119**

**Chapter 6**

**Abstract**

Conditions

*and V.B. Sameer Kumar*

osteogenesis, nerve tissue degeneration

**1. Introduction**

Regulation of Angiogenesis Using

Nanomaterial Based Formulations:

An Emerging Therapeutic Strategy

to Manage Multiple Pathological

*Aswini Poyyakkara, Sruthi Thekkeveedu, Sharath S. Shankar* 

Angiogenesis is an indispensable biological process, any aberrancy associated with which can lead to pathological manifestations. To manage different pathological conditions associated with abnormal angiogenesis, Nanomaterial based formulations have been tested in *in vitro* and *in vivo* models by different groups. The research advancements pertaining to the applications of major candidate nanomaterials for the treatment of pathologies like tumor, cardiovascular diseases, diabetic retinopathy, age related macular degeneration, chronic wounds, impaired osteogen-

Angiogenesis is an important biological process which involves the development of new capillary network from the pre-existing vasculature [1, 2]. The process of angiogenesis is indispensable in supplying oxygen and nutrients to cells under hypoxia, and it has been implicated in different physiological processes such as wound healing, embryogenesis etc. It has also been reported to play key role in many pathologies including diabetic retinopathy and cancer [3]. Angiogenesis is a multi-step process, which commences when the primary, pro angiogenic cytokine, VEGF, is secreted by the cells experiencing hypoxia. Thereafter the interaction of VEGF with its receptor (VEGFR2) on the nearby endothelial cells (EC), leads to EC activation, proliferation, migration, extra cellular matrix (ECM) remodeling, tube formation followed by loop formation leading finally to neo vessel formation and vascular stabilization [4, 5]. The process of angiogenesis is regulated by multiple factors, which may be pro- or anti-angiogenic in nature. The endogenous pro angiogenic factors include growth factors like VEGF, PDGF, FGF, EGF, angiopoietin-1, interleukin-8, placental growth

esis and nerve tissue degeneration, have been briefed in this chapter.

**Keywords:** angiogenesis, nanomaterials, tumor, cardiovascular diseases, diabetic retinopathy, age related macular degeneration, chronic wounds,

#### **Chapter 6**

## Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic Strategy to Manage Multiple Pathological Conditions

*Aswini Poyyakkara, Sruthi Thekkeveedu, Sharath S. Shankar and V.B. Sameer Kumar*

#### **Abstract**

Angiogenesis is an indispensable biological process, any aberrancy associated with which can lead to pathological manifestations. To manage different pathological conditions associated with abnormal angiogenesis, Nanomaterial based formulations have been tested in *in vitro* and *in vivo* models by different groups. The research advancements pertaining to the applications of major candidate nanomaterials for the treatment of pathologies like tumor, cardiovascular diseases, diabetic retinopathy, age related macular degeneration, chronic wounds, impaired osteogenesis and nerve tissue degeneration, have been briefed in this chapter.

**Keywords:** angiogenesis, nanomaterials, tumor, cardiovascular diseases, diabetic retinopathy, age related macular degeneration, chronic wounds, osteogenesis, nerve tissue degeneration

#### **1. Introduction**

Angiogenesis is an important biological process which involves the development of new capillary network from the pre-existing vasculature [1, 2]. The process of angiogenesis is indispensable in supplying oxygen and nutrients to cells under hypoxia, and it has been implicated in different physiological processes such as wound healing, embryogenesis etc. It has also been reported to play key role in many pathologies including diabetic retinopathy and cancer [3]. Angiogenesis is a multi-step process, which commences when the primary, pro angiogenic cytokine, VEGF, is secreted by the cells experiencing hypoxia. Thereafter the interaction of VEGF with its receptor (VEGFR2) on the nearby endothelial cells (EC), leads to EC activation, proliferation, migration, extra cellular matrix (ECM) remodeling, tube formation followed by loop formation leading finally to neo vessel formation and vascular stabilization [4, 5].

The process of angiogenesis is regulated by multiple factors, which may be pro- or anti-angiogenic in nature. The endogenous pro angiogenic factors include growth factors like VEGF, PDGF, FGF, EGF, angiopoietin-1, interleukin-8, placental growth

factor, angiogenin etc. The anti- angiogenic factors include endostatin, angiostatin, prolactin, fibronectin, vasostatin, interleukin-12, platelet factor 4 etc. [6, 7]. An equilibrium exists between the pro- and anti-angiogenic factors under physiological conditions, and any disturbance in that equilibrium would result in pathological manifestations [3]. Targeting angiogenesis therefore has drawn huge attention with respect to the therapeutics of pathologies were excessive or insufficient angiogenesis prevails [7]. One of the major approaches in angiogenesis targeted therapy involves targeting VEGF signaling pathway. Humanized monoclonal antibody targeting VEGFA, namely, Bevacizumab, with the approval of US Food and Drug Administration (FDA), has been employed in a combination therapy for the treatment of metastatic colorectal cancer [8]. In addition, an aptamer which inhibits VEGF 165, namely, Pegaptanib has been approved by FDA to treat Age related macular degeneration [9]*.* In spite of all such interventions, targeting angiogenesis demands much more explorations due to a variety of unresolved issues such as development of resistance to antiangiogenic therapy, lack of adequate treatment for ischaemic disorders etc. [10].

In an urge to overcome the limitations of conventional angiogenic therapy, researchers globally have focused on developing 'nanomedicines' for the treatment and diagnosis of various diseases associated with aberrant angiogenesis [11]. The field of nanomedicine involves the use of nanomaterials for biological and medicinal applications by virtue of their ability to interact with nucleic acids, proteins and membrane receptors effortlessly [10]*.* In this chapter, we have therefore focused on various research achievements pertaining to candidate nanomaterials that can be developed as potential drugs for angiogenic therapy.

#### **2. Nanomaterials**

The class of substances having at least one dimension less than 100 nano meters are called nanoscale materials and the field of science that deals with the synthesis, study of structure, physical and chemical properties and applications of various types of nanoscale materials is referred as Nanotechnology [12]*.* Nanomaterials usually occur as zero, one, two and three-dimensional structures. Generally, the nanoparticles are comprised of three layers called the surface layer, the shell layer and the core. The core is the central portion of the materials surrounded by the shell and surface layer. The shell layer is chemically different from the core and the outer layer. The surface layer permits surface modification with a variety of moieties like polymers, metal ions, and surfactants [13]. The physical and chemical properties of bulk materials are independent of their size, however, when converted into nano scale materials their optical, physical, mechanical and chemical properties vary according to their size [14]. Such properties include solubility, color, toxicity etc. The major reason for these improved properties of nanomaterials are due to their high surface mass ratio as compared with the bulk [15]. Due to their unique size, shape, structure and solubility they have found application in the biomedical, optical, sensor, electric and energy harvesting fields. Many nanomaterials are already being explored for their use in biomedical imaging [16], bio/chemical sensing [17], targeted gene and drug delivery [18]. We here focus on candidate nanomaterials which are potential nanomedicines in the field of therapeutic angiogenesis.

#### **2.1 Classification of nanomaterials according to chemical composition**

Based on the origin, size, morphology and chemical composition, nanomaterials are divided into various categories. In the present chapter we are focusing on some of the important classes that have found applications in biological field.

**121**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

Metal nanoparticles are those particles which may be the pure metal or metal compounds like metal oxide, hydroxides, sulphides etc., exhibit size in the submicron scale. A variety of metal nanoparticles has been synthesized with varied structural morphology, size and compositions [19]. These metal nanoparticles can be synthesized from various metal precursors and can be functionalized with several groups [20]. The metal nanoparticles permit surface modification with various chemical functional groups and further allow them to be conjugated with polymers, ligands, antibodies etc. The improved surface mass ratio, shape, morphology and functionality, quantum confinement and plasmon excitation make them suitable for the applications in the field of energy, catalysis, electronics, and medicine [21]. However, they show some demerits such as tendency to get agglomerate and chances of formation of impurities due to their high reactivity. Many of the nano-

Among the various carbonaceous nanomaterials, the zero-dimensional carbon-

 hybridized carbon atoms rolled up to design a cylindrical shape. They exist as both single-walled CNTs and multi-walled CNTs depending on the number rolled-up graphene sheets. Due to their exceptional structural, mechanical, and electrical diversities, they deliver remarkable flexibility, strength, and electrical properties suitable for various biological applications like medical diagnostics, sensing and treatment of diseases. Graphene represents the 2D nano allotrope of carbon

surpassingly large surface area, easy functionalization and chemical purity makes it a potential candidate for drug delivery. Moreover, it is also widely explored for *in* 

Polymeric nanoparticles are constructed with the aid of natural or synthetic polymers. As compared to other nanoparticles, they offer advantages like non-toxicity and biocompatibility suited for specific biological applications. Although they are used for biosensing and bioimaging, the major purpose of polymeric nanoparticles lies in the field of drug delivery [25]. Biomolecules or drugs are encapsulated into polymeric nanoparticles to obtain a gradual and continuous release of the drugs

Nanoscale ceramics, which include various ceramic nanoparticles of zirconia, hydroxyapatite, alumina and titanium oxide have also found potential biological applications. Some of the distinct features like high load capacity, stability and effortless incorporation to hydrophilic and hydrophobic systems enhance

hybridized carbon network. Its

based quantum dots (CQDs and GQDs), one-dimensional carbon nanotubes (CNTs) and two-dimensional graphene (GR) are currently the most popular nanocarbon representatives in biological applications [22]. Carbon-based QDs are the recent extension in the nano carbon family with fascinating properties like biocompatibility, resistance to photobleaching and attractive photoluminescence. These outstanding properties make them smart candidates for bioimaging, sensing, drug delivery and cancer therapy [23, 24]. CNTs have a unique 1D nanostructure,

materials except gold, silver, and platinum exhibits high cyto-toxicity.

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

*2.1.1 Metal nanoparticles*

*2.1.2 Carbon-based nanomaterials*

illustrating a planar graphitic structure with sp2

*vivo* imaging and cancer detection.

*2.1.3 Polymeric nanoparticles*

at the specifically targeted sites.

*2.1.4 Ceramic nanoparticles*

with sp2

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

#### *2.1.1 Metal nanoparticles*

*Theranostics - An Old Concept in New Clothing*

factor, angiogenin etc. The anti- angiogenic factors include endostatin, angiostatin, prolactin, fibronectin, vasostatin, interleukin-12, platelet factor 4 etc. [6, 7]. An equilibrium exists between the pro- and anti-angiogenic factors under physiological conditions, and any disturbance in that equilibrium would result in pathological manifestations [3]. Targeting angiogenesis therefore has drawn huge attention with respect to the therapeutics of pathologies were excessive or insufficient angiogenesis prevails [7]. One of the major approaches in angiogenesis targeted therapy involves targeting VEGF signaling pathway. Humanized monoclonal antibody targeting VEGFA, namely, Bevacizumab, with the approval of US Food and Drug Administration (FDA), has been employed in a combination therapy for the treatment of metastatic colorectal cancer [8]. In addition, an aptamer which inhibits VEGF 165, namely, Pegaptanib has been approved by FDA to treat Age related macular degeneration [9]*.* In spite of all such interventions, targeting angiogenesis demands much more explorations due to a variety of unresolved issues such as development of resistance to antiangiogenic

therapy, lack of adequate treatment for ischaemic disorders etc. [10].

developed as potential drugs for angiogenic therapy.

**2. Nanomaterials**

In an urge to overcome the limitations of conventional angiogenic therapy, researchers globally have focused on developing 'nanomedicines' for the treatment and diagnosis of various diseases associated with aberrant angiogenesis [11]. The field of nanomedicine involves the use of nanomaterials for biological and medicinal applications by virtue of their ability to interact with nucleic acids, proteins and membrane receptors effortlessly [10]*.* In this chapter, we have therefore focused on various research achievements pertaining to candidate nanomaterials that can be

The class of substances having at least one dimension less than 100 nano meters are called nanoscale materials and the field of science that deals with the synthesis, study of structure, physical and chemical properties and applications of various types of nanoscale materials is referred as Nanotechnology [12]*.* Nanomaterials usually occur as zero, one, two and three-dimensional structures. Generally, the nanoparticles are comprised of three layers called the surface layer, the shell layer and the core. The core is the central portion of the materials surrounded by the shell and surface layer. The shell layer is chemically different from the core and the outer layer. The surface layer permits surface modification with a variety of moieties like polymers, metal ions, and surfactants [13]. The physical and chemical properties of bulk materials are independent of their size, however, when converted into nano scale materials their optical, physical, mechanical and chemical properties vary according to their size [14]. Such properties include solubility, color, toxicity etc. The major reason for these improved properties of nanomaterials are due to their high surface mass ratio as compared with the bulk [15]. Due to their unique size, shape, structure and solubility they have found application in the biomedical, optical, sensor, electric and energy harvesting fields. Many nanomaterials are already being explored for their use in biomedical imaging [16], bio/chemical sensing [17], targeted gene and drug delivery [18]. We here focus on candidate nanomaterials which are potential nanomedicines in the field of therapeutic angiogenesis.

**2.1 Classification of nanomaterials according to chemical composition**

of the important classes that have found applications in biological field.

Based on the origin, size, morphology and chemical composition, nanomaterials are divided into various categories. In the present chapter we are focusing on some

**120**

Metal nanoparticles are those particles which may be the pure metal or metal compounds like metal oxide, hydroxides, sulphides etc., exhibit size in the submicron scale. A variety of metal nanoparticles has been synthesized with varied structural morphology, size and compositions [19]. These metal nanoparticles can be synthesized from various metal precursors and can be functionalized with several groups [20]. The metal nanoparticles permit surface modification with various chemical functional groups and further allow them to be conjugated with polymers, ligands, antibodies etc. The improved surface mass ratio, shape, morphology and functionality, quantum confinement and plasmon excitation make them suitable for the applications in the field of energy, catalysis, electronics, and medicine [21]. However, they show some demerits such as tendency to get agglomerate and chances of formation of impurities due to their high reactivity. Many of the nanomaterials except gold, silver, and platinum exhibits high cyto-toxicity.

#### *2.1.2 Carbon-based nanomaterials*

Among the various carbonaceous nanomaterials, the zero-dimensional carbonbased quantum dots (CQDs and GQDs), one-dimensional carbon nanotubes (CNTs) and two-dimensional graphene (GR) are currently the most popular nanocarbon representatives in biological applications [22]. Carbon-based QDs are the recent extension in the nano carbon family with fascinating properties like biocompatibility, resistance to photobleaching and attractive photoluminescence. These outstanding properties make them smart candidates for bioimaging, sensing, drug delivery and cancer therapy [23, 24]. CNTs have a unique 1D nanostructure, with sp2 hybridized carbon atoms rolled up to design a cylindrical shape. They exist as both single-walled CNTs and multi-walled CNTs depending on the number rolled-up graphene sheets. Due to their exceptional structural, mechanical, and electrical diversities, they deliver remarkable flexibility, strength, and electrical properties suitable for various biological applications like medical diagnostics, sensing and treatment of diseases. Graphene represents the 2D nano allotrope of carbon illustrating a planar graphitic structure with sp2 hybridized carbon network. Its surpassingly large surface area, easy functionalization and chemical purity makes it a potential candidate for drug delivery. Moreover, it is also widely explored for *in vivo* imaging and cancer detection.

#### *2.1.3 Polymeric nanoparticles*

Polymeric nanoparticles are constructed with the aid of natural or synthetic polymers. As compared to other nanoparticles, they offer advantages like non-toxicity and biocompatibility suited for specific biological applications. Although they are used for biosensing and bioimaging, the major purpose of polymeric nanoparticles lies in the field of drug delivery [25]. Biomolecules or drugs are encapsulated into polymeric nanoparticles to obtain a gradual and continuous release of the drugs at the specifically targeted sites.

#### *2.1.4 Ceramic nanoparticles*

Nanoscale ceramics, which include various ceramic nanoparticles of zirconia, hydroxyapatite, alumina and titanium oxide have also found potential biological applications. Some of the distinct features like high load capacity, stability and effortless incorporation to hydrophilic and hydrophobic systems enhance

their efficiency in the field of biomedicine, however, work on scaling down its cytotoxicity remains to be addressed before its full-fledged use in the biological system [26].

#### *2.1.5 Semiconductor nanoparticles*

Semiconductor nanoparticles, particularly QDs have been heavily explored for a wide variety of biological applications like biosensing, molecular imaging, livecell labelling and drug delivery. They possess unique optical properties like a long fluorescence lifetime and low photobleaching when correlated with conventional organic dyes and fluorescent polymers [27]. Although, the toxicity of the traditional semiconductor QDs is a typical concern that has to be addressed for *in vivo* applications.

#### *2.1.6 Lipid-based nanoparticles*

Lipid-based nanoparticles, consisting of liposomes, nanostructured lipid carriers and solid lipid nanoparticles have gained tremendous attention in the field of cancer treatment and drug delivery. These nanoparticles exhibit very low toxicity, can act as a carrier for both hydrophilic and hydrophobic molecules and ensures controlled release of drugs. Due to its versatility and biocompatibility, liposomes are the extensively utilized lipid-based nanoparticles [28].

#### **3. Nanomaterial mediated therapy for pathologies with aberrant angiogenesis**

Abnormal or excessive angiogenesis has been reported to be involved in the progression of a wide variety of diseases affecting different organs. For example, aberrant angiogenesis has been implicated to promote diseases like tumor, auto immune disorders and infectious diseases caused by the pathogens inducing angiogenesis and such diseases have been reported to affect multiple organ systems [29]. Further, it has also been reported to be involved in the advancement of skin tissue associated diseases like psoriasis, allergic dermatitis, blistering disease, scar keloids etc. In addition, it has been reported to be the major cause for diabetic retinopathy and choroidal neovascularization associated with wet type AMD, which affect the eyes [29]*.* Abnormal angiogenesis has also been reported to be involved in the progression of blood vessel associated disorders like atherosclerosis, transplant arteriopathy etc*.* [30]*.* The involvement of angiogenesis has also been reported in the progression of primary pulmonary hypertension, asthma and nasal polyps [29]. In addition, it has also been reported in the progression of diseases that affect the reproductive system, which include ovarian hyper stimulation, endometriosis etc. [31]*.* Aberrant angiogenesis has also been the leading cause for the progression of diseases like osteomyelitis which is characterized by impaired osteogenesis [29]*.* It has also been reported to promote nerve system associated diseases like diabetic neuropathy and amyotrophic lateral sclerosis, which are characterized by nerve tissue degeneration [32]*.* The process of angiogenesis has also been reported to promote physiological processes like wound healing and discrepancy associated with that could lead to complications like development of chronic wounds [33]*.* Different candidate disorders associated with aberrant angiogenesis and the candidate nanomaterials that can be developed as potential drugs for the treatment of such disorders have been detailed below.

**123**

expression of SIRT1 gene [50]*.*

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

The essentiality of angiogenesis in the progression of tumor growth was a breakthrough finding by Judah Folkman way back in 1971, which opened up an era of investigations, concerned with targeting angiogenesis for cancer therapeutics. It has been established that a tumor cannot grow beyond 2 mm in diameter without a steady supply of oxygen and nutrients by means of angiogenesis [34–36]*.* Therefore, preventing the neovascularisation has been suggested as one of the key strategies for cancer therapeutics. Angiogenesis in a tumor micro environment, unlike that under physiological conditions, is characterized by the formation of immature, leaky blood vessels, resulting in a continual state of inflammation. This happens mainly due to the increased expression of a variety of pro angiogenic factors including VEGF, angiopoietin, integrins etc. and such factors are being targeted for anti-angiogenic therapy. Anti-angiogenic agents targeting VEGF, such as Bevacizumab has been approved by FDA, however, release of other pro angiogenic factors over ruled the efficiency of such mono-therapies [37–40]. Therefore, combination therapies using multiple anti-angiogenic agents were more appreciated

Nanoparticles (NPs) could be employed as a vehicle to deliver multiple drugs, targeting different molecules and pathways associated with tumor angiogenesis [37, 41]. The therapeutic drugs are generally loaded on to the NPs either by chemical conjugation or by encapsulation [38]. The NP-based drug delivery can either be passive or active in mode. The presence of leaky blood vessels in the vicinity of tumors facilitates the passive extravasation of NPs with size less than 200 nm into the tumor site by the Enhanced Permeability and Retention effect (EPR) and such NPs are later on cleared by the liver [39, 42]. In addition, limited lymphatic drainage facilitates the retention of NPs at the site of tumors which in turn promotes sustained drug delivery [39]. It has been reported that NP conjugated Doxorubicin [43, 44] and small molecule inhibitors of angiogenesis [45] could accumulate in the tumor micro environment by EPR effect, which lead to the stoppage of tumor angiogenesis and tumor growth [38]. Further, Caplostatin (TNP-470), an angiogenic inhibitor, has been reported to get selectively piled up in the blood vessels associated with tumors by EPR effect which in turn blocked tumor associated vascular hyperpermeability [46, 47]. The Active targeting of tumor vasculature by NPs is achieved by means of ligands presented on NP surfaces. The ligands would selectively bind to receptors which are over expressed on tumor cells as well as on tumor associated ECs, such receptors include VEGFRs, αvβ3 integrins etc. [38, 48]*.* NP mediated targeting of different miRNAs have also been tested for their therapeutic efficacy [49]. For instance, treatment with NP containing anti-miR-21 (CTX-SNALP-anti miR-21) has been reported to silence miR-21 in patients with glioblastoma resulting in an increase in the levels of its target gene RhoB both at mRNA and protein levels. Further, NP mediated administration of anti-miR-21 has been reported to inhibit tumor proliferation, induce apoptosis and promote survival rate in the animal model [49]. Exosomes are endogenous lipid-based NPs which are involved in the transfer of biomolecules like RNA and proteins between cells. It has been reported that miR-23a encapsulated exosomes could effectively induce angiogenesis in CAM model as well as in *in ovo* xenograft model by regulating the

Different metal NPs like gold and silver NPs have been reported to be effective for anti-angiogenic therapy. It has been reported that gold NPs (AuNPs) are capable of binding to the heparin binding domains of various growth factors like VEGF165 and bFGF leading to the conformational changes associated with the impaired functioning of such growth factors. AuNP mediated inhibition of VEGF was found

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

to quick fix resistance to angiogenic monotherapy.

**3.1 Tumor**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

#### **3.1 Tumor**

*Theranostics - An Old Concept in New Clothing*

*2.1.5 Semiconductor nanoparticles*

*2.1.6 Lipid-based nanoparticles*

the extensively utilized lipid-based nanoparticles [28].

system [26].

applications.

**angiogenesis**

their efficiency in the field of biomedicine, however, work on scaling down its cytotoxicity remains to be addressed before its full-fledged use in the biological

Semiconductor nanoparticles, particularly QDs have been heavily explored for a

Lipid-based nanoparticles, consisting of liposomes, nanostructured lipid carriers and solid lipid nanoparticles have gained tremendous attention in the field of cancer treatment and drug delivery. These nanoparticles exhibit very low toxicity, can act as a carrier for both hydrophilic and hydrophobic molecules and ensures controlled release of drugs. Due to its versatility and biocompatibility, liposomes are

Abnormal or excessive angiogenesis has been reported to be involved in the progression of a wide variety of diseases affecting different organs. For example, aberrant angiogenesis has been implicated to promote diseases like tumor, auto immune disorders and infectious diseases caused by the pathogens inducing angiogenesis and such diseases have been reported to affect multiple organ systems [29]. Further, it has also been reported to be involved in the advancement of skin tissue associated diseases like psoriasis, allergic dermatitis, blistering disease, scar keloids etc. In addition, it has been reported to be the major cause for diabetic retinopathy and choroidal neovascularization associated with wet type AMD, which affect the eyes [29]*.* Abnormal angiogenesis has also been reported to be involved in the progression of blood vessel associated disorders like atherosclerosis, transplant arteriopathy etc*.* [30]*.* The involvement of angiogenesis has also been reported in the progression of primary pulmonary hypertension, asthma and nasal polyps [29]. In addition, it has also been reported in the progression of diseases that affect the reproductive system, which include ovarian hyper stimulation, endometriosis etc. [31]*.* Aberrant angiogenesis has also been the leading cause for the progression of diseases like osteomyelitis which is characterized by impaired osteogenesis [29]*.* It has also been reported to promote nerve system associated diseases like diabetic neuropathy and amyotrophic lateral sclerosis, which are characterized by nerve tissue degeneration [32]*.* The process of angiogenesis has also been reported to promote physiological processes like wound healing and discrepancy associated with that could lead to complications like development of chronic wounds [33]*.* Different candidate disorders associated with aberrant angiogenesis and the candidate nanomaterials that can be developed as potential drugs for the treatment of such disorders have been detailed

**3. Nanomaterial mediated therapy for pathologies with aberrant** 

wide variety of biological applications like biosensing, molecular imaging, livecell labelling and drug delivery. They possess unique optical properties like a long fluorescence lifetime and low photobleaching when correlated with conventional organic dyes and fluorescent polymers [27]. Although, the toxicity of the traditional semiconductor QDs is a typical concern that has to be addressed for *in vivo*

**122**

below.

The essentiality of angiogenesis in the progression of tumor growth was a breakthrough finding by Judah Folkman way back in 1971, which opened up an era of investigations, concerned with targeting angiogenesis for cancer therapeutics. It has been established that a tumor cannot grow beyond 2 mm in diameter without a steady supply of oxygen and nutrients by means of angiogenesis [34–36]*.* Therefore, preventing the neovascularisation has been suggested as one of the key strategies for cancer therapeutics. Angiogenesis in a tumor micro environment, unlike that under physiological conditions, is characterized by the formation of immature, leaky blood vessels, resulting in a continual state of inflammation. This happens mainly due to the increased expression of a variety of pro angiogenic factors including VEGF, angiopoietin, integrins etc. and such factors are being targeted for anti-angiogenic therapy. Anti-angiogenic agents targeting VEGF, such as Bevacizumab has been approved by FDA, however, release of other pro angiogenic factors over ruled the efficiency of such mono-therapies [37–40]. Therefore, combination therapies using multiple anti-angiogenic agents were more appreciated to quick fix resistance to angiogenic monotherapy.

Nanoparticles (NPs) could be employed as a vehicle to deliver multiple drugs, targeting different molecules and pathways associated with tumor angiogenesis [37, 41]. The therapeutic drugs are generally loaded on to the NPs either by chemical conjugation or by encapsulation [38]. The NP-based drug delivery can either be passive or active in mode. The presence of leaky blood vessels in the vicinity of tumors facilitates the passive extravasation of NPs with size less than 200 nm into the tumor site by the Enhanced Permeability and Retention effect (EPR) and such NPs are later on cleared by the liver [39, 42]. In addition, limited lymphatic drainage facilitates the retention of NPs at the site of tumors which in turn promotes sustained drug delivery [39]. It has been reported that NP conjugated Doxorubicin [43, 44] and small molecule inhibitors of angiogenesis [45] could accumulate in the tumor micro environment by EPR effect, which lead to the stoppage of tumor angiogenesis and tumor growth [38]. Further, Caplostatin (TNP-470), an angiogenic inhibitor, has been reported to get selectively piled up in the blood vessels associated with tumors by EPR effect which in turn blocked tumor associated vascular hyperpermeability [46, 47]. The Active targeting of tumor vasculature by NPs is achieved by means of ligands presented on NP surfaces. The ligands would selectively bind to receptors which are over expressed on tumor cells as well as on tumor associated ECs, such receptors include VEGFRs, αvβ3 integrins etc. [38, 48]*.*

NP mediated targeting of different miRNAs have also been tested for their therapeutic efficacy [49]. For instance, treatment with NP containing anti-miR-21 (CTX-SNALP-anti miR-21) has been reported to silence miR-21 in patients with glioblastoma resulting in an increase in the levels of its target gene RhoB both at mRNA and protein levels. Further, NP mediated administration of anti-miR-21 has been reported to inhibit tumor proliferation, induce apoptosis and promote survival rate in the animal model [49]. Exosomes are endogenous lipid-based NPs which are involved in the transfer of biomolecules like RNA and proteins between cells. It has been reported that miR-23a encapsulated exosomes could effectively induce angiogenesis in CAM model as well as in *in ovo* xenograft model by regulating the expression of SIRT1 gene [50]*.*

Different metal NPs like gold and silver NPs have been reported to be effective for anti-angiogenic therapy. It has been reported that gold NPs (AuNPs) are capable of binding to the heparin binding domains of various growth factors like VEGF165 and bFGF leading to the conformational changes associated with the impaired functioning of such growth factors. AuNP mediated inhibition of VEGF was found

to be negatively regulating the phosphorylation of VEGFR2. The inhibitory effect of AuNPs on Heparin binding growth factors (HB-GFs) was found to be greatly depended on the size of AuNPs, further, AuNPs with 20 nm in diameter exhibited maximum inhibitory effect. In addition, AuNP with bare surface was found to be essential for the inhibitory effect on HB-GFs. Further, AuNPs have been reported to block of MAPK pathway in tumor cells which lead to the inhibition of epithelial to mesenchymal transition (EMT) and thence, the process of metastasis [51, 52]*.*

AuNP has also been used as the carrier tool for drug delivery. It has been used to deliver an anti-EMT agent, Quercetin (Qu) and AuNP-Qu was found to be more effective when compared to free Qu, in inhibiting cell migration in MDA-MB-23 and MCF-7 cell lines [53]. In addition, recombinant human endostatin (rhES), an anti- angiogenic molecule, which in conjugation with AuNP-PEG (rhES-AuNPs-PEG), when administrated, targeted tumor cells more efficiently and exhibited better performance when compared to rhES. Moreover, the administration of rhES-AuNPs-PEG in combination with 5-flouro uracil (5-FU) facilitated improved localization of 5-FU on to the tumor site with subsequent reduction in tumor size than that in case of mono therapeutic administration of 5FU [54].

Silver NPs (AgNPs) have been reported to inhibit VEGF induced cell proliferation, migration and tube formation in bovine retinal endothelial cells (BRECs). It has also been reported to inhibit vessel formation in matrigel plug assay system. AgNP mediated anti angiogenic effect was found to involve negative regulation of PI3K/Akt pathway [55, 56]*.* According to a different study, AgNP has been reported to exert anti angiogenic effect by inhibiting HIF-1 in a dose dependant manner [57]*.*

In addition to metal NPs, NPs based on cationic polysaccharides like chitosan has also been explored for biomedical applications taking an advantage of their relatively low toxic nature and high biodegradability and biocompatibility. Chitosan NPs (CNPs) showed anti-cancer effect in the xenograft model of hepatocellular carcinoma by inhibiting the expression of VEGFR2 and thereby negatively regulating the process of tumor angiogenesis [58]. Further, CNPs in conjugation with Ursolic acid (CH-UA-NPs) have been shown to inhibit cell migration and tube formation in human umbilical vein endothelial cells (HUVECs) *in-vitro*. In addition, CH-UA-NPs have also been reported to inhibit the expression of VEGF in hepatoma cell xenografts [59]. CNPs have also been utilized as a vehicle for the co delivery of psiRNA VEGF and pIL-4 in MCF-7 cells which caused relatively huge reduction in the levels of VEGF protein when compared to the cases where the plasmids were used individually [60]*.*

Ruthenium modified selenium NPs (Ru-SeNPs) have also been reported to exhibit anti angiogenic properties, in CAM model as well as in HUVEC cells, mainly by inhibiting the phosphorylation of Akt, FGFR1 and Erk1/2. Further, it has been shown that SeNPs protected with Ru (II)-thiols (Ru-MUA@Se) was endocytosed by the cells by clathrin mediated mechanism [61]. SeNPs have also been used as a carrier tool for siRNA delivery. A pH sensitive, modified SeNP carrying VEGF-siRNA, namely, G2/PAH-Cit/SeNPs@siRNA, has been shown to exhibit high efficiency in terms of cellular uptake, drug release and gene silencing [62]*.*

The cerium oxide NPs (CONPs) have been reported to exhibit anti-oxidant activity and they are characterized by a cerium core and a shield with an oxygen lattice. Chen et al., have shown that CONPs are capable of inhibiting reactive oxygen species (ROS) induced angiogenic signaling pathways [63]*.* In addition, the nanoceria conjugated with heparin was reported to inhibit the proliferation of human coronary artery endothelial cells (HCAECs) in a better way than that by unconjugated nanoceria [64]*.* Nanoceria has also been reported to inhibit the

**125**

**Figure 1.**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

proliferation of ovarian cancer cells in xenograft model *in-vivo* [65]*.* Further, the nanoceria conjugated with folic acid has also been reported to inhibit proliferation and angiogenesis in xenografts of ovarian cancer cells *in vivo* [66]*.* The anti-angiogenic effect imparted by nanoceria was reported to involve the inhibition of VEGF signaling pathway leading to the decreased phosphorylation of VEGFR2 at Tyr1173 and Y951 [65]*.* However, a report by Das et al., have suggested that nanoceria might exhibit pro angiogenic effect also [67]*,* making the use of these NPs as anti-angio-

Silica based NPs have also been reported to exhibit anti angiogenic properties.

Silicate NPs (SiO2 NPs) have been reported to inhibit VEGFR2 phosphorylation and ERK1/2 activation in human micro vascular retinal endothelial cells (HMRECs), thereby inhibiting angiogenesis [68]. Mesoporous silica based nanoparticles (MSNs) have been used as a vehicle for the targeted delivery of chemotherapeutic agent, doxorubicin hydrochloride (MSNs@DOX). MSNs@ DOX has been reported to suppress the metastasis of lung cancer cells by inhibiting VEGF induced angiogenesis [69]*.* Further RGD (Arg-Gly-Asp) modified MSN has been used as a carrier tool for the targeted delivery of anti-angiogenic agent,

Further, MoS2 nanoflakes containing ZnO NPs were found to inhibit tumor

*Applications of nanomaterials in anti-tumor therapy. Many candidate nanomaterials possess intrinsic antiangiogenic property and few could be used as vehicles for targeted drug delivery. Nanoparticles encapsulated/ conjugated with anti- angiogenic drugs or nanoparticle based anti-angiogenic scaffolds, when administrated in* 

*in vivo models, precisely target tumor vasculature and inhibit tumor growth.*

growth in *in-ovo* xenograft model by inducing apoptosis and by negatively regulating the processes of angiogenesis as well as EMT [71]. Similarly, the Tetraiodothyroacetic acid (Tetrac) based NPs have also been reported to be antiangiogenic in nature in CAM model and in xenograft model of renal cancer cells [72]*.* Shereema et al., have formulated a green luminescent CQDs, which inhibited angiogenesis in CAM model by negatively regulating the expression levels of pro angiogenic factors including VEGF and FGF. The CQDs showed anti-cancer property *in vitro*, suggesting it to be a potential drug candidate for targeting tumor angiogenesis [73]. The applications of nanomaterials for anti tumor therapy have

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

genic molecules doubtful under clinical setup.

been represented schematically in **Figure 1**.

NAMI-A [70]*.*

#### *Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

proliferation of ovarian cancer cells in xenograft model *in-vivo* [65]*.* Further, the nanoceria conjugated with folic acid has also been reported to inhibit proliferation and angiogenesis in xenografts of ovarian cancer cells *in vivo* [66]*.* The anti-angiogenic effect imparted by nanoceria was reported to involve the inhibition of VEGF signaling pathway leading to the decreased phosphorylation of VEGFR2 at Tyr1173 and Y951 [65]*.* However, a report by Das et al., have suggested that nanoceria might exhibit pro angiogenic effect also [67]*,* making the use of these NPs as anti-angiogenic molecules doubtful under clinical setup.

Silica based NPs have also been reported to exhibit anti angiogenic properties. Silicate NPs (SiO2 NPs) have been reported to inhibit VEGFR2 phosphorylation and ERK1/2 activation in human micro vascular retinal endothelial cells (HMRECs), thereby inhibiting angiogenesis [68]. Mesoporous silica based nanoparticles (MSNs) have been used as a vehicle for the targeted delivery of chemotherapeutic agent, doxorubicin hydrochloride (MSNs@DOX). MSNs@ DOX has been reported to suppress the metastasis of lung cancer cells by inhibiting VEGF induced angiogenesis [69]*.* Further RGD (Arg-Gly-Asp) modified MSN has been used as a carrier tool for the targeted delivery of anti-angiogenic agent, NAMI-A [70]*.*

Further, MoS2 nanoflakes containing ZnO NPs were found to inhibit tumor growth in *in-ovo* xenograft model by inducing apoptosis and by negatively regulating the processes of angiogenesis as well as EMT [71]. Similarly, the Tetraiodothyroacetic acid (Tetrac) based NPs have also been reported to be antiangiogenic in nature in CAM model and in xenograft model of renal cancer cells [72]*.* Shereema et al., have formulated a green luminescent CQDs, which inhibited angiogenesis in CAM model by negatively regulating the expression levels of pro angiogenic factors including VEGF and FGF. The CQDs showed anti-cancer property *in vitro*, suggesting it to be a potential drug candidate for targeting tumor angiogenesis [73]. The applications of nanomaterials for anti tumor therapy have been represented schematically in **Figure 1**.

#### **Figure 1.**

*Theranostics - An Old Concept in New Clothing*

dose dependant manner [57]*.*

vidually [60]*.*

to be negatively regulating the phosphorylation of VEGFR2. The inhibitory effect of AuNPs on Heparin binding growth factors (HB-GFs) was found to be greatly depended on the size of AuNPs, further, AuNPs with 20 nm in diameter exhibited maximum inhibitory effect. In addition, AuNP with bare surface was found to be essential for the inhibitory effect on HB-GFs. Further, AuNPs have been reported to block of MAPK pathway in tumor cells which lead to the inhibition of epithelial to mesenchymal transition (EMT) and thence, the process of metastasis [51, 52]*.* AuNP has also been used as the carrier tool for drug delivery. It has been used to deliver an anti-EMT agent, Quercetin (Qu) and AuNP-Qu was found to be more effective when compared to free Qu, in inhibiting cell migration in MDA-MB-23 and MCF-7 cell lines [53]. In addition, recombinant human endostatin (rhES), an anti- angiogenic molecule, which in conjugation with AuNP-PEG (rhES-AuNPs-PEG), when administrated, targeted tumor cells more efficiently and exhibited better performance when compared to rhES. Moreover, the administration of rhES-AuNPs-PEG in combination with 5-flouro uracil (5-FU) facilitated improved localization of 5-FU on to the tumor site with subsequent reduction in tumor size

than that in case of mono therapeutic administration of 5FU [54].

Silver NPs (AgNPs) have been reported to inhibit VEGF induced cell proliferation, migration and tube formation in bovine retinal endothelial cells (BRECs). It has also been reported to inhibit vessel formation in matrigel plug assay system. AgNP mediated anti angiogenic effect was found to involve negative regulation of PI3K/Akt pathway [55, 56]*.* According to a different study, AgNP has been reported to exert anti angiogenic effect by inhibiting HIF-1 in a

In addition to metal NPs, NPs based on cationic polysaccharides like chitosan has also been explored for biomedical applications taking an advantage of their relatively low toxic nature and high biodegradability and biocompatibility. Chitosan NPs (CNPs) showed anti-cancer effect in the xenograft model of hepatocellular carcinoma by inhibiting the expression of VEGFR2 and thereby negatively regulating the process of tumor angiogenesis [58]. Further, CNPs in conjugation with Ursolic acid (CH-UA-NPs) have been shown to inhibit cell migration and tube formation in human umbilical vein endothelial cells (HUVECs) *in-vitro*. In addition, CH-UA-NPs have also been reported to inhibit the expression of VEGF in hepatoma cell xenografts [59]. CNPs have also been utilized as a vehicle for the co delivery of psiRNA VEGF and pIL-4 in MCF-7 cells which caused relatively huge reduction in the levels of VEGF protein when compared to the cases where the plasmids were used indi-

Ruthenium modified selenium NPs (Ru-SeNPs) have also been reported to exhibit anti angiogenic properties, in CAM model as well as in HUVEC cells, mainly by inhibiting the phosphorylation of Akt, FGFR1 and Erk1/2. Further, it has been shown that SeNPs protected with Ru (II)-thiols (Ru-MUA@Se) was endocytosed by the cells by clathrin mediated mechanism [61]. SeNPs have also been used as a carrier tool for siRNA delivery. A pH sensitive, modified SeNP carrying VEGF-siRNA, namely, G2/PAH-Cit/SeNPs@siRNA, has been shown to exhibit high efficiency in

The cerium oxide NPs (CONPs) have been reported to exhibit anti-oxidant activity and they are characterized by a cerium core and a shield with an oxygen lattice. Chen et al., have shown that CONPs are capable of inhibiting reactive oxygen species (ROS) induced angiogenic signaling pathways [63]*.* In addition, the nanoceria conjugated with heparin was reported to inhibit the proliferation of human coronary artery endothelial cells (HCAECs) in a better way than that by unconjugated nanoceria [64]*.* Nanoceria has also been reported to inhibit the

terms of cellular uptake, drug release and gene silencing [62]*.*

**124**

*Applications of nanomaterials in anti-tumor therapy. Many candidate nanomaterials possess intrinsic antiangiogenic property and few could be used as vehicles for targeted drug delivery. Nanoparticles encapsulated/ conjugated with anti- angiogenic drugs or nanoparticle based anti-angiogenic scaffolds, when administrated in in vivo models, precisely target tumor vasculature and inhibit tumor growth.*

#### **3.2 Cardio vascular diseases**

Cardio vascular diseases (CVDs), which refer to a class of ailments encompassing coronary artery disease (CHD), peripheral arterial disease, cerebrovascular disease etc., account for the leading cause of death worldwide [74, 75]*.* Atherosclerosis is the most prevalent pathology behind CVDs, which involves the local accumulation of cholesterol within the walls of medium and large arteries leading to the emergence of atherosclerotic plaque [76, 77]*.* The process of angiogenesis has been implicated to play key role in plaque growth and intra plaque hemorrhage leading to plaque rapture [78, 79]. The application of nanomaterials has found its way in the diagnosis as well as treatment of CVDs. Integrin αvβ3 has been found to be over expressed in ECs actively involved in angiogenesis, thus, it has been targeted using NPs for CVD diagnosis [80]. For instance, in a murine model of hind limb ischemia, 76Br- labeled multivalent dendrimers conjugated with integrin αvβ3 targeting peptides, were utilized for the detection of angiogenesis by positron emission tomography-computed tomography (PET-CT) [81]. In a different experiment using murine model of hind limb ischemia, a natriuretic peptide receptor C- targeted, 64Cu labeled NP probe was used for the detection of angiogenesis [82]. Further, gadolinium-loaded perfluorocarbon (PFC) NP conjugated with a vitronectin antagonist peptide mimic, has been suggested to be a promising candidate for the detection of atherosclerotic lesions [83]. In addition, PFC NPs incorporated with anti-angiogenic drug, Fumagillin, have been implicated for the treatment of plaque angiogenesis [84].

#### **3.3 Chronic wounds**

Wounds are the disruption of the normal physiology of the skin, mucosal surfaces or organs, which occur as a part of a disease or etiology. The process of wound healing is divided into four distinct stages: hemostasis, inflammation, proliferation, and tissue remodeling. Injuries that show delayed healing up to 12 weeks after the initial insult are termed chronic wounds, often it happens because of various reasons such as persistent pathological inflammation [85], complications of ischemia, diabetes mellitus, or chronic venous insufficiency [86]. The application of growth factors has been employed to improve wound healing by promoting angiogenesis, but it possessed some drawbacks like rapid degradation of the candidate growth factors and the lack of controlled and localized delivery system.

Different NPs have been reported to promote wound healing, and many of them were implicated as drug carriers. Studies have shown that different metal ions-based nanomaterials possess the ability to promote angiogenesis and thereby induce wound healing [87, 88]. The metal ions such as Sr2+ and Co2+ when combined with nano bioactive glass showed pro angiogenic activity [89]*.* Colloidal AuNPs have been widely studied for biomedical applications due to their unique surface characteristics as well as optical and electronic properties [90]*.* AuNPs combined with epigallocatechin gallate and α-lipoic acid, reduced oxidative stress and inflammation and augmented angiogenesis, which led to cutaneous wound healing in rodent models [91]*.* The increased surface area of spherical AuNP helps in electron acceptance and also in scavenging reactive oxygen species that cause oxidative stress and impaired wound healing [92]*.* Formulation of AuNPs and scrambled peptides were reported to be suitable for angiogenic modulation in *in vivo* and *in vitro* models [93]*.* Moreover, NPs encapsulated in a microparticle developed by the microfluidic method provided a way to introduce a wide range of proteins including pro angiogenic agents to the injury site [94]*.*

**127**

**Figure 2.**

*models by promoting the process of angiogenesis.*

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

Low expression levels of angiogenic growth factors lead to impaired angiogenesis and wound healing. Heparin mimetic peptide nanofiber scaffolds have been used to overcome this situation, which showed improved vascular development associated with enhanced VEGF production in the treated animals. Also, hierarchically micro-patterned nanofibrous scaffolds with a surface modified nanosized bio-glass have been implicated in improving wound healing [95]. Xie et al. have developed an electrospun fiber nano composites containing different components such as antibacterial polymer chitosan, poly (ethylene oxide), VEGF and PDGF-BB loaded poly (lactic-co-glycolic acid) NPs. They have demonstrated that the application of such a nano composite would prevent bacterial attack in the vicinity of wound. In addition, they have demonstrated that the nano composite facilitated the early delivery of VEGF from the nanofiber and sustained delivery of PDGF-BB from the NPs, thereby accelerating tissue regeneration and remodeling in a full-thickness rat skin wound model [96]*.* Lino et al. have shown that light-responsive plasmonic gold nanocarrier could be used as a carrier vehicle for the delivery of microRNAs such as miR-302a and miR-155, which regulated the proliferation and survival of ECs

Carbon nanotubes were functionalized with different side-chain moieties and they were applied for diagnosis as well as drug delivery purposes [98]*.* It has been shown that the Multi-Walled Carbon Nanotube (MWCNT) supports angiogenesis as the macrophages engulfing MWCNT, produce angiogenic cytokines such as VEGF and MMP9 [99]. Liu et al. have constructed a composite scaffold of VEGF165 loaded functionalized MWCNT, for the prolonged and sustained delivery of VEGF165, and it promoted tissue remodeling and repairing in the *in vivo*

Graphene based NPs have also been implicated to have massive applications in angiogenesis-based therapeutics [101]*.* Graphene, graphene oxide (GO) and reduced graphene oxide (rGO) have received great attraction as inorganic additive in biopolymers for developing biomaterial composites [102]*.* The Gelatinmethacryloyl (GelMA) hydrogel containing rGO has been indicated to promote cell proliferation and migration in *in-vitro* model of wound healing and it has also been implicated to promote angiogenesis in chick embryo model [103]. In addition, ZnO nanoflower based nanomaterials [104] and water-soluble CONPs [105] were also implicated to exhibit wound healing properties by modulating the process of angiogenesis. The candidate nanomaterials which possess the ability to promote wound healing, by promoting angiogenesis have been indicated schematically in **Figure 2**.

*Pro-angiogenic nanomaterials promote wound healing. Nanomaterials like cerium oxide nanoparticles, zinc oxide nanoflowers, multi walled carbon nanotubes, reduced graphene oxide nanoparticles and metal ion based nanoparticles like strontium ions and cobalt ions, promote wound healing in different in vitro and in vivo* 

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

thereby promoting wound healing [97]*.*

models [100]*.*

#### *Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

Low expression levels of angiogenic growth factors lead to impaired angiogenesis and wound healing. Heparin mimetic peptide nanofiber scaffolds have been used to overcome this situation, which showed improved vascular development associated with enhanced VEGF production in the treated animals. Also, hierarchically micro-patterned nanofibrous scaffolds with a surface modified nanosized bio-glass have been implicated in improving wound healing [95]. Xie et al. have developed an electrospun fiber nano composites containing different components such as antibacterial polymer chitosan, poly (ethylene oxide), VEGF and PDGF-BB loaded poly (lactic-co-glycolic acid) NPs. They have demonstrated that the application of such a nano composite would prevent bacterial attack in the vicinity of wound. In addition, they have demonstrated that the nano composite facilitated the early delivery of VEGF from the nanofiber and sustained delivery of PDGF-BB from the NPs, thereby accelerating tissue regeneration and remodeling in a full-thickness rat skin wound model [96]*.* Lino et al. have shown that light-responsive plasmonic gold nanocarrier could be used as a carrier vehicle for the delivery of microRNAs such as miR-302a and miR-155, which regulated the proliferation and survival of ECs thereby promoting wound healing [97]*.*

Carbon nanotubes were functionalized with different side-chain moieties and they were applied for diagnosis as well as drug delivery purposes [98]*.* It has been shown that the Multi-Walled Carbon Nanotube (MWCNT) supports angiogenesis as the macrophages engulfing MWCNT, produce angiogenic cytokines such as VEGF and MMP9 [99]. Liu et al. have constructed a composite scaffold of VEGF165 loaded functionalized MWCNT, for the prolonged and sustained delivery of VEGF165, and it promoted tissue remodeling and repairing in the *in vivo* models [100]*.*

Graphene based NPs have also been implicated to have massive applications in angiogenesis-based therapeutics [101]*.* Graphene, graphene oxide (GO) and reduced graphene oxide (rGO) have received great attraction as inorganic additive in biopolymers for developing biomaterial composites [102]*.* The Gelatinmethacryloyl (GelMA) hydrogel containing rGO has been indicated to promote cell proliferation and migration in *in-vitro* model of wound healing and it has also been implicated to promote angiogenesis in chick embryo model [103]. In addition, ZnO nanoflower based nanomaterials [104] and water-soluble CONPs [105] were also implicated to exhibit wound healing properties by modulating the process of angiogenesis. The candidate nanomaterials which possess the ability to promote wound healing, by promoting angiogenesis have been indicated schematically in **Figure 2**.

#### **Figure 2.**

*Theranostics - An Old Concept in New Clothing*

Cardio vascular diseases (CVDs), which refer to a class of ailments encompassing coronary artery disease (CHD), peripheral arterial disease, cerebrovascular disease etc., account for the leading cause of death worldwide [74, 75]*.* Atherosclerosis is the most prevalent pathology behind CVDs, which involves the local accumulation of cholesterol within the walls of medium and large arteries leading to the emergence of atherosclerotic plaque [76, 77]*.* The process of angiogenesis has been implicated to play key role in plaque growth and intra plaque hemorrhage leading to plaque rapture [78, 79]. The application of nanomaterials has found its way in the diagnosis as well as treatment of CVDs. Integrin αvβ3 has been found to be over expressed in ECs actively involved in angiogenesis, thus, it has been targeted using NPs for CVD diagnosis [80]. For instance, in a murine model of hind limb ischemia, 76Br- labeled multivalent dendrimers conjugated with integrin αvβ3 targeting peptides, were utilized for the detection of angiogenesis by positron emission tomography-computed tomography (PET-CT) [81]. In a different experiment using murine model of hind limb ischemia, a natriuretic peptide receptor C- targeted, 64Cu labeled NP probe was used for the detection of angiogenesis [82]. Further, gadolinium-loaded perfluorocarbon (PFC) NP conjugated with a vitronectin antagonist peptide mimic, has been suggested to be a promising candidate for the detection of atherosclerotic lesions [83]. In addition, PFC NPs incorporated with anti-angiogenic drug, Fumagillin, have been implicated for the treatment of plaque

Wounds are the disruption of the normal physiology of the skin, mucosal surfaces or organs, which occur as a part of a disease or etiology. The process of wound healing is divided into four distinct stages: hemostasis, inflammation, proliferation, and tissue remodeling. Injuries that show delayed healing up to 12 weeks after the initial insult are termed chronic wounds, often it happens because of various reasons such as persistent pathological inflammation [85], complications of ischemia, diabetes mellitus, or chronic venous insufficiency [86]. The application of growth factors has been employed to improve wound healing by promoting angiogenesis, but it possessed some drawbacks like rapid degradation of the candidate growth

Different NPs have been reported to promote wound healing, and many of them were implicated as drug carriers. Studies have shown that different metal ions-based nanomaterials possess the ability to promote angiogenesis and thereby induce wound healing [87, 88]. The metal ions such as Sr2+ and Co2+ when combined with nano bioactive glass showed pro angiogenic activity [89]*.* Colloidal AuNPs have been widely studied for biomedical applications due to their unique surface characteristics as well as optical and electronic properties [90]*.* AuNPs combined with epigallocatechin gallate and α-lipoic acid, reduced oxidative stress and inflammation and augmented angiogenesis, which led to cutaneous wound healing in rodent models [91]*.* The increased surface area of spherical AuNP helps in electron acceptance and also in scavenging reactive oxygen species that cause oxidative stress and impaired wound healing [92]*.* Formulation of AuNPs and scrambled peptides were reported to be suitable for angiogenic modulation in *in vivo* and *in vitro* models [93]*.* Moreover, NPs encapsulated in a microparticle developed by the microfluidic method provided a way to introduce a wide range of proteins including pro angio-

factors and the lack of controlled and localized delivery system.

**3.2 Cardio vascular diseases**

angiogenesis [84].

**3.3 Chronic wounds**

**126**

genic agents to the injury site [94]*.*

*Pro-angiogenic nanomaterials promote wound healing. Nanomaterials like cerium oxide nanoparticles, zinc oxide nanoflowers, multi walled carbon nanotubes, reduced graphene oxide nanoparticles and metal ion based nanoparticles like strontium ions and cobalt ions, promote wound healing in different in vitro and in vivo models by promoting the process of angiogenesis.*

#### **3.4 Diabetic retinopathy and age-related macular degeneration**

Diabetic retinopathy (DR) is one of the critical leading causes of blindness and it is a secondary complication associated with Diabetic Mellitus. Diabetes affects the entire neurovascular regions of the retina, with ongoing neurodegeneration, gliosis, neuroinflammation, edema, angiogenesis, and fibrosis [106]. The changes in the vasculature cause perceptible abnormality in vision and lead to blindness. VEGFA, which gets upregulated in response to hypoxia, plays a central role in the initiation of DR. In addition to that, MMP9 has also been implicated to play key role in the onset and severity of DR [107].

The Age-related macular degeneration (AMD) is another complication where pathological angiogenesis is involved. AMD has been classified into two types. The type of AMD which is characterized by yellowish deposits in the macula is known as the Dry AMD, whereas, the AMD with characteristic choroidal neovascularisation (CNV) is termed as the wet type or neovascular AMD [108]*.*

Laser photocoagulation and multiple intra ocular injections are the treatment strategies adopted for the diseases that affect the vascular structure of the posterior eye. It has complications like the destruction of healthy tissues. Though 'introducing protein drugs', was put forth as one of the treatment strategies, it possessed drawbacks like drug instability due to proteases action followed by drug injection. It therefore warranted novel treatment strategies to conquer these drawbacks. So, in an effort to develop alternative therapeutic strategies for ocular diseases, the efficacy of different candidate NPs, exhibiting innate anti angiogenic property or possessing the ability to carry drug, growth factors etc., to specific tissue sites, have been tested by different groups [109, 110]*.*

The AuNPs, as mentioned earlier, possess anti angiogenic properties in addition to their unique electronic, biocompatible, and molecular-recognition properties [111]. It has been reported to induce the nano structural reorganization of VEGFR2 in HUVECs and consequently suppressed angiogenesis [112]. AuNPs have also been reported to suppress VEGF induced cell migration by negatively regulating the phosphorylation of Akt and eNOS in retinal endothelial cells [113]*.* It has also been reported to obstruct the proliferation of VEGF treated retinal endothelial cells by suppressing Src signaling pathways [114]*.*

Kringle 5 (K5), a proteolytic fragment of plasminogen possessing 80 amino acids, has been shown to be highly effective in the inhibition of EC growth [115]. It has also been reported to inhibit ischemia-stimulated retinal neovascularization in the oxygen-induced retinopathy (OIR) model [116]. But it possessed the drawback of a short life span. An expression plasmid of K5 was encapsulated with PLGA polymer to form nanoparticles (K5-NP) which effectively inhibited VEGF expression and attenuated ischemia-induced retinal vascular leakage and retinal neovascularization in the OIR rat model [117]*.* Biodegradable NPs loaded with Fenofibrate (Feno-NPs) have been reported to be particularly useful for the targeted delivery and treatment of DR and neovascular AMD. Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist, which is effective against DR. In diabetic rat models, at 8 weeks after the administration of Feno-NP by one intravitreal injection, the vascular leakage in the retina was found to be reduced. In addition to that the retinal leukostasis was inhibited, and further, the expression of VEGF and ICAM-1 were down regulated [118].

Octreotide (OCT), an analog of somatostatin, is an established neuroprotective and anti-angiogenic agent that targets VEGF. The intra ocular delivery of OCT combined with Magnetic NPs (MNP-OCT) has been suggested to improve the half-life and bio activity of OCT [119]. Polliner et al. have checked the possibility of receptor mediated targeting of NPs to capillary endothelial cells in the retina, and

**129**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

they have demonstrated that Cyclo (RGDfC)-modified QDs specifically bind to the αvβ3 integrin receptors on the ECs and the cellular uptake mediated by receptor binding led to the accumulation of the NPs in the choriocapillaris and intraretinal

Likewise, Luo et al. have used, biodegradable PLGA nanoparticles conjugated with integrin-binding linear RGD peptide, as a carrier tool for the delivery of recombinant tFlt23k intraceptor plasmid possessing VEGF binding domains. The nontoxic RGD-functionalized NP delivery system was observed to be getting targeted directly to the choroidal neovascularization lesions after intravenous injection, and exhibited excellent vision restoration in both primate and murine AMD

Celecoxib is a cyclooxygenase-2 inhibitor, exhibiting anti-inflammatory and anti-angiogenic properties. Celecoxib-loaded poly (ortho ester) NPs were found to be highly effective against AMD and DR [123]*.* Interleukin-12 (IL-12) has been reported to exhibit anti-angiogenic property by reducing the levels of MMP9 and VEGFA [124]. Zheng and colleagues combined IL-12 with PLGA nanoparticles (IL-12-PNP) and proved it to be exhibiting better efficacy in terms of inhibition of VEGFA and MMP9 expressions in DR mouse retina and rat ECs. Further, the intra ocular administration of IL-12-PNPs showed reduced retinal damage in mice model

Osteogenesis is referred to the process of regeneration of bones, which involves multiple steps such as the activation, migration and differentiation of different cell types [126]*.* The process of angiogenesis is crucial for the supply of growth factors, hormones, cytokines, chemokines, and metabolites required for osteogenesis. Any aberrancy associated with the vascular supply to the bone tissues would lead to different pathologies such as osteonecrosis [127], osteomyelitis [128], and osteoporosis [129, 130]. Discrepancy in angiogenesis has also been reported as one of the main reasons for the failure of osteogenesis after implantation. VEGF and HIFα are the major angiogenesis related factors that promote osteoblast differentiation and osteogenesis. So, it has been suggested that restoring angiogenesis would promote

bone function and defect repair in pathologies with impaired osteogenesis.

Many candidate nanomaterials have been reported to be effective in improving the repair of bone tissues [131]. For example, synthesized chitin–CaSO4–nanofibrin based injectable gel system showed enhanced osteo-regeneration via enhanced angiogenesis [132]. Further, the β CaSiO3/PDLGA composite has been reported to induce the phosphorylation and activation of Akt and eNOS respectively in HUVECs with a resultant increase in the synthesis and release of NO and VEGF. Further the bone regeneration study in the rabbit femur defect model using β CaSiO3/PDLGA composite has shown enhanced angiogenesis and osteogenesis [133]. Nano-hydroxyapatite has been reported to regulate the PI3K/Akt pathway for inhibiting migration and tube formation in HUVECs via inhibiting NO synthesis

Yandrapu et al. have formulated 'Nanoparticles in Porous Micropaticles (NPinPMP)', by encapsulating bevacizumab coated poly lactic acid NPs into porousifying PLGA microparticles (NPinPMP) using supercritical carbon dioxide (SC CO2). Bevacizumab is a protein drug used to treat neovascular AMD and it was necessary to inject once in a month intravitreally. The *in vitro* studies revealed that, NPinPMP showed a sustained release of bevacizumab for a period of 4 months. In addition, bevacizumab has been detected for a period of 2 months after intravitreal injection of NPinPMP in rat model, while it was detected only for 2 weeks upon its

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

intravitreal administration in individual form [121]*.*

capillaries [120]*.*

models [122]*.*

with DR [125]*.*

**3.5 Impaired osteogenesis**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

they have demonstrated that Cyclo (RGDfC)-modified QDs specifically bind to the αvβ3 integrin receptors on the ECs and the cellular uptake mediated by receptor binding led to the accumulation of the NPs in the choriocapillaris and intraretinal capillaries [120]*.*

Yandrapu et al. have formulated 'Nanoparticles in Porous Micropaticles (NPinPMP)', by encapsulating bevacizumab coated poly lactic acid NPs into porousifying PLGA microparticles (NPinPMP) using supercritical carbon dioxide (SC CO2). Bevacizumab is a protein drug used to treat neovascular AMD and it was necessary to inject once in a month intravitreally. The *in vitro* studies revealed that, NPinPMP showed a sustained release of bevacizumab for a period of 4 months. In addition, bevacizumab has been detected for a period of 2 months after intravitreal injection of NPinPMP in rat model, while it was detected only for 2 weeks upon its intravitreal administration in individual form [121]*.*

Likewise, Luo et al. have used, biodegradable PLGA nanoparticles conjugated with integrin-binding linear RGD peptide, as a carrier tool for the delivery of recombinant tFlt23k intraceptor plasmid possessing VEGF binding domains. The nontoxic RGD-functionalized NP delivery system was observed to be getting targeted directly to the choroidal neovascularization lesions after intravenous injection, and exhibited excellent vision restoration in both primate and murine AMD models [122]*.*

Celecoxib is a cyclooxygenase-2 inhibitor, exhibiting anti-inflammatory and anti-angiogenic properties. Celecoxib-loaded poly (ortho ester) NPs were found to be highly effective against AMD and DR [123]*.* Interleukin-12 (IL-12) has been reported to exhibit anti-angiogenic property by reducing the levels of MMP9 and VEGFA [124]. Zheng and colleagues combined IL-12 with PLGA nanoparticles (IL-12-PNP) and proved it to be exhibiting better efficacy in terms of inhibition of VEGFA and MMP9 expressions in DR mouse retina and rat ECs. Further, the intra ocular administration of IL-12-PNPs showed reduced retinal damage in mice model with DR [125]*.*

#### **3.5 Impaired osteogenesis**

*Theranostics - An Old Concept in New Clothing*

onset and severity of DR [107].

been tested by different groups [109, 110]*.*

suppressing Src signaling pathways [114]*.*

ICAM-1 were down regulated [118].

**3.4 Diabetic retinopathy and age-related macular degeneration**

(CNV) is termed as the wet type or neovascular AMD [108]*.*

Diabetic retinopathy (DR) is one of the critical leading causes of blindness and it is a secondary complication associated with Diabetic Mellitus. Diabetes affects the entire neurovascular regions of the retina, with ongoing neurodegeneration, gliosis, neuroinflammation, edema, angiogenesis, and fibrosis [106]. The changes in the vasculature cause perceptible abnormality in vision and lead to blindness. VEGFA, which gets upregulated in response to hypoxia, plays a central role in the initiation of DR. In addition to that, MMP9 has also been implicated to play key role in the

The Age-related macular degeneration (AMD) is another complication where pathological angiogenesis is involved. AMD has been classified into two types. The type of AMD which is characterized by yellowish deposits in the macula is known as the Dry AMD, whereas, the AMD with characteristic choroidal neovascularisation

Laser photocoagulation and multiple intra ocular injections are the treatment strategies adopted for the diseases that affect the vascular structure of the posterior eye. It has complications like the destruction of healthy tissues. Though 'introducing protein drugs', was put forth as one of the treatment strategies, it possessed drawbacks like drug instability due to proteases action followed by drug injection. It therefore warranted novel treatment strategies to conquer these drawbacks. So, in an effort to develop alternative therapeutic strategies for ocular diseases, the efficacy of different candidate NPs, exhibiting innate anti angiogenic property or possessing the ability to carry drug, growth factors etc., to specific tissue sites, have

The AuNPs, as mentioned earlier, possess anti angiogenic properties in addition to their unique electronic, biocompatible, and molecular-recognition properties [111]. It has been reported to induce the nano structural reorganization of VEGFR2 in HUVECs and consequently suppressed angiogenesis [112]. AuNPs have also been reported to suppress VEGF induced cell migration by negatively regulating the phosphorylation of Akt and eNOS in retinal endothelial cells [113]*.* It has also been reported to obstruct the proliferation of VEGF treated retinal endothelial cells by

Kringle 5 (K5), a proteolytic fragment of plasminogen possessing 80 amino acids, has been shown to be highly effective in the inhibition of EC growth [115]. It has also been reported to inhibit ischemia-stimulated retinal neovascularization in the oxygen-induced retinopathy (OIR) model [116]. But it possessed the drawback of a short life span. An expression plasmid of K5 was encapsulated with PLGA polymer to form nanoparticles (K5-NP) which effectively inhibited VEGF expression and attenuated ischemia-induced retinal vascular leakage and retinal neovascularization in the OIR rat model [117]*.* Biodegradable NPs loaded with Fenofibrate (Feno-NPs) have been reported to be particularly useful for the targeted delivery and treatment of DR and neovascular AMD. Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist, which is effective against DR. In diabetic rat models, at 8 weeks after the administration of Feno-NP by one intravitreal injection, the vascular leakage in the retina was found to be reduced. In addition to that the retinal leukostasis was inhibited, and further, the expression of VEGF and

Octreotide (OCT), an analog of somatostatin, is an established neuroprotective

and anti-angiogenic agent that targets VEGF. The intra ocular delivery of OCT combined with Magnetic NPs (MNP-OCT) has been suggested to improve the half-life and bio activity of OCT [119]. Polliner et al. have checked the possibility of receptor mediated targeting of NPs to capillary endothelial cells in the retina, and

**128**

Osteogenesis is referred to the process of regeneration of bones, which involves multiple steps such as the activation, migration and differentiation of different cell types [126]*.* The process of angiogenesis is crucial for the supply of growth factors, hormones, cytokines, chemokines, and metabolites required for osteogenesis. Any aberrancy associated with the vascular supply to the bone tissues would lead to different pathologies such as osteonecrosis [127], osteomyelitis [128], and osteoporosis [129, 130]. Discrepancy in angiogenesis has also been reported as one of the main reasons for the failure of osteogenesis after implantation. VEGF and HIFα are the major angiogenesis related factors that promote osteoblast differentiation and osteogenesis. So, it has been suggested that restoring angiogenesis would promote bone function and defect repair in pathologies with impaired osteogenesis.

Many candidate nanomaterials have been reported to be effective in improving the repair of bone tissues [131]. For example, synthesized chitin–CaSO4–nanofibrin based injectable gel system showed enhanced osteo-regeneration via enhanced angiogenesis [132]. Further, the β CaSiO3/PDLGA composite has been reported to induce the phosphorylation and activation of Akt and eNOS respectively in HUVECs with a resultant increase in the synthesis and release of NO and VEGF. Further the bone regeneration study in the rabbit femur defect model using β CaSiO3/PDLGA composite has shown enhanced angiogenesis and osteogenesis [133]. Nano-hydroxyapatite has been reported to regulate the PI3K/Akt pathway for inhibiting migration and tube formation in HUVECs via inhibiting NO synthesis

and eNOS phosphorylation [134]. Similarly, calcium phosphate combined with electro spun poly (lactic acid) has been reported to promote VEGF expression in endothelial cells. It has also been reported to support vascular development and bone regeneration when injected subcutaneously in mice, by promoting the expression of proangiogenic factors like VEGF, IGF-2, GM-CSF, IL-1 beta, IL-6, IL-12p70 etc. [135]. Similarly, Nano bioactive glass, characterized by higher surface area and three-dimensional channel structure, is another material that could promote angiogenesis and bone regeneration [136, 137]*.*

Nanomaterials can also act as carrier tools for different pro angiogenic small molecules and proteins like deferoxamine, adrenomedullin, VEGF etc. For example, Mesoporous silicate nanoparticles (MSNs) incorporated-3D nanofibrous gelatin (GF) scaffold has been employed for the dual-delivery of bone morphogenetic protein-2 (BMP2) and deferoxamine (DFO). DFO, being a hypoxia-mimetic drug, could trigger the stabilization of HIF-1α, and initiate subsequent angiogenesis. Further, it has been shown that DFO could significantly enhance BMP2 induced osteogenic differentiation in mouse and human stem cell models [138]*.*

Ionic components have been utilized for the modification of vascularized bone tissue engineering scaffold. The Copper based nanomaterials could promote the expression level of VEGF, which in turn promoted the proliferation of ECs. Nano-structured surfaces on the Hydroxyapatite scaffolds in copper ion (Cu2+) containing solutions under hydrothermal conditions could affect EC proliferation. Further, the nano-structured surfaces on the Hydroxyapatite scaffolds, promoted angiogenesis and bone regeneration. Dexamethasone (DEX), an osteogenic inducer combined with biphasic calcium phosphate nanoparticle (BCP NPs) scaffold, was found to induce the expression of VEGF and VEGFR2 and supported bone regeneration. The micro-grooves present in the scaffolds managed the assembly of HUVECs into tubular structures and promoted angiogenesis [139]. The gene encapsulated magnetic microspheres have also been used as a promising delivery system. For instance, introduction of VEGF165 with superparamagnetic (nano-Fe3O4) chitosan, induced *in vitro* and *in vivo* angiogenesis and bone regeneration [140].

The AuNPs have also been reported to induce angiogenesis during osteogenesis. AuNPs exhibited differences in angiogenic activity based on their surface charges and the presence of functional groups. The Gene profiling data revealed that in comparison with the cells (hMSCs) treated with AuNPs possessing amine or hydroxyl functional groups (AuNPeNH2 or AuNPeOH), the cells treated with carboxyl group containing AuNPs (AuNPeCOOH) showed augmented expression levels of TGFβ and FGF-2, which in turn promoted cell proliferation over osteogenic differentiation [141].

#### **3.6 Nerve tissue degeneration**

Nerve tissue degeneration is a critical clinical challenge that leads to diseases like trauma or permanent paralysis, so research advancement in the field of nerve tissue regeneration is quite necessary. In the recent years, the applications of nanomaterials have received much attention from the research community focusing on nerve tissue repair.

The process of angiogenesis plays key role in supplying nutrients to the nerve tissue which in turn helps to repair segmental nerve defects. Recently, Lopez-Dolado et al. have designed a 3D scaffold containing partially reduced graphene oxide, which when implanted in the injured site in the spinal cord of a rat model, a remarkable induction in angiogenesis and axon regeneration was observed [142]*.*

**131**

schematically in **Figure 3**.

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

Further, GO/polycaprolactone (PCL) nano scaffolds have been implicated to promote angiogenesis by modulating Akt-eNOS-VEGF signaling pathway and it

In addition, Xu et al. have formulated an acellular spinal cord scaffold (ASCS), namely, V-ASCS, for the sustained delivery of VEGF, and it was composed of VEGF165 encapsulated PLGA nanoparticles conjugated with ASCS. When V-ASCS was implanted at the injury site in a rat spinal cord hemisection model, it rendered significant progress in neovascularization [144]. Wen et al. fabricated a hyaluronic acid scaffold with brain-derived neurotrophic factor and VEGF loaded PLGA microspheres, which promoted angiogenesis and nerve fiber regeneration when implanted at the injured site in the spinal cord of rat model [145]*.* Yu and his co-workers have formulated PLGA microspheres encapsulated with VEGF, angiopoietin-1 and bFGF, and these angiogenic microspheres could release the angiogenic factors in a sustained fashion, which then induced angiogenesis and neurogenesis when administered at the injured site in the spinal cord

Jian et al. have fabricated a nanohybrid hydrogel containing sulfated glycosaminoglycan-based polyelectrolyte complex nanoparticles (PCN), and it could accelerate neurogenesis and angiogenesis in *in-vivo* ischemic stroke model [147]. Amorphous non-fibrous hydrogel comprised of hyaluronic acid containing high cluster VEGF, when injected directly within the stroke cavity, stimulated the formation of a vascular and neuronal structures, that preceded to behavioral improve-

Delivery of superparamagnetic iron oxide nanoparticle labeled Endothelial progenitor cells (EPCs) was found to induce the formation of vessel-like structures by the production of VEGF and FGF [149]*.* Similarly, superparamagnetic iron oxide (SPIO)-Au core-shell NPs incorporated with nerve growth factor (NGF) have been

Aberrancy associated with angiogenesis pave the way for the progression of a number of diseases like tumor, cardio vascular diseases, diabetic retinopathy, age related macular degeneration etc. So, targeting angiogenesis presents itself as one of the key therapeutic strategies to tackle such complications. The currently available therapies though beneficial, do possess some limitations like acquisition of drug resistance by cells, fast decay of protein drugs by protease action, off target effects leading to decreased drug efficacy etc. Different candidate nanomaterials were implicated to possess anti- angiogenic properties, which were tested *in vitro* and *in vivo* to explore their additional properties like precise targeting of pathological angiogenesis, cellular uptake, efficacy etc. Nanoparticles have also been utilized as carrier tools for drug delivery. Surface modification of nanoparticles with RGD, VEGF etc. has reinforced them with specific targeting, internalization and sustained drug delivery. Growth factor encapsulated nanoparticle-based scaffolds were fabricated by different groups, to effectuate wound healing, osteogenesis and nerve tissue regeneration in *in vivo* models. On the whole, the application of nanomaterial-based formulations in pro or anti angiogenic therapy is a rewarding strategy for the treatment of complications associated with aberrant angiogenesis, which however, requires more explorations for translating from bench to bedside. The candidate disorders associated with aberrant angiogenesis and various applications of nanomaterials for the treatment of such disorders have been represented

implicated to promote neuron growth and differentiation [150]*.*

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

of rat model [146]*.*

ment *in vivo* [148]*.*

**4. Conclusion**

facilitated peripheral nerve regeneration *in-vivo* [143]*.*

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

Further, GO/polycaprolactone (PCL) nano scaffolds have been implicated to promote angiogenesis by modulating Akt-eNOS-VEGF signaling pathway and it facilitated peripheral nerve regeneration *in-vivo* [143]*.*

In addition, Xu et al. have formulated an acellular spinal cord scaffold (ASCS), namely, V-ASCS, for the sustained delivery of VEGF, and it was composed of VEGF165 encapsulated PLGA nanoparticles conjugated with ASCS. When V-ASCS was implanted at the injury site in a rat spinal cord hemisection model, it rendered significant progress in neovascularization [144]. Wen et al. fabricated a hyaluronic acid scaffold with brain-derived neurotrophic factor and VEGF loaded PLGA microspheres, which promoted angiogenesis and nerve fiber regeneration when implanted at the injured site in the spinal cord of rat model [145]*.* Yu and his co-workers have formulated PLGA microspheres encapsulated with VEGF, angiopoietin-1 and bFGF, and these angiogenic microspheres could release the angiogenic factors in a sustained fashion, which then induced angiogenesis and neurogenesis when administered at the injured site in the spinal cord of rat model [146]*.*

Jian et al. have fabricated a nanohybrid hydrogel containing sulfated glycosaminoglycan-based polyelectrolyte complex nanoparticles (PCN), and it could accelerate neurogenesis and angiogenesis in *in-vivo* ischemic stroke model [147]. Amorphous non-fibrous hydrogel comprised of hyaluronic acid containing high cluster VEGF, when injected directly within the stroke cavity, stimulated the formation of a vascular and neuronal structures, that preceded to behavioral improvement *in vivo* [148]*.*

Delivery of superparamagnetic iron oxide nanoparticle labeled Endothelial progenitor cells (EPCs) was found to induce the formation of vessel-like structures by the production of VEGF and FGF [149]*.* Similarly, superparamagnetic iron oxide (SPIO)-Au core-shell NPs incorporated with nerve growth factor (NGF) have been implicated to promote neuron growth and differentiation [150]*.*

#### **4. Conclusion**

*Theranostics - An Old Concept in New Clothing*

angiogenesis and bone regeneration [136, 137]*.*

stem cell models [138]*.*

esis and bone regeneration [140].

differentiation [141].

tissue repair.

**3.6 Nerve tissue degeneration**

and eNOS phosphorylation [134]. Similarly, calcium phosphate combined with electro spun poly (lactic acid) has been reported to promote VEGF expression in endothelial cells. It has also been reported to support vascular development and bone regeneration when injected subcutaneously in mice, by promoting the expression of proangiogenic factors like VEGF, IGF-2, GM-CSF, IL-1 beta, IL-6, IL-12p70 etc. [135]. Similarly, Nano bioactive glass, characterized by higher surface area and three-dimensional channel structure, is another material that could promote

Nanomaterials can also act as carrier tools for different pro angiogenic small molecules and proteins like deferoxamine, adrenomedullin, VEGF etc. For example, Mesoporous silicate nanoparticles (MSNs) incorporated-3D nanofibrous gelatin (GF) scaffold has been employed for the dual-delivery of bone morphogenetic protein-2 (BMP2) and deferoxamine (DFO). DFO, being a hypoxia-mimetic drug, could trigger the stabilization of HIF-1α, and initiate subsequent angiogenesis. Further, it has been shown that DFO could significantly enhance BMP2 induced osteogenic differentiation in mouse and human

Ionic components have been utilized for the modification of vascularized bone tissue engineering scaffold. The Copper based nanomaterials could promote the expression level of VEGF, which in turn promoted the proliferation of ECs. Nano-structured surfaces on the Hydroxyapatite scaffolds in copper ion (Cu2+) containing solutions under hydrothermal conditions could affect EC proliferation. Further, the nano-structured surfaces on the Hydroxyapatite scaffolds, promoted angiogenesis and bone regeneration. Dexamethasone (DEX), an osteogenic inducer combined with biphasic calcium phosphate nanoparticle (BCP NPs) scaffold, was found to induce the expression of VEGF and VEGFR2 and supported bone regeneration. The micro-grooves present in the scaffolds managed the assembly of HUVECs into tubular structures and promoted angiogenesis [139]. The gene encapsulated magnetic microspheres have also been used as a promising delivery system. For instance, introduction of VEGF165 with superparamagnetic (nano-Fe3O4) chitosan, induced *in vitro* and *in vivo* angiogen-

The AuNPs have also been reported to induce angiogenesis during osteogenesis. AuNPs exhibited differences in angiogenic activity based on their surface charges and the presence of functional groups. The Gene profiling data revealed that in comparison with the cells (hMSCs) treated with AuNPs possessing amine or hydroxyl functional groups (AuNPeNH2 or AuNPeOH), the cells treated with carboxyl group containing AuNPs (AuNPeCOOH) showed augmented expression levels of TGFβ and FGF-2, which in turn promoted cell proliferation over osteogenic

Nerve tissue degeneration is a critical clinical challenge that leads to diseases like trauma or permanent paralysis, so research advancement in the field of nerve tissue regeneration is quite necessary. In the recent years, the applications of nanomaterials have received much attention from the research community focusing on nerve

The process of angiogenesis plays key role in supplying nutrients to the nerve tissue which in turn helps to repair segmental nerve defects. Recently, Lopez-Dolado et al. have designed a 3D scaffold containing partially reduced graphene oxide, which when implanted in the injured site in the spinal cord of a rat model, a remarkable induction in angiogenesis and axon regeneration was observed [142]*.*

**130**

Aberrancy associated with angiogenesis pave the way for the progression of a number of diseases like tumor, cardio vascular diseases, diabetic retinopathy, age related macular degeneration etc. So, targeting angiogenesis presents itself as one of the key therapeutic strategies to tackle such complications. The currently available therapies though beneficial, do possess some limitations like acquisition of drug resistance by cells, fast decay of protein drugs by protease action, off target effects leading to decreased drug efficacy etc. Different candidate nanomaterials were implicated to possess anti- angiogenic properties, which were tested *in vitro* and *in vivo* to explore their additional properties like precise targeting of pathological angiogenesis, cellular uptake, efficacy etc. Nanoparticles have also been utilized as carrier tools for drug delivery. Surface modification of nanoparticles with RGD, VEGF etc. has reinforced them with specific targeting, internalization and sustained drug delivery. Growth factor encapsulated nanoparticle-based scaffolds were fabricated by different groups, to effectuate wound healing, osteogenesis and nerve tissue regeneration in *in vivo* models. On the whole, the application of nanomaterial-based formulations in pro or anti angiogenic therapy is a rewarding strategy for the treatment of complications associated with aberrant angiogenesis, which however, requires more explorations for translating from bench to bedside. The candidate disorders associated with aberrant angiogenesis and various applications of nanomaterials for the treatment of such disorders have been represented schematically in **Figure 3**.

#### **Figure 3.**

*Nanomaterial based formulations for the treatment of pathological conditions with aberrant angiogenesis. Abnormal angiogenesis promotes the progression of different diseases like tumor, cardiovascular disease, chronic wounds, diabetic retinopathy, wet type age related macular regeneration, bone and nerve tissue degeneration etc. nanomaterials possessing intrinsic pro- or anti- angiogenic property could be utilized individually or as a part of biodegradable polymer based-scaffolds for the treatment of such disorders. Different candidate nanoparticles with surface modifications with peptides like arginine-glycine-aspartate (RGD) and vascular endothelial growth factor (VEGF), could be utilized as carrier tools for targeted drug delivery.*

#### **Author details**

Aswini Poyyakkara1†, Sruthi Thekkeveedu2†, Sharath S. Shankar1 \* and V.B. Sameer Kumar1 \*

1 Department of Biochemistry and Molecular Biology, Central University of Kerala, India

2 Thomas Jefferson University, Philadelphia, USA

\*Address all correspondence to: sharathshankar82@gmail.com; sameerkumarvb@gmail.com

† These authors have contributed equally.

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**133**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

macular degeneration. N. Engl. J. Med.

[10] Barui AK, Nethi SK, Haque S, Basuthakur P, Patra CR. Recent Development of Metal Nanoparticles for Angiogenesis Study and Their Therapeutic Applications. ACS Appl.

Bio Mater. 2019; 2:5492-5511.

115:11147-11190.

1050-1074.

[11] Min YZ, Caster JM, Eblan MJ, Wang AZ. Clinical Translation of Nanomedicine. Chem. Rev. 2015;

[12] Jeevanandam J, Barhoum A, ChanYS, Dufresne A, Danquah MK. Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations. Beilstein J Nanotechnol. 2018; 9:

[13] El-Toni AM, Habila MA, Labis JP, Alothman ZA,

Nanoscale*.* 2016;8: 2510-2531.

2018;10: 12871-12934.

1175-1194.

[15] Khan I, Saeed K, Khan I.

Nanoparticles: Properties, applications and toxicities. Arabian Journal of Chemistry*.*2019*;*12: 908-931.

[16] Nune SK, Gunda P, Thallapally PK, Lin Y-Y, Forrest mL, Berkland CJ. Nanoparticles for biomedical imaging. Expert Opin Drug Deliv. 2009;6:

[17] Shereema RM, Sankar V, Raghu KG, Rao TP, Shankar SS. One step green

[14] Mourdikoudis S, Pallares RM, Thanh NTK. Characterization techniques for nanoparticles: comparison and complementarity upon studying nanoparticle properties. Nanoscale.

Alhoshan M, Elzatahry AA, Zhang F. Design, synthesis and applications of core–shell, hollow core, and nano rattle multifunctional nanostructures.

2004; 351: 2805-2816.

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

[1] Ribatti D. The discovery of tumor angiogenesis factors: a historical overview. In: Ribatti. D, editor. Tumor Angiogenesis Assays: Methods and Protocols. New York: Springer New

**References**

York. 2016; 1464:1-12.

Med. 2000; 6:389-395.

Cancer.1996; 32: 2451-2460

22:251-256.

2013:55-75.

[2] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat

[3] Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003; 9:653-660.

[4] Ellis LM and Fidler IJ. Angiogenesis and metastasis. European Journal of

[5] Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci.1997;

[6] Mousa SA, Arias HR, Davis PJ. Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis modulation. In: Mousa SA, Davis PJ, editors. Angiogenesis Modulations in Health and Disease: Practical Applications of Pro- and Anti-angiogenesis Targets. Dordrecht: Springer Netherlands.

[7] Gacche RN and Meshram RJ. Angiogenic factors as potential drug target: efficacy and limitations of antiangiogenic therapy. Biochim Biophys Acta Rev Cancer. 2014; 1846:161-179.

[8] Hurwitz H, Fehrenbacher L,

J. Med. 2004; 350:2335-2342.

[9] Gragoudas ES, Adamis AP,

Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl.

Cunningham ET, Feinsod M, Guyer DR. Pegaptanib for neovascular age-related

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

#### **References**

*Theranostics - An Old Concept in New Clothing*

**132**

**Author details**

India

**Figure 3.**

V.B. Sameer Kumar1

sameerkumarvb@gmail.com

\*

† These authors have contributed equally.

provided the original work is properly cited.

2 Thomas Jefferson University, Philadelphia, USA

Aswini Poyyakkara1†, Sruthi Thekkeveedu2†, Sharath S. Shankar1

\*Address all correspondence to: sharathshankar82@gmail.com;

1 Department of Biochemistry and Molecular Biology, Central University of Kerala,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Nanomaterial based formulations for the treatment of pathological conditions with aberrant angiogenesis. Abnormal angiogenesis promotes the progression of different diseases like tumor, cardiovascular disease, chronic wounds, diabetic retinopathy, wet type age related macular regeneration, bone and nerve tissue degeneration etc. nanomaterials possessing intrinsic pro- or anti- angiogenic property could be utilized individually or as a part of biodegradable polymer based-scaffolds for the treatment of such disorders. Different candidate nanoparticles with surface modifications with peptides like arginine-glycine-aspartate (RGD) and vascular* 

*endothelial growth factor (VEGF), could be utilized as carrier tools for targeted drug delivery.*

\* and

[1] Ribatti D. The discovery of tumor angiogenesis factors: a historical overview. In: Ribatti. D, editor. Tumor Angiogenesis Assays: Methods and Protocols. New York: Springer New York. 2016; 1464:1-12.

[2] Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6:389-395.

[3] Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003; 9:653-660.

[4] Ellis LM and Fidler IJ. Angiogenesis and metastasis. European Journal of Cancer.1996; 32: 2451-2460

[5] Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci.1997; 22:251-256.

[6] Mousa SA, Arias HR, Davis PJ. Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis modulation. In: Mousa SA, Davis PJ, editors. Angiogenesis Modulations in Health and Disease: Practical Applications of Pro- and Anti-angiogenesis Targets. Dordrecht: Springer Netherlands. 2013:55-75.

[7] Gacche RN and Meshram RJ. Angiogenic factors as potential drug target: efficacy and limitations of antiangiogenic therapy. Biochim Biophys Acta Rev Cancer. 2014; 1846:161-179.

[8] Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J, Baron A, Griffing S, Holmgren E, Ferrara N, Fyfe G, Rogers B, Ross R, Kabbinavar F. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N. Engl. J. Med. 2004; 350:2335-2342.

[9] Gragoudas ES, Adamis AP, Cunningham ET, Feinsod M, Guyer DR. Pegaptanib for neovascular age-related

macular degeneration. N. Engl. J. Med. 2004; 351: 2805-2816.

[10] Barui AK, Nethi SK, Haque S, Basuthakur P, Patra CR. Recent Development of Metal Nanoparticles for Angiogenesis Study and Their Therapeutic Applications. ACS Appl. Bio Mater. 2019; 2:5492-5511.

[11] Min YZ, Caster JM, Eblan MJ, Wang AZ. Clinical Translation of Nanomedicine. Chem. Rev. 2015; 115:11147-11190.

[12] Jeevanandam J, Barhoum A, ChanYS, Dufresne A, Danquah MK. Review on nanoparticles and nanostructured materials: history, sources, toxicity and regulations. Beilstein J Nanotechnol. 2018; 9: 1050-1074.

[13] El-Toni AM, Habila MA, Labis JP, Alothman ZA, Alhoshan M, Elzatahry AA, Zhang F. Design, synthesis and applications of core–shell, hollow core, and nano rattle multifunctional nanostructures. Nanoscale*.* 2016;8: 2510-2531.

[14] Mourdikoudis S, Pallares RM, Thanh NTK. Characterization techniques for nanoparticles: comparison and complementarity upon studying nanoparticle properties. Nanoscale. 2018;10: 12871-12934.

[15] Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arabian Journal of Chemistry*.*2019*;*12: 908-931.

[16] Nune SK, Gunda P, Thallapally PK, Lin Y-Y, Forrest mL, Berkland CJ. Nanoparticles for biomedical imaging. Expert Opin Drug Deliv. 2009;6: 1175-1194.

[17] Shereema RM, Sankar V, Raghu KG, Rao TP, Shankar SS. One step green

synthesis of carbon quantum dots and its application towards the bioelectroanalytical and biolabeling studies, Electrochimica Acta. 2015; 182: 588-595.

[18] Mohammadi MR, Nojoomi A, Mozafari M, Dubnika A, Inayathullah M, Rajadas J. Nanomaterials engineering for drug delivery: a hybridization approach, J. Mater. Chem. B. 2017;**5**: 3995-4018.

[19] Shereema RM, Nambiar SR, Shankar SS, Rao TP. ceo2–MWCNT nanocomposite based electrochemical sensor for acetaldehyde. Anal. Methods. 2015; 7: 4912-4918.

[20] Neouze M-A and Schubert U. Surface Modification and Functionalization of Metal and Metal Oxide Nanoparticles by Organic Ligands, Monatshefte für Chemie. 2008; 139:183-195.

[21] Ali A, Zafar H, Zia M, Haq I, Phull AR, Ali JS, Hussain A. Synthesis, characterization, applications, and challenges of iron oxide nanoparticles. Nanotechnology, Science and Applications*.* 2016; 9: 49-67.

[22] Patel KD, Singh RK, Kim HW, Carbon-based nanomaterials as an emerging platform for theranostics. Mater. Horiz. 2019; **6**: 434-469.

[23] Notarianni M, Liu J, Vernon K, Motta N. Synthesis and applications of carbon nanomaterials for energy generation and storage. Beilstein J. Nanotechnol. 2016; 7: 149-196.

[24] Shereema RM, Rao TP, Kumar SVB, Sruthi TV, Vishnu R, Prabhu GRD, Shankar SS. Individual and simultaneous electrochemical determination of metanil yellow and curcumin on carbon quantum dots based glassy carbon electrode. Materials Science & Engineering C*.* 2018*;* 93:21-27.

[25] Banik BL, Fattahi P, Brown JL. Polymeric nanoparticles: the future of nanomedicine. Nanomed Nanobiotechnol*.* 2016, 8:271-299.

[26] Thomas SC, Harshita, Mishra PK, Talegaonkar S. Ceramic nanoparticles: fabrication methods and applications in drug delivery. Curr Pharm Des*.* 2015; 21: 6165-6188.

[27] Jung D-R, Kim J, Nahm C, Choi H, Nam S, Park B. Review Paper: Semiconductor Nanoparticles with Surface Passivation and Surface Plasmon. Electronic Materials Letters. 2011; 7:185-194.

[28] Feng L and Mumper RJ. A critical review of lipid-based nanoparticles for taxane delivery. Cancer Letters*.* 2012; 334:157-175.

[29] Carmeliet P. Angiogenesis in health and disease. Nature Medicine. 2003; 9: 653-660.

[30] Van Belle E, Rivard A, Chen D, Silver M, Bunting F, Ferrara N, Symes JF, Bauters C, Isner JM. Hypercholesterolemia Attenuates Angiogenesis but Does Not Preclude Augmentation by Angiogenic Cytokines. Circulation. 1997; 96: 2667-2674.

[31] LeCouter J, Kowalski J, Foster J, Hass P, Zhang Z, Dillard-Telm L, Frantz G, Rangell L, DeGuzman L, Keller GA, Peale F, Gurney A, Hillan KJ, Ferrara N. Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature. 2001; 412: 877-884.

[32] Oosthuyse B, Moons L, Storkebaum E *et al.* Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet.2001; 28:131-138.

**135**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

[44] Chaudhuri P, Harfouche R, Soni S, Hentschel DM, Sengupta S: Shape effect of carbon nanovectors on angiogenesis.

Soni S, Hentschel DM, Mashelkar RA, Sengupta S. Nanoparticle-mediated targeting of phosphatidylinositol-3 kinase signaling inhibits angiogenesis. Angiogenesis. 2009; 12:325-338.

[46] Satchi-Fainaro R, Mamluk R, Wang L, Short SM, Nagy JA, Feng D, Dvorak AM, Dvorak HF, Puder M, Mukhopadhyay D, Folkman J: Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin.

Cancer Cell. 2005; 7:251-261.

Med. 2004; 10:255-261.

2:479-485.

[47] Satchi-Fainaro R, Puder M, Davies JW, Tran HT, Sampson DA, Greene AK, Corfas G, Folkman J: Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470. Nat

[48] Xie J, Shen Z, Li KC, Danthi N: Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle. Int J Nanomedicine. 2007;

[49] Costa PM, Cardoso AL, Custodia C, Cunha P, Pereira de Almeida L, Pedroso de Lima MC. MiRNA-21 silencing mediated by tumortargeted

nanoparticles combined with sunitinib: A new multimodal gene therapy approach for glioblastoma. Journal of Control Release. 2015; 207:31-39.

Poyyakkara A, Kumar SVB. Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis. J Cell Physiol. 2018; 233:3498-3514.

Rotello VM, Mukherjee P. Mechanism

[50] Sruthi TV, Edatt L, Raji GR, Kunhiraman H, Shankar SS, Shankar V, Ramachandran V,

[51] Arvizo RR, Rana S, Miranda OR, Bhattacharya R,

ACS Nano. 2010; 4:574-582.

[45] Harfouche R, Basu S,

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

pylori prevents proliferative stage of angiogenesis *in vitro*: role of cytokines. Dig.Dis.Sci. 2002; 47: 1857-1862.

[34] Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med.

[35] Folkman J: Incipient angiogenesis 1. J Natl Cancer Inst. 2000; 92:94-95

[36] Naumov GN, Akslen LA, Folkman J: Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch. Cell Cycle. 2006;

1971; 285:1182-1186.

5:1779-1787

[37] Hashemi Goradel N,

2018; 233:2902-2910.

Cell. 2011; 3: 3.

2007; 2:751-760.

69:11-16.

23:1417-1450.

2005; 436:568-572.

Ghiyami-Hour F, Jahangiri S, et al. Nanoparticles as new tools for inhibition of cancer angiogenesis. J Cell Physiol.

[38] Banerjee D, Harfouche R,

[39] Peer D, Karp JM, Hong S,

Sengupta S. Nanotechnology-mediated targeting of tumor angiogenesis. Vasc

Farokhzad OC, Margalit R, Langer R: Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol.

[40] Ferrara N: VEGF as a therapeutic target in cancer. Oncology. 2005;

[41] Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Advanced Drug Delivery

Nanotechnology: intelligent design to treat complex disease. Pharm Res. 2006;

[43] Sengupta S, Eavarone D, Capila I, Zhao G, Watson N, Kiziltepe T, Sasisekharan R: Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system. Nature.

Reviews. 2002; 54:631-651.

[42] Couvreur P, Vauthier C:

[33] Jenkinson L, Bardhan KD, Atherton J, Kalia N. Helicobacter *Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

pylori prevents proliferative stage of angiogenesis *in vitro*: role of cytokines. Dig.Dis.Sci. 2002; 47: 1857-1862.

*Theranostics - An Old Concept in New Clothing*

[25] Banik BL, Fattahi P,

6165-6188.

2011; 7:185-194.

334:157-175.

653-660.

877-884.

28:131-138.

Brown JL. Polymeric nanoparticles: the future of nanomedicine. Nanomed Nanobiotechnol*.* 2016, 8:271-299.

[26] Thomas SC, Harshita, Mishra PK, Talegaonkar S. Ceramic nanoparticles: fabrication methods and applications in drug delivery. Curr Pharm Des*.* 2015; 21:

Choi H, Nam S, Park B. Review Paper: Semiconductor Nanoparticles with Surface Passivation and Surface Plasmon. Electronic Materials Letters.

[28] Feng L and Mumper RJ. A critical review of lipid-based nanoparticles for taxane delivery. Cancer Letters*.* 2012;

[29] Carmeliet P. Angiogenesis in health and disease. Nature Medicine. 2003; 9:

[30] Van Belle E, Rivard A, Chen D, Silver M, Bunting F, Ferrara N, Symes JF, Bauters C, Isner JM. Hypercholesterolemia Attenuates Angiogenesis but Does Not Preclude Augmentation by Angiogenic Cytokines.

Circulation. 1997; 96: 2667-2674.

Foster J, Hass P, Zhang Z, Dillard-Telm L, Frantz G, Rangell L, DeGuzman L, Keller GA, Peale F, Gurney A, Hillan KJ, Ferrara N. Identification of an angiogenic mitogen selective for endocrine gland endothelium. Nature. 2001; 412:

[32] Oosthuyse B, Moons L, Storkebaum E *et al.* Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet.2001;

[33] Jenkinson L, Bardhan KD, Atherton J, Kalia N. Helicobacter

[31] LeCouter J, Kowalski J,

[27] Jung D-R, Kim J, Nahm C,

synthesis of carbon quantum dots and its application towards the bioelectroanalytical and biolabeling studies, Electrochimica Acta. 2015; 182:

Nojoomi A, Mozafari M, Dubnika A, Inayathullah M, Rajadas J. Nanomaterials

hybridization approach, J. Mater. Chem.

engineering for drug delivery: a

[19] Shereema RM, Nambiar SR, Shankar SS, Rao TP. ceo2–MWCNT nanocomposite based electrochemical sensor for acetaldehyde. Anal. Methods.

Schubert U. Surface Modification and Functionalization of Metal and Metal Oxide Nanoparticles by Organic Ligands, Monatshefte für Chemie. 2008;

[21] Ali A, Zafar H, Zia M, Haq I, Phull AR, Ali JS, Hussain A. Synthesis, characterization, applications, and challenges of iron oxide nanoparticles.

Nanotechnology, Science and Applications*.* 2016; 9: 49-67.

[22] Patel KD, Singh RK, Kim HW, Carbon-based nanomaterials as an emerging platform for theranostics. Mater. Horiz. 2019; **6**: 434-469.

[23] Notarianni M, Liu J, Vernon K, Motta N. Synthesis and applications of carbon nanomaterials for energy generation and storage. Beilstein J. Nanotechnol. 2016; 7: 149-196.

[24] Shereema RM, Rao TP, Kumar SVB, Sruthi TV, Vishnu R, Prabhu GRD, Shankar SS. Individual and simultaneous electrochemical determination of metanil yellow and curcumin on carbon quantum dots based glassy carbon electrode. Materials

Science & Engineering C*.* 2018*;*

588-595.

[18] Mohammadi MR,

B. 2017;**5**: 3995-4018.

2015; 7: 4912-4918.

139:183-195.

[20] Neouze M-A and

**134**

93:21-27.

[34] Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971; 285:1182-1186.

[35] Folkman J: Incipient angiogenesis 1. J Natl Cancer Inst. 2000; 92:94-95

[36] Naumov GN, Akslen LA, Folkman J: Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch. Cell Cycle. 2006; 5:1779-1787

[37] Hashemi Goradel N, Ghiyami-Hour F, Jahangiri S, et al. Nanoparticles as new tools for inhibition of cancer angiogenesis. J Cell Physiol. 2018; 233:2902-2910.

[38] Banerjee D, Harfouche R, Sengupta S. Nanotechnology-mediated targeting of tumor angiogenesis. Vasc Cell. 2011; 3: 3.

[39] Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R: Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol. 2007; 2:751-760.

[40] Ferrara N: VEGF as a therapeutic target in cancer. Oncology. 2005; 69:11-16.

[41] Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer therapy and diagnosis. Advanced Drug Delivery Reviews. 2002; 54:631-651.

[42] Couvreur P, Vauthier C: Nanotechnology: intelligent design to treat complex disease. Pharm Res. 2006; 23:1417-1450.

[43] Sengupta S, Eavarone D, Capila I, Zhao G, Watson N, Kiziltepe T, Sasisekharan R: Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system. Nature. 2005; 436:568-572.

[44] Chaudhuri P, Harfouche R, Soni S, Hentschel DM, Sengupta S: Shape effect of carbon nanovectors on angiogenesis. ACS Nano. 2010; 4:574-582.

[45] Harfouche R, Basu S, Soni S, Hentschel DM, Mashelkar RA, Sengupta S. Nanoparticle-mediated targeting of phosphatidylinositol-3 kinase signaling inhibits angiogenesis. Angiogenesis. 2009; 12:325-338.

[46] Satchi-Fainaro R, Mamluk R, Wang L, Short SM, Nagy JA, Feng D, Dvorak AM, Dvorak HF, Puder M, Mukhopadhyay D, Folkman J: Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin. Cancer Cell. 2005; 7:251-261.

[47] Satchi-Fainaro R, Puder M, Davies JW, Tran HT, Sampson DA, Greene AK, Corfas G, Folkman J: Targeting angiogenesis with a conjugate of HPMA copolymer and TNP-470. Nat Med. 2004; 10:255-261.

[48] Xie J, Shen Z, Li KC, Danthi N: Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle. Int J Nanomedicine. 2007; 2:479-485.

[49] Costa PM, Cardoso AL, Custodia C, Cunha P, Pereira de Almeida L, Pedroso de Lima MC. MiRNA-21 silencing mediated by tumortargeted nanoparticles combined with sunitinib: A new multimodal gene therapy approach for glioblastoma. Journal of Control Release. 2015; 207:31-39.

[50] Sruthi TV, Edatt L, Raji GR, Kunhiraman H, Shankar SS, Shankar V, Ramachandran V, Poyyakkara A, Kumar SVB. Horizontal transfer of miR-23a from hypoxic tumor cell colonies can induce angiogenesis. J Cell Physiol. 2018; 233:3498-3514.

[51] Arvizo RR, Rana S, Miranda OR, Bhattacharya R, Rotello VM, Mukherjee P. Mechanism of anti-angiogenic property of gold nanoparticles: Role of nanoparticle size and surface charge. Nanomedicine: Nanotechnology, Biology and Medicine. 2011; 7:580-587.

[52] Arvizo, RR, Saha S, Wang E, Robertson JD, Bhattacharya R, Mukherjee P. Inhibition of tumor growth and metastasis by a selftherapeutic nanoparticle. Proceedings of the National Academy of Sciences. 2013; 110:6700-6705.

[53] Balakrishnan S, Bhat F, Raja Singh P, et al. Gold nanoparticle– conjugated quercetin inhibits epithelial mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR-2 mediated pathway in breast cancer. Cell Proliferation. 2016; 49:678-697.

[54] Li W, Zhao X, Du B, et al. Gold nanoparticle-mediated targeted delivery of recombinant human endostatin normalizes tumour vasculature and improves cancer therapy. Scientific Reports. 2016; 6: 30619.

[55] Baharara J, Namvar F, Mousavi M, Ramezani T, Mohamad R. Antiangiogenesis effect of biogenic silver nanoparticles synthesized using saliva officinalis on chick chorioalantoic membrane (CAM). Molecules. 2014; 19:13498-13508.

[56] Khandia R, Munjal A, Bangrey R, Mehra R, Dhama K, Sharma N. Evaluation of silver nanoparticle mediated reduction of neovascularisation (angiogenesis) in chicken model. Advances in Animal and Veterinary Sciences. 2015; 3:372-376.

[57] Yang T, Yao Q, Cao F, Liu Q, Liu B, Wang X-H. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: Insight into the cytotoxicity and antiangiogenesis. International Journal of Nanomedicine. 2016; 11:6679.

[58] Xu Y, Wen Z, Xu Z. Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism. Anticancer Research. 2009; 29:5103-5109.

[59] Jin H, Pi J, Yang F, et al. Ursolic acid-loaded chitosan nanoparticles induce potent antiangiogenesis in tumor. Applied Microbiology and Biotechnology. 2016; 100:6643-6652.

[60] Şalva E, Turan SO, Kabasakal L, Alan S, Özkan N, Eren F, Akbuğa J. Investigation of the therapeutic efficacy of codelivery of psiRNA-Vascular endothelial growth factor and pIL-4 into chitosan nanoparticles in the Breast tumor model. Journal of Pharmaceutical Sciences. 2014; 103:785-795.

[61] Sun D, Liu Y, Yu Q, et al. Inhibition of tumor growth and vasculature and fluorescence imaging using functionalized ruthenium-thiol protected selenium nanoparticles. Biomaterials. 2014; 35:1572-1583.

[62] Yu Q, Liu Y, Cao C, Le F, Qin X, Sun D, Liu J. The use of Ph sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging. Nanoscale. 2014; 6: 9279-9292.

[63] Chen J, Patil S, Seal S, McGinnis JF. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nature Nanotechnology. 2006; 1:142-150.

[64] Lord MS, Tsoi B, Gunawan C, Teoh WY, Amal R, Whitelock JM. Anti-angiogenic activity of heparin functionalised cerium oxide nanoparticles. Biomaterials. 2013; 34:8808-8818.

[65] Giri S, Karakoti A, Graham RP, et al. Nanoceria: A rare-earth nanoparticle as a novel antiangiogenic therapeutic agent

**137**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

Angiogenic profiling of synthesized Carbon Quantum Dotes. Biochemistry.

[74] Namara KM, Alzubaidi H, Jackson JK. Cardiovascular disease as a leading cause of death: how are pharmacists getting involved? Integr Pharm Res Pract. 2019; 8: 1-11

[75] Stewart J, Manmathan G, Wilkinson P. Primary prevention of cardiovascular disease: A review of contemporary guidance and literature.

JRSM Cardiovasc Dis. 2017; 6:

[77] Ross R. Atherosclerosis–an inflammatory disease.N Engl J Med.

[78] Sueishi K, Yonemitsu Y,

Nakagawa K, Kaneda Y, Kumamoto M, Nakashima Y. Atherosclerosis and angiogenesis. Its pathophysiological significance in humans as well as in an animal model induced by the gene transfer of vascular endothelial growth factor. Ann. NY Acad. Sci.1997; 811:

[79] Moreno PR, Purushothaman KR, Sirol M, Levy AP, Fuster V. Neovascularization in human

atherosclerosis. Circulation. 2006; 113:

[80] Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr. Top. Dev. Biol.

[81] Almutairi A, Rossin R, Shokeen M, et al. Biodegradable dendritic positronemitting nanoprobes for the noninvasive imaging of angiogenesis. Proc Natl Acad

Sci USA. 2009; 106:685-690.

[76] Gallino A, Aboyans V, Diehm C, et al. European Society of Cardiology Working Group on Peripheral Circulation. Non-coronary atherosclerosis. Eur Heart J. 2014;

2048004016687211.

35:1112-1119.

322-324.

2245-2252.

2004; 64: 207-238.

1999; 340:115-126.

2015; 54:6352-6356.

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

in ovarian cancer. PLoS ONE. 2013; 8:

[66] Hijaz M, Das S, Mert I, et al. Folic acid tagged nanoceria as a novel therapeutic agent in ovarian cancer.

[67] Das S, Singh S, Dowding JM, et al. The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments. Biomaterials. 2012; 33:

[68] Jo DH, Kim JH, Yu YS, Lee TG, Kim JH. Antiangiogenic effect of silicate nanoparticle on retinal neovascularization induced by vascular endothelial growth factor. Nanomedicine: Nanotechnology, Biology and Medicine. 2012; 8:784-791.

[69] Zhang M and Jiang L. Doxorubicin hydrochloride-loaded mesoporous silica nanoparticles inhibit non-Small cell lung cancer metastasis by suppressing VEGF-Mediated angiogenesis. Journal of Biomedical Nanotechnology. 2016;

[70] Hu H, You Y, He L, Chen, T. The rational design of NAMI-A loaded mesoporous silica nanoparticles as antiangiogenic nanosystems. Journal of Materials Chemistry B. 2015;

Kumar VBS, Aneesh PM. MoS2-ZnO nano composites as highly functional agents for anti-angiogenic and anticancer theranostics. J. Mater. Chem. B.

[72] Yalcin M, Dyskin E, Lansing L, et al. Tetraiodothyroacetic acid (tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid. The Journal of Clinical Endocrinology & Metabolism. 2010; 95:1972-1980.

[71] Chako L, Poyyakkara A,

[73] Shereema RM, Sruthi TV, Kumar VBS, Rao TP, Shankar SS.

BMC Cancer. 2016; 16:220.

e54578.

7746-7755.

12:1975-1986.

3:6338-6346.

2018; 6:3048-3057.

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

in ovarian cancer. PLoS ONE. 2013; 8: e54578.

*Theranostics - An Old Concept in New Clothing*

[58] Xu Y, Wen Z, Xu Z. Chitosan nanoparticles inhibit the growth of human hepatocellular carcinoma xenografts through an antiangiogenic mechanism. Anticancer Research. 2009;

[59] Jin H, Pi J, Yang F, et al. Ursolic acid-loaded chitosan nanoparticles induce potent antiangiogenesis in tumor. Applied

Sciences. 2014; 103:785-795.

Microbiology and Biotechnology. 2016;

[60] Şalva E, Turan SO, Kabasakal L, Alan S, Özkan N, Eren F, Akbuğa J. Investigation of the therapeutic efficacy of codelivery of psiRNA-Vascular endothelial growth factor and pIL-4 into chitosan nanoparticles in the Breast tumor model. Journal of Pharmaceutical

[61] Sun D, Liu Y, Yu Q, et al. Inhibition of tumor growth and vasculature and fluorescence imaging using functionalized ruthenium-thiol protected selenium nanoparticles. Biomaterials. 2014; 35:1572-1583.

[62] Yu Q, Liu Y, Cao C, Le F, Qin X, Sun D, Liu J. The use of Ph sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging. Nanoscale. 2014; 6: 9279-9292.

[63] Chen J, Patil S, Seal S, McGinnis JF. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nature Nanotechnology.

[64] Lord MS, Tsoi B, Gunawan C, Teoh WY, Amal R, Whitelock JM. Anti-angiogenic activity of heparin functionalised cerium oxide nanoparticles. Biomaterials. 2013;

[65] Giri S, Karakoti A, Graham RP, et al. Nanoceria: A rare-earth nanoparticle as a novel antiangiogenic therapeutic agent

2006; 1:142-150.

34:8808-8818.

29:5103-5109.

100:6643-6652.

of anti-angiogenic property of gold nanoparticles: Role of nanoparticle size and surface charge. Nanomedicine: Nanotechnology, Biology and Medicine.

[52] Arvizo, RR, Saha S, Wang E, Robertson JD, Bhattacharya R, Mukherjee P. Inhibition of tumor growth and metastasis by a selftherapeutic nanoparticle. Proceedings of the National Academy of Sciences.

[53] Balakrishnan S, Bhat F, Raja Singh P, et al. Gold nanoparticle– conjugated quercetin inhibits epithelial mesenchymal transition, angiogenesis and invasiveness via EGFR/VEGFR-2 mediated pathway in breast cancer. Cell

Proliferation. 2016; 49:678-697.

Reports. 2016; 6: 30619.

19:13498-13508.

[54] Li W, Zhao X, Du B, et al. Gold nanoparticle-mediated targeted delivery of recombinant human endostatin normalizes tumour vasculature and improves cancer therapy. Scientific

[55] Baharara J, Namvar F, Mousavi M, Ramezani T, Mohamad R. Antiangiogenesis effect of biogenic silver nanoparticles synthesized using saliva officinalis on chick chorioalantoic membrane (CAM). Molecules. 2014;

[56] Khandia R, Munjal A, Bangrey R, Mehra R, Dhama K, Sharma N. Evaluation

[57] Yang T, Yao Q, Cao F, Liu Q, Liu B, Wang X-H. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: Insight into the cytotoxicity and antiangiogenesis. International Journal of Nanomedicine.

of silver nanoparticle mediated reduction of neovascularisation (angiogenesis) in chicken model. Advances in Animal and Veterinary

Sciences. 2015; 3:372-376.

2011; 7:580-587.

2013; 110:6700-6705.

**136**

2016; 11:6679.

[66] Hijaz M, Das S, Mert I, et al. Folic acid tagged nanoceria as a novel therapeutic agent in ovarian cancer. BMC Cancer. 2016; 16:220.

[67] Das S, Singh S, Dowding JM, et al. The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments. Biomaterials. 2012; 33: 7746-7755.

[68] Jo DH, Kim JH, Yu YS, Lee TG, Kim JH. Antiangiogenic effect of silicate nanoparticle on retinal neovascularization induced by vascular endothelial growth factor. Nanomedicine: Nanotechnology, Biology and Medicine. 2012; 8:784-791.

[69] Zhang M and Jiang L. Doxorubicin hydrochloride-loaded mesoporous silica nanoparticles inhibit non-Small cell lung cancer metastasis by suppressing VEGF-Mediated angiogenesis. Journal of Biomedical Nanotechnology. 2016; 12:1975-1986.

[70] Hu H, You Y, He L, Chen, T. The rational design of NAMI-A loaded mesoporous silica nanoparticles as antiangiogenic nanosystems. Journal of Materials Chemistry B. 2015; 3:6338-6346.

[71] Chako L, Poyyakkara A, Kumar VBS, Aneesh PM. MoS2-ZnO nano composites as highly functional agents for anti-angiogenic and anticancer theranostics. J. Mater. Chem. B. 2018; 6:3048-3057.

[72] Yalcin M, Dyskin E, Lansing L, et al. Tetraiodothyroacetic acid (tetrac) and nanoparticulate tetrac arrest growth of medullary carcinoma of the thyroid. The Journal of Clinical Endocrinology & Metabolism. 2010; 95:1972-1980.

[73] Shereema RM, Sruthi TV, Kumar VBS, Rao TP, Shankar SS. Angiogenic profiling of synthesized Carbon Quantum Dotes. Biochemistry. 2015; 54:6352-6356.

[74] Namara KM, Alzubaidi H, Jackson JK. Cardiovascular disease as a leading cause of death: how are pharmacists getting involved? Integr Pharm Res Pract. 2019; 8: 1-11

[75] Stewart J, Manmathan G, Wilkinson P. Primary prevention of cardiovascular disease: A review of contemporary guidance and literature. JRSM Cardiovasc Dis. 2017; 6: 2048004016687211.

[76] Gallino A, Aboyans V, Diehm C, et al. European Society of Cardiology Working Group on Peripheral Circulation. Non-coronary atherosclerosis. Eur Heart J. 2014; 35:1112-1119.

[77] Ross R. Atherosclerosis–an inflammatory disease.N Engl J Med. 1999; 340:115-126.

[78] Sueishi K, Yonemitsu Y, Nakagawa K, Kaneda Y, Kumamoto M, Nakashima Y. Atherosclerosis and angiogenesis. Its pathophysiological significance in humans as well as in an animal model induced by the gene transfer of vascular endothelial growth factor. Ann. NY Acad. Sci.1997; 811: 322-324.

[79] Moreno PR, Purushothaman KR, Sirol M, Levy AP, Fuster V. Neovascularization in human atherosclerosis. Circulation. 2006; 113: 2245-2252.

[80] Stupack DG, Cheresh DA. Integrins and angiogenesis. Curr. Top. Dev. Biol. 2004; 64: 207-238.

[81] Almutairi A, Rossin R, Shokeen M, et al. Biodegradable dendritic positronemitting nanoprobes for the noninvasive imaging of angiogenesis. Proc Natl Acad Sci USA. 2009; 106:685-690.

[82] Liu Y, Pressly ED, Abendschein DR, et al. Targeting angiogenesis using a C-type atrial natriuretic factorconjugated nanoprobe and PET. J Nucl Med. 2011; 52:1956-1963.

[83] Winter PM, Morawski AM, Caruthers SD, Fuhrhop RW, Zhang H, Williams TA, Allen JS, Lacy EK, Robertson JD, Lanza GM, Wickline SA. Molecular imaging of angiogenesis in early-stage atherosclerosis with alpha(v)beta3 integrin-targeted nanoparticles. Circulation. 2003; 108: 2270-2274.

[84] Winter PM, Neubauer AM, Caruthers SD, Harris TD, Robertson JD, Williams TA, Schmieder AH, Hu G, Allen JS, Lacy EK, Zhang H, Wickline SA, Lanza GM. Endothelial alpha(v) beta3 integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2103-2109.

[85] Singh S, Young A, McNaught CE. The physiology of wound healing. Surgery. 2017; 35:473-477.

[86] Patel S, Srivastava S, Singh MR, Singh D. Mechanistic insight into diabetic wounds: Pathogenesis, molecular targets and treatment strategies to pace wound healing. Biomedicine & Pharmacotherapy. 2019; 112:108615.

[87] Vargas GE, Durand LAH, Cadena V, et al. Effect of nano-sized bioactive glass particles on the angiogenic properties of collagen based composites. J Mater Sci Mater Med. 2013; 24:1261-1269.

[88] Kargozar S, Baino F, Hamzehlou S, Hill RG, Mozafari M. Bioactive glasses: sprouting angiogenesis in tissue engineering. Trends Biotechnol. 2018; 36:430-444.

[89] Kargozar S, Lotfibakhshaiesh N, Ai J, et al. Strontium and cobaltsubstituted bioactive glasses seeded

with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities. Acta Biomater. 2017; 58: 502-514.

[90] Danieland MC and Astruc D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev. 2004; 104: 293-346.

[91] Leu JG, Chen SA, Chen HM, et al. The effects of gold nanoparticles in wound healing with antioxidant epigallocatechin gallate and alpha-lipoic acid. Nanomedicine. 2012; 8:767-775.

[92] Poljsak B, Šuput D, Milisav I. Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants. Oxid Med Cell Longev. 2013; 2013:956792.

[93] Roma-Rodrigues C, Heuer-Jungemann A, Fernandes AR, Kanaras AG, Baptista PV. Peptidecoated gold nanoparticles for modulation of angiogenesis in vivo. International journal of nanomedicine. 2016; 11:2633-2639.

[94] Zarubova J, Hasani-Sadrabadi MM, Bacakova L, Li S. Nano-in-Micro Dual Delivery Platform for Chronic Wound Healing Applications. Micromachines. 2020; 11:158.

[95] Xu H, Lv F, Zhang Y, Yi Z, Ke Q, Wu C, Liu M, Chang J. Hierarchically micro-patterned nanofibrous scaffolds with a nanosized bio-glass surface for accelerating wound healing. Nanoscale*.* 2015; *7*:18446-18452.

[96] Xie Z, Paras CB, Weng H, Punnakitikashem P, Su LC, Vu K, Tang L, Yang J, Nguyen KT. Dual growth factor releasing multifunctional nanofibers for wound healing. Acta biomaterialia*.* 2013; 9:9351-9359.

**139**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

4:7861-7869.

34:2194-2201.

Murali S, Rana RK, Chatterjee S, Patra CR . Zinc oxide nanoflowers make new blood vessels. Nanoscale. 2012;

[105] Chigurupati S, Mughal MR, Okun E, Das S, Kumar A, McCaffery M, Seal S, Mattson MP. Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing. Biomaterials. 2013;

[106] Abcouwer SF. Angiogenic factors and cytokines in diabetic retinopathy. J Clin Cell Immunol. 2013; 1: 1-12.

[107] Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME. Vascular endothelial growth factor in eye disease. Progress in retinal and eye

[108] Salvi SM, Akhtar S, Currie Z. Ageing changes in the eye. Postgraduate Medical Journal. 2006;82:581-587.

[109] Amato R, Catalani E, Dal Monte M, et al. Autophagy-mediated neuroprotection induced by octreotide in an ex vivo model of early diabetic retinopathy. Pharmacol. 2018;

[110] Bisht R, Mandal A, Jaiswal JK, Rupenthal ID. Nanocarrier mediated retinal drug delivery: overcoming ocular barriers to treat posterior eye diseases. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol*.* 2018; 10:10.1002/

[111] Jahangirian H, Kalantari K, Izadiyan Z, Rafiee-Moghaddam R, Shameli K, Webster TJ. A review of small molecules and drug delivery applications using gold and iron nanoparticles. Int. J. Nanomedicine.

[112] Cai J and Du B. Gold nanoparticles induce nanostructural reorganization

128:167-178.

wnan.1473.

2019; 14:1633-1657.

research. 2008; 27:331-371.

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

[98] Battigelli A, Menard-Moyon C, Da Ros T, Prato M, Bianco A. Endowing Carbon Nanotubes with Biological and Biomedical Properties by Chemical Modifications. Adv. Drug Delivery Rev.

[99] Meng J, Li X, Wang C, Guo H, Liu J, Xu H. Carbon nanotubes activate macrophages into a M1/M2 mixed status: recruiting naive macrophages and supporting angiogenesis. ACS applied materials & interfaces. 2015;

[100] Liu Z, Feng X, Wang H, Ma J, Liu W, Cui D, Gu Y, Tang R. Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair.

International journal of nanomedicine.

[101] Zhao H, Ding R, Zhao X, et al. Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering. Drug Discov Today.

[102] Terzopoulou Z, Kyzas GZ, Bikiaris DN. Recent advances in nanocomposite materials of graphene derivatives with polysaccharides. Materials. 2015; 8:652-683.

[103] Ur Rehman SR, Augustine R, Zahid AA, Ahmed R, Tariq M, Hasan A. Reduced Graphene Oxide Incorporated

[104] Barui AK, Veeriah V, Mukherjee S, Manna J, Patel AK, Patra S, Pal K,

GelMA Hydrogel Promotes Angiogenesis For Wound Healing Applications. International Journal of Nanomedicine. 2019; 14:9603-9617.

[97] Lino MM, Simões S, Vilaça A, Antunes H, Zonari A, Ferreira L. Modulation of angiogenic activity by light-activatable miRNA-loaded nanocarriers. ACS nano. 2018;

12:5207-5220.

2013; 65:1899-1920.

7:3180-3188.

2014; 9:1275

2017; 22: 1302-1317.

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

[97] Lino MM, Simões S, Vilaça A, Antunes H, Zonari A, Ferreira L. Modulation of angiogenic activity by light-activatable miRNA-loaded nanocarriers. ACS nano. 2018; 12:5207-5220.

*Theranostics - An Old Concept in New Clothing*

[82] Liu Y, Pressly ED, Abendschein DR, et al. Targeting angiogenesis using a C-type atrial natriuretic factorconjugated nanoprobe and PET. J Nucl

with human umbilical cord perivascular cells to promote bone regeneration via enhanced osteogenic and angiogenic activities. Acta Biomater. 2017; 58:

[90] Danieland MC and Astruc D. Gold nanoparticles: assembly, supramolecular

chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology.

Chem Rev. 2004; 104: 293-346.

[91] Leu JG, Chen SA, Chen HM, et al. The effects of gold nanoparticles in wound healing with antioxidant epigallocatechin gallate and alpha-lipoic acid. Nanomedicine. 2012; 8:767-775.

[92] Poljsak B, Šuput D, Milisav I.

2013; 2013:956792.

2016; 11:2633-2639.

2020; 11:158.

9:9351-9359.

2015; *7*:18446-18452.

[96] Xie Z, Paras CB, Weng H, Punnakitikashem P, Su LC, Vu K, Tang L, Yang J, Nguyen KT.

Dual growth factor releasing multifunctional nanofibers for wound healing. Acta biomaterialia*.* 2013;

[93] Roma-Rodrigues C,

Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants. Oxid Med Cell Longev.

Heuer-Jungemann A, Fernandes AR, Kanaras AG, Baptista PV. Peptidecoated gold nanoparticles for modulation of angiogenesis in vivo. International journal of nanomedicine.

[94] Zarubova J, Hasani-Sadrabadi MM, Bacakova L, Li S. Nano-in-Micro Dual Delivery Platform for Chronic Wound Healing Applications. Micromachines.

[95] Xu H, Lv F, Zhang Y, Yi Z, Ke Q, Wu C, Liu M, Chang J. Hierarchically micro-patterned nanofibrous scaffolds with a nanosized bio-glass surface for accelerating wound healing. Nanoscale*.*

502-514.

Med. 2011; 52:1956-1963.

[83] Winter PM, Morawski AM, Caruthers SD, Fuhrhop RW, Zhang H, Williams TA, Allen JS, Lacy EK, Robertson JD, Lanza GM, Wickline SA. Molecular imaging of angiogenesis in early-stage atherosclerosis with alpha(v)beta3 integrin-targeted nanoparticles. Circulation. 2003; 108: 2270-2274.

[84] Winter PM, Neubauer AM,

Lanza GM. Endothelial alpha(v) beta3 integrin-targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2103-2109.

[85] Singh S, Young A, McNaught CE. The physiology of wound healing.

[86] Patel S, Srivastava S, Singh MR, Singh D. Mechanistic insight into diabetic wounds: Pathogenesis, molecular targets and treatment strategies to pace wound healing. Biomedicine & Pharmacotherapy. 2019;

[87] Vargas GE, Durand LAH, Cadena V, et al. Effect of nano-sized bioactive glass particles on the angiogenic properties of collagen based composites. J Mater Sci Mater Med. 2013; 24:1261-1269.

[88] Kargozar S, Baino F, Hamzehlou S, Hill RG, Mozafari M. Bioactive glasses: sprouting angiogenesis in tissue engineering. Trends Biotechnol. 2018;

[89] Kargozar S, Lotfibakhshaiesh N, Ai J, et al. Strontium and cobaltsubstituted bioactive glasses seeded

Surgery. 2017; 35:473-477.

112:108615.

36:430-444.

Caruthers SD, Harris TD, Robertson JD, Williams TA, Schmieder AH, Hu G, Allen JS, Lacy EK, Zhang H, Wickline SA,

**138**

[98] Battigelli A, Menard-Moyon C, Da Ros T, Prato M, Bianco A. Endowing Carbon Nanotubes with Biological and Biomedical Properties by Chemical Modifications. Adv. Drug Delivery Rev. 2013; 65:1899-1920.

[99] Meng J, Li X, Wang C, Guo H, Liu J, Xu H. Carbon nanotubes activate macrophages into a M1/M2 mixed status: recruiting naive macrophages and supporting angiogenesis. ACS applied materials & interfaces. 2015; 7:3180-3188.

[100] Liu Z, Feng X, Wang H, Ma J, Liu W, Cui D, Gu Y, Tang R. Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair. International journal of nanomedicine. 2014; 9:1275

[101] Zhao H, Ding R, Zhao X, et al. Graphene-based nanomaterials for drug and/or gene delivery, bioimaging, and tissue engineering. Drug Discov Today. 2017; 22: 1302-1317.

[102] Terzopoulou Z, Kyzas GZ, Bikiaris DN. Recent advances in nanocomposite materials of graphene derivatives with polysaccharides. Materials. 2015; 8:652-683.

[103] Ur Rehman SR, Augustine R, Zahid AA, Ahmed R, Tariq M, Hasan A. Reduced Graphene Oxide Incorporated GelMA Hydrogel Promotes Angiogenesis For Wound Healing Applications. International Journal of Nanomedicine. 2019; 14:9603-9617.

[104] Barui AK, Veeriah V, Mukherjee S, Manna J, Patel AK, Patra S, Pal K,

Murali S, Rana RK, Chatterjee S, Patra CR . Zinc oxide nanoflowers make new blood vessels. Nanoscale. 2012; 4:7861-7869.

[105] Chigurupati S, Mughal MR, Okun E, Das S, Kumar A, McCaffery M, Seal S, Mattson MP. Effects of cerium oxide nanoparticles on the growth of keratinocytes, fibroblasts and vascular endothelial cells in cutaneous wound healing. Biomaterials. 2013; 34:2194-2201.

[106] Abcouwer SF. Angiogenic factors and cytokines in diabetic retinopathy. J Clin Cell Immunol. 2013; 1: 1-12.

[107] Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME. Vascular endothelial growth factor in eye disease. Progress in retinal and eye research. 2008; 27:331-371.

[108] Salvi SM, Akhtar S, Currie Z. Ageing changes in the eye. Postgraduate Medical Journal. 2006;82:581-587.

[109] Amato R, Catalani E, Dal Monte M, et al. Autophagy-mediated neuroprotection induced by octreotide in an ex vivo model of early diabetic retinopathy. Pharmacol. 2018; 128:167-178.

[110] Bisht R, Mandal A, Jaiswal JK, Rupenthal ID. Nanocarrier mediated retinal drug delivery: overcoming ocular barriers to treat posterior eye diseases. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol*.* 2018; 10:10.1002/ wnan.1473.

[111] Jahangirian H, Kalantari K, Izadiyan Z, Rafiee-Moghaddam R, Shameli K, Webster TJ. A review of small molecules and drug delivery applications using gold and iron nanoparticles. Int. J. Nanomedicine. 2019; 14:1633-1657.

[112] Cai J and Du B. Gold nanoparticles induce nanostructural reorganization

of VEGFR2 to repress angiogenesis. J. Biomed. Nanotechnol. 2013; 9:1746-1756.

[113] Chan CM, Hsiao CY, Li HJ, Fang JY, Chang DC, Hung CF. The Inhibitory Effects of Gold Nanoparticles on VEGF-A-Induced Cell Migration in Choroid-Retina Endothelial Cells. International Journal of Molecular Sciences. 2020; 21:109.

[114] Kim JH, Kim MH, Jo DH, Yu YS, Lee TG, Kim JH. The inhibition of retinal neovascularization by gold nanoparticles via suppression of VEGFR-2 activation. Biomaterials. 2011; 32:1865-1871.

[115] Cao Y, Chen A, An SS, Ji RW, Davidson D, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem. 1997; 272:22924-22928.

[116] Zhang D, Kaufman PL, Gao G, Saunders RA, Ma JX: Intravitreal injection of plasminogen kringle 5, an endogenous angiogenic inhibitor, arrests retinal neovascularization in rats. Diabetologia. 2001; 44:757-765.

[117] Park K, Chen Y, Hu Y, Mayo AS, Kompella UB, Longeras R, Ma JX. Nanoparticle-mediated expression of an angiogenic inhibitor ameliorates ischemia-induced retinal neovascularization and diabetesinduced retinal vascular leakage. Diabetes. 2009; 58:1902-1913.

[118] Qiu F, Meng T, Chen Q, Zhou K, Shao Y, Matlock G, Ma X, Wu W, Du Y, Wang X, Deng G. Fenofibrate-loaded biodegradable nanoparticles for the treatment of experimental diabetic retinopathy and neovascular age-related macular degeneration. Molecular pharmaceutics. 2019; 16:1958-1970.

[119] Amato R, Giannaccini M, Dal Monte M, Cammalleri M, Pini A, Raffa V, Lulli M, Casini G. Association of the Somatostatin Analog Octreotide with Magnetic Nanoparticles for Intraocular Delivery: A Possible Approach for the Treatment of Diabetic Retinopathy. Frontiers in Bioengineering and Biotechnology. 2020; 8:144.

[120] Pollinger K, Hennig R, Ohlmann A, Fuchshofer R, Wenzel R, Breunig M, Tessmar J, Tamm ER, Goepferich A. Ligand-functionalized nanoparticles target endothelial cells in retinal capillaries after systemic application. Proceedings of the National Academy of Sciences. 2013; 110:6115-6120.

[121] Yandrapu SK, Upadhyay AK, Petrash JM, Kompella UB. Nanoparticles in porous microparticles prepared by supercritical infusion and pressure quench technology for sustained delivery of bevacizumab. Molecular pharmaceutics. 2013; 10:4676-4686.

[122] Luo L, Zhang X, Hirano Y, Tyagi P, Barabás P, Uehara H, Miya TR, Singh N, Archer B, Qazi Y, Jackman K. Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration. ACS nano. 2013; 7:3264-3275.

[123] Palamoor M and Jablonski MM. Synthesis, characterization and in vitro studies of celecoxib-loaded poly (ortho ester) nanoparticles targeted for intraocular drug delivery. Colloids and Surfaces B: Biointerfaces. 2013; 112: 474-482.

[124] Roupakia E, Markopoulos GS, Kolettas E. IL-12-mediated transcriptional regulation of matrix metalloproteinases. Biosci Rep. 2018; 38:BSR20171420.

[125] Zeng L, Ma W, Shi L, Chen X, Wu R, Zhang Y, Chen H, Chen H. Poly (lactic-co-glycolic acid) nanoparticlemediated interleukin-12 delivery for the treatment of diabetic retinopathy.

**141**

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic…*

2013; 34: 64-77.

by porous β-CaSiO3/PDLGA composite scaffold via activation of AMPK/ERK1/2 and PI3K/Akt pathways. Biomaterials.

Wang C. Interaction of hydroxyapatite nanoparticles with endothelial cells: internalization and inhibition of angiogenesis in vitro through the PI3K/ Akt pathway. International journal of nanomedicine. 2017; 12:5781-5795.

[135] Oliveira H, Catros S, Boiziau C, Siadous R, Marti-Munoz J, Bareille R, Rey S, Castano O, Planell J, Amédée J, Engel E. The proangiogenic potential of a novel calcium releasing biomaterial: Impact on cell recruitment. Acta biomaterialia. 2016; 29:435-445.

[136] Kang MS, Lee NH, Singh RK, Mandakhbayar N, Perez RA, Lee JH, Kim HW. Nanocements produced from mesoporous bioactive glass nanoparticles. Biomaterials. 2018;

[137] Tian T, Xie W, Gao W, Wang G, Zeng L, Miao G, Lei B, Lin Z, Chen X. Micro-nano bioactive glass particles incorporated porous scaffold for promoting osteogenesis and angiogenesis in vitro. Frontiers in

[138] Yao Q, Liu Y, Selvaratnam B, Koodali RT, Sun H. Mesoporous silicate nanoparticles/3D nanofibrous scaffoldmediated dual-drug delivery for bone tissue engineering. Journal of Controlled

[139] Chen Y, Chen S, Kawazoe N, Chen G. Promoted angiogenesis and osteogenesis by dexamethasone-loaded calcium phosphatenanoparticles/ collagen composite scaffolds with microgroove networks. Scientific

[140] Luo C, Yang X, Li M, Huang H, Kang Q, Zhang X, Hui H, Zhang X,

162:183-199.

chemistry. 2019; 7:186.

Release. 2018; 279: 69-78.

reports. 2018; *8*:1-12.

[134] Shi X, Zhou K, Huang F,

*DOI: http://dx.doi.org/10.5772/intechopen.94151*

International journal of nanomedicine.

[126] Colnot C, Romero DM, Huang S, Helms JA. Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects. Clin Orthop

[127] Childs SG. Osteonecrosis: death of bone cells. Orthopaedic Nursing. 2005;

[128] Lazzarini L, De Lalla F, Mader JT. Long bone osteomyelitis. Current infectious disease reports. 2002;

[129] Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Gilg T. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone. 1987;

[130] Alagiakrishnan K, Juby A, Hanley D, Tymchak W, Sclater A. Role of vascular factors in osteoporosis. J Gerontol A Biol Sci Med Sci. 2003;

[131] Holmes B, Bulusu K, Plesniak M, Zhang LG. A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured

scaffolds for vascularized bone tissue repair. Nanotechnology. 2016;

chitin composite hydrogel for

Carbohydrate polymers. 2016;

[132] Kumar RA, Sivashanmugam A, Deepthi S, Bumgardner JD, Nair SV, Jayakumar R. Nano-fibrin stabilized CaSO4 crystals incorporated injectable

enhanced angiogenesis & osteogenesis.

[133] Wang C, Lin K, Chang J, Sun J. Osteogenesis and angiogenesis induced

2019; 14: 6357-6369.

Relat Res.2005; 435: 69-78.

24:295-301.

4:439-445.

8:157-164.

58:362-366.

27:064001.

140:144-153.

*Regulation of Angiogenesis Using Nanomaterial Based Formulations: An Emerging Therapeutic… DOI: http://dx.doi.org/10.5772/intechopen.94151*

International journal of nanomedicine. 2019; 14: 6357-6369.

*Theranostics - An Old Concept in New Clothing*

of the Somatostatin Analog Octreotide with Magnetic Nanoparticles for Intraocular Delivery: A Possible Approach for the Treatment of Diabetic Retinopathy. Frontiers in Bioengineering and Biotechnology.

[120] Pollinger K, Hennig R, Ohlmann A, Fuchshofer R, Wenzel R, Breunig M, Tessmar J, Tamm ER, Goepferich A. Ligand-functionalized nanoparticles target endothelial cells in retinal capillaries after systemic application. Proceedings of the National Academy of

Sciences. 2013; 110:6115-6120.

[121] Yandrapu SK, Upadhyay AK, Petrash JM, Kompella UB. Nanoparticles in porous microparticles prepared by supercritical infusion and pressure quench technology for sustained delivery of bevacizumab. Molecular pharmaceutics. 2013; 10:4676-4686.

[122] Luo L, Zhang X, Hirano Y,

degeneration. ACS nano. 2013;

[123] Palamoor M and Jablonski MM. Synthesis, characterization and in vitro studies of celecoxib-loaded poly (ortho ester) nanoparticles targeted for intraocular drug delivery. Colloids and Surfaces B: Biointerfaces. 2013; 112:

[124] Roupakia E, Markopoulos GS,

transcriptional regulation of matrix metalloproteinases. Biosci Rep. 2018;

[125] Zeng L, Ma W, Shi L, Chen X, Wu R, Zhang Y, Chen H, Chen H. Poly (lactic-co-glycolic acid) nanoparticlemediated interleukin-12 delivery for the treatment of diabetic retinopathy.

Kolettas E. IL-12-mediated

38:BSR20171420.

7:3264-3275.

474-482.

Tyagi P, Barabás P, Uehara H, Miya TR, Singh N, Archer B, Qazi Y, Jackman K. Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular

2020; 8:144.

[113] Chan CM, Hsiao CY, Li HJ, Fang JY, Chang DC, Hung CF. The Inhibitory Effects of Gold Nanoparticles on VEGF-A-Induced Cell Migration in Choroid-Retina Endothelial Cells. International Journal of Molecular Sciences. 2020;

[114] Kim JH, Kim MH, Jo DH, Yu YS, Lee TG, Kim JH. The inhibition of retinal neovascularization by gold nanoparticles via suppression of

VEGFR-2 activation. Biomaterials. 2011;

Gao G, Saunders RA, Ma JX: Intravitreal injection of plasminogen kringle 5, an endogenous angiogenic inhibitor, arrests retinal neovascularization in rats. Diabetologia. 2001; 44:757-765.

[117] Park K, Chen Y, Hu Y, Mayo AS, Kompella UB, Longeras R, Ma JX. Nanoparticle-mediated expression

ameliorates ischemia-induced retinal neovascularization and diabetesinduced retinal vascular leakage. Diabetes. 2009; 58:1902-1913.

[118] Qiu F, Meng T, Chen Q, Zhou K, Shao Y, Matlock G, Ma X, Wu W, Du Y, Wang X, Deng G. Fenofibrate-loaded biodegradable nanoparticles for the treatment of experimental diabetic retinopathy and neovascular age-related macular degeneration. Molecular pharmaceutics. 2019; 16:1958-1970.

[119] Amato R, Giannaccini M, Dal Monte M, Cammalleri M, Pini A, Raffa V, Lulli M, Casini G. Association

[115] Cao Y, Chen A, An SS, Ji RW, Davidson D, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem.

1997; 272:22924-22928.

[116] Zhang D, Kaufman PL,

of an angiogenic inhibitor

of VEGFR2 to repress angiogenesis. J. Biomed. Nanotechnol. 2013;

9:1746-1756.

21:109.

32:1865-1871.

**140**

[126] Colnot C, Romero DM, Huang S, Helms JA. Mechanisms of action of demineralized bone matrix in the repair of cortical bone defects. Clin Orthop Relat Res.2005; 435: 69-78.

[127] Childs SG. Osteonecrosis: death of bone cells. Orthopaedic Nursing. 2005; 24:295-301.

[128] Lazzarini L, De Lalla F, Mader JT. Long bone osteomyelitis. Current infectious disease reports. 2002; 4:439-445.

[129] Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Gilg T. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone. 1987; 8:157-164.

[130] Alagiakrishnan K, Juby A, Hanley D, Tymchak W, Sclater A. Role of vascular factors in osteoporosis. J Gerontol A Biol Sci Med Sci. 2003; 58:362-366.

[131] Holmes B, Bulusu K, Plesniak M, Zhang LG. A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair. Nanotechnology. 2016; 27:064001.

[132] Kumar RA, Sivashanmugam A, Deepthi S, Bumgardner JD, Nair SV, Jayakumar R. Nano-fibrin stabilized CaSO4 crystals incorporated injectable chitin composite hydrogel for enhanced angiogenesis & osteogenesis. Carbohydrate polymers. 2016; 140:144-153.

[133] Wang C, Lin K, Chang J, Sun J. Osteogenesis and angiogenesis induced by porous β-CaSiO3/PDLGA composite scaffold via activation of AMPK/ERK1/2 and PI3K/Akt pathways. Biomaterials. 2013; 34: 64-77.

[134] Shi X, Zhou K, Huang F, Wang C. Interaction of hydroxyapatite nanoparticles with endothelial cells: internalization and inhibition of angiogenesis in vitro through the PI3K/ Akt pathway. International journal of nanomedicine. 2017; 12:5781-5795.

[135] Oliveira H, Catros S, Boiziau C, Siadous R, Marti-Munoz J, Bareille R, Rey S, Castano O, Planell J, Amédée J, Engel E. The proangiogenic potential of a novel calcium releasing biomaterial: Impact on cell recruitment. Acta biomaterialia. 2016; 29:435-445.

[136] Kang MS, Lee NH, Singh RK, Mandakhbayar N, Perez RA, Lee JH, Kim HW. Nanocements produced from mesoporous bioactive glass nanoparticles. Biomaterials. 2018; 162:183-199.

[137] Tian T, Xie W, Gao W, Wang G, Zeng L, Miao G, Lei B, Lin Z, Chen X. Micro-nano bioactive glass particles incorporated porous scaffold for promoting osteogenesis and angiogenesis in vitro. Frontiers in chemistry. 2019; 7:186.

[138] Yao Q, Liu Y, Selvaratnam B, Koodali RT, Sun H. Mesoporous silicate nanoparticles/3D nanofibrous scaffoldmediated dual-drug delivery for bone tissue engineering. Journal of Controlled Release. 2018; 279: 69-78.

[139] Chen Y, Chen S, Kawazoe N, Chen G. Promoted angiogenesis and osteogenesis by dexamethasone-loaded calcium phosphatenanoparticles/ collagen composite scaffolds with microgroove networks. Scientific reports. 2018; *8*:1-12.

[140] Luo C, Yang X, Li M, Huang H, Kang Q, Zhang X, Hui H, Zhang X,

Cen C, Luo Y, Xie L. A novel strategy for in vivo angiogenesis and osteogenesis: magnetic micro-movement in a bone scaffold. Artificial cells, nanomedicine, and biotechnology. 2018; 46:636-645.

[141] Kawazoe N and Chen G. Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis. Biomaterials. 2015; 54:226-236.

[142] López-Dolado E,

González-Mayorga A, Gutiérrez MC, Serrano MC. Immunomodulatory and angiogenic responses induced by graphene oxide scaffolds in chronic spinal hemisected rats. Biomaterials. 2016; 99:72-81.

[143] Qian Y, Song J, Zhao X, et al. 3D fabrication with integration molding of a graphene oxide/polycaprolactone nanoscaffold for neurite regeneration and angiogenesis. Adv Sci. 2018; 5:1700499.

[144] Xu ZX, Zhang LQ, Wang CS, Chen RS, Li GS, Guo Y, Xu WH. Acellular spinal cord scaffold implantation promotes vascular remodeling with sustained delivery of VEGF in a rat spinal cord hemisection model. Current Neurovascular Research. 2017; 14:274-289.

[145] Wen Y, Yu S, Wu Y, et al*.* Spinal cord injury repair by implantation of structured hyaluronic acid scaffold with PLGA microspheres in the rat. Cell Tissue Res. 2016; 364:17-28.

[146] Yu S, Yao S, Wen Y, Wang Y, Wang H, Xu Q. Angiogenic microspheres promote neural regeneration and motor function recovery after spinal cord injury in rats. Scientific reports. 2016; 6:1-13.

[147] Jian W-H, Wang H-C, Kuan C-H, Chen M-H, Wu H-C, Sun J-S, Wang T-W. Glycosaminoglycan-based hybrid hydrogel encapsulated with

polyelectrolyte complex nanoparticles for endogenous stem cell regulation in central nervous system regeneration. Biomaterials. 2018; 174:17-30.

[148] Nih LR, Gojgini S, Carmichael ST, Segura T. Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Nature materials. 2018; 17:642-651.

[149] Carenza E, BarcelóV, Morancho A, Levander L, Boada C, Laromaine A, Roig A, Montaner J, Rosell A. In vitro angiogenic performance and in vivo brain targeting of magnetized endothelial progenitor cells for neurorepair therapies. Nanomedicine: Nanotechnology, Biology and Medicine. 2014; 10:225-234.

[150] Yuan M, Wang Y, Qin Y-X. Promoting neuroregeneration by applying dynamic magnetic fields to a novel nanomedicine: Superparamagnetic iron oxide (SPIO) gold nanoparticles bounded with nerve growth factor (NGF). Nanomedicine. 2018; 14:1337-1347.

*Theranostics - An Old Concept in New Clothing*

Cen C, Luo Y, Xie L. A novel strategy for in vivo angiogenesis and osteogenesis: magnetic micro-movement in a bone scaffold. Artificial cells, nanomedicine, and biotechnology. 2018; 46:636-645.

polyelectrolyte complex nanoparticles for endogenous stem cell regulation in central nervous system regeneration.

[148] Nih LR, Gojgini S, Carmichael ST, Segura T. Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Nature

Biomaterials. 2018; 174:17-30.

materials. 2018; 17:642-651.

[149] Carenza E, BarcelóV,

and in vivo brain targeting of magnetized endothelial progenitor cells for neurorepair therapies. Nanomedicine: Nanotechnology, Biology and Medicine. 2014; 10:225-234.

[150] Yuan M, Wang Y, Qin Y-X. Promoting neuroregeneration by applying dynamic magnetic fields to a novel nanomedicine: Superparamagnetic iron oxide (SPIO) gold nanoparticles bounded with nerve growth factor (NGF). Nanomedicine.

2018; 14:1337-1347.

Morancho A, Levander L, Boada C, Laromaine A, Roig A, Montaner J, Rosell A. In vitro angiogenic performance

[141] Kawazoe N and Chen G. Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis.

Biomaterials. 2015; 54:226-236.

González-Mayorga A, Gutiérrez MC, Serrano MC. Immunomodulatory and angiogenic responses induced by graphene oxide scaffolds in chronic spinal hemisected rats. Biomaterials.

[143] Qian Y, Song J, Zhao X, et al. 3D fabrication with integration molding of a graphene oxide/polycaprolactone nanoscaffold for neurite regeneration and angiogenesis. Adv Sci. 2018;

[142] López-Dolado E,

2016; 99:72-81.

5:1700499.

[144] Xu ZX, Zhang LQ,

Wang CS, Chen RS, Li GS, Guo Y, Xu WH. Acellular spinal cord scaffold implantation promotes vascular remodeling with sustained delivery of VEGF in a rat spinal cord hemisection model. Current Neurovascular Research. 2017; 14:274-289.

[145] Wen Y, Yu S, Wu Y, et al*.* Spinal cord injury repair by implantation of structured hyaluronic acid scaffold with PLGA microspheres in the rat. Cell

Wang Y, Wang H, Xu Q. Angiogenic microspheres promote neural regeneration and motor function recovery after spinal cord injury in rats.

Scientific reports. 2016; 6:1-13.

[147] Jian W-H, Wang H-C, Kuan C-H, Chen M-H, Wu H-C, Sun J-S, Wang T-W. Glycosaminoglycan-based hybrid hydrogel encapsulated with

Tissue Res. 2016; 364:17-28.

[146] Yu S, Yao S, Wen Y,

**142**

### *Edited by Elisabeth Eppard*

In recent years, due to advancing technology and diagnostic and therapeutic techniques, medicine and health care have become more patient-oriented. This concept of personalized medicine or theranostics can be traced back to the beginnings of nuclear medicine when radioisotopes were uncovered as diagnostic and therapeutic tools. Nowadays, the field of theranostics is in flux, as new techniques and materials allow a growing range of applications beneficial for patients. This book examines new developments in theranostics and provides a comprehensive overview of the state of the art in this exciting discipline.

Published in London, UK © 2021 IntechOpen © SorinVidis / iStock

Theranostics - An Old Concept in New Clothing

Theranostics

An Old Concept in New Clothing

*Edited by Elisabeth Eppard*