**1. Introduction**

Structurally, Carbon nanotubes (CNTs) can be viewed as wrapped from graphene sheets. Single-walled carbon nanotubes (SWNTs) have one layer of graphene sheet, whereas, the multiwalled carbon nanotubes (MWNTs) contain multi layers of graphene sheets. The wellordered molecular structure brings CNTs many remarkable physical properties, such as, ex‐ cellent mechanic strength, ultrahigh surface area, high aspect ratio, distinct optical properties [1], and excellent electrical conductivity [2]. In last decade, CNTs are intensively explored for in-vitro and in-vivo delivery of therapeutics, which was inspired by an impor‐ tant finding that CNTs can penetrate cells by themselves without apparent cytotoxic effect to the cells [3]. The high aspect ratio makes CNTs outstanding from other types of round nanoparticles in that the needle-like CNTs allow loading large quantities of payloads along the longitude of tubes without affecting their cell penetration capability. With the adequate loading capacity, the CNTs can carry multifunctional therapeutics, including drugs, genes and targeting molecules, into one cell to exert multi-valence effects. In the other side, owing to the ultrahigh surface area along with the strong mechanical properties and electrically conductive nature, CNTs are excellent material for nanoscaffolds and three dimensional nanocomposites. In recent year, CNT-based devices have been successfully utilized in tissue engineering and stem cell based therapeutic applications, including myocardial therapy, bone formation, muscle and neuronal regeneration. Furthermore, owing to the distinct opti‐ cal properties of CNTs, such as, high absorption in the near-infrared (NIR) range, photolu‐ minescence, and strong Raman shift [4], CNTs are excellent agents for biology detection and imaging. Combined with high surface area of CNTs for attaching molecular recognition molecules, CNT-based, targeted nanodevices have been developed for selective imaging

© 2013 Shao et al.; licensee InTech. This is an open access article 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. © 2013 Shao et al.; licensee InTech. This is a paper 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.

and sensing. There are many areas where CNTs are extremely useful. Given the scope in this chapter, We describe strategies for preparation of CNTs for their use in medicine. Specifical‐ ly, we focus and highlight the important biomedical applications of CNTs in the field of drug delivery, gene delivery, stem cell therapy, thermal therapy, biological detection and imaging (figure 1). The methods for formulating CNT-based therapeutics to suit different routes of drug administration are also described. The limitations with emphasis on toxicity and over all future directions are discussed.

**2.1. Non-covalent modification of carbon nanotube surface**

ystyrene-block-polyacrylic acid.

out destruction of their *sp* <sup>2</sup>

The non-covalent modification approaches typically use amphiphilic molecules ranged from small molecules to polymers. The amphiphilic molecules associate with CNTs by either ad‐ sorbing onto or wrapping the CNTs [5]. The non-covalent modifications of CNTs are easy to perform. The process is only involved in sonication of CNTs with amphiphilic molecules in solvent at room temperature. Since it is a mild condition, CNTs molecular structure is not affected, and therefore their optical and electrical conductive properties are conserved.

**Figure 2.** Schematic representation of adsorption of amphiphilic molecules onto carbon nanotube surface by π*-*π stacking and other hydrophobic interactions. Abbreviations: PL-PEG, phospholipid-polyethylene glycol; PS-b-PAA, pol‐

Adsorption of amphiphilic molecules, such as surfactants, amphiphilic copolymers or oth‐ ers, onto CNT surfaces is one of the simplest and most effective way to disperse CNTs with‐

interact with the polar solvent molecules, whereas, the hydrophobic portions adsorb onto the nanotube surface [5, 6]. The dispersity depends strongly on the length of the hydropho‐ bic regions and the types of hydrophilic groups in the amphiphilic molecule. For example, surfactants with ionic hydrophilic head groups, such as sodiumdodecylsulfate (SDS) [7] or cetyltrimethyl ammonium bromide (CTAB) [8, 9], can stabilize a nanotube by electrostatic repulsion between micellar domains [7]. Nonionic surfactants, such as Triton X-100 [8], dis‐ perse CNTs mainly by forming a large solvation shell around a nanotube [8]. Figure 2 illus‐ trates the manor of adsorbing amphiphilic molecules onto CNT surfaces, in which, hydrophobic alkyl chains or aromatic rings lay flat on graphitic tube surfaces. For example, an synthetic biocompatible lipid-polymer conjugate, phospholipid-polyethylene glycol (PL-PEG) has been applied for surface modification of CNTs, which gives rise to a variety of bio‐ medical applications ranged from drug delivery, biomedical imaging, detection and biosensors [10]. The ionic surfactants, particularly those based on alkyl-substituted imidazo‐ lium cationic surfactants [11], can effectively disperse CNTs in organic or aqueous media by the counter anion [12, 13]. Polyaromatic derivatives carrying hydrophilic moieties can also effectively disperse CNTs in aqueous media by forming specific directional π*–*π stacking

hybridization [5]. The hydrophilic portions of surfactants

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287

**Figure 1.** Functionalized CNTs in major biomedical applications.

#### **2. Preparation of CNTs for use in medicine**

Raw CNTs, persisting metallic nature, are highly hydrophobic. Therefore, surface modifica‐ tion of CNTs, or CNT functionalization, so as to disperse them into aqueous solutions be‐ comes a key step for their biomedical applications. The CNT modification methods are involved in non-covalent and covalent strategies. The non-covalent modification utilizes the hydropho‐ bic nature of CNTs, especially, π*-*π interactions for coating of amphiphilic molecules. The covalent modification generates chemical bonds on carbon atoms on CNT surface via chemi‐ cal reactions followed by further conjugation of hydrophilic organic molecules or polymers rendering CNTs better solubility. These modifications not only offer CNTs water solubility, but also produce functional moieties that enable linking of therapeutic agents, such as genes , drugs, and recognition molecules for biomedical applications.

#### **2.1. Non-covalent modification of carbon nanotube surface**

and sensing. There are many areas where CNTs are extremely useful. Given the scope in this chapter, We describe strategies for preparation of CNTs for their use in medicine. Specifical‐ ly, we focus and highlight the important biomedical applications of CNTs in the field of drug delivery, gene delivery, stem cell therapy, thermal therapy, biological detection and imaging (figure 1). The methods for formulating CNT-based therapeutics to suit different routes of drug administration are also described. The limitations with emphasis on toxicity

Raw CNTs, persisting metallic nature, are highly hydrophobic. Therefore, surface modifica‐ tion of CNTs, or CNT functionalization, so as to disperse them into aqueous solutions be‐ comes a key step for their biomedical applications. The CNT modification methods are involved in non-covalent and covalent strategies. The non-covalent modification utilizes the hydropho‐ bic nature of CNTs, especially, π*-*π interactions for coating of amphiphilic molecules. The covalent modification generates chemical bonds on carbon atoms on CNT surface via chemi‐ cal reactions followed by further conjugation of hydrophilic organic molecules or polymers rendering CNTs better solubility. These modifications not only offer CNTs water solubility, but also produce functional moieties that enable linking of therapeutic agents, such as genes ,

and over all future directions are discussed.

286 Syntheses and Applications of Carbon Nanotubes and Their Composites

**Figure 1.** Functionalized CNTs in major biomedical applications.

**2. Preparation of CNTs for use in medicine**

drugs, and recognition molecules for biomedical applications.

The non-covalent modification approaches typically use amphiphilic molecules ranged from small molecules to polymers. The amphiphilic molecules associate with CNTs by either ad‐ sorbing onto or wrapping the CNTs [5]. The non-covalent modifications of CNTs are easy to perform. The process is only involved in sonication of CNTs with amphiphilic molecules in solvent at room temperature. Since it is a mild condition, CNTs molecular structure is not affected, and therefore their optical and electrical conductive properties are conserved.

**Figure 2.** Schematic representation of adsorption of amphiphilic molecules onto carbon nanotube surface by π*-*π stacking and other hydrophobic interactions. Abbreviations: PL-PEG, phospholipid-polyethylene glycol; PS-b-PAA, pol‐ ystyrene-block-polyacrylic acid.

Adsorption of amphiphilic molecules, such as surfactants, amphiphilic copolymers or oth‐ ers, onto CNT surfaces is one of the simplest and most effective way to disperse CNTs with‐ out destruction of their *sp* <sup>2</sup> hybridization [5]. The hydrophilic portions of surfactants interact with the polar solvent molecules, whereas, the hydrophobic portions adsorb onto the nanotube surface [5, 6]. The dispersity depends strongly on the length of the hydropho‐ bic regions and the types of hydrophilic groups in the amphiphilic molecule. For example, surfactants with ionic hydrophilic head groups, such as sodiumdodecylsulfate (SDS) [7] or cetyltrimethyl ammonium bromide (CTAB) [8, 9], can stabilize a nanotube by electrostatic repulsion between micellar domains [7]. Nonionic surfactants, such as Triton X-100 [8], dis‐ perse CNTs mainly by forming a large solvation shell around a nanotube [8]. Figure 2 illus‐ trates the manor of adsorbing amphiphilic molecules onto CNT surfaces, in which, hydrophobic alkyl chains or aromatic rings lay flat on graphitic tube surfaces. For example, an synthetic biocompatible lipid-polymer conjugate, phospholipid-polyethylene glycol (PL-PEG) has been applied for surface modification of CNTs, which gives rise to a variety of bio‐ medical applications ranged from drug delivery, biomedical imaging, detection and biosensors [10]. The ionic surfactants, particularly those based on alkyl-substituted imidazo‐ lium cationic surfactants [11], can effectively disperse CNTs in organic or aqueous media by the counter anion [12, 13]. Polyaromatic derivatives carrying hydrophilic moieties can also effectively disperse CNTs in aqueous media by forming specific directional π*–*π stacking with the graphitic surfaces of nanotubes [6, 14]. In this context, pyrene, a polyaromatic mole‐ cule, demonstrated a high affinity toward CNT surfaces [6]. Interactions of the polyaromatic‐ moitie of pyrene with CNTs are strong enough to be irreversible, and therefore, the pyrene derivatives are used to anchor proteins or biomolecules on nanotube surface [6, 14, 15]. Oth‐ er classes of polyaromatic molecules, such as substituted anthracenes, heterocyclic polyaro‐ maticporphyrins [16] and phthalocyanines [17], disperse CNTs via the same mechanism.

The polymers containing hydrophobic backbone and hydrophilic side groups, eg. poly[p- {2,5-bis(3-propoxysulfonic acid sodium salt)}phenylene] ethynylene (PPES), can effectively disperse CNTs in water, in which, the strong π*–* π interactions between CNTs and aromatic backbone of the polymers drive the wrapping of CNTs, and the water-soluble side groups impart solubility of CNTs in water [18]. DNA and siRNA can disperse CNTs by wrapping. DNA or siRNA are made of hydrophobic bases and alternative hydrophilic phosphates and riboses. Such structure facilitates CNT dispersion by the bases wrapping to CNTs and the hydrophilic sugar-phosphate groups extending to water phase [19].

**Figure 3.** Covalent modification of carbon nanotubesby a) Oxidation reaction of cap end of CNTs and b) further at‐

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In contrast to non-covalent surface modifications, which do not locally disrupt *sp* <sup>2</sup> hybridi‐

**Figure 4.** Sidewall covalent modification of carbon nanotubes a) a general scheme of 1,3-dipolar cycloaddition reac‐

Cancer is one of the most common causes of death worldwide. Chemotherapy in addition to the surgical removal of tumors is a conventional treatment for cancers. However, the effec‐

tion b) preparation of amino-functionalized CNTs by 1,3-dipolar cycloaddition.

**3. Carbon nanotube based therapeutics**

**3.1. Carbon nanotubes for chemotherapy drug delivery**


zation, or create defects, the covalent surface modifications disrupt CNT *sp* <sup>2</sup>

structures and therefore, could affect the electronic and optical performances [5].

taching hydrophilic molecules by amidation reactions.

#### **2.2. Covalent modification of CNT surface**

The covalent modification, namely the chemical modification of CNTs is an emerging area in materials science. Among the various strategies, the most common ones are:


Oxidation of CNTs is a purification method for raw CNTs. Oxidation of CNTs is carried out by reflexing raw CNTs in strong acidic media, e.g. HNO3/H2SO4. Under this condition, the end caps of the CNTs are opened, and carboxylic groups are formed at these ends caps and at some defect sites on nanotube sidewalls (Figure 3a) [20]. The carboxylic groups provide opportunities for further derivatization of the CNTs through esterification or amidation re‐ actions. For example, some organic molecules with amine groups can be directly condensed with the carboxylic groups present on the surface of the CNTs [6, 14]. Alternatively, the car‐ boxyl moieties can be activated with thionyl chloride and subsequent react with amine groups (Figure 3b) [6, 14]. These reactions are widely applied for conjugation of water-solu‐ ble organic molecules, hydrophilic polymers, nucleic acid (DNA or RNA), or peptides to the oxidized CNTs, which result in multifunctional CNTs [6, 14]. In most cases, the length of nanotubes is often shortened [20] ,but the electronic properties of such functionalized CNTs remain intact. Oxidation reaction only generates carboxyl groups on cap ends and defect sites on CNTs. To generate chemical bonds on sidewall and cap ends of CNTs, cycloaddition reactions are used [21](Figure 4). Cycloaddition reaction is a very powerful methodology, in which the 1,3-dipolar cycloaddition of azomethineylides can easily attach a large amount of pyrrolidine rings on sidewalls of nanotubes. Thus, the resulting functionalized CNTs are highly soluble in water [22]. In addition, pyrrolidine ring can be substituted with many functional groups for different applications.

**Figure 3.** Covalent modification of carbon nanotubesby a) Oxidation reaction of cap end of CNTs and b) further at‐ taching hydrophilic molecules by amidation reactions.

In contrast to non-covalent surface modifications, which do not locally disrupt *sp* <sup>2</sup> hybridi‐ zation, or create defects, the covalent surface modifications disrupt CNT *sp* <sup>2</sup> -conjugated structures and therefore, could affect the electronic and optical performances [5].

**Figure 4.** Sidewall covalent modification of carbon nanotubes a) a general scheme of 1,3-dipolar cycloaddition reac‐ tion b) preparation of amino-functionalized CNTs by 1,3-dipolar cycloaddition.

### **3. Carbon nanotube based therapeutics**

with the graphitic surfaces of nanotubes [6, 14]. In this context, pyrene, a polyaromatic mole‐ cule, demonstrated a high affinity toward CNT surfaces [6]. Interactions of the polyaromatic‐ moitie of pyrene with CNTs are strong enough to be irreversible, and therefore, the pyrene derivatives are used to anchor proteins or biomolecules on nanotube surface [6, 14, 15]. Oth‐ er classes of polyaromatic molecules, such as substituted anthracenes, heterocyclic polyaro‐ maticporphyrins [16] and phthalocyanines [17], disperse CNTs via the same mechanism.

The polymers containing hydrophobic backbone and hydrophilic side groups, eg. poly[p- {2,5-bis(3-propoxysulfonic acid sodium salt)}phenylene] ethynylene (PPES), can effectively disperse CNTs in water, in which, the strong π*–* π interactions between CNTs and aromatic backbone of the polymers drive the wrapping of CNTs, and the water-soluble side groups impart solubility of CNTs in water [18]. DNA and siRNA can disperse CNTs by wrapping. DNA or siRNA are made of hydrophobic bases and alternative hydrophilic phosphates and riboses. Such structure facilitates CNT dispersion by the bases wrapping to CNTs and the

The covalent modification, namely the chemical modification of CNTs is an emerging area

Oxidation of CNTs is a purification method for raw CNTs. Oxidation of CNTs is carried out by reflexing raw CNTs in strong acidic media, e.g. HNO3/H2SO4. Under this condition, the end caps of the CNTs are opened, and carboxylic groups are formed at these ends caps and at some defect sites on nanotube sidewalls (Figure 3a) [20]. The carboxylic groups provide opportunities for further derivatization of the CNTs through esterification or amidation re‐ actions. For example, some organic molecules with amine groups can be directly condensed with the carboxylic groups present on the surface of the CNTs [6, 14]. Alternatively, the car‐ boxyl moieties can be activated with thionyl chloride and subsequent react with amine groups (Figure 3b) [6, 14]. These reactions are widely applied for conjugation of water-solu‐ ble organic molecules, hydrophilic polymers, nucleic acid (DNA or RNA), or peptides to the oxidized CNTs, which result in multifunctional CNTs [6, 14]. In most cases, the length of nanotubes is often shortened [20] ,but the electronic properties of such functionalized CNTs remain intact. Oxidation reaction only generates carboxyl groups on cap ends and defect sites on CNTs. To generate chemical bonds on sidewall and cap ends of CNTs, cycloaddition reactions are used [21](Figure 4). Cycloaddition reaction is a very powerful methodology, in which the 1,3-dipolar cycloaddition of azomethineylides can easily attach a large amount of pyrrolidine rings on sidewalls of nanotubes. Thus, the resulting functionalized CNTs are highly soluble in water [22]. In addition, pyrrolidine ring can be substituted with many

**ii.** generation of functional groups on CNT sidewalls by cycloaddition reactions.

in materials science. Among the various strategies, the most common ones are:

hydrophilic sugar-phosphate groups extending to water phase [19].

**2.2. Covalent modification of CNT surface**

288 Syntheses and Applications of Carbon Nanotubes and Their Composites

functional groups for different applications.

**i.** esterification and amidation of oxidized CNTs,

#### **3.1. Carbon nanotubes for chemotherapy drug delivery**

Cancer is one of the most common causes of death worldwide. Chemotherapy in addition to the surgical removal of tumors is a conventional treatment for cancers. However, the effec‐ tiveness of chemotherapy drugs is often limited by the toxicity to other tissues in the body. This is because most chemotherapy drugs do not specifically kill cancer cells, they act to kill all cells undergoing fast division. Nanoparticles have been applied to drug delivery and showed improved drug efficiency and reduced off-target tissue toxicity due to accumulation in tumor tissues. Nanoparticles target tumor tissues by two mechanisms: passive targeting and active targeting. As fast growing tissues, tumors display enhanced vascular permeabili‐ ty due to high demand for nutrients and possible oxygen. The features of the leaky vascula‐ ture are employed for delivery of nanoparticle drugs since the size of nanoparticle allows them to accumulate in tumor tissues [23]. The phenomenon is termed as tumor-selective *en‐ hanced permeability and retention* (EPR) effect. More efficient tumor targeting can be achieved through active targeting approaches, in which, targeting molecules can recognize tumor bio‐ markers on cancer cell surface. The properties of CNTs are beneficial for cancer drug deliv‐ ery, firstly, like other nanoparticles, the size of functionalized CNTs is preferable for accumulation in tumor tissues; secondly, CNTs contain ultrahigh surface area of CNTs facil‐ itate loading of drugs and targeting molecules; thirdly, the hydrophobic benzene ring struc‐ ture of CNTs can be used for loading drugs that contain benzene ring structure, eg. doxorubicin (DOX), epirubicin (EPI), and daunorubicin (DAU).

been applied for construction of CNT-based, which will be discussed separate in the follow‐

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Supermolecular benzene ring structure of CNTs affords surprisingly high degree of aromat‐ ic molecules by π-π stacking. DOX, an important chemotherapeutic agent, has been effi‐ ciently loaded onto SWNTs-PL-PEG for tumor-targeted delivery [30, 31]. Binding to and release of drug molecules from nanotubes could be controlled by adjusting pH.The appro‐ priate diameter of nanotube for drug loading was used because the strength of π-π stacking of aromatic molecules was dependent on nanotube diameter. In-vivo study with SWNTs-PL-PEG/DOX showed significantly enhanced therapeutic efficacy in a murine breast cancer model [30]. With further attaching tumor targeting molecules, eg. folic acid (FA) or RGD, the targeted SWNTs-DOX could more effectively inhibit the growth of cancer cells in-vitro and in-vivo [31-34]. Similar physical absorption method was applied for drug DAU using SWNTs, in which, sgc8c Aptamer was used to target leukemia biomarker protein tyrosine kinase-7 [33]. It has been shown that Aptamer-SWNTs-DAU was able to selectively target leukemia cells. The release of DAU was pH-dependent.Other hydrophobic drug molecules, such as paclitaxel (PTX), docetaxel (DTX), can also be absorbed on CNTs surface for delivery [32, 35], however, their loading efficiency and stability were much lower due to their compa‐

*Targeted delivery of chemotherapy drugs by covalently linked to functionalized carbon nanotubes*

Non-aromatic small molecule drugs can be chemically conjugated to CNTs for delivery. However, the drugmolecules have to be released from the CNTs to take effect, so the linkag‐ es between the drugs and CNTs have to be cleavable. Preferably, the active drugs are re‐ leased inside of the target cells to reduce toxic effect to the neighbouring healthy cells. The common linkers that are used for drug delivery include ester, peptide, and disulfide bonds. These linkers can be cleaved by the 7enzymes present in the routes of delivery. Specifically designed linkers allow controlled release of drug into desired sites. For example, drug cis‐ platin has been conjugated directly to oxidized SWNTs via a peptide linker [36]. This specif‐ ic peptide linkage has been shown tobe selectively cleaved by proteases overexpressed in tumor cells. Further conjugation of epidermal growth factor (EGF), a growth factor that se‐ lective binding to EGF receptor overexpressed on cancer cells, to SWNTs-cisplatin led to more efficient tumor inhibition compared to both free cisplatin and non-targeted SWNTscisplatin [36]. Alternative to conjugation of drugs to CNTs directly, drugs can also be conju‐ gated to the molecules, eg. polymers, that are used to disperse CNTs. The end functional groups in the polymers are used for drug linkage. This method is very useful for delivery of bulky, hydrophobic drug molecules. In one example, SWNTs was dispersed using a biocom‐ patible polymer PL-PEG-NH2[10] and drug PTX was conjugated to SWNT-PL-PEG-NH2 via via ester bonding for delivery [37]. PTX is one of the most important drugs for metastatic breast cancer. However, currently available formulations for PTX have to be infused intrave‐ nously over long periods of time due to the side effects. In addition, due to poor water solu‐ bility of the drug, necessity of use organic solvent, such as Cremophor in clinical formulation Taxol® causes sever side effects and hypersensitivity reactions [38, 39]. Conju‐

*Targeted delivery of chemotherapy drugs by physically absorbed on carbon nanotubes*

ing sub-sections.

ratively bulky structure.

#### *Preparation of tumor-targeted devices using carbon nanotubes*

A range of tumor targeting molecules has been discovered, including tumor specific anti‐ bodies, peptides, and others. Antibodies have been developed to specifically binding to bio‐ markers on cancer cell surface, eg, Trastuzumab recognizes Human Epidermal Growth Factor Receptor 2 (HER-2) positive cancer cells[24] and anti-CD20 for CD20 biomarker on B cell lymphoma [25]. These antibodies have therapeutic effects on their own, and can also serve as tumor targeting probes. Alpha V beta 3 (αvβ3) integrin is a heterodimerictrans‐ membrane glycoprotein found on a variety of tumor cells, including osteosarcomas, neuro‐ blastomas, glioblastomas, melanomas, lung, breast, prostate cancers. αvβ3) integrin is a wellrecognized target for cancers. The amino acid sequence of Arg-Gly-Asp (RGD) is identi‐ fied to be responsible for tight binding to αvβ3 integrin, which leads to the development of short tumor targeting peptide RGD [26]. Similar to RGD, another type of peptide contains Asn-Gly-Asp (NGR) triad that binds to the endothelium cells on neoangiogenic vessels. NGR-tagged delivery systems have been developed to deliver cytokines, nanoparticles, and imaging agents to tumor blood vessels [27]. Folic acid, a small molecule vitamin, binds to folate receptor overexpressed in a variety of cancer cells, including breast, colon, renal and lung tumors[28]. As described in section 2 of this chapter, a variety of chemical and physical methods have been developed for functionalization of CNTs. The above listed tumor target‐ ing molecules are mostly proteins or peptides, which contains sulfhydryl groups that can be easily conjugated to amino-functionalized CNTs [14] using heterbifunctional linker mole‐ cules that contain NHS ester on one end and Maleimide on the other end [29]. These conju‐ gation reactions are usually carried out under mild conditions [10]. Thus, the molecular structure of CNTs is not disturbed, and therefore, the optical properties are preserved. The CNT-based targeted devices developed by this methods are good for potential tumor detec‐ tion and imaging applications. To date, all above-mentioned tumor-targeting strategies have been applied for construction of CNT-based, which will be discussed separate in the follow‐ ing sub-sections.

#### *Targeted delivery of chemotherapy drugs by physically absorbed on carbon nanotubes*

tiveness of chemotherapy drugs is often limited by the toxicity to other tissues in the body. This is because most chemotherapy drugs do not specifically kill cancer cells, they act to kill all cells undergoing fast division. Nanoparticles have been applied to drug delivery and showed improved drug efficiency and reduced off-target tissue toxicity due to accumulation in tumor tissues. Nanoparticles target tumor tissues by two mechanisms: passive targeting and active targeting. As fast growing tissues, tumors display enhanced vascular permeabili‐ ty due to high demand for nutrients and possible oxygen. The features of the leaky vascula‐ ture are employed for delivery of nanoparticle drugs since the size of nanoparticle allows them to accumulate in tumor tissues [23]. The phenomenon is termed as tumor-selective *en‐ hanced permeability and retention* (EPR) effect. More efficient tumor targeting can be achieved through active targeting approaches, in which, targeting molecules can recognize tumor bio‐ markers on cancer cell surface. The properties of CNTs are beneficial for cancer drug deliv‐ ery, firstly, like other nanoparticles, the size of functionalized CNTs is preferable for accumulation in tumor tissues; secondly, CNTs contain ultrahigh surface area of CNTs facil‐ itate loading of drugs and targeting molecules; thirdly, the hydrophobic benzene ring struc‐ ture of CNTs can be used for loading drugs that contain benzene ring structure, eg.

A range of tumor targeting molecules has been discovered, including tumor specific anti‐ bodies, peptides, and others. Antibodies have been developed to specifically binding to bio‐ markers on cancer cell surface, eg, Trastuzumab recognizes Human Epidermal Growth Factor Receptor 2 (HER-2) positive cancer cells[24] and anti-CD20 for CD20 biomarker on B cell lymphoma [25]. These antibodies have therapeutic effects on their own, and can also serve as tumor targeting probes. Alpha V beta 3 (αvβ3) integrin is a heterodimerictrans‐ membrane glycoprotein found on a variety of tumor cells, including osteosarcomas, neuro‐ blastomas, glioblastomas, melanomas, lung, breast, prostate cancers. αvβ3) integrin is a wellrecognized target for cancers. The amino acid sequence of Arg-Gly-Asp (RGD) is identi‐ fied to be responsible for tight binding to αvβ3 integrin, which leads to the development of short tumor targeting peptide RGD [26]. Similar to RGD, another type of peptide contains Asn-Gly-Asp (NGR) triad that binds to the endothelium cells on neoangiogenic vessels. NGR-tagged delivery systems have been developed to deliver cytokines, nanoparticles, and imaging agents to tumor blood vessels [27]. Folic acid, a small molecule vitamin, binds to folate receptor overexpressed in a variety of cancer cells, including breast, colon, renal and lung tumors[28]. As described in section 2 of this chapter, a variety of chemical and physical methods have been developed for functionalization of CNTs. The above listed tumor target‐ ing molecules are mostly proteins or peptides, which contains sulfhydryl groups that can be easily conjugated to amino-functionalized CNTs [14] using heterbifunctional linker mole‐ cules that contain NHS ester on one end and Maleimide on the other end [29]. These conju‐ gation reactions are usually carried out under mild conditions [10]. Thus, the molecular structure of CNTs is not disturbed, and therefore, the optical properties are preserved. The CNT-based targeted devices developed by this methods are good for potential tumor detec‐ tion and imaging applications. To date, all above-mentioned tumor-targeting strategies have

doxorubicin (DOX), epirubicin (EPI), and daunorubicin (DAU).

*Preparation of tumor-targeted devices using carbon nanotubes*

290 Syntheses and Applications of Carbon Nanotubes and Their Composites

Supermolecular benzene ring structure of CNTs affords surprisingly high degree of aromat‐ ic molecules by π-π stacking. DOX, an important chemotherapeutic agent, has been effi‐ ciently loaded onto SWNTs-PL-PEG for tumor-targeted delivery [30, 31]. Binding to and release of drug molecules from nanotubes could be controlled by adjusting pH.The appro‐ priate diameter of nanotube for drug loading was used because the strength of π-π stacking of aromatic molecules was dependent on nanotube diameter. In-vivo study with SWNTs-PL-PEG/DOX showed significantly enhanced therapeutic efficacy in a murine breast cancer model [30]. With further attaching tumor targeting molecules, eg. folic acid (FA) or RGD, the targeted SWNTs-DOX could more effectively inhibit the growth of cancer cells in-vitro and in-vivo [31-34]. Similar physical absorption method was applied for drug DAU using SWNTs, in which, sgc8c Aptamer was used to target leukemia biomarker protein tyrosine kinase-7 [33]. It has been shown that Aptamer-SWNTs-DAU was able to selectively target leukemia cells. The release of DAU was pH-dependent.Other hydrophobic drug molecules, such as paclitaxel (PTX), docetaxel (DTX), can also be absorbed on CNTs surface for delivery [32, 35], however, their loading efficiency and stability were much lower due to their compa‐ ratively bulky structure.

#### *Targeted delivery of chemotherapy drugs by covalently linked to functionalized carbon nanotubes*

Non-aromatic small molecule drugs can be chemically conjugated to CNTs for delivery. However, the drugmolecules have to be released from the CNTs to take effect, so the linkag‐ es between the drugs and CNTs have to be cleavable. Preferably, the active drugs are re‐ leased inside of the target cells to reduce toxic effect to the neighbouring healthy cells. The common linkers that are used for drug delivery include ester, peptide, and disulfide bonds. These linkers can be cleaved by the 7enzymes present in the routes of delivery. Specifically designed linkers allow controlled release of drug into desired sites. For example, drug cis‐ platin has been conjugated directly to oxidized SWNTs via a peptide linker [36]. This specif‐ ic peptide linkage has been shown tobe selectively cleaved by proteases overexpressed in tumor cells. Further conjugation of epidermal growth factor (EGF), a growth factor that se‐ lective binding to EGF receptor overexpressed on cancer cells, to SWNTs-cisplatin led to more efficient tumor inhibition compared to both free cisplatin and non-targeted SWNTscisplatin [36]. Alternative to conjugation of drugs to CNTs directly, drugs can also be conju‐ gated to the molecules, eg. polymers, that are used to disperse CNTs. The end functional groups in the polymers are used for drug linkage. This method is very useful for delivery of bulky, hydrophobic drug molecules. In one example, SWNTs was dispersed using a biocom‐ patible polymer PL-PEG-NH2[10] and drug PTX was conjugated to SWNT-PL-PEG-NH2 via via ester bonding for delivery [37]. PTX is one of the most important drugs for metastatic breast cancer. However, currently available formulations for PTX have to be infused intrave‐ nously over long periods of time due to the side effects. In addition, due to poor water solu‐ bility of the drug, necessity of use organic solvent, such as Cremophor in clinical formulation Taxol® causes sever side effects and hypersensitivity reactions [38, 39]. Conju‐ gation of PTX to SWNTs-PL-PEG-NH2 enable removing of solvent in delivery. Indeed, the SWNTs-PL-PEG-PTX displayed increased tumor inhibition effect and reduced side effects in a murine breast cancer model compared with Taxol® formulation [37].

48]. Using this deliver vehicle, expression of test plasmid pCMV-βgal was examined in-vi‐ tro.Result showed that the transfection efficiency of CNTs carrier was 5-10 times higher than naked DNA; but, much lower than that of liposome [47]. It has been shown that charge ratio (ammonium groups on CNTs *vs* phosphate groups of the DNA backbone) is a determination factor for gene expression [48]. In contrast to DNA delivery, the same CNTs carrier for deliv‐ ery of cyclin A2 siRNA demonstrated pronounced silencing effect in-vitro[51]. Surprisingly, In-vivo delivery of SOCS1 significantly inhibited SOCS1 expression and retarded the tumor growth in murine B16 tumor model [52]. The studies with PEI functionalized CNTs also showed very positive results. PEI is an efficient gene delivery reagent by its own, however, high amount of PEI is toxic to cells. The siRNA delivery by PEI-grafted MWNTs showed im‐ proved gene expression to the equivalent amounts of PEI polymer alone but with reduced

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*Gene delivery by covalently conjugation to carbon nanotubes via cleavable chemical bonds*

system in hard-to-transfect human T cells and primary cells lines [54].

*Gene delivery by wrapping directly on carbon nanotubes*

**3.3. Carbon nanotubes for stem cell related therapies**

*Carbon nanotubes for stem cell based heart therapy*

Alternatively, genes can be conjugated to amphiphilic polymers that are used for non-cova‐ lent CNT functionalization [10, 54,55]. Incorporation of cleavable chemical bonds facilitates releasing of DNA or siRNA cargos from CNTs in a controlled manner [54]. Thiol-modified DNA or siRNA were covalently conjugated to amino group of SWNT-PL-PEG- NH2via cleav‐ able disulfide bond [55]. The genes were released by the cleavage of disulfide bonds by thiol digesting enzymes upon cellular internalization of CNT-PL-PEG-siRNA. The CNT-mediat‐ ed siRNA delivery showed better gene transfection efficiency than liposome-based delivery

Nucleic acids, DNA or siRNA, contains alternative amphiphilic motifs, which can be used to disperse CNTs in water. The nucleic acids form helical wrapping around the CNTs with the bases binding to the hydrophobic CNTs and the hydrophilic sugar-phosphate groups ex‐ tending to the water phase [19]. In this way, DNA or siRNA serves both CNT dispersing agent and the cargo. It has been shown that the siRNA functionalized SWNTs readily enter cells and exerts its biological activity in cell culture [19]. Studies with intratumoralinjection

There has been an increasing trend in attempts to design and develop different CNT based tools and devices for tissue engineering and stem cell therapy applications. In particular, CNT impregnated nanoscaffolds have shown multiple advantages over currently available scaffolds. This includes its strong mechanical properties, resemblance of structure with col‐ lagen fibrils and extracellular matrix and electrically conductive nature. These attributes of the CNT based scaffolds and three dimensional nanocomposites have led to their diverse therapeutic applications in the field of myocardial therapy, bone formation, muscle and neu‐ ronal regeneration. These applications are mainly based on one principle and that is to mod‐ ulate the stem cell growth and differentiation in a more controlled and desirable manner.

of siRNA functionalized SWNTs showed significantly inhibition effect in-vivo [56].

cytotoxicity [46, 53].


**Table 1.** . Abbreviations: SWNTs, single walled carbon nanotubes; MWNTs, multiwalled carbon nanotubes, EGF, epidermal growth factor; RGD, peptide with arginine-glycine-aspartate sequence; NGR, peptide with asparagineglycine - arginine sequence; Sgc8c, oligonucleotide sequence.

#### **3.2. Carbon nanotubes for gene delivery**

Gene therapy is an important treatment for cancer and other genetic diseases. However, the effects of gene therapy are limited by the efficiencies of transfection and system delivery. Since DNA and siRNA are macromolecules, they cannot pass through cell membrane by themselves, carriers are needed to take them inside of cells to take effects. Structurally, both DNA and siRNA contain anionic phosphodiester backbone that and be complexed with cati‐ onic reagents, such as cationic lipids and polymers, etc. For system delivery, the DNA or siRNA can be loaded into cationic nanoparticles made from cationic lipids or polymers [43, 44]. The nanoparticles could protect them from nucleases degradation. Since CNTs are able to penetrate cells [3], they are investigated for gene delivery. Typically, two methods are used for loading nucleic acids to CNTs:


#### *Gene delivery using cationic molecule functionalized carbon nanotubes via electrostatic interactions*

As discussed early, cationic molecules, such as, ammonium-containing molecules and poly‐ ethylene imine (PEI), can be covalently linked to chemically modified CNTs by oxidation or 1,3-cycloadditions reactions[47-50]. In one application, DNA was loaded into CNTs conju‐ gated with ammonium-terminated oligoethylene glycol(CNTs-OEG-NH3 <sup>+</sup> ) for delivery [47, 48]. Using this deliver vehicle, expression of test plasmid pCMV-βgal was examined in-vi‐ tro.Result showed that the transfection efficiency of CNTs carrier was 5-10 times higher than naked DNA; but, much lower than that of liposome [47]. It has been shown that charge ratio (ammonium groups on CNTs *vs* phosphate groups of the DNA backbone) is a determination factor for gene expression [48]. In contrast to DNA delivery, the same CNTs carrier for deliv‐ ery of cyclin A2 siRNA demonstrated pronounced silencing effect in-vitro[51]. Surprisingly, In-vivo delivery of SOCS1 significantly inhibited SOCS1 expression and retarded the tumor growth in murine B16 tumor model [52]. The studies with PEI functionalized CNTs also showed very positive results. PEI is an efficient gene delivery reagent by its own, however, high amount of PEI is toxic to cells. The siRNA delivery by PEI-grafted MWNTs showed im‐ proved gene expression to the equivalent amounts of PEI polymer alone but with reduced cytotoxicity [46, 53].

#### *Gene delivery by covalently conjugation to carbon nanotubes via cleavable chemical bonds*

Alternatively, genes can be conjugated to amphiphilic polymers that are used for non-cova‐ lent CNT functionalization [10, 54,55]. Incorporation of cleavable chemical bonds facilitates releasing of DNA or siRNA cargos from CNTs in a controlled manner [54]. Thiol-modified DNA or siRNA were covalently conjugated to amino group of SWNT-PL-PEG- NH2via cleav‐ able disulfide bond [55]. The genes were released by the cleavage of disulfide bonds by thiol digesting enzymes upon cellular internalization of CNT-PL-PEG-siRNA. The CNT-mediat‐ ed siRNA delivery showed better gene transfection efficiency than liposome-based delivery system in hard-to-transfect human T cells and primary cells lines [54].

#### *Gene delivery by wrapping directly on carbon nanotubes*

gation of PTX to SWNTs-PL-PEG-NH2 enable removing of solvent in delivery. Indeed, the SWNTs-PL-PEG-PTX displayed increased tumor inhibition effect and reduced side effects in

**Drugs Targeting Moieties Cancer Biomarkers Type of CNTs References** Cisplatin EGF EGF Receptor SWNTs [36] ] Daunorubicin Sgc8c Aptamer Tyrosine Kinase-7 SWNTs [33] Docetaxel NGR Endothelial Cells SWNTs [32] Doxorubicin Folate /Magnetic Folate Receptor MWNTs [34] Doxorubicin RGD Integrin αvβ3 SWNTs [30] Doxorubicin Folate Folate Receptor SWNTs [31, 40] Gemcitabine Magnetic / MWNTs [41] Platinum (IV) Folate Folate Receptor SWNTs [42]

**Table 1.** . Abbreviations: SWNTs, single walled carbon nanotubes; MWNTs, multiwalled carbon nanotubes, EGF,

Gene therapy is an important treatment for cancer and other genetic diseases. However, the effects of gene therapy are limited by the efficiencies of transfection and system delivery. Since DNA and siRNA are macromolecules, they cannot pass through cell membrane by themselves, carriers are needed to take them inside of cells to take effects. Structurally, both DNA and siRNA contain anionic phosphodiester backbone that and be complexed with cati‐ onic reagents, such as cationic lipids and polymers, etc. For system delivery, the DNA or siRNA can be loaded into cationic nanoparticles made from cationic lipids or polymers [43, 44]. The nanoparticles could protect them from nucleases degradation. Since CNTs are able to penetrate cells [3], they are investigated for gene delivery. Typically, two methods are

**i.** electrostatic association with cationic molecule functionalized CNTs [45, 46];

**iii.** DNA or siRNA are directly wrap to raw or oxidized CNTs.

gated with ammonium-terminated oligoethylene glycol(CNTs-OEG-NH3 <sup>+</sup>

**ii.** chemical conjugation of nucleic acids to functionalized CNTs via cleavable chemi‐

*Gene delivery using cationic molecule functionalized carbon nanotubes via electrostatic interactions*

As discussed early, cationic molecules, such as, ammonium-containing molecules and poly‐ ethylene imine (PEI), can be covalently linked to chemically modified CNTs by oxidation or 1,3-cycloadditions reactions[47-50]. In one application, DNA was loaded into CNTs conju‐

) for delivery [47,

epidermal growth factor; RGD, peptide with arginine-glycine-aspartate sequence; NGR, peptide with

asparagineglycine - arginine sequence; Sgc8c, oligonucleotide sequence.

**3.2. Carbon nanotubes for gene delivery**

used for loading nucleic acids to CNTs:

cal bonds [47];

a murine breast cancer model compared with Taxol® formulation [37].

292 Syntheses and Applications of Carbon Nanotubes and Their Composites

Nucleic acids, DNA or siRNA, contains alternative amphiphilic motifs, which can be used to disperse CNTs in water. The nucleic acids form helical wrapping around the CNTs with the bases binding to the hydrophobic CNTs and the hydrophilic sugar-phosphate groups ex‐ tending to the water phase [19]. In this way, DNA or siRNA serves both CNT dispersing agent and the cargo. It has been shown that the siRNA functionalized SWNTs readily enter cells and exerts its biological activity in cell culture [19]. Studies with intratumoralinjection of siRNA functionalized SWNTs showed significantly inhibition effect in-vivo [56].

#### **3.3. Carbon nanotubes for stem cell related therapies**

There has been an increasing trend in attempts to design and develop different CNT based tools and devices for tissue engineering and stem cell therapy applications. In particular, CNT impregnated nanoscaffolds have shown multiple advantages over currently available scaffolds. This includes its strong mechanical properties, resemblance of structure with col‐ lagen fibrils and extracellular matrix and electrically conductive nature. These attributes of the CNT based scaffolds and three dimensional nanocomposites have led to their diverse therapeutic applications in the field of myocardial therapy, bone formation, muscle and neu‐ ronal regeneration. These applications are mainly based on one principle and that is to mod‐ ulate the stem cell growth and differentiation in a more controlled and desirable manner.

*Carbon nanotubes for stem cell based heart therapy*

Over the past two decades there has been significant advancement in stem cell therapy to repair and replace damaged tissues, such as heart muscle[57]. This is because of their ability to divide and differentiate into diverse specialized cell types. Recently, there has been in‐ creasing body of evidence indicating that the extracellular matrix plays a critical role in stem cell viability, proliferation and differentiation [58, 59]. Hence, designing a microenvironment prepared from polymeric scaffolds which imitate the physical characteristics of natural bio‐ matrix has been the central strategy in tissue engineering. The emergence of nanomaterials such as nanotubes provide opportunities to design such biocompatible scaffolds for hosting and directing stem cell differentiation [60].

Surface functionalizing the nanotube surface with bone morphogenetic protein-2 (BMP-2) further accelerates chondrogenic and osteogenic differentiation of MSCs [65, 66]. This stimu‐ lation is a combined effect of the surface nanoscale geometry of the substrate nanostructures and their BMP-2 coating efficiency. In a similar kind of study, the system also exhibited higher cell proliferation rate, apart from enhanced differentiation [66]. Nanotubes can also be used for extended drug release as has been demonstrated by Hu et al, where drug loaded nanotubes, in combination with multilayers of gelatin and chitosan, have been shown as a new way to use nanotubes as reservoir for storing drugs [67]. The system effectively pro‐ moted osteoblastic differentiation of MSCs. Further studies in this direction can be beneficial

Carbon Nanotubes for Use in Medicine: Potentials and Limitations

http://dx.doi.org/10.5772/51785

295

The unique abilities of human embryonic stem cells (hESCs), such as their self-renewal and potency, hold great promise in the field of regenerative medicine and stem cell based ther‐ apy. The derivation of neuronal lineages from hESCs holds promise to treat neurological pathologies of the central and peripheral nervous system such as Parkinson's disease, spi‐ nal cord injury, multiple sclerosis and glaucoma [68, 69]. CNT based substrates have been shown to promote neuronal differentiation [70]. It has also been proposed that neurons grown on a CNT meshwork displayed better signal transmission, due to tight contacts between the CNTs and neural membranes conducible to electrical shortcuts [71]. It was demonstrated that the MSCs and the neurosphere of cortex-derived neural stem cells (NSCs) can grow on the CNT array and both MSCs and NSCs interacted with the aligned CNTs. The results suggest that CNTs assist in the proliferation of MSCs and aid differentiation of cortex-derived NSCs [72]. However, due to the harsh external environment in the host body and lack of supportive substrates during transplantation, much of the transplanted cells lose its viability resulting in reduced therapeutic efficacy [73]. It has been reported that two dimensional thin film scaffolds, composed of biocompatible poly(acrylic acid) polymer graft‐ ed carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells in‐ to neuron cells while maintaining the viability of transplanted cells [74]. Even multiwalled carbon nanotube (MWNT) sheets showed to significantly enhance neural differentiation of hMSCs grown on the CNT sheets. Axon outgrowth was also controlled using nanoscale patterning of CNTs [75]. Recently, silk-CNT-based nanocomposite scaffolds are shown to protect and promote neuronal differentiation of hESCs [76]. Silks are natural polymers (pro‐ tein) that have been widely used as biomaterials for many years. Fibroin, comprising the major portion of the silk protein fibre, consists of 90% of amino acids including glycine, alanine, and serine. Due to its strong mechanical and flexible nature in thin film form, biocompatibility, and *in-vivo*bioresorbable properties, fibroin protein has been used as the building block for scaffolds. As confirmed by scanning electron microscope (Figure5 A-C), similar results were obtained with the developed silk-CNT scaffold where cells grown on the silk substrate exhibited denser complex three-dimensional axonal bundle networks as well as better spatial density distribution of the networks compared to other scaffolds. Over‐ all, the silk-CNT nanocomposite provided an efficient three-dimensional supporting ma‐ trix for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for patients with neurological disorder. In-vitro analysis showed that β-

in order to develop potential bone implants for improved bone osteointegration.

*Carbon nanotubes for stem cell based neuronal regeneration*

Preliminary studies demonstrate that neonatal rat ventricular myocytes cultured on sub‐ strates of multiwall carbon nanotubes can interact with the nanofibres by forming tight con‐ tacts and show significantly improved mitotic and chemotactic effects [61]. Moreover, such mode of culture also altered the electrophysiological properties of cardiomyocytes, indicat‐ ing that CNts are able to promote cardiomyocyte maturation. Further investigations with a nanocomposite of PLGA:CNF show that cardiomyocyte density increases with greater amounts of CNF in PLGA [62]. The study also showed similar trends with neurons. The im‐ mense potential of this technology for myocardial therapy roots from the fact that this car‐ diac patch can not only promote myocardial cells, but also induce the nerve cell growth that help the cardiac cells to contract. In addition, it also supports endothelial cells that make the inner lining of the blood vessels supplying oxygen to the heart.

#### *Carbon nanotubes for stem cell based bone regeneration*

In order to direct stem cell differentiation towards bone regeneration, there has been in‐ creasing interest by the researchers to explore topographical features of the cell culture sub‐ strate. Physical factors, such as rigidity of the extracellular environment, can influence stem cell growth and differentiation. Such differentiation of human stem cells can be detected by altering the size of the nanotubes on which the cells are grown [63]. It has been reported that 70- to 100-nm diameter nanotubes can initiate rapid stem cell elongations, which induce cy‐ toskeletal stress and selective differentiation into osteoblast-like cells, offering a promising route for quicker and better recovery, for example, for patients who undergo orthopedic sur‐ gery. The group also showed that the differentiated stem cells express osteopontin and os‐ teocalcin, the two important osteogenetic protein markers.

Moreover CNTs are promising materials for nanaoscaffold and implantation purposes due to the fact that CNTs are conductive, have excellent mechanical properties and their nano‐ structured dimensions mimic the 3D structure of proteins found in extracellular matrices. Their dimensions resembles closely with that of the triple helix of collagen fibrils which can promote for nucleation and growth of hydroxyapatite, the major inorganic component of bone. A newly developed nanocomposite scaffold of CNFs/CNTs has been shown to influ‐ ence the cell behaviour [64]. In-vitro study demonstrated that, smaller dimension CNFs dis‐ persed in polycarbonate urethane promoted osteoblast adhesion but did not promote the adhesion of fibroblasts, chondrocytes, and smooth muscle cells. But the mechanisms that guide such cell functions are yet to be understood.

Surface functionalizing the nanotube surface with bone morphogenetic protein-2 (BMP-2) further accelerates chondrogenic and osteogenic differentiation of MSCs [65, 66]. This stimu‐ lation is a combined effect of the surface nanoscale geometry of the substrate nanostructures and their BMP-2 coating efficiency. In a similar kind of study, the system also exhibited higher cell proliferation rate, apart from enhanced differentiation [66]. Nanotubes can also be used for extended drug release as has been demonstrated by Hu et al, where drug loaded nanotubes, in combination with multilayers of gelatin and chitosan, have been shown as a new way to use nanotubes as reservoir for storing drugs [67]. The system effectively pro‐ moted osteoblastic differentiation of MSCs. Further studies in this direction can be beneficial in order to develop potential bone implants for improved bone osteointegration.

#### *Carbon nanotubes for stem cell based neuronal regeneration*

Over the past two decades there has been significant advancement in stem cell therapy to repair and replace damaged tissues, such as heart muscle[57]. This is because of their ability to divide and differentiate into diverse specialized cell types. Recently, there has been in‐ creasing body of evidence indicating that the extracellular matrix plays a critical role in stem cell viability, proliferation and differentiation [58, 59]. Hence, designing a microenvironment prepared from polymeric scaffolds which imitate the physical characteristics of natural bio‐ matrix has been the central strategy in tissue engineering. The emergence of nanomaterials such as nanotubes provide opportunities to design such biocompatible scaffolds for hosting

Preliminary studies demonstrate that neonatal rat ventricular myocytes cultured on sub‐ strates of multiwall carbon nanotubes can interact with the nanofibres by forming tight con‐ tacts and show significantly improved mitotic and chemotactic effects [61]. Moreover, such mode of culture also altered the electrophysiological properties of cardiomyocytes, indicat‐ ing that CNts are able to promote cardiomyocyte maturation. Further investigations with a nanocomposite of PLGA:CNF show that cardiomyocyte density increases with greater amounts of CNF in PLGA [62]. The study also showed similar trends with neurons. The im‐ mense potential of this technology for myocardial therapy roots from the fact that this car‐ diac patch can not only promote myocardial cells, but also induce the nerve cell growth that help the cardiac cells to contract. In addition, it also supports endothelial cells that make the

In order to direct stem cell differentiation towards bone regeneration, there has been in‐ creasing interest by the researchers to explore topographical features of the cell culture sub‐ strate. Physical factors, such as rigidity of the extracellular environment, can influence stem cell growth and differentiation. Such differentiation of human stem cells can be detected by altering the size of the nanotubes on which the cells are grown [63]. It has been reported that 70- to 100-nm diameter nanotubes can initiate rapid stem cell elongations, which induce cy‐ toskeletal stress and selective differentiation into osteoblast-like cells, offering a promising route for quicker and better recovery, for example, for patients who undergo orthopedic sur‐ gery. The group also showed that the differentiated stem cells express osteopontin and os‐

Moreover CNTs are promising materials for nanaoscaffold and implantation purposes due to the fact that CNTs are conductive, have excellent mechanical properties and their nano‐ structured dimensions mimic the 3D structure of proteins found in extracellular matrices. Their dimensions resembles closely with that of the triple helix of collagen fibrils which can promote for nucleation and growth of hydroxyapatite, the major inorganic component of bone. A newly developed nanocomposite scaffold of CNFs/CNTs has been shown to influ‐ ence the cell behaviour [64]. In-vitro study demonstrated that, smaller dimension CNFs dis‐ persed in polycarbonate urethane promoted osteoblast adhesion but did not promote the adhesion of fibroblasts, chondrocytes, and smooth muscle cells. But the mechanisms that

and directing stem cell differentiation [60].

294 Syntheses and Applications of Carbon Nanotubes and Their Composites

inner lining of the blood vessels supplying oxygen to the heart.

teocalcin, the two important osteogenetic protein markers.

guide such cell functions are yet to be understood.

*Carbon nanotubes for stem cell based bone regeneration*

The unique abilities of human embryonic stem cells (hESCs), such as their self-renewal and potency, hold great promise in the field of regenerative medicine and stem cell based ther‐ apy. The derivation of neuronal lineages from hESCs holds promise to treat neurological pathologies of the central and peripheral nervous system such as Parkinson's disease, spi‐ nal cord injury, multiple sclerosis and glaucoma [68, 69]. CNT based substrates have been shown to promote neuronal differentiation [70]. It has also been proposed that neurons grown on a CNT meshwork displayed better signal transmission, due to tight contacts between the CNTs and neural membranes conducible to electrical shortcuts [71]. It was demonstrated that the MSCs and the neurosphere of cortex-derived neural stem cells (NSCs) can grow on the CNT array and both MSCs and NSCs interacted with the aligned CNTs. The results suggest that CNTs assist in the proliferation of MSCs and aid differentiation of cortex-derived NSCs [72]. However, due to the harsh external environment in the host body and lack of supportive substrates during transplantation, much of the transplanted cells lose its viability resulting in reduced therapeutic efficacy [73]. It has been reported that two dimensional thin film scaffolds, composed of biocompatible poly(acrylic acid) polymer graft‐ ed carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells in‐ to neuron cells while maintaining the viability of transplanted cells [74]. Even multiwalled carbon nanotube (MWNT) sheets showed to significantly enhance neural differentiation of hMSCs grown on the CNT sheets. Axon outgrowth was also controlled using nanoscale patterning of CNTs [75]. Recently, silk-CNT-based nanocomposite scaffolds are shown to protect and promote neuronal differentiation of hESCs [76]. Silks are natural polymers (pro‐ tein) that have been widely used as biomaterials for many years. Fibroin, comprising the major portion of the silk protein fibre, consists of 90% of amino acids including glycine, alanine, and serine. Due to its strong mechanical and flexible nature in thin film form, biocompatibility, and *in-vivo*bioresorbable properties, fibroin protein has been used as the building block for scaffolds. As confirmed by scanning electron microscope (Figure5 A-C), similar results were obtained with the developed silk-CNT scaffold where cells grown on the silk substrate exhibited denser complex three-dimensional axonal bundle networks as well as better spatial density distribution of the networks compared to other scaffolds. Over‐ all, the silk-CNT nanocomposite provided an efficient three-dimensional supporting ma‐ trix for stem cell-derived neuronal transplants, offering a promising opportunity for nerve repair treatments for patients with neurological disorder. In-vitro analysis showed that βIII tubulin, representing the mature differentiated neurons and nestin, representing the neu‐ ron precursors, were highly expressed in hESCs grown on the silk-CNT substrate compared to the expression level of cells grown on the control poly-L-ornithine substrate (figure 5D). In addition, hESCs cultured on the silk-CNT scaffold exhibited higher maturity along with dense axonal projections.

human CD22 conjugated SWNTsonly targeted CD22(+)CD25(-) Daudi cells; whereas, anti-CD25 mAb coupled SWNTs only target CD22(-)CD25(+) activated peripheral blood mono‐ nuclear cells [81]. The thermal ablation effects can be combined with other therapies, eg.

Carbon Nanotubes for Use in Medicine: Potentials and Limitations

http://dx.doi.org/10.5772/51785

297

Tumors, in general, contain a small population of tumor initiating stem-like cells, termed as cancer stem cells. These cells are unmanageable by standard treatment modalities such as chemotherapy and radiotherapy and tend to persist after treatment [84]. Heat-based cancer treatments are increasingly becoming a potential alternative to approach this problem. Com‐ bining CNTs with such hyperthermia based therapies can further enhance its efficacy by si‐ multaneously eliminating both the stem cells and bulk cancer cells that constitute a tumor. In fact, CNTs offer several properties that make them promising candidates for such thermal therapy. This includes their ability for thermal conductance and strong absorbance of elec‐ tromagnetic radiation. It generates significant amounts of heat upon excitation with near-in‐ frared light which is transparent to biological systems including skins. Such a photothermal effect can be employed to induce thermal cell death in a noninvasive manner. Thus, if CNTs can be localized to tumors, they can be stimulated with near-infrared radiation or radiofre‐ quency energy to generate site-specific heat [85]. Preliminary in-vivo results show that a combination of multiwalled carbon nanotubes (MWNTs) and NIR can be useful for tumor regression and long-term survival in a mouse model [86]. Such CNT-mediated thermal ther‐ apy addresses the limitations of presently available medical strategies. This includes the minimally invasive site-specific heating which will greatly diminish the off-target toxicities, generation of uniform temperature distribution throughout the tumor mass by the activated CNTs, its compatibility with concurrent MRI temperature mapping techniques. It has also been recently reported that breast cancer stem cells, highly resistant to conventional thermal treatments, can be successfully treated with CNT-based photothermal therapies by promot‐ ing necrotic cell death [84]. Further studies in this direction shows that DNA-encased MWNTs are more efficient at converting NIR irradiation into heat compared to non-encased MWNTs and that this method can be effectively used in-vivo for the selective thermal abla‐

Glioblastomamultiforme is the most common and aggressive malignant primary brain tu‐ mor involving glial cells and accounting for a large percentage of brain and intracranial tu‐ mor [88, 89]. It is also known for its recurrence and overall resistance to therapy. CD133+ stem cells occurring among GBM cells are responsible for such huge recurrence risk [90]. Re‐ search has been focused on developing strategies to efficiently deliver CNTs to these target sites, harboring CD133+ cancer stem cells [80]. In-vitro studies show that such targeted elim‐ ination of CD133 (+) cancer stem cells are possible by adding SWNTs functionalized with CD133 monoclonal antibody, followed by irradiation with NIR laser light. In a separate study, embryonic stem cells, once administered with MWNTs, have shown to induce an en‐ hanced immune boost and provide subsequent anticancer protection in mice with colon can‐ cer by suppressing the proliferation and development of malignant colon tumors [91].

chemotherapy, by loading drugs on CNTs for synergic effect [32].

tion of cancer cells [87].

**Figure 5.** Scanning Electron Microscope images of hESCs on various substrates. SEM images of (A) cells cultured on PLO exhibiting a flat morphology and two-dimensional axonal connections, (B) cells cultured on silk scaffolds demon‐ strating three-dimensional structures and cell migration, and (C) cells cultured on silk-CNT scaffolds demonstrating three-dimensional axonal connections and silk-CNT matrix degradation. (D) Two neuronal markers (β-III tubulin and nestin) were used to further determine the hESC differentiation efficiency.Expression intensity of β-III tubulin and nes‐ tin was observed with fluorescence microscopy. Silk-CNT scaffolds exhibited maximum β-III tubulin expression, while nestin expression exhibited a similar trend. \*=*P*< 0.01, and \*\*= *P*< 0.001[76]. Abbreviations: PLO, Poly-L-ornithine; hESCs, human embryonic stem cells; CNT, carbon nanotube.

#### **3.4. Carbon nanotubes for thermal destruction of tumors**

Tissues are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light, whereas, SWNTs display strong optical absorbance in this special spectral window. When constantly absorb energy in NIR region, SWNTs emit heat [77]. Continuous heating leads to killing of the cells. SWNTs have been engineered with tumor recognition molecules for se‐ lective enteringcancer cells. Upon NIR radiation, the cancer cells were killed by thermal ablation [78-83]. Previous studies have shown that folic acid decorated SWNTs more effec‐ tively killed folate receptor positive cancer cells [83]; monoclonal antibody (mAb) against human CD22 conjugated SWNTsonly targeted CD22(+)CD25(-) Daudi cells; whereas, anti-CD25 mAb coupled SWNTs only target CD22(-)CD25(+) activated peripheral blood mono‐ nuclear cells [81]. The thermal ablation effects can be combined with other therapies, eg. chemotherapy, by loading drugs on CNTs for synergic effect [32].

III tubulin, representing the mature differentiated neurons and nestin, representing the neu‐ ron precursors, were highly expressed in hESCs grown on the silk-CNT substrate compared to the expression level of cells grown on the control poly-L-ornithine substrate (figure 5D). In addition, hESCs cultured on the silk-CNT scaffold exhibited higher maturity along with

**Figure 5.** Scanning Electron Microscope images of hESCs on various substrates. SEM images of (A) cells cultured on PLO exhibiting a flat morphology and two-dimensional axonal connections, (B) cells cultured on silk scaffolds demon‐ strating three-dimensional structures and cell migration, and (C) cells cultured on silk-CNT scaffolds demonstrating three-dimensional axonal connections and silk-CNT matrix degradation. (D) Two neuronal markers (β-III tubulin and nestin) were used to further determine the hESC differentiation efficiency.Expression intensity of β-III tubulin and nes‐ tin was observed with fluorescence microscopy. Silk-CNT scaffolds exhibited maximum β-III tubulin expression, while nestin expression exhibited a similar trend. \*=*P*< 0.01, and \*\*= *P*< 0.001[76]. Abbreviations: PLO, Poly-L-ornithine;

Tissues are known to be highly transparent to 700- to 1,100-nm near-infrared (NIR) light, whereas, SWNTs display strong optical absorbance in this special spectral window. When constantly absorb energy in NIR region, SWNTs emit heat [77]. Continuous heating leads to killing of the cells. SWNTs have been engineered with tumor recognition molecules for se‐ lective enteringcancer cells. Upon NIR radiation, the cancer cells were killed by thermal ablation [78-83]. Previous studies have shown that folic acid decorated SWNTs more effec‐ tively killed folate receptor positive cancer cells [83]; monoclonal antibody (mAb) against

hESCs, human embryonic stem cells; CNT, carbon nanotube.

**3.4. Carbon nanotubes for thermal destruction of tumors**

dense axonal projections.

296 Syntheses and Applications of Carbon Nanotubes and Their Composites

Tumors, in general, contain a small population of tumor initiating stem-like cells, termed as cancer stem cells. These cells are unmanageable by standard treatment modalities such as chemotherapy and radiotherapy and tend to persist after treatment [84]. Heat-based cancer treatments are increasingly becoming a potential alternative to approach this problem. Com‐ bining CNTs with such hyperthermia based therapies can further enhance its efficacy by si‐ multaneously eliminating both the stem cells and bulk cancer cells that constitute a tumor. In fact, CNTs offer several properties that make them promising candidates for such thermal therapy. This includes their ability for thermal conductance and strong absorbance of elec‐ tromagnetic radiation. It generates significant amounts of heat upon excitation with near-in‐ frared light which is transparent to biological systems including skins. Such a photothermal effect can be employed to induce thermal cell death in a noninvasive manner. Thus, if CNTs can be localized to tumors, they can be stimulated with near-infrared radiation or radiofre‐ quency energy to generate site-specific heat [85]. Preliminary in-vivo results show that a combination of multiwalled carbon nanotubes (MWNTs) and NIR can be useful for tumor regression and long-term survival in a mouse model [86]. Such CNT-mediated thermal ther‐ apy addresses the limitations of presently available medical strategies. This includes the minimally invasive site-specific heating which will greatly diminish the off-target toxicities, generation of uniform temperature distribution throughout the tumor mass by the activated CNTs, its compatibility with concurrent MRI temperature mapping techniques. It has also been recently reported that breast cancer stem cells, highly resistant to conventional thermal treatments, can be successfully treated with CNT-based photothermal therapies by promot‐ ing necrotic cell death [84]. Further studies in this direction shows that DNA-encased MWNTs are more efficient at converting NIR irradiation into heat compared to non-encased MWNTs and that this method can be effectively used in-vivo for the selective thermal abla‐ tion of cancer cells [87].

Glioblastomamultiforme is the most common and aggressive malignant primary brain tu‐ mor involving glial cells and accounting for a large percentage of brain and intracranial tu‐ mor [88, 89]. It is also known for its recurrence and overall resistance to therapy. CD133+ stem cells occurring among GBM cells are responsible for such huge recurrence risk [90]. Re‐ search has been focused on developing strategies to efficiently deliver CNTs to these target sites, harboring CD133+ cancer stem cells [80]. In-vitro studies show that such targeted elim‐ ination of CD133 (+) cancer stem cells are possible by adding SWNTs functionalized with CD133 monoclonal antibody, followed by irradiation with NIR laser light. In a separate study, embryonic stem cells, once administered with MWNTs, have shown to induce an en‐ hanced immune boost and provide subsequent anticancer protection in mice with colon can‐ cer by suppressing the proliferation and development of malignant colon tumors [91].
