Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple Organisms: From Prokaryotes to Mammalians Cells

*Betty Revon Liu, Yue-Wern Huang and Han-Jung Lee*

#### **Abstract**

Nanotechnology is the study of materials in the nanoscale. By its nature, nanotechnology is interdisciplinary. Nanotechnology has made a significant stride in recent two decades in various industries. Numerous nanomaterials are devised for biomedical applications which include intracellular tracking and labeling, gene detection and hybridization, tumor or tissue targeting, pharmaceutical therapies, pathogenic inhibiting, and medical instrument coating for disinfections. High photostability and quantum yield of fluorescent nanoparticles are ideal for long-term monitoring of molecular events in living organisms. Here, we discuss delivery of three fluorescent nanoparticles in A549 cells, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea. As these nanoparticles cannot enter cells, arginine-rich cell-penetrating peptides (CPPs) were used to enhance their internalization at the cellular or organismal level. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and sulforhodamine B (SRB) assay demonstrated that CPP complexed fluorescent nanoparticles did not produce lethal effect in all organisms tested. The discussion of these nanomaterials in this chapter intends to broaden our understanding of their biocompatibility in organisms of various hierarchical levels.

**Keywords:** fluorescent nanoparticles, cell-penetrating peptides, hypotoxicity, rotifer, prokaryotes

#### **1. Introduction**

Nanoscience and nanotechnology are fast growing multidisciplinary fields in the past two decades [1]. Nanomaterials are the foundation of devices and systems in various industries that revolutionize functionalities of end-user products [2]. Nanomaterials range from simple zero-dimensional structures such as nanodots [3], wire-like nanocomposites in one-dimension nanoscales [4], to two-dimensional nanosheets, and to three-dimensional structures [5, 6]. Furthermore, anisotropy and unique nano level physical and chemical properties can result in nanomaterials of the same elemental compositions having totally different functionalities [6, 7].

Nanomaterials have contributed to various biomedical applications, including molecular labeling and tracking, DNA/RNA/proteins probing, tumor or tissue targeting, drug delivery and therapies, pathogenic intervention, and biomedical imaging [2, 8–10]. In general, nanomaterials are classified into four categories: carbon-based nanomaterials, metal and metal oxide related nanomaterials, organicbased nanopolymers, and composite nanomaterials with complicated structures [11]. Carbon-based nanomaterials such as fullerenes, carbon nanotubes (CNTs), and graphenes have been used as tissue scaffolds, biosensors, targeted drug delivery, and cosmetic additives [10, 12]. The studies and applications of metal- or metal oxide-nanocomposites are commonly found in toxicology, cancer therapies, and antimicrobial infections [13–15]. Organic-based nanomaterials such as liposomes, micelles, microemulsions, and dendrimers are mainly used in pharmaceuticals and drug delivery systems [16]. These organic nanopolymers can be combined with metal or carbon-based nanoparticles for controlled release of drug delivery and antitumor targeting [11]. There is an alternative classification of nanomaterials based upon their applications: drugs and medications, manufacturing and materials, the environment, electronics, energy harvest, and mechanical industries [17]. The first category can be further divided into cell specificity enhancement [18, 19], drugs, peptides, genes, vaccine delivery [16, 18, 20–25], diagnostics and imaging [2, 24, 26–28], antiinfectious agents or germicides [15, 21, 29], cancer therapy [24, 28–31], tissue/organ/ tumor targeting [19, 24–26, 28, 31], scavengers for free radical or thrombosis [32, 33], DNA/RNA/PNA sensor and sequencing [34, 35], and angiogenesis inhibition [36, 37]. Toxicities and health implications become a dominant issue, even though most nanomaterials are hypotoxic for cell/tissue separation or identification, pharmacotherapeutic molecules delivery, diagnostics, as well as imaging [2, 24–26, 28].

In this chapter, we focus on fluorescent nanomaterials which are used as a probe for detection, biomedical imaging, and diagnosis. Their properties and applications in different species will be discussed; their toxicity to the test organisms will be evaluated. Mammalian cell lines, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea are the choices of our topic. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and sulforhodamine B (SRB) assays are indicative of cytotoxicity.

#### **1.1 Fluorescent nanoparticles**

Bioimaging is increasingly popular and important because of its noninvasive, dynamic, and real-time properties. Fluorescent molecules play a pivotal role in optical imaging of life science research and biomedical applications. Fluorescent probes can be used to detect RNA and DNA; analyze proteins, hormones, and viral antigens; and identify organelles, specific proteins, tissues, or tumors via antibody conjugation [38]. However, traditional fluorescent probes made by organic compounds have a lot of room for improvement such as sensitivity, fluorescent intensity, water-solubility, and photo-bleaching [38]. Another critical issue is their asymmetric excitation spectrum with a long tail leading to significant overlapping with other fluorescent dyes [38]. Various fluorescent nanomaterials have high quantum yield and permanent fluorescence with the potential to 1 day replacing the traditional probes [39]. Advantages of fluorescent nanoparticles include, but not limited to, tunable in both sizes and compositions, ultra-bright with narrow spectrum of emission but broad spectrum of excitation, resistant to chemical degradation and photobleaching for long-term observation [40, 41]. Semiconductor nanoparticles such as CdSe/ZnS quantum dots (QDs) are the best example in bioimaging, diagnostics, and therapeutic molecules delivery [42]. Additional fluorescent nanomaterials have been developed to serve as bioimaging probes. We describe some popular fluorescent nanoparticles as follows.

**35**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

Cadmium-based QDs are the first colloidal semiconductor which started the new era of nanotechnology in bioimaging [43]. Extreme brightness and sharp peak of emission wavelength are their signature properties [40–43]. High stability in the biological environment makes them applicable for long-term bioimaging [40–43]. The versatile surface modifications of their outer shells greatly expand their func-

Indium phosphate/zinc sulfate (InP/ZnS) QDs which belongs to the III-V groups of semiconductors replace the toxic cadmium core as a new generation of nanoparticles in biomedical imaging [44]. InP/ZnS QDs modified with PEG-containing negative charges enable them to interact with cationic peptides [44]. PEG modification also increase stability in the aqueous environment. These new complexes

Graphene is a two-dimensional sheet structure with single-atom thickness. GQDs have attracted plenty of attention due to their low toxicity, water solubility, high stability, stable emission spectrum, better surface grafting, and high electrical and thermal conductivity [45]. There are a variety of synthesis methods for GQDs

The fluorescent property of carbon dots were found accidentally [39]. CDs are low cytotoxic, eco-friendly, and highly biocompatible. These features allow them to overtake cadmium-cored QDs in bioimaging [39]. Safety studies have shown that they neither enter nucleus and damage chromosomes nor accumulate in mice

*1.1.5 Zirconium porphyrinic metal-organic framework nanoparticles (ZrMOF)*

Metal–organic frameworks (MOF) combined the inorganic and organic materials and the distinguished features are tunable pore size, high surface areas, and alterable internal surface properties. MOFs do not emit fluorescence, but can be loaded with organic dyes for biomedical sensing. Quenching and therefore became the target-induced sensor [46]. ZrMOF nanoparticles can be unquenched in the target site, thereby facilitating the effect of photodynamic

Soft fluorescent nanomaterials include dye-doped polymer, semiconducting polymer, organic-complex nanoparticles, micelles, and nanogels [47]. This group of fluorescent nanomaterials is a complicated convention. For example, semiconductor QDs are composed of V-III or VI-II groups of elements in their cores and shells, but they can also be functionized by organic materials via chemical linkage on the surface [41, 44]. Modified QDs are classified as soft fluorescent nanomaterials while FDA approved molecules are

used and these complexes are fabricated under a mild condition [47].

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

tionalities and biocompatibilities [40–43].

entered cells via endocytosis without toxicity [44].

that can control the shape, size, and yield [45].

*1.1.3 Graphene quantum dots (GQDs)*

*1.1.1 Cadmium-based QDs*

*1.1.2 Indium-based QDs*

*1.1.4 Carbon dots (CDs)*

cancer therapies [46].

*1.1.6 Soft fluorescent nanomaterials*

bodies [39].

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

#### *1.1.1 Cadmium-based QDs*

*Biotechnology and Bioengineering*

B (SRB) assays are indicative of cytotoxicity.

Bioimaging is increasingly popular and important because of its noninvasive, dynamic, and real-time properties. Fluorescent molecules play a pivotal role in optical imaging of life science research and biomedical applications. Fluorescent probes can be used to detect RNA and DNA; analyze proteins, hormones, and viral antigens; and identify organelles, specific proteins, tissues, or tumors via antibody conjugation [38]. However, traditional fluorescent probes made by organic compounds have a lot of room for improvement such as sensitivity, fluorescent intensity, water-solubility, and photo-bleaching [38]. Another critical issue is their asymmetric excitation spectrum with a long tail leading to significant overlapping with other fluorescent dyes [38]. Various fluorescent nanomaterials have high quantum yield and permanent fluorescence with the potential to 1 day replacing the traditional probes [39]. Advantages of fluorescent nanoparticles include, but not limited to, tunable in both sizes and compositions, ultra-bright with narrow spectrum of emission but broad spectrum of excitation, resistant to chemical degradation and photobleaching for long-term observation [40, 41]. Semiconductor nanoparticles such as CdSe/ZnS quantum dots (QDs) are the best example in bioimaging, diagnostics, and therapeutic molecules delivery [42]. Additional fluorescent nanomaterials have been developed to serve as bioimaging probes. We describe some popular fluorescent nanoparticles as follows.

**1.1 Fluorescent nanoparticles**

Nanomaterials have contributed to various biomedical applications, including molecular labeling and tracking, DNA/RNA/proteins probing, tumor or tissue targeting, drug delivery and therapies, pathogenic intervention, and biomedical imaging [2, 8–10]. In general, nanomaterials are classified into four categories: carbon-based nanomaterials, metal and metal oxide related nanomaterials, organicbased nanopolymers, and composite nanomaterials with complicated structures [11]. Carbon-based nanomaterials such as fullerenes, carbon nanotubes (CNTs), and graphenes have been used as tissue scaffolds, biosensors, targeted drug delivery, and cosmetic additives [10, 12]. The studies and applications of metal- or metal oxide-nanocomposites are commonly found in toxicology, cancer therapies, and antimicrobial infections [13–15]. Organic-based nanomaterials such as liposomes, micelles, microemulsions, and dendrimers are mainly used in pharmaceuticals and drug delivery systems [16]. These organic nanopolymers can be combined with metal or carbon-based nanoparticles for controlled release of drug delivery and antitumor targeting [11]. There is an alternative classification of nanomaterials based upon their applications: drugs and medications, manufacturing and materials, the environment, electronics, energy harvest, and mechanical industries [17]. The first category can be further divided into cell specificity enhancement [18, 19], drugs, peptides, genes, vaccine delivery [16, 18, 20–25], diagnostics and imaging [2, 24, 26–28], antiinfectious agents or germicides [15, 21, 29], cancer therapy [24, 28–31], tissue/organ/ tumor targeting [19, 24–26, 28, 31], scavengers for free radical or thrombosis [32, 33], DNA/RNA/PNA sensor and sequencing [34, 35], and angiogenesis inhibition [36, 37]. Toxicities and health implications become a dominant issue, even though most nanomaterials are hypotoxic for cell/tissue separation or identification, pharmacotherapeutic molecules delivery, diagnostics, as well as imaging [2, 24–26, 28]. In this chapter, we focus on fluorescent nanomaterials which are used as a probe for detection, biomedical imaging, and diagnosis. Their properties and applications in different species will be discussed; their toxicity to the test organisms will be evaluated. Mammalian cell lines, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea are the choices of our topic. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and sulforhodamine

**34**

Cadmium-based QDs are the first colloidal semiconductor which started the new era of nanotechnology in bioimaging [43]. Extreme brightness and sharp peak of emission wavelength are their signature properties [40–43]. High stability in the biological environment makes them applicable for long-term bioimaging [40–43]. The versatile surface modifications of their outer shells greatly expand their functionalities and biocompatibilities [40–43].

#### *1.1.2 Indium-based QDs*

Indium phosphate/zinc sulfate (InP/ZnS) QDs which belongs to the III-V groups of semiconductors replace the toxic cadmium core as a new generation of nanoparticles in biomedical imaging [44]. InP/ZnS QDs modified with PEG-containing negative charges enable them to interact with cationic peptides [44]. PEG modification also increase stability in the aqueous environment. These new complexes entered cells via endocytosis without toxicity [44].

#### *1.1.3 Graphene quantum dots (GQDs)*

Graphene is a two-dimensional sheet structure with single-atom thickness. GQDs have attracted plenty of attention due to their low toxicity, water solubility, high stability, stable emission spectrum, better surface grafting, and high electrical and thermal conductivity [45]. There are a variety of synthesis methods for GQDs that can control the shape, size, and yield [45].

#### *1.1.4 Carbon dots (CDs)*

The fluorescent property of carbon dots were found accidentally [39]. CDs are low cytotoxic, eco-friendly, and highly biocompatible. These features allow them to overtake cadmium-cored QDs in bioimaging [39]. Safety studies have shown that they neither enter nucleus and damage chromosomes nor accumulate in mice bodies [39].

#### *1.1.5 Zirconium porphyrinic metal-organic framework nanoparticles (ZrMOF)*

Metal–organic frameworks (MOF) combined the inorganic and organic materials and the distinguished features are tunable pore size, high surface areas, and alterable internal surface properties. MOFs do not emit fluorescence, but can be loaded with organic dyes for biomedical sensing. Quenching and therefore became the target-induced sensor [46]. ZrMOF nanoparticles can be unquenched in the target site, thereby facilitating the effect of photodynamic cancer therapies [46].

#### *1.1.6 Soft fluorescent nanomaterials*

Soft fluorescent nanomaterials include dye-doped polymer, semiconducting polymer, organic-complex nanoparticles, micelles, and nanogels [47]. This group of fluorescent nanomaterials is a complicated convention. For example, semiconductor QDs are composed of V-III or VI-II groups of elements in their cores and shells, but they can also be functionized by organic materials via chemical linkage on the surface [41, 44]. Modified QDs are classified as soft fluorescent nanomaterials while FDA approved molecules are used and these complexes are fabricated under a mild condition [47].

#### **1.2 Biocompatible enhancer for nanoparticle delivery**

Most organic and inorganic nanomaterials including the nanoparticles of the six categories described above are either hydrophobic or water-insoluble, which means they are difficult to be delivered into cells or organisms. Lipid-derived or peptide-based modifications of shells on the surfaces of nanoparticles enhance the biocompatibility and increase the efficiency of cellular uptake [48, 49]. The routes of cellular deliveries in these nanoparticles functionized by lipid-derived or peptide-based linking chains are related to endocytosis [50, 51]. Antibody-functionalized nanoparticles are advantageous in targeting [51]; however, lysosomal degradation of the delivered drugs or bioactive molecules was a major concern. Several strategies have been developed to circumvent this issue. Among them are cell-penetrating peptides (CPPs) [52–54].

CPPs (a.k.a., PTDs), are proteins that possess the ability to penetrate cell membranes. In recent decades, they have attracted immense popularity in delivering genes, bioactive macromolecules, and drugs due to their effect intracellular translocation. The first CPPs were identified in the human immunodeficiency virus type I (HIV-1) transcriptional activator Tat which consists of 11-amino acids (YGRKKRRQRRR) [55, 56]. Later, various natural CPPs were found in many organisms and numerous synthetic CPPs were designed [57]. To date, there are more than 1700 CPPs in the databank [58]. CPPs can be classified into three categories: cationic, amphipathic, and hydrophobic [57]. Modifications such as cyclization, branches, D-form alteration, and non-primary amino-acid utilization have been performed to improve their transduction efficiency [59–62]. The mechanisms by which CPPs internalized have been vigorously studied. Numerous studies suggest that depending on the physicochemical properties and secondary structures of CPPs, energy-dependent endocytosis or direct membrane translocation is employed [52, 57]. Four major endocytoses are: clathrin-dependent, caveolae-dependent, clathrin and caveolaeindependent, and macropinocytosis [57]. Entry of molecules via these four pathways is trapped in lysosomes and their bioactive and therapeutic characteristics would be lost in the low pH environment [63]. CPP-mediated direct membrane location is an option for delivering drugs and other bioactive molecules [52, 53, 64].

CPPs can interact with cargoes in a covalent, noncovalent, or covalent and noncovalent protein transduction (CNPT) manner. Cargoes are various including proteins, DNA, siRNA, and semiconductors QDs [44, 64–68]. CPP-mediated cellular uptake can be found from prokaryotic to eukaryotic organisms including mammalian cells, insect cells, aquatic microorganism, yeasts, plant tissue, mice dermis, Gram-negative and Gram-positive bacteria, and archaea [53, 65, 69–73]. Uptake mechanisms vary depending upon peptide sequences. IR9 consists of the INF7 fusion peptide and nona-arginine CPP. IR9 without taking any cargoes penetrated cell membranes via macropinocytosis. However, when IR9 was mixed with DNAs or QDs, classical endocytosis was utilized [74]. HR9 contains nona-arginine in the center, flanks with five histidines on either side, and caps with a cysteine in both ends. Out study showed that HR9-mediated cellular entry involves direct membrane translocation [53, 75].

PR9 which consists of nona-arginine and a penetration accelerating peptide sequence has been used to deliver ODs. The complexes entered cells by classical endocytosis [54]. Subsequently, the PR9/QD complexes escaped from lysosomes and entered nucleus [54]. The fluorescent quantum yield and complexes properties were unaltered indicating that CPP/QD complexes were suitable for long-term intracellular imaging and tracking [54, 77, 78].

In the following sections, we discuss biocompatibility of CPP-mediated delivery in various systems including mammalian cells, rotifers, Gram-negative bacteria, Gram-positive bacteria, and archaea.

**37**

**Figure 1.**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

In our studies, human bronchoalveolar carcinoma A549 cells were used as a model cell line to investigate CPP-mediated uptake of inorganic fluorescent nanoparticles which are CdSe/ZnS QD with green fluorescence, InP/ZnS emitted green fluorescence, and CdSe/ZnS QD with red fluorescence. Synthetic nona-arginine (named SR9) CPP were premixed with these three QDs respectively and incubated

*Semiconductor nanoparticles treatments in the mammalian A549 cell line. (A) Penetrations of various semiconductor nanoparticles and CPP/nanoparticles complexes in mammalian cells. A549 cells were treated with green fluorescent CdSe/ZnS QD, green fluorescent InP/ZnS QD, red fluorescent CdSe/ZnS QD, SR9/CdSe/ ZnS QDgreen complexes, SR9/InP/ZnS QDgreen complexes, and SR9/CdSe/ZnS QDred for 1 h at 37°C, respectively. Protein transductions were recorded using a BD pathway 435 system. Green and red fluorescence revealed the distribution of nanoparticles, and blue fluorescence indicated the nuclei. Images were taken at a magnification of 600×. (B) Cell viabilities in A549 cells treated with either nanoparticles alone or CPP/nanoparticles complexes. Cells were treated as previous description shown in (A) and the SRB assay was performed for cytotoxic analysis. Cells without any treatments and treated with 100% DMSO were served as the negative and positive groups, respectively. Histogram of cell viability was represented by mean ± SD from three independent experiments in each treatment group. Significant differences at P < 0.01 (\*\*,††) are indicated [44, 53, 78].*

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

**2. Cell viability in mammalian cells**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

#### **2. Cell viability in mammalian cells**

*Biotechnology and Bioengineering*

**1.2 Biocompatible enhancer for nanoparticle delivery**

option for delivering drugs and other bioactive molecules [52, 53, 64].

CPPs can interact with cargoes in a covalent, noncovalent, or covalent and noncovalent protein transduction (CNPT) manner. Cargoes are various including proteins, DNA, siRNA, and semiconductors QDs [44, 64–68]. CPP-mediated cellular uptake can be found from prokaryotic to eukaryotic organisms including mammalian cells, insect cells, aquatic microorganism, yeasts, plant tissue, mice dermis, Gram-negative and Gram-positive bacteria, and archaea [53, 65, 69–73]. Uptake mechanisms vary depending upon peptide sequences. IR9 consists of the INF7 fusion peptide and nona-arginine CPP. IR9 without taking any cargoes penetrated cell membranes via macropinocytosis. However, when IR9 was mixed with DNAs or QDs, classical endocytosis was utilized [74]. HR9 contains nona-arginine in the center, flanks with five histidines on either side, and caps with a cysteine in both ends. Out study showed that HR9-mediated cellular entry involves direct

PR9 which consists of nona-arginine and a penetration accelerating peptide sequence has been used to deliver ODs. The complexes entered cells by classical endocytosis [54]. Subsequently, the PR9/QD complexes escaped from lysosomes and entered nucleus [54]. The fluorescent quantum yield and complexes properties were unaltered indicating that CPP/QD complexes were suitable for long-term

In the following sections, we discuss biocompatibility of CPP-mediated delivery in various systems including mammalian cells, rotifers, Gram-negative bacteria,

Most organic and inorganic nanomaterials including the nanoparticles of the six categories described above are either hydrophobic or water-insoluble, which means they are difficult to be delivered into cells or organisms. Lipid-derived or peptide-based modifications of shells on the surfaces of nanoparticles enhance the biocompatibility and increase the efficiency of cellular uptake [48, 49]. The routes of cellular deliveries in these nanoparticles functionized by lipid-derived or peptide-based linking chains are related to endocytosis [50, 51]. Antibody-functionalized nanoparticles are advantageous in targeting [51]; however, lysosomal degradation of the delivered drugs or bioactive molecules was a major concern. Several strategies have been developed to circumvent this issue. Among them are cell-penetrating peptides (CPPs) [52–54]. CPPs (a.k.a., PTDs), are proteins that possess the ability to penetrate cell membranes. In recent decades, they have attracted immense popularity in delivering genes, bioactive macromolecules, and drugs due to their effect intracellular translocation. The first CPPs were identified in the human immunodeficiency virus type I (HIV-1) transcriptional activator Tat which consists of 11-amino acids (YGRKKRRQRRR) [55, 56]. Later, various natural CPPs were found in many organisms and numerous synthetic CPPs were designed [57]. To date, there are more than 1700 CPPs in the databank [58]. CPPs can be classified into three categories: cationic, amphipathic, and hydrophobic [57]. Modifications such as cyclization, branches, D-form alteration, and non-primary amino-acid utilization have been performed to improve their transduction efficiency [59–62]. The mechanisms by which CPPs internalized have been vigorously studied. Numerous studies suggest that depending on the physicochemical properties and secondary structures of CPPs, energy-dependent endocytosis or direct membrane translocation is employed [52, 57]. Four major endocytoses are: clathrin-dependent, caveolae-dependent, clathrin and caveolaeindependent, and macropinocytosis [57]. Entry of molecules via these four pathways is trapped in lysosomes and their bioactive and therapeutic characteristics would be lost in the low pH environment [63]. CPP-mediated direct membrane location is an

**36**

membrane translocation [53, 75].

intracellular imaging and tracking [54, 77, 78].

Gram-positive bacteria, and archaea.

In our studies, human bronchoalveolar carcinoma A549 cells were used as a model cell line to investigate CPP-mediated uptake of inorganic fluorescent nanoparticles which are CdSe/ZnS QD with green fluorescence, InP/ZnS emitted green fluorescence, and CdSe/ZnS QD with red fluorescence. Synthetic nona-arginine (named SR9) CPP were premixed with these three QDs respectively and incubated

#### **Figure 1.**

*Semiconductor nanoparticles treatments in the mammalian A549 cell line. (A) Penetrations of various semiconductor nanoparticles and CPP/nanoparticles complexes in mammalian cells. A549 cells were treated with green fluorescent CdSe/ZnS QD, green fluorescent InP/ZnS QD, red fluorescent CdSe/ZnS QD, SR9/CdSe/ ZnS QDgreen complexes, SR9/InP/ZnS QDgreen complexes, and SR9/CdSe/ZnS QDred for 1 h at 37°C, respectively. Protein transductions were recorded using a BD pathway 435 system. Green and red fluorescence revealed the distribution of nanoparticles, and blue fluorescence indicated the nuclei. Images were taken at a magnification of 600×. (B) Cell viabilities in A549 cells treated with either nanoparticles alone or CPP/nanoparticles complexes. Cells were treated as previous description shown in (A) and the SRB assay was performed for cytotoxic analysis. Cells without any treatments and treated with 100% DMSO were served as the negative and positive groups, respectively. Histogram of cell viability was represented by mean ± SD from three independent experiments in each treatment group. Significant differences at P < 0.01 (\*\*,††) are indicated [44, 53, 78].*

with A549 cells for 1 h followed by Hoechst 33342 nuclear staining. Internalizations of QDs and SR9/QD complexes were determined using confocal microscopy. Rare green fluorescence emitted from CdSe/ZnS QDgreen and InP/ZnS QDgreen as well as red fluorescence from CdSe/ZnS QDred were observed while cells were treated with these semiconductors alone (**Figure 1A**). However, strong green and red fluorescence were observed in the groups of SR9-mediated QDs delivery, which meant SR9 facilitated the internalizations of nanoparticles (**Figure 1A**).

To understand toxicity of these CPP-QDs complexes, the SRB assay was conducted for viability analysis. A549 cells were treated with the materials for 24 h and then stained with SRB. Cells without any treatment or with 100% DMSO served as a negative control or a positive control, respectively. Cell viability of CdSe/ZnS QDgreen, InP/ZnS QDgreen, CdSe/ZnS QDred, and SR9-modified CdSe/ZnS QDgreen, InP/ZnS QDgreen, and CdSe/ZnS QDred complexes did not differ from the negative control (**Figure 1B**). Collectively, semiconductor fluorescent nanoparticles and their CPP-modified complexes did not reduce cell viability.

#### **3. Survival rate in rotifers**

Rotifers are non-arthropoda, metazoan aquatic invertebrates with a completed digestive systems. They form the basis of the microzooplankton community in the plankton food web and link the energy flow to higher organisms. Recently, a growing number of studies considers rotifers as an indicator of marine pollution and toxicity of plastic nanoparticles [79–81], as well as a model species for pharmaceutical and toxicological studies [80, 82]. To test the toxicity and uptake efficiency of fluorescent nanoparticles, *Brachionus calyciflorus* were treated with CdSe/ZnS QDred and IR9-FITC mixed CdSe/ZnS QDred complexes respectively (**Figure 2**). Low red fluorescent intensity was detected in rotifers which meant CdSe/ZnS QDred were difficult to enter rotifers without any help. Contrarily, CdSe/ZnS QDred enter rotifers easily by forming complexes with IR9-FITC (**Figure 2A**).

To investigate potential cytotoxicity of CPP-associated quantum dots on rotifers, the MTT assay was performed (**Figure 1B**). *Brachionus calyciflorus* were treated with CdSe/ZnS QDred alone, IR9-FITC alone, and IR9-FITC/QDred complexes for 24 h. Rotifers without treatment served as a negative control, while rotifers treated with 100% DMSO as a positive control. Hypotoxicity was observed in the QDred, IR9- FITC, and IR9-FITC/QDred complexes groups (**Figure 2B**). In contrast, DMSO significantly reduced the survival of rotifers (**Figure 2B**). Collectively, CPP-mediated cellular entry of quantum dots resulted in relatively harmless in rotifers.

#### **4. Hypotoxicity shown in prokaryotic organisms**

Microorganisms are regarded as a vital member in the ecosystem as they play an important role in the natural recycling, elements and energy transforming, and environmental balancing of living materials [83, 84]. Disruption of microorganisms cause reduction of microbial diversity and obliquely influence our natural world [85]. Prokaryotic organisms are major microorganisms; the prokaryotic domain include bacteria and archaea [70, 86, 87]. Here, *Arthrobacter ilicis* D50–1 (Grampositive bacteria), *Escherichia coli* DH5α (Gram-negative bacteria), and *Thermus aquaticus* (archaea) were studied for protein transduction and cytotoxicity. They were treated with Cd-core green semiconductor nanoparticles; hardly any green fluorescence detected (**Figure 3A**). Bright green fluorescence was observed in the SR9-mediated uptake of CdSe/ZnS QDgreen in all three organisms (**Figure 3B**).

**39**

**Figure 2.**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

Toxicological studies of nanomaterials on prokaryotic organisms are important. Bactericidal nanomaterials can affect nonpathogenic bacteria leading to imbalance of a microbiome community and, to the greatest extent, ecological disasters [88]. The toxicity of CdSe/ZnS QDgreen and SR9-modified QDgreen complexes were studied using archaea, Gram-positive bacteria, and Gram-negative bacteria (**Figure 3B**). Organisms were treated for 1 h at room temperature. The MTT assay showed no reduction of viability

*Semiconductor nanoparticles treatments in the rotifer (Brachionus calyciflorus). (A) Protein transduction in rotifer treated with semiconductor nanoparticles CdSe/ZnS QDred and IR9-FITC carpeted QDred complexes. Rotifers were treated with QDred alone or IR9-FITC/QDred complexes for 1 h at 25–28°C. Green fluorescence referred to IR9-FITC and red fluorescence indicated QDred. Merged fluorescent images and bright-field images were recorded at a magnification of 200× using a BD pathway 435 system. (B) Histogram of rotifer survival rate. Rotifers were treated with CdSe/ZnS QDred alone, IR9-FITC alone, and IR9-FITC/QDred complexes, respectively and the survival rate was analyzed by MTT assay. Rotifers treated with water and 100% DMSO were served as negative and positive controls, respectively. Each treatment group was compared with the negative control. Significant differences at P < 0.01 (\*\*) were indicated. Data were presented as mean ± standard deviation from three independent experiments in each treatment groups [74].*

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

Toxicological studies of nanomaterials on prokaryotic organisms are important. Bactericidal nanomaterials can affect nonpathogenic bacteria leading to imbalance of a microbiome community and, to the greatest extent, ecological disasters [88]. The toxicity of CdSe/ZnS QDgreen and SR9-modified QDgreen complexes were studied using archaea, Gram-positive bacteria, and Gram-negative bacteria (**Figure 3B**). Organisms were treated for 1 h at room temperature. The MTT assay showed no reduction of viability

#### **Figure 2.**

*Biotechnology and Bioengineering*

**3. Survival rate in rotifers**

with A549 cells for 1 h followed by Hoechst 33342 nuclear staining. Internalizations of QDs and SR9/QD complexes were determined using confocal microscopy. Rare green fluorescence emitted from CdSe/ZnS QDgreen and InP/ZnS QDgreen as well as red fluorescence from CdSe/ZnS QDred were observed while cells were treated with these semiconductors alone (**Figure 1A**). However, strong green and red fluorescence were observed in the groups of SR9-mediated QDs delivery, which meant SR9

To understand toxicity of these CPP-QDs complexes, the SRB assay was conducted for viability analysis. A549 cells were treated with the materials for 24 h and then stained with SRB. Cells without any treatment or with 100% DMSO served as a negative control or a positive control, respectively. Cell viability of CdSe/ZnS QDgreen, InP/ZnS QDgreen, CdSe/ZnS QDred, and SR9-modified CdSe/ZnS QDgreen, InP/ZnS QDgreen, and CdSe/ZnS QDred complexes did not differ from the negative control (**Figure 1B**). Collectively, semiconductor fluorescent nanoparticles and

Rotifers are non-arthropoda, metazoan aquatic invertebrates with a completed digestive systems. They form the basis of the microzooplankton community in the plankton food web and link the energy flow to higher organisms. Recently, a growing number of studies considers rotifers as an indicator of marine pollution and toxicity of plastic nanoparticles [79–81], as well as a model species for pharmaceutical and toxicological studies [80, 82]. To test the toxicity and uptake efficiency of fluorescent nanoparticles, *Brachionus calyciflorus* were treated with CdSe/ZnS QDred and IR9-FITC mixed CdSe/ZnS QDred complexes respectively (**Figure 2**). Low red fluorescent intensity was detected in rotifers which meant CdSe/ZnS QDred were difficult to enter rotifers without any help. Contrarily, CdSe/ZnS QDred enter

To investigate potential cytotoxicity of CPP-associated quantum dots on rotifers, the MTT assay was performed (**Figure 1B**). *Brachionus calyciflorus* were treated with CdSe/ZnS QDred alone, IR9-FITC alone, and IR9-FITC/QDred complexes for 24 h. Rotifers without treatment served as a negative control, while rotifers treated with 100% DMSO as a positive control. Hypotoxicity was observed in the QDred, IR9- FITC, and IR9-FITC/QDred complexes groups (**Figure 2B**). In contrast, DMSO significantly reduced the survival of rotifers (**Figure 2B**). Collectively, CPP-mediated

Microorganisms are regarded as a vital member in the ecosystem as they play an important role in the natural recycling, elements and energy transforming, and environmental balancing of living materials [83, 84]. Disruption of microorganisms cause reduction of microbial diversity and obliquely influence our natural world [85]. Prokaryotic organisms are major microorganisms; the prokaryotic domain include bacteria and archaea [70, 86, 87]. Here, *Arthrobacter ilicis* D50–1 (Grampositive bacteria), *Escherichia coli* DH5α (Gram-negative bacteria), and *Thermus aquaticus* (archaea) were studied for protein transduction and cytotoxicity. They were treated with Cd-core green semiconductor nanoparticles; hardly any green fluorescence detected (**Figure 3A**). Bright green fluorescence was observed in the SR9-mediated uptake of CdSe/ZnS QDgreen in all three organisms (**Figure 3B**).

facilitated the internalizations of nanoparticles (**Figure 1A**).

their CPP-modified complexes did not reduce cell viability.

rotifers easily by forming complexes with IR9-FITC (**Figure 2A**).

cellular entry of quantum dots resulted in relatively harmless in rotifers.

**4. Hypotoxicity shown in prokaryotic organisms**

**38**

*Semiconductor nanoparticles treatments in the rotifer (Brachionus calyciflorus). (A) Protein transduction in rotifer treated with semiconductor nanoparticles CdSe/ZnS QDred and IR9-FITC carpeted QDred complexes. Rotifers were treated with QDred alone or IR9-FITC/QDred complexes for 1 h at 25–28°C. Green fluorescence referred to IR9-FITC and red fluorescence indicated QDred. Merged fluorescent images and bright-field images were recorded at a magnification of 200× using a BD pathway 435 system. (B) Histogram of rotifer survival rate. Rotifers were treated with CdSe/ZnS QDred alone, IR9-FITC alone, and IR9-FITC/QDred complexes, respectively and the survival rate was analyzed by MTT assay. Rotifers treated with water and 100% DMSO were served as negative and positive controls, respectively. Each treatment group was compared with the negative control. Significant differences at P < 0.01 (\*\*) were indicated. Data were presented as mean ± standard deviation from three independent experiments in each treatment groups [74].*

#### **Figure 3.**

*Treatments of CdSe cored QD with green fluorescence in three types of prokaryocytes. (A) Fluorescent microscopy of Thermus aquaticus (archaea), Arthrobacter ilicis D50-1 (Gram-positive bacteria), and Escherichia coli DH5α (Gram-negative bacteria) treated with QDgreen alone or SR9/QDgreen complexes. Three prokaryocytes were incubated with QDgreen alone or SR9/QDgreen complexes for 1 h at room temperature. Protein transductions were detected in GFP channel and cell morphologies were observed in bright-field with an AE31 fluorescent microscope. (B) Cytotoxicity of nanoparticles in archaea, Gram-positive bacteria, and Gram-negative bacteria. Archaea, D50–1, and DH5α were treated with QDgreen, SR9, and SR9/QDgreen complexes. Cells treated with their specific media and 75% alcohol as negative control and positive control, respectively. Significant differences from negative control at P < 0.01 (\*\*) were indicated. Data were presented as mean ± standard deviation from three independent experiments [89].*

**41**

**Author details**

**5. Conclusion**

Betty Revon Liu1

Hualien, Taiwan

Technology, Rolla, MO, USA

Hwa University, Hualien, Taiwan

provided the original work is properly cited.

\*, Yue-Wern Huang2

\*Address all correspondence to: brliu7447@gms.tcu.edu.tw

© 2019 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,

and Han-Jung Lee3

1 Department of Laboratory Medicine and Biotechnology, Tzu Chi University,

3 Department of Natural Resources and Environmental Studies, National Dong

2 Department of Biological Sciences, Missouri University of Science and

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

in the groups of QDgreen, SR9, and SR9/QDgreen complexes (**Figure 3B**). This result indicated that Cd-core nanoparticles did not cause lethal effect to prokaryotic organisms. We reasonably ratiocinated that fluorescent nanoparticles applied in bioimaging and biotechnologies might not provoke natural imbalance and environmental problems.

We discussed applications and safety issues of various fluorescent nanoparticles. The cellular entry of particles of interest can be facilitated by CPPs. The particles did not produce lethal effects in mammalian cells, rotifers, archaea, Gram-positive bacteria, and Gram-negative bacteria. The outcome from assessing nanoparticle safety in mammalian cells suggests their potential medical applications. Hypotoxicity in rotifers and prokaryotes infers their environmental safety and eco-friendliness. In summary, these fluorescent nanoparticles and their CPP-modified complexes can be potent tools

in various biological, environmental, and medical applications in the future.

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

in the groups of QDgreen, SR9, and SR9/QDgreen complexes (**Figure 3B**). This result indicated that Cd-core nanoparticles did not cause lethal effect to prokaryotic organisms. We reasonably ratiocinated that fluorescent nanoparticles applied in bioimaging and biotechnologies might not provoke natural imbalance and environmental problems.

### **5. Conclusion**

*Biotechnology and Bioengineering*

**40**

**Figure 3.**

*Treatments of CdSe cored QD with green fluorescence in three types of prokaryocytes. (A) Fluorescent microscopy of Thermus aquaticus (archaea), Arthrobacter ilicis D50-1 (Gram-positive bacteria), and Escherichia coli DH5α (Gram-negative bacteria) treated with QDgreen alone or SR9/QDgreen complexes. Three prokaryocytes were incubated with QDgreen alone or SR9/QDgreen complexes for 1 h at room temperature. Protein transductions were detected in GFP channel and cell morphologies were observed in bright-field with an AE31 fluorescent microscope. (B) Cytotoxicity of nanoparticles in archaea, Gram-positive bacteria, and Gram-negative bacteria. Archaea, D50–1, and DH5α were treated with QDgreen, SR9, and SR9/QDgreen complexes. Cells treated with their specific media and 75% alcohol as negative control and positive control, respectively. Significant differences from negative control at P < 0.01 (\*\*) were indicated. Data were presented as mean ± standard deviation from three independent experiments [89].*

We discussed applications and safety issues of various fluorescent nanoparticles. The cellular entry of particles of interest can be facilitated by CPPs. The particles did not produce lethal effects in mammalian cells, rotifers, archaea, Gram-positive bacteria, and Gram-negative bacteria. The outcome from assessing nanoparticle safety in mammalian cells suggests their potential medical applications. Hypotoxicity in rotifers and prokaryotes infers their environmental safety and eco-friendliness. In summary, these fluorescent nanoparticles and their CPP-modified complexes can be potent tools in various biological, environmental, and medical applications in the future.

### **Author details**

Betty Revon Liu1 \*, Yue-Wern Huang2 and Han-Jung Lee3

1 Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien, Taiwan

2 Department of Biological Sciences, Missouri University of Science and Technology, Rolla, MO, USA

3 Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien, Taiwan

\*Address all correspondence to: brliu7447@gms.tcu.edu.tw

© 2019 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] Kagan CR, Fernandez LE, Gogotsi Y, Hammond PT, Hersam MC, et al. Nano day: Celebrating the next decade of nanoscience and nanotechnology. ACS Nano. 2016;**10**:9093-9103

[2] Salata OV. Applications of nanoparticles in biology and medicine. Journal of Nanobiotechnology. 2004;**2**:3-3

[3] Ostadhossein F, Pan D. Functional carbon nanodots for multiscale imaging and therapy. Wiley Interdisciplinary Reviews. Nanomedicine and Nanobiotechnology. 2017:9

[4] Lu X, Jia W, Chai H, Hu J, Wang S, et al. Solid-state chemical fabrication of one-dimensional mesoporous β-nickel molybdate nanorods as remarkable electrode material for supercapacitors. Journal of Colloid and Interface Science. 2019;**534**:322-331

[5] Patra S, Roy E, Tiwari A, Madhuri R, Sharma PK. 2-Dimensional graphene as a route for emergence of additional dimension nanomaterials. Biosensors and Bioelectronics. 2017;**89**:8-27

[6] Chimene D, Alge DL, Gaharwar AK. Two-dimensional nanomaterials for biomedical applications: Emerging trends and future prospects. Advanced Materials. 2015;**27**:7261-7284

[7] Ray PC. Size and shape dependent second order nonlinear optical properties of nanomaterials and their application in biological and chemical sensing. Chemical Reviews. 2010;**110**:5332-5365

[8] Usman MS, Zowalaty MEE, Shameli K, Zainuddin N, Salama M, et al. Synthesis, characterization, and antimicrobial properties of copper nanoparticles. International Journal of Nanomedicine. 2013;**8**:4467-4479

[9] Ganju A, Khan S, Hafeez BB, Behrman SW, Yallapu MM, et al. miRNA nanotherapeutics for cancer. Drug Discovery Today. 2017;**22**:424-432

[10] Lalwani G, Patel SC, Sitharaman B. Two- and three-dimensional allcarbon nanomaterial assemblies for tissue engineering and regenerative medicine. Annals of Biomedical Engineering. 2016;**44**:2020-2035

[11] Jeevanandam J, Barhoum A, Chan YS, Dufresne A, Danquah MK. Review on nanoparticles and nanostructured materials: History, sources, toxicity and regulations. Beilstein Journal of Nanotechnology. 2018;**9**:1050-1074

[12] Cha C, Shin SR, Annabi N, Dokmeci MR, Khademhosseini A. Carbonbased nanomaterials: Multifunctional materials for biomedical engineering. ACS Nano. 2013;**7**:2891-2897

[13] Golbamaki A, Golbamaki N, Sizochenko N, Rasulev B, Leszczynski J, et al. Genotoxicity induced by metal oxide nanoparticles: A weight of evidence study and effect of particle surface and electronic properties. Nanotoxicology. 2018:1-17

[14] Wu L, Zhang F, Wei Z, Li X, Zhao H, et al. Magnetic delivery of Fe3O4@ polydopamine nanoparticle-loaded natural killer cells suggest a promising anticancer treatment. Biomaterials Science. 2018;**6**:2714-2725

[15] Radhakrishnan VS, Reddy Mudiam MK, Kumar M, Dwivedi SP, Singh SP, et al. Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (*Candida albicans*). International Journal of Nanomedicine. 2018;**13**:2647-2663

**43**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

efficiency of gene delivery system. Cancer Research. 2007;**67**:8156-8163

[24] Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose

[25] Elzoghby AO, Abd-Elwakil MM, Abd-Elsalam K, Elsayed MT, Hashem Y, et al. Natural polymeric nanoparticles for brain-targeting: Implications on drug and gene delivery. Current Pharmaceutical Design.

[26] Amiot CL, Xu S, Liang S, Pan L, Zhao JX. Near-infrared fluorescent materials for sensing of biological targets. Sensors (Basel).

[27] Moore A, Weissleder R, Bogdanov

[28] Gao X, Cui Y, Levenson RM, Chung LW, Nie S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology.

A Jr. Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. Journal of Magnetic Resonance Imaging.

[29] Tegos GP, Demidova TN, Arcila-Lopez D, Lee H, Wharton T, et al. Cationic fullerenes are effective and selective antimicrobial photosensitizers. Chemistry & Biology.

[30] Markovic Z, Trajkovic V. Biomedical potential of the reactive oxygen species generation and quenching by fullerenes (C60). Biomaterials. 2008;**29**:3561-3573

[31] Freitas RA Jr. Pharmacytes: An ideal vehicle for targeted drug delivery. Journal of Nanoscience and Nanotechnology. 2006;**6**:2769-2775

2016;**22**:3305-3323

2008;**8**:3082-3105

1997;**7**:1140-1145

2004;**22**:969-976

2005;**12**:1127-1135

nanodevices for oncology drug delivery and diagnostic imaging. Biochemical Society Transactions. 2007;**35**:61-67

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

biopharmaceuticals. Part II: Liposomes,

[16] Ana CS, Carla ML, José MSL, Maria HA. Delivery systems for

micelles, microemulsions and dendrimers. Current Pharmaceutical Biotechnology. 2015;**16**:955-965

[17] Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arabian

Journal of Chemistry. 2017

pp. 33-93

2007;**48**:1180-1189

2008;**28**:243-246

2018;**10**:27578-27583

[18] Bhatia S. Nanoparticles Types, Classification, Characterization, Fabrication Methods and Drug Delivery Applications. Natural Polymer Drug Delivery Systems. Cham: Springer; 2016.

[19] McDevitt MR, Chattopadhyay D, Kappel BJ, Jaggi JS, Schiffman SR, et al. Tumor targeting with antibodyfunctionalized, radiolabeled carbon nanotubes. Journal of Nuclear Medicine.

[20] Zhang Z, Yang X, Zhang Y, Zeng B, Wang S, et al. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged

single-walled carbon nanotubes suppresses tumor growth. Clinical Cancer Research. 2006;**12**:4933-4939

[21] Ji H, Yang Z, Jiang W, Geng C, Gong M, et al. Antiviral activity of nano carbon fullerene lipidosome against influenza virus in vitro. Journal of Huazhong University of Science and Technology. Medical Sciences.

[22] Levin A, Michaels TCT, Mason TO, Muller T, Adler-Abramovich L, et al. Self-assembly-mediated release of peptide nanoparticles through jets across microdroplet interfaces. ACS Applied Materials & Interfaces.

[23] Pan B, Cui D, Sheng Y, Ozkan C, Gao F, et al. Dendrimer-modified magnetic nanoparticles enhance

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

[16] Ana CS, Carla ML, José MSL, Maria HA. Delivery systems for biopharmaceuticals. Part II: Liposomes, micelles, microemulsions and dendrimers. Current Pharmaceutical Biotechnology. 2015;**16**:955-965

[17] Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arabian Journal of Chemistry. 2017

[18] Bhatia S. Nanoparticles Types, Classification, Characterization, Fabrication Methods and Drug Delivery Applications. Natural Polymer Drug Delivery Systems. Cham: Springer; 2016. pp. 33-93

[19] McDevitt MR, Chattopadhyay D, Kappel BJ, Jaggi JS, Schiffman SR, et al. Tumor targeting with antibodyfunctionalized, radiolabeled carbon nanotubes. Journal of Nuclear Medicine. 2007;**48**:1180-1189

[20] Zhang Z, Yang X, Zhang Y, Zeng B, Wang S, et al. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clinical Cancer Research. 2006;**12**:4933-4939

[21] Ji H, Yang Z, Jiang W, Geng C, Gong M, et al. Antiviral activity of nano carbon fullerene lipidosome against influenza virus in vitro. Journal of Huazhong University of Science and Technology. Medical Sciences. 2008;**28**:243-246

[22] Levin A, Michaels TCT, Mason TO, Muller T, Adler-Abramovich L, et al. Self-assembly-mediated release of peptide nanoparticles through jets across microdroplet interfaces. ACS Applied Materials & Interfaces. 2018;**10**:27578-27583

[23] Pan B, Cui D, Sheng Y, Ozkan C, Gao F, et al. Dendrimer-modified magnetic nanoparticles enhance

efficiency of gene delivery system. Cancer Research. 2007;**67**:8156-8163

[24] Tomalia DA, Reyna LA, Svenson S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochemical Society Transactions. 2007;**35**:61-67

[25] Elzoghby AO, Abd-Elwakil MM, Abd-Elsalam K, Elsayed MT, Hashem Y, et al. Natural polymeric nanoparticles for brain-targeting: Implications on drug and gene delivery. Current Pharmaceutical Design. 2016;**22**:3305-3323

[26] Amiot CL, Xu S, Liang S, Pan L, Zhao JX. Near-infrared fluorescent materials for sensing of biological targets. Sensors (Basel). 2008;**8**:3082-3105

[27] Moore A, Weissleder R, Bogdanov A Jr. Uptake of dextran-coated monocrystalline iron oxides in tumor cells and macrophages. Journal of Magnetic Resonance Imaging. 1997;**7**:1140-1145

[28] Gao X, Cui Y, Levenson RM, Chung LW, Nie S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology. 2004;**22**:969-976

[29] Tegos GP, Demidova TN, Arcila-Lopez D, Lee H, Wharton T, et al. Cationic fullerenes are effective and selective antimicrobial photosensitizers. Chemistry & Biology. 2005;**12**:1127-1135

[30] Markovic Z, Trajkovic V. Biomedical potential of the reactive oxygen species generation and quenching by fullerenes (C60). Biomaterials. 2008;**29**:3561-3573

[31] Freitas RA Jr. Pharmacytes: An ideal vehicle for targeted drug delivery. Journal of Nanoscience and Nanotechnology. 2006;**6**:2769-2775

**42**

*Biotechnology and Bioengineering*

Nano. 2016;**10**:9093-9103

2004;**2**:3-3

**References**

[2] Salata OV. Applications of

Journal of Nanobiotechnology.

Reviews. Nanomedicine and Nanobiotechnology. 2017:9

[4] Lu X, Jia W, Chai H, Hu J, Wang S, et al. Solid-state chemical fabrication of one-dimensional mesoporous β-nickel molybdate nanorods as remarkable electrode material for supercapacitors. Journal of Colloid and Interface Science.

2019;**534**:322-331

[1] Kagan CR, Fernandez LE, Gogotsi Y, Hammond PT, Hersam MC, et al. Nano day: Celebrating the next decade of nanoscience and nanotechnology. ACS

[9] Ganju A, Khan S, Hafeez BB, Behrman SW, Yallapu MM, et al. miRNA nanotherapeutics for cancer. Drug Discovery Today.

[10] Lalwani G, Patel SC, Sitharaman B. Two- and three-dimensional allcarbon nanomaterial assemblies for tissue engineering and regenerative medicine. Annals of Biomedical Engineering. 2016;**44**:2020-2035

[11] Jeevanandam J, Barhoum A, Chan YS, Dufresne A, Danquah MK. Review on nanoparticles and nanostructured materials: History, sources, toxicity and regulations. Beilstein Journal of Nanotechnology. 2018;**9**:1050-1074

[12] Cha C, Shin SR, Annabi N, Dokmeci

MR, Khademhosseini A. Carbonbased nanomaterials: Multifunctional materials for biomedical engineering.

[13] Golbamaki A, Golbamaki N, Sizochenko N, Rasulev B, Leszczynski J, et al. Genotoxicity induced by metal oxide nanoparticles: A weight of evidence study and effect of particle surface and electronic properties.

[14] Wu L, Zhang F, Wei Z, Li X, Zhao H, et al. Magnetic delivery of Fe3O4@ polydopamine nanoparticle-loaded natural killer cells suggest a promising anticancer treatment. Biomaterials

ACS Nano. 2013;**7**:2891-2897

Nanotoxicology. 2018:1-17

Science. 2018;**6**:2714-2725

Journal of Nanomedicine.

2018;**13**:2647-2663

[15] Radhakrishnan VS, Reddy Mudiam MK, Kumar M, Dwivedi SP, Singh SP, et al. Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (*Candida albicans*). International

2017;**22**:424-432

nanoparticles in biology and medicine.

[3] Ostadhossein F, Pan D. Functional carbon nanodots for multiscale imaging and therapy. Wiley Interdisciplinary

[5] Patra S, Roy E, Tiwari A, Madhuri R, Sharma PK. 2-Dimensional graphene as a route for emergence of additional dimension nanomaterials. Biosensors and Bioelectronics. 2017;**89**:8-27

[6] Chimene D, Alge DL, Gaharwar AK. Two-dimensional nanomaterials for biomedical applications: Emerging trends and future prospects. Advanced

[7] Ray PC. Size and shape dependent second order nonlinear optical properties of nanomaterials and their application in biological and chemical sensing. Chemical Reviews.

Materials. 2015;**27**:7261-7284

[8] Usman MS, Zowalaty MEE, Shameli K, Zainuddin N, Salama M, et al. Synthesis, characterization, and antimicrobial properties of copper nanoparticles. International Journal of Nanomedicine. 2013;**8**:4467-4479

2010;**110**:5332-5365

[32] Cai X, Jia H, Liu Z, Hou B, Luo C, et al. Polyhydroxylated fullerene derivative C(60)(OH)(24) prevents mitochondrial dysfunction and oxidative damage in an MPP(+) -induced cellular model of Parkinson's disease. Journal of Neuroscience Research. 2008;**86**:3622-3634

[33] Iverson N, Plourde N, Chnari E, Nackman GB, Moghe PV. Convergence of nanotechnology and cardiovascular medicine: Progress and emerging prospects. Bio Drugs. 2008;**22**:1-10

[34] Heerema SJ, Dekker C. Graphene nanodevices for DNA sequencing. Nature Nanotechnology. 2016;**11**:127-136

[35] Li BR, Chen CC, Kumar UR, Chen YT. Advances in nanowire transistors for biological analysis and cellular investigation. Analyst. 2014;**139**:1589-1608

[36] Zhang L, Liu Z, Yang K, Kong C, Liu C, et al. Tumor progression of non-small cell lung cancer controlled by albumin and micellar nanoparticles of itraconazole, a multitarget angiogenesis inhibitor. Molecular Pharmaceutics. 2017;**14**:4705-4713

[37] Kim GH, Won JE, Byeon Y, Kim MG, Wi TI, et al. Selective delivery of PLXDC1 small interfering RNA to endothelial cells for anti-angiogenesis tumor therapy using CD44-targeted chitosan nanoparticles for epithelial ovarian cancer. Drug Delivery. 2018;**25**:1394-1402

[38] Fei X, Gu Y. Progress in modifications and applications of fluorescent dye probe. Progress in Natural Science. 2009;**19**:1-7

[39] Zuo J, Jiang T, Zhao X, Xiong X, Xiao S, et al. Preparation and application of fluorescent carbon dots. Journal of Nanomaterials. 2015, 2015:13

[40] Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science. 2005;**307**:538-544

[41] Chen F, Gerion D. Fluorescent CdSe/ZnS nanocrystal−peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Letters. 2004;**4**:1827-1832

[42] Bilan R, Nabiev I, Sukhanova A. Quantum dot-based nanotools for bioimaging, diagnostics, and drug delivery. Chembiochem. 2016;**17**:2103-2114

[43] Rizvi SB, Ghaderi S, Keshtgar M, Seifalian AM. Semiconductor quantum dots as fluorescent probes for in vitro and in vivo bio-molecular and cellular imaging. Nano Reviews. 2010;**1**. DOI: 10.3402/nano.v3401i3400.5161

[44] Liu BR, Winiarz JG, Moon JS, Lo SY, Huang YW, et al. Synthesis, characterization and applications of carboxylated and polyethyleneglycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides. Colloids and Surfaces. B, Biointerfaces. 2013;**111**:162-170

[45] Liu Q , Zhang J, He H, Huang G, Xing B, et al. Green preparation of high yield fluorescent graphene quantum dots from coal-tar-pitch by mild oxidation. Nanomaterials. 2018;**8**:844

[46] Liu Y, Hou W, Xia L, Cui C, Wan S, et al. ZrMOF nanoparticles as quenchers to conjugate DNA aptamers for target-induced bioimaging and photodynamic therapy. Chemical Science. 2018;**9**:7505-7509

[47] Peng H-S, Chiu DT. Soft fluorescent nanomaterials for biological and biomedical imaging. Chemical Society Reviews. 2015;**44**:4699-4722

**45**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

[56] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science.

[57] Guidotti G, Brambilla L, Rossi D. Cell-penetrating peptides: From basic research to clinics. Trends in Pharmacological Sciences.

[58] Gautam A, Singh H, Tyagi A, Chaudhary K, Kumar R, et al. CPPsite: A curated database of cell penetrating peptides. Database: The Journal of Biological Databases and Curation.

[59] Qian Z, Liu T, Liu YY, Briesewitz R, Barrios AM, et al. Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS Chemical Biology. 2013;**8**:423-431

[60] Ezzat K, Andaloussi SE, Zaghloul EM, Lehto T, Lindberg S, et al. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids

Research. 2011;**39**:5284-5298

2005;**127**:9459-9468

2010;**21**:2164-2167

[61] Farrera-Sinfreu J, Giralt E, Castel S, Albericio F, Royo M. Cellpenetrating cis-gamma-amino-lproline-derived peptides. Journal of the American Chemical Society.

[62] Angeles-Boza AM, Erazo-Oliveras A, Lee YJ, Pellois JP. Generation of endosomolytic reagents by branching of cell-penetrating peptides: Tools for the delivery of bioactive compounds to live cells in cis or trans. Bioconjugate Chemistry.

[63] Hogset A, Prasmickaite L, Selbo PK, Hellum M, Engesaeter BO, et al. Photochemical internalisation in drug and gene delivery. Advanced Drug Delivery Reviews. 2004;**56**:95-115

1999;**285**:1569-1572

2017;**38**:406-424

2012, bas015

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

[48] Ranalli A, Santi M, Capriotti L, Voliani V, Porciani D, et al. Peptidebased stealth nanoparticles for targeted and pH-triggered delivery. Bioconjugate

Chemistry. 2017;**28**:627-635

[50] Oh N, Park J-H. Endocytosis and exocytosis of nanoparticles in mammalian cells. International Journal of Nanomedicine. 2014;**9**(Suppl 1):51-63

[51] Wu FL, Zhang J, Li W, Bian BX, Hong YD, et al. Enhanced antiproliferative activity of antibodyfunctionalized polymeric nanoparticles for targeted delivery of anti-miR-21 to HER2 positive gastric cancer. Oncotarget. 2017;**8**:67189-67202

[52] Liu BR, Huang YW, Korivi M, Lo SY, Aronstam RS, et al. The primary mechanism of cellular internalization for a short cell-penetrating peptide as a nano-scale delivery system. Current Pharmaceutical Biotechnology.

[53] Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ. Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cellpenetrating peptide through the direct membrane translocation mechanism. Biomaterials. 2011;**32**:3520-3537

[54] Liu BR, Lo SY, Liu CC, Chyan CL, Huang YW, et al. Endocytic trafficking of nanoparticles delivered by cell-penetrating peptides comprised of nona-arginine and a penetration accelerating sequence. PLoS One.

[55] Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell.

2017;**18**:569-584

2013;**8**:e67100

1988;**55**:1189-1193

[49] Andonova V, Peneva P. Characterization methods for solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). Current Pharmaceutical Design. 2017

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

[48] Ranalli A, Santi M, Capriotti L, Voliani V, Porciani D, et al. Peptidebased stealth nanoparticles for targeted and pH-triggered delivery. Bioconjugate Chemistry. 2017;**28**:627-635

*Biotechnology and Bioengineering*

[32] Cai X, Jia H, Liu Z, Hou B, Luo C, et al. Polyhydroxylated fullerene derivative C(60)(OH)(24) prevents mitochondrial dysfunction and oxidative damage in an MPP(+) -induced cellular model of Parkinson's disease. Journal of Neuroscience Research. 2008;**86**:3622-3634

[40] Michalet X, Pinaud FF, Bentolila

Quantum dots for live cells, in vivo imaging, and diagnostics. Science.

[41] Chen F, Gerion D. Fluorescent CdSe/ZnS nanocrystal−peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells. Nano Letters.

[42] Bilan R, Nabiev I, Sukhanova A. Quantum dot-based nanotools for bioimaging, diagnostics, and drug delivery. Chembiochem.

[43] Rizvi SB, Ghaderi S, Keshtgar M, Seifalian AM. Semiconductor quantum dots as fluorescent probes for in vitro and in vivo bio-molecular and cellular imaging. Nano Reviews. 2010;**1**. DOI: 10.3402/nano.v3401i3400.5161

[44] Liu BR, Winiarz JG, Moon JS, Lo SY, Huang YW, et al. Synthesis, characterization and applications of carboxylated and polyethyleneglycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides. Colloids and Surfaces. B, Biointerfaces.

[45] Liu Q , Zhang J, He H, Huang G, Xing B, et al. Green preparation of high yield fluorescent graphene quantum dots from coal-tar-pitch by mild oxidation. Nanomaterials. 2018;**8**:844

[46] Liu Y, Hou W, Xia L, Cui C, Wan S, et al. ZrMOF nanoparticles as quenchers to conjugate DNA aptamers for target-induced bioimaging and photodynamic therapy. Chemical

[47] Peng H-S, Chiu DT. Soft fluorescent

nanomaterials for biological and biomedical imaging. Chemical Society

Reviews. 2015;**44**:4699-4722

Science. 2018;**9**:7505-7509

LA, Tsay JM, Doose S, et al.

2005;**307**:538-544

2004;**4**:1827-1832

2016;**17**:2103-2114

2013;**111**:162-170

[33] Iverson N, Plourde N, Chnari E, Nackman GB, Moghe PV. Convergence of nanotechnology and cardiovascular medicine: Progress and emerging prospects. Bio Drugs. 2008;**22**:1-10

[34] Heerema SJ, Dekker C. Graphene nanodevices for DNA sequencing.

[35] Li BR, Chen CC, Kumar UR, Chen YT. Advances in nanowire transistors for biological analysis and cellular investigation. Analyst.

[36] Zhang L, Liu Z, Yang K, Kong C, Liu C, et al. Tumor progression of non-small cell lung cancer controlled by albumin and micellar nanoparticles of itraconazole, a multitarget angiogenesis inhibitor. Molecular Pharmaceutics.

[37] Kim GH, Won JE, Byeon Y, Kim MG, Wi TI, et al. Selective delivery of PLXDC1 small interfering RNA to endothelial cells for anti-angiogenesis tumor therapy using CD44-targeted chitosan nanoparticles for epithelial ovarian cancer. Drug Delivery.

Nature Nanotechnology.

2016;**11**:127-136

2014;**139**:1589-1608

2017;**14**:4705-4713

2018;**25**:1394-1402

[38] Fei X, Gu Y. Progress in modifications and applications of fluorescent dye probe. Progress in Natural Science. 2009;**19**:1-7

[39] Zuo J, Jiang T, Zhao X, Xiong X, Xiao S, et al. Preparation and application of fluorescent carbon dots. Journal of Nanomaterials. 2015,

**44**

2015:13

[49] Andonova V, Peneva P. Characterization methods for solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC). Current Pharmaceutical Design. 2017

[50] Oh N, Park J-H. Endocytosis and exocytosis of nanoparticles in mammalian cells. International Journal of Nanomedicine. 2014;**9**(Suppl 1):51-63

[51] Wu FL, Zhang J, Li W, Bian BX, Hong YD, et al. Enhanced antiproliferative activity of antibodyfunctionalized polymeric nanoparticles for targeted delivery of anti-miR-21 to HER2 positive gastric cancer. Oncotarget. 2017;**8**:67189-67202

[52] Liu BR, Huang YW, Korivi M, Lo SY, Aronstam RS, et al. The primary mechanism of cellular internalization for a short cell-penetrating peptide as a nano-scale delivery system. Current Pharmaceutical Biotechnology. 2017;**18**:569-584

[53] Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ. Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cellpenetrating peptide through the direct membrane translocation mechanism. Biomaterials. 2011;**32**:3520-3537

[54] Liu BR, Lo SY, Liu CC, Chyan CL, Huang YW, et al. Endocytic trafficking of nanoparticles delivered by cell-penetrating peptides comprised of nona-arginine and a penetration accelerating sequence. PLoS One. 2013;**8**:e67100

[55] Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988;**55**:1189-1193

[56] Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science. 1999;**285**:1569-1572

[57] Guidotti G, Brambilla L, Rossi D. Cell-penetrating peptides: From basic research to clinics. Trends in Pharmacological Sciences. 2017;**38**:406-424

[58] Gautam A, Singh H, Tyagi A, Chaudhary K, Kumar R, et al. CPPsite: A curated database of cell penetrating peptides. Database: The Journal of Biological Databases and Curation. 2012, bas015

[59] Qian Z, Liu T, Liu YY, Briesewitz R, Barrios AM, et al. Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS Chemical Biology. 2013;**8**:423-431

[60] Ezzat K, Andaloussi SE, Zaghloul EM, Lehto T, Lindberg S, et al. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Research. 2011;**39**:5284-5298

[61] Farrera-Sinfreu J, Giralt E, Castel S, Albericio F, Royo M. Cellpenetrating cis-gamma-amino-lproline-derived peptides. Journal of the American Chemical Society. 2005;**127**:9459-9468

[62] Angeles-Boza AM, Erazo-Oliveras A, Lee YJ, Pellois JP. Generation of endosomolytic reagents by branching of cell-penetrating peptides: Tools for the delivery of bioactive compounds to live cells in cis or trans. Bioconjugate Chemistry. 2010;**21**:2164-2167

[63] Hogset A, Prasmickaite L, Selbo PK, Hellum M, Engesaeter BO, et al. Photochemical internalisation in drug and gene delivery. Advanced Drug Delivery Reviews. 2004;**56**:95-115

[64] Liu BR, Huang YW, Aronstam RS, Lee HJ. Identification of a short cell-penetrating peptide from bovine Lactoferricin for intracellular delivery of DNA in human A549 cells. PLoS One. 2016;**11**:e0150439

[65] Chang M, Chou JC, Chen CP, Liu BR, Lee HJ. Noncovalent protein transduction in plant cells by macropinocytosis. The New Phytologist. 2007;**174**:46-56

[66] Chang M, Chou JC, Lee HJ. Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant & Cell Physiology. 2005;**46**:482-488

[67] Hu JW, Liu BR, Wu CY, Lu SW, Lee HJ. Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides. Peptides. 2009;**30**:1669-1678

[68] Wang YH, Hou YW, Lee HJ. An intracellular delivery method for siRNA by an arginine-rich peptide. Journal of Biochemical and Biophysical Methods. 2007;**70**:579-586

[69] Hou YW, Chan MH, Hsu HR, Liu BR, Chen CP, et al. Transdermal delivery of proteins mediated by non-covalently associated argininerich intracellular delivery peptides. Experimental Dermatology. 2007;**16**:999-1006

[70] Liu BR, Chou JC, Lee HJ. Cell membrane diversity in noncovalent protein transduction. The Journal of Membrane Biology. 2008;**222**:1-15

[71] Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, et al. A gene delivery system for insect cells mediated by argininerich cell-penetrating peptides. Gene. 2012;**493**:201-210

[72] Dai YH, Liu BR, Chiang HJ, Lee HJ. Gene transport and expression by arginine-rich cell-penetrating peptides in paramecium. Gene. 2011;**489**:89-97

[73] Liu BR, Liou JS, Chen YJ, Huang YW, Lee HJ. Delivery of nucleic acids, proteins, and nanoparticles by argininerich cell-penetrating peptides in rotifers. Marine Biotechnology (New York, N.Y.). 2013;**15**:584-595

[74] Liu BR, Liou JS, Huang YW, Aronstam RS, Lee HJ. Intracellular delivery of nanoparticles and DNAs by IR9 cell-penetrating peptides. PLoS One. 2013;**8**:e64205

[75] Liu BR, Lin MD, Chiang HJ, Lee HJ. Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene. 2012;**505**:37-45

[76] Sangtani A, Petryayeva E, Wu M, Susumu K, Oh E, et al. Intracellularly actuated quantum dot-peptidedoxorubicin nanobioconjugates for controlled drug delivery via the endocytic pathway. Bioconjugate Chemistry. 2018;**29**:136-148

[77] Huang YW, Lee HJ, Liu BR, Chiang HJ, Wu CH. Cellular internalization of quantum dots. Methods in Molecular Biology. 2013;**991**:249-259

[78] Liu BR, Chen HH, Chan MH, Huang YW, Aronstam RS, et al. Three argininerich cell-penetrating peptides facilitate cellular internalization of red-emitting quantum dots. Journal of Nanoscience and Nanotechnology. 2015;**15**:2067-2078

[79] Anjusha A, Jyothibabu R, Jagadeesan L, Arunpandi N. Role of rotifers in microzooplankton community in a large monsoonal estuary (Cochin backwaters) along the west coast of India. Environmental Monitoring and Assessment. 2018;**190**:295

[80] Jeong CB, Kang HM, Lee YH, Kim MS, Lee JS, et al. Nanoplastic ingestion enhances toxicity of persistent

**47**

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple…*

[89] Liu BR, Huang YW, Aronstam RS, Lee HJ. Comparative mechanisms of protein transduction mediated by cellpenetrating peptides in prokaryotes. Journal of Membrane Biology.

2015;**248**:355-368

*DOI: http://dx.doi.org/10.5772/intechopen.83818*

organic pollutants (POPs) in the monogonont rotifer *Brachionus koreanus* via multixenobiotic resistance (MXR) disruption. Environmental Science & Technology. 2018;**52**:11411-11418

[81] Manfra L, Rotini A, Bergami E, Grassi G, Faleri C, et al. Comparative

ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer *Brachionus plicatilis*. Ecotoxicology and Environmental

Safety. 2017;**145**:557-563

2018;**6**:138

2015;**35**:66-72

2001;**4**:103-109

1977;**74**:5088-5090

[82] Dahms HU, Hagiwara A, Lee JS. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquatic Toxicology. 2011;**101**:1-12

[83] Dalcin Martins P, Danczak RE, Roux S, Frank J, Borton MA, et al. Viral and metabolic controls on high rates of microbial sulfur and carbon cycling in wetland ecosystems. Microbiome.

[84] Barberan A, Casamayor EO, Fierer N. The microbial contribution

[85] Gibbons SM, Gilbert JA. Microbial diversity—Exploration of natural ecosystems and microbiomes. Current Opinion in Genetics & Development.

[86] Guerrero R. Bergey's manuals and the classification of prokaryotes.

[87] Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America.

International Microbiology.

[88] Wilson N. Nanoparticles: Environmental problems or problem solvers? Bioscience. 2018;**68**:241-246

to macroecology. Frontiers in Microbiology. 2014;**5**:203

*Hypotoxic Fluorescent Nanoparticles Delivery by Cell-Penetrating Peptides in Multiple… DOI: http://dx.doi.org/10.5772/intechopen.83818*

organic pollutants (POPs) in the monogonont rotifer *Brachionus koreanus* via multixenobiotic resistance (MXR) disruption. Environmental Science & Technology. 2018;**52**:11411-11418

*Biotechnology and Bioengineering*

2016;**11**:e0150439

2007;**174**:46-56

2009;**30**:1669-1678

2007;**70**:579-586

2007;**16**:999-1006

2012;**493**:201-210

[64] Liu BR, Huang YW, Aronstam RS, Lee HJ. Identification of a short cell-penetrating peptide from bovine Lactoferricin for intracellular delivery of DNA in human A549 cells. PLoS One. arginine-rich cell-penetrating peptides in paramecium. Gene. 2011;**489**:89-97

[73] Liu BR, Liou JS, Chen YJ, Huang YW, Lee HJ. Delivery of nucleic acids, proteins, and nanoparticles by argininerich cell-penetrating peptides in rotifers. Marine Biotechnology (New York, N.Y.).

[74] Liu BR, Liou JS, Huang YW, Aronstam RS, Lee HJ. Intracellular delivery of nanoparticles and DNAs by IR9 cell-penetrating peptides. PLoS

[75] Liu BR, Lin MD, Chiang HJ, Lee HJ. Arginine-rich cell-penetrating peptides deliver gene into living human

[76] Sangtani A, Petryayeva E, Wu M, Susumu K, Oh E, et al. Intracellularly actuated quantum dot-peptidedoxorubicin nanobioconjugates for controlled drug delivery via the endocytic pathway. Bioconjugate Chemistry. 2018;**29**:136-148

[77] Huang YW, Lee HJ, Liu BR, Chiang HJ, Wu CH. Cellular internalization of quantum dots. Methods in Molecular

[78] Liu BR, Chen HH, Chan MH, Huang YW, Aronstam RS, et al. Three argininerich cell-penetrating peptides facilitate cellular internalization of red-emitting quantum dots. Journal of Nanoscience and Nanotechnology. 2015;**15**:2067-2078

cells. Gene. 2012;**505**:37-45

Biology. 2013;**991**:249-259

[79] Anjusha A, Jyothibabu R, Jagadeesan L, Arunpandi N. Role of rotifers in microzooplankton community in a large monsoonal estuary (Cochin backwaters) along the west coast of India. Environmental

Monitoring and Assessment.

[80] Jeong CB, Kang HM, Lee YH, Kim MS, Lee JS, et al. Nanoplastic ingestion enhances toxicity of persistent

2018;**190**:295

2013;**15**:584-595

One. 2013;**8**:e64205

[65] Chang M, Chou JC, Chen CP, Liu BR, Lee HJ. Noncovalent protein transduction in plant cells by

macropinocytosis. The New Phytologist.

[66] Chang M, Chou JC, Lee HJ. Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant & Cell Physiology. 2005;**46**:482-488

[67] Hu JW, Liu BR, Wu CY, Lu SW, Lee HJ. Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides. Peptides.

[68] Wang YH, Hou YW, Lee HJ. An intracellular delivery method for siRNA by an arginine-rich peptide. Journal of Biochemical and Biophysical Methods.

[69] Hou YW, Chan MH, Hsu HR, Liu BR, Chen CP, et al. Transdermal delivery of proteins mediated by non-covalently associated argininerich intracellular delivery peptides. Experimental Dermatology.

[70] Liu BR, Chou JC, Lee HJ. Cell membrane diversity in noncovalent protein transduction. The Journal of Membrane Biology. 2008;**222**:1-15

[71] Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, et al. A gene delivery system for insect cells mediated by argininerich cell-penetrating peptides. Gene.

[72] Dai YH, Liu BR, Chiang HJ, Lee HJ. Gene transport and expression by

**46**

[81] Manfra L, Rotini A, Bergami E, Grassi G, Faleri C, et al. Comparative ecotoxicity of polystyrene nanoparticles in natural seawater and reconstituted seawater using the rotifer *Brachionus plicatilis*. Ecotoxicology and Environmental Safety. 2017;**145**:557-563

[82] Dahms HU, Hagiwara A, Lee JS. Ecotoxicology, ecophysiology, and mechanistic studies with rotifers. Aquatic Toxicology. 2011;**101**:1-12

[83] Dalcin Martins P, Danczak RE, Roux S, Frank J, Borton MA, et al. Viral and metabolic controls on high rates of microbial sulfur and carbon cycling in wetland ecosystems. Microbiome. 2018;**6**:138

[84] Barberan A, Casamayor EO, Fierer N. The microbial contribution to macroecology. Frontiers in Microbiology. 2014;**5**:203

[85] Gibbons SM, Gilbert JA. Microbial diversity—Exploration of natural ecosystems and microbiomes. Current Opinion in Genetics & Development. 2015;**35**:66-72

[86] Guerrero R. Bergey's manuals and the classification of prokaryotes. International Microbiology. 2001;**4**:103-109

[87] Woese CR, Fox GE. Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences of the United States of America. 1977;**74**:5088-5090

[88] Wilson N. Nanoparticles: Environmental problems or problem solvers? Bioscience. 2018;**68**:241-246

[89] Liu BR, Huang YW, Aronstam RS, Lee HJ. Comparative mechanisms of protein transduction mediated by cellpenetrating peptides in prokaryotes. Journal of Membrane Biology. 2015;**248**:355-368

**49**

**Chapter 5**

**Abstract**

**1. Introduction**

Repair Research

rhBMP-2-Coated Acellular Dermal

A rotator cuff tear is a common shoulder injury in sports medicine. However, a

) rotator

rotator cuff repair still has the high failure rate (57%) in large torn (>8 cm2

cuff cases. One of the main reasons is failing at suture-tendon cause of continuous tensional and torsional stresses even after surgery, and thus, an ideal biologic augmentation to overcome large tears is an essential challenge. The ECM graft, the biological material can be useful for augment repair of large torn rotator cuff. Recombinant human bone morphogenetic protein 2 (rhBMP-2), which belongs to transforming growth factor-β superfamily, is well known as an osteoinductive growth factor. It plays an important role in the development of bone and cartilage. rhBMP-2 also facilitates chemotaxis in the host tissue. In this study, rhBMP-2 coated acellular dermal graft, which is isolated from human cadaveric donor, was transplanted in the rabbit with the chronic rotator cuff injury. The radiologic image, histomorphometric, histologic image analyses, and tensile test were performed to evaluate the effectiveness. The results showed the enhancement of increased host cell infiltration, new bone formation, and tensile mechanical property. The rhBMP-2-coated acellular dermal graft will be promising for chronic rotator cuff healing.

**Keywords:** rotator cuff, rhBMP-2, acellular dermis, tendon-bone healing, enthesis

A rotator cuff plays an important role in shoulder movements and maintaining shoulder joint stability [1]. The rotator cuff is composed of supraspinatus muscle, subscapularis muscle, infraspinatus muscle, and teres minor muscle. The rotator cuff is connected between the scapula and the humeral bone head, forming a cuff at the shoulder joint. The rotator cuff tear, which is partial tear and full-thickness tear, mainly occurs in the supraspinatus tendon under the acromion [2]. A torn rotator cuff weakens shoulder's physical function. Almost 2 million people in the United States visit to see doctors cause of the painful rotator cuff at every year [3]. The rotator cuff repair requires reattachment of the rotator cuff tendon back to the humeral bone by using suture and fixation tools, which is non-biological technique [4, 5]. Rotator cuff repair has showed good long-term follow-up results so far. However, it still shows the high failure rate (57%) in the case of large tear

Graft for Chronic Rotator Cuff

Healing: Translational Tendon

*Kwang-Il Lee, Ju-Woong Jang and Kwang-Won Lee*

#### **Chapter 5**
