2. Carbon nanostructures

1. Introduction

234 Cytotoxicity

Nanostructured materials are defined as possessing one of their dimensions ranging from 1 to 100 nm, according to the American Society for Testing and Materials (ASTM) international standards definition [1]. For nanoparticles (NPs), which can be of more or less spherical or cubical shape, two dimensions are required to be within this range. In contrast, the shape of nanorods (NRs) is in one dimension much larger than in the others. For a small aspect ratio (<10) both their length and diameter are in the nanoscale, whereas NRs with a large aspect ratio (>10) only have their diameter within this scale, and they are often called "nanowires" (NWs). Nanostructures within this specific size scale show unique size-dependent optical, magnetic, catalytic and electrochemical properties, among others, as well as high surface to volume ratios. Moreover, their shape, surface chemistry and chemical composition can be used to tailor-specific

The size scale of nanostructures is within the range of several biomolecules, such as proteins and antibodies, allowing specific interactions to occur between them. This, when coupled with the high surface to volume ratios and tunable sizes and properties, makes nanostructures prime candidates for biomedical applications such as imaging, drug delivery and therapy [4–6]. Examples of applications include the use of NPs as magnetic resonance imaging (MRI) contrast agents [7, 8], tissue engineering [9–11], as well as the recent focus on hyperthermia and cancer cell eradication with the use of NPs and NRs [12–17]. Such applications, if they are aimed for a clinical setting, ultimately require a direct NP/NR exposure in the form of ingestion or intravenous delivery into the body. Naturally, there is a rigorous testing required before any new drug formulation is approved for clinical use in order to ensure their safety and effectiveness. Currently, very few NPs-based drugs have been approved by the Food and Drug Administration and are commercially available. Examples include GastroMARK, used as an MRI contrast agent to enhance the delineation of the bowel, and ferumoxytol, an iron-replacement formulation

Within this scope, biocompatibility and cytotoxicity data are of paramount importance to evaluate the potential of nanostructures for biomedical applications. Nanostructures are normally engineered to interface and target-specific cells or tissues to execute their functions, raising questions about their toxicological effects. For instance, there are several characteristics involved in the toxicity of fiber-like nanomaterials, such as shape, length, chemical composition, agglomeration and purity, making them suitable to fit the "fiber toxicological paradigm" according to the World Health Organization (WHO) criteria used to describe the toxicity of asbestos fibers [19]. Further, nanostructures are usually tuned for biocompatibility on top of the desired biomedical function, with the most relevant aspects that influence their toxicity being the material [20], size and shape [21], surface charge [22] and surface functionalization [23]. In vitro studies, while not able to give a complete insight into the biocompatibility of nanostructures, have a high importance, due to their easy implementation, and provide valuable cytotoxicology data regarding the safety of the use of nanostructures in biomedical applications. Previously published reviews regarding the biosafety of nanostructures include that of Lewinski et al. [24] and Zhao et al. [25]. The former focuses mostly on the cytotoxicity of

properties, making nanostructures highly versatile for different applications [2, 3].

approved for adults with chronic kidney disease with an iron deficiency [18].

Carbon nanostructures include a broad diversity of carbon allotropes that differ from pristine diamond and graphite. Carbon has been used in many technological applications, exploiting its capability of forming networks composed exclusively of C-atoms with the same electronic configuration or hybridizing configurations sp3 -, sp2 - and sp-, expanding the possible allotropes that can be constructed [26]. Since the synthesis of the first carbon nanostructures, such as fullerene C60 (0D) [27] and CNTs (1D) [28] (Figure 2), there has been a tremendous effort for understanding the properties of these nanomaterials and for exploring the broad range of applications in which they can be used. Carbon-based nanomaterials (CNMs) have created a great deal of interest in various applications such as optical imaging [29], drug and gene delivery [30], and nanotherapeutics [31, 32] due to their excellent mechanical, optical and electrical

Figure 2. Chemical structure of representative carbon-based nanomaterials. Structure of fullerene C60 (A) and carbon nanotube (B).

properties [33–35], as well as due to their ability to translocate through the cell membrane or be internalized via energy-dependent endocytic pathways [36]. Similarly, CNMs possess an extraordinary ability to be loaded with drugs or different chemical agents that are either attached to the surface or, in the case of CNTs, they can be packed into the interior cores [37].

These widespread applications of CNMs are also accompanied by increasing concerns regarding their interactions with tissues, cells, and biomolecules as well as degradation pathways, and at a macroscale, the potential deleterious effects on human health and the environment.

## 2.1. Fullerene C60

The structure of fullerene C60, which has a van der Walls diameter of approximately 1 nm, is formed from 60 carbon atoms arranged in a spherical, cage-like structure consisting of 60 vertices, 12 pentagonal faces and 20 hexagonal faces [38]. Fullerenes and their derivatives are probably the most extensively studied NPs with several properties and applications including MRI [39], drug delivery [40, 41], photodynamic therapy (PDT) [40] and photothermal therapy (PTT) [42].

dRLh-84, HepG2 and rat hepatocytes as shown in Figure 3 [46]. C60(OH)6–<sup>12</sup> and C60(OH)12 were found to induce cytotoxic effects after 3 days of exposure in dRLh-84 cells at a concentration of 10 μg/mL reducing the cell viability 30 and 40%, respectively, in the form of inhibition of mitochondrial activity. Similarly, to Nakawaga's findings, these results indicate that the number of hydroxyl groups on C60(OH)<sup>x</sup> contributes to the cytotoxic potential and mitochon-

Figure 3. Cytotoxicity of fullerene and hydroxylated fullerenes in liver cells. HepG2 (A); dRLh-84 (B); and primary cultured rat hepatocytes (C) were exposed to C60, C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36 for 3 days. Data are represented as mean SD (n = 3). (\*) statistically significant from control (p < 0.05). Adapted from Shimizu et al. [46].

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Copyright 2013 by the authors. Licensee MDPI, Basel, Switzerland. CC BY 3.0.

Other fullerene derivatives have also been tested for cytotoxic effects in human epithelial HEp-2 cells, such as C60-PVP, C60-NO2-proline and sodium salt of polycarboxylic C60 [47]. However, the PVP and NO2-proline derivatives did not have an effect on cell viability, and the sodium salt of polycarboxylic derivative induced a drastic decrease in cell number of about 80% at a

Further, the molecular mechanisms underlying the cytotoxic effects of two similar fullerene derivatives (C60-1,3-dipolar cycloaddition of azomethine ylides) on human MCF-7 cells were analyzed by RNA-seq-based gene expression [44]. It was found that whereas one derivative had a negligible effect, the addition of an extra trifluoroacetate group induced a significant, time-dependent alteration of gene expression, mainly in biological processes involving protein synthesis, cell cycle progression and cell adhesion, with the authors suggesting an inhibition

drial damage.

concentration around 0.1 mg/mL.

effect of the mTOR pathway.

Although fullerenes are generally hydrophobic molecules, many strategies have been developed for improving their solubilization in water that is, synthesized water-soluble derivatives of fullerenes by chemical modifications through the addition of functional groups such as hydroxyl-, carboxyl-, amino- and alkyl-groups and other side-chain/cyclic moieties to the C60 structure [43]. The different methods employed to increase C60 water solubility profoundly influence the physiochemical properties and the toxicological effects of these compounds, raising uncertainties about the possible consequences on human health and potential medical uses [44]. Nakagawa et al. studied the effects of the hydroxylated fullerenes (fullerenols) C60(OH)24 and C60(OH)12 0.125 mM in rat hepatocytes, observing a concentration and timedependent cell death accompanied by mitochondrial dysfunction, with C60(OH)24 found to be more cytotoxic with almost 100% of cell death after 30 min. The authors concluded that the toxic effects of fullerenols may depend on the number of hydroxyl groups [38]. C60(OH)24 at a concentration of 0.1 mM caused cell blebbing, loss of cellular ATP and lipid peroxidation in rat hepatocytes [45]. Similarly, the cytotoxic effects of fullerene C60 and the derivatives C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36, were evaluated in three different types of liver cells: Review of In Vitro Toxicity of Nanoparticles and Nanorods—Part 2 http://dx.doi.org/10.5772/intechopen.78616 237

properties [33–35], as well as due to their ability to translocate through the cell membrane or be internalized via energy-dependent endocytic pathways [36]. Similarly, CNMs possess an extraordinary ability to be loaded with drugs or different chemical agents that are either attached to the

Figure 2. Chemical structure of representative carbon-based nanomaterials. Structure of fullerene C60 (A) and carbon

These widespread applications of CNMs are also accompanied by increasing concerns regarding their interactions with tissues, cells, and biomolecules as well as degradation pathways, and at a macroscale, the potential deleterious effects on human health and the environment.

The structure of fullerene C60, which has a van der Walls diameter of approximately 1 nm, is formed from 60 carbon atoms arranged in a spherical, cage-like structure consisting of 60 vertices, 12 pentagonal faces and 20 hexagonal faces [38]. Fullerenes and their derivatives are probably the most extensively studied NPs with several properties and applications including MRI [39], drug delivery [40, 41], photodynamic therapy (PDT) [40] and photothermal therapy

Although fullerenes are generally hydrophobic molecules, many strategies have been developed for improving their solubilization in water that is, synthesized water-soluble derivatives of fullerenes by chemical modifications through the addition of functional groups such as hydroxyl-, carboxyl-, amino- and alkyl-groups and other side-chain/cyclic moieties to the C60 structure [43]. The different methods employed to increase C60 water solubility profoundly influence the physiochemical properties and the toxicological effects of these compounds, raising uncertainties about the possible consequences on human health and potential medical uses [44]. Nakagawa et al. studied the effects of the hydroxylated fullerenes (fullerenols) C60(OH)24 and C60(OH)12 0.125 mM in rat hepatocytes, observing a concentration and timedependent cell death accompanied by mitochondrial dysfunction, with C60(OH)24 found to be more cytotoxic with almost 100% of cell death after 30 min. The authors concluded that the toxic effects of fullerenols may depend on the number of hydroxyl groups [38]. C60(OH)24 at a concentration of 0.1 mM caused cell blebbing, loss of cellular ATP and lipid peroxidation in rat hepatocytes [45]. Similarly, the cytotoxic effects of fullerene C60 and the derivatives C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36, were evaluated in three different types of liver cells:

surface or, in the case of CNTs, they can be packed into the interior cores [37].

2.1. Fullerene C60

nanotube (B).

236 Cytotoxicity

(PTT) [42].

Figure 3. Cytotoxicity of fullerene and hydroxylated fullerenes in liver cells. HepG2 (A); dRLh-84 (B); and primary cultured rat hepatocytes (C) were exposed to C60, C60(OH)2, C60(OH)6–12, C60(OH)12 and C60(OH)36 for 3 days. Data are represented as mean SD (n = 3). (\*) statistically significant from control (p < 0.05). Adapted from Shimizu et al. [46]. Copyright 2013 by the authors. Licensee MDPI, Basel, Switzerland. CC BY 3.0.

dRLh-84, HepG2 and rat hepatocytes as shown in Figure 3 [46]. C60(OH)6–<sup>12</sup> and C60(OH)12 were found to induce cytotoxic effects after 3 days of exposure in dRLh-84 cells at a concentration of 10 μg/mL reducing the cell viability 30 and 40%, respectively, in the form of inhibition of mitochondrial activity. Similarly, to Nakawaga's findings, these results indicate that the number of hydroxyl groups on C60(OH)<sup>x</sup> contributes to the cytotoxic potential and mitochondrial damage.

Other fullerene derivatives have also been tested for cytotoxic effects in human epithelial HEp-2 cells, such as C60-PVP, C60-NO2-proline and sodium salt of polycarboxylic C60 [47]. However, the PVP and NO2-proline derivatives did not have an effect on cell viability, and the sodium salt of polycarboxylic derivative induced a drastic decrease in cell number of about 80% at a concentration around 0.1 mg/mL.

Further, the molecular mechanisms underlying the cytotoxic effects of two similar fullerene derivatives (C60-1,3-dipolar cycloaddition of azomethine ylides) on human MCF-7 cells were analyzed by RNA-seq-based gene expression [44]. It was found that whereas one derivative had a negligible effect, the addition of an extra trifluoroacetate group induced a significant, time-dependent alteration of gene expression, mainly in biological processes involving protein synthesis, cell cycle progression and cell adhesion, with the authors suggesting an inhibition effect of the mTOR pathway.

In a recent study performed by Canape et al., C60 fullerenes were covalently functionalized with PEG of various sizes, Full–PEG2000, Full–PEG5000 and Full–PEG10000, and viability was studied on a variety of cell lines 24 h after exposure, evaluating mitochondrial activity, cell membrane integrity and hemolysis [48]. However, all the tested compounds were found to reduce, to some extent, the cellular metabolic activity, only two affected the cell membrane integrity, and none induced hemolysis. It was concluded that fullerenes C60 functionalized with higher molecular weight PEGs possess a higher biocompatibility and that side toxicity can be alleviated using proper surface coating. Together, all these findings support that the surface functionalization of fullerenes plays an important role with regard to their interaction with biological systems.

SWCNTs functionalized with PEG has been evaluated with neuronal PC12 cells at the biochemical, cellular and gene expression levels by Zhang et al. [61]. Cytotoxicity increased with the concentration, whereby SWCNT-PEGs exhibited less cytotoxic potency than bare SWCNTs at the highest concentration tested by reducing the cell viability in approximately 70 and 50%, respectively (Figure 4). Morphological changes appeared in PC12 cells treated with both SWCNTs and SWCNTs-PEG as shown in Figure 5. Cells exposed to SWCNTs showed an elongated shape, which was related to higher toxic effects induced by the untreated CNTs. ROS were generated as a function of both concentration and surface coating after exposure, whereas gene expression analysis showed that the genes involved in oxidoreductases and antioxidant activity, nucleic acid or lipid metabolism and mitochondria dysfunction were highly altered. Interestingly, alteration of the genes was also surface coating-dependent. The authors concluded that surface functionalization of SWCNTs decreases the ROS-mediated toxicological response in vitro, corroborating the relevance of surface functionalization in the interaction between nanostructures and biological systems. Likewise, proteins such as type I

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Figure 4. Cytotoxic effect of SWCNTs and SWCNTs-PEG in PC12 cells. Mitochondrial toxicity and membrane damage of neuronal cells incubated with different concentrations of pristine SWCNTs and PEG-coated SWCNTs for 24 h evaluated by MTT (A), XTT (B) and LDH (C) assays. Data are expressed as mean standard error (n = 3). (\*) statistically significant from control; (#) indicates statistically significant within the same concentration group (p < 0.05). Adapted with permis-

sion from Zhang et al. [61]. Copyright 2011 American Chemical Society.

The interaction of CNMs with lipid membranes is of great interest because biological activity requires crossing or breaking lipid membranes. In a study concerning the interaction of fullerenes with the lipid bilayer and the possibility of fullerene crossing it, it was observed that hydrophobic molecules of C60 were localized within the inner part of the membrane, whereas hydrophilic C60(OH)n fullerenols molecules were adsorbed on the heads of membrane phospholipids [49], where they can interact with membrane proteins, such as ATPases and influence their activity [50, 51]. Similarly, Raoof et al. showed that the internalization of a watersoluble derivatized C60 malonodiserinolamide takes place through multiple energy-dependent pathways, and they escape endocytotic vesicles to eventually localize and accumulate in the nucleus through the nuclear pore complex [41].

#### 2.2. Single-walled carbon nanotubes

CNTs are classified in single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). The first ones are formed from a single layer of graphene (0.4–10 nm in diameter), whereas the second ones consist of multiple concentric cylinders of graphene with increasing diameters (10–100 nm) [52]. The length of CNTs can range from nanometers to centimeters [53], and they possess unique physical and chemical properties such as a lightweight, high tensile strength, high electrical and thermal conductivities, unique optical properties and extreme chemical stability, as well as high surface-to-volume ratios with reactive surface chemistries. Such properties have made CNTs an interesting material for biomedical applications, where they have been used as drug, protein and nucleic acid delivery tools [54–56], cancer cell destruction [57, 64, 91], diagnostics [59] and as noninvasive and highly sensitive imaging aids [31, 58]. Naturally, biosafety concerns of CNTs are rapidly emerging with numerous reports indicating their potential hazards to the public health.

The graphene sheets can be wrapped in a variety of ways that are denoted by a pair of indices (n, m), which define both the diameter and the chirality of SWCNTs, which can be either metallic (M) or semiconducting (S). As synthesized, SWNTs have a wide range of diameters and chiral angles, which leads to a polydisperse sample of discrete properties [59, 60]. SWCNTs possess small diameters and the large aspect ratios that render them ideal onedimensional quantum wires that elicit different biological behavior compared to spherical NPs, when introduced in biological systems [26]. The cytotoxicity of pristine SWCNTs and SWCNTs functionalized with PEG has been evaluated with neuronal PC12 cells at the biochemical, cellular and gene expression levels by Zhang et al. [61]. Cytotoxicity increased with the concentration, whereby SWCNT-PEGs exhibited less cytotoxic potency than bare SWCNTs at the highest concentration tested by reducing the cell viability in approximately 70 and 50%, respectively (Figure 4). Morphological changes appeared in PC12 cells treated with both SWCNTs and SWCNTs-PEG as shown in Figure 5. Cells exposed to SWCNTs showed an elongated shape, which was related to higher toxic effects induced by the untreated CNTs. ROS were generated as a function of both concentration and surface coating after exposure, whereas gene expression analysis showed that the genes involved in oxidoreductases and antioxidant activity, nucleic acid or lipid metabolism and mitochondria dysfunction were highly altered. Interestingly, alteration of the genes was also surface coating-dependent. The authors concluded that surface functionalization of SWCNTs decreases the ROS-mediated toxicological response in vitro, corroborating the relevance of surface functionalization in the interaction between nanostructures and biological systems. Likewise, proteins such as type I

In a recent study performed by Canape et al., C60 fullerenes were covalently functionalized with PEG of various sizes, Full–PEG2000, Full–PEG5000 and Full–PEG10000, and viability was studied on a variety of cell lines 24 h after exposure, evaluating mitochondrial activity, cell membrane integrity and hemolysis [48]. However, all the tested compounds were found to reduce, to some extent, the cellular metabolic activity, only two affected the cell membrane integrity, and none induced hemolysis. It was concluded that fullerenes C60 functionalized with higher molecular weight PEGs possess a higher biocompatibility and that side toxicity can be alleviated using proper surface coating. Together, all these findings support that the surface functionalization of fullerenes plays an important role with regard to their interaction

The interaction of CNMs with lipid membranes is of great interest because biological activity requires crossing or breaking lipid membranes. In a study concerning the interaction of fullerenes with the lipid bilayer and the possibility of fullerene crossing it, it was observed that hydrophobic molecules of C60 were localized within the inner part of the membrane, whereas hydrophilic C60(OH)n fullerenols molecules were adsorbed on the heads of membrane phospholipids [49], where they can interact with membrane proteins, such as ATPases and influence their activity [50, 51]. Similarly, Raoof et al. showed that the internalization of a watersoluble derivatized C60 malonodiserinolamide takes place through multiple energy-dependent pathways, and they escape endocytotic vesicles to eventually localize and accumulate in the

CNTs are classified in single-walled carbon nanotubes (SWCNTs) and multiwalled carbon nanotubes (MWCNTs). The first ones are formed from a single layer of graphene (0.4–10 nm in diameter), whereas the second ones consist of multiple concentric cylinders of graphene with increasing diameters (10–100 nm) [52]. The length of CNTs can range from nanometers to centimeters [53], and they possess unique physical and chemical properties such as a lightweight, high tensile strength, high electrical and thermal conductivities, unique optical properties and extreme chemical stability, as well as high surface-to-volume ratios with reactive surface chemistries. Such properties have made CNTs an interesting material for biomedical applications, where they have been used as drug, protein and nucleic acid delivery tools [54–56], cancer cell destruction [57, 64, 91], diagnostics [59] and as noninvasive and highly sensitive imaging aids [31, 58]. Naturally, biosafety concerns of CNTs are rapidly emerging with numerous reports

The graphene sheets can be wrapped in a variety of ways that are denoted by a pair of indices (n, m), which define both the diameter and the chirality of SWCNTs, which can be either metallic (M) or semiconducting (S). As synthesized, SWNTs have a wide range of diameters and chiral angles, which leads to a polydisperse sample of discrete properties [59, 60]. SWCNTs possess small diameters and the large aspect ratios that render them ideal onedimensional quantum wires that elicit different biological behavior compared to spherical NPs, when introduced in biological systems [26]. The cytotoxicity of pristine SWCNTs and

with biological systems.

238 Cytotoxicity

nucleus through the nuclear pore complex [41].

indicating their potential hazards to the public health.

2.2. Single-walled carbon nanotubes

Figure 4. Cytotoxic effect of SWCNTs and SWCNTs-PEG in PC12 cells. Mitochondrial toxicity and membrane damage of neuronal cells incubated with different concentrations of pristine SWCNTs and PEG-coated SWCNTs for 24 h evaluated by MTT (A), XTT (B) and LDH (C) assays. Data are expressed as mean standard error (n = 3). (\*) statistically significant from control; (#) indicates statistically significant within the same concentration group (p < 0.05). Adapted with permission from Zhang et al. [61]. Copyright 2011 American Chemical Society.

2.3. Multiwalled carbon nanotubes

ation state and charge of the CNTs [73].

ibility in various biological environments [65].

MWCNTs are defined as a nested coaxial array of SWCNTs, each nanotube being formed by a

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It has been postulated that MWCNTs can provide an innovative and promising alternative to conventional drug formulations for cancer therapy, as they can be conjugated with various bioactive molecules such as drugs, surfactants, diagnostic agents and antibodies in order to

The generation of carboxyl groups by oxidation on the surface of CNTs is one of the most used strategies for introducing hydrophilic moieties onto the CNT hydrophobic surface and in order to conquer a lack of solubility and to improve their biocompatibility [69–71]. Thus, Liu et al. have studied the effects of carboxylated c-MWCNTs on the human normal liver cell line L02 and found a reduction in the toxicity, when compared to pristine MWCNTs with a reduction of around 60% of cell viability at the highest concentration tested after 72 h and concluded that this effect is probably due to a reduced activation of the mitochondria mediated apoptotic pathway [72]. Moreover, as charged entities, c-MWCNTs bind to proteins in the bloodstream through noncovalent interactions to form a protein corona. De Paoli et al. have characterized the interactions of c-MWCNTs with common human proteins such as albumin, fibrinogen, gimmunoglobulins and histone H1 and found that the association of proteins to c-MWCNTs depends on the protein's charge, size and structural flexibility and that it affects the agglomer-

As with SWCNTs, molecules can be covalently and noncovalently attached to the surface of MWCNTs [74]. The main disadvantage of noncovalent attachment is the lack of biomolecule specificity upon adsorption, which affects the CNTs dispersion stability by replacing the functional surface coating with proteins and molecules contained in all physiological fluids (cell culture media or blood) [65]. Heister et al. have compared five types of CNTs, varying in their dimensions and surface properties, for a multidimensional analysis of dispersion stability and their toxicity toward cancer cells (Figure 6), from which it was emphasized that the covalent link between PEG and oxidized MWCNTs leads to stable dispersion and biocompat-

It has been proposed that the metal impurities trapped inside the MWCNTs may be responsible for their toxicity that partially occurs through the generation of ROS [75]. Fe impurities trapped inside the MWCNTs may be partially responsible for neurotoxicity, as postulated by Meng et al., who investigated and compared the effects of two kinds of MWCNTs with different concentrations of Fe impurities in rat pheochromocytoma PC-12 cells [76]. They found that the exposure to Fe MWCNTs can reduce cell viability up to 80% after 72 h exposure and increase cytoskeletal disruption of undifferentiated PC-12 cells, diminish the ability to form mature neurites and then

Additionally, MWCNTs have been shown to affect the immune system. Pescatori et al. used a whole-genome expression approach to assess whether functionalized MWCNTs could stimulate

adversely influence the neuronal dopaminergic phenotype in NGF-treated cells.

graphene sheet rolled into a cylinder of nanometer size diameter [65].

target receptors that are overexpressed in cancer cells [66–68].

Figure 5. Morphological changes of PC12 cells after 24 h incubation with SWCNTs and SWCNTs-PEG. (A) Normal morphology of the PC12 cells. (B) PC12 cells incubated with SWCNTs present a spindle shape (arrows). (C) SWCNT-PEGs inhibit the dendrite growth (arrows). Adapted with permission from Zhang et al. [61]. Copyright 2011 American Chemical Society.

collagen have shown great potential as surface coting agents in SWCNTs, showing no obvious negative cellular effects and with a high level of internalization taking place through adsorption by the extracellular matrix in bovine articular chondrocytes [62].

Avti et al. showed that SWCNTs synthesized using Gd3+ NPs as catalysts induced no structural damage to NIH/3T3 fibroblasts or decreased their viability at concentrations between 1 and 10 μg/mL [53]. In contrast, highly pure SWCNTs triggered similar amounts of pulmonary fibrosis-related compounds interleukin 1β (IL-1β) and transforming growth factor (TGF-β1) in THP-1 and BEAS-2B pulmonary cells without affecting cell viability [63]. Similarly, Di Giorgio et al. studied the cyto- and genotoxic effects, as well as the inflammatory response and ROS production, of SWCNTs on the mouse macrophage cell line RAW 264.7 [64]. There, the authors reported that SWCNTs induced ROS release, cell ultrastructural damage, necrosis and chromosomal aberrations, but did not cause an inflammatory response.

## 2.3. Multiwalled carbon nanotubes

collagen have shown great potential as surface coting agents in SWCNTs, showing no obvious negative cellular effects and with a high level of internalization taking place through adsorp-

Figure 5. Morphological changes of PC12 cells after 24 h incubation with SWCNTs and SWCNTs-PEG. (A) Normal morphology of the PC12 cells. (B) PC12 cells incubated with SWCNTs present a spindle shape (arrows). (C) SWCNT-PEGs inhibit the dendrite growth (arrows). Adapted with permission from Zhang et al. [61]. Copyright 2011 American

Avti et al. showed that SWCNTs synthesized using Gd3+ NPs as catalysts induced no structural damage to NIH/3T3 fibroblasts or decreased their viability at concentrations between 1 and 10 μg/mL [53]. In contrast, highly pure SWCNTs triggered similar amounts of pulmonary fibrosis-related compounds interleukin 1β (IL-1β) and transforming growth factor (TGF-β1) in THP-1 and BEAS-2B pulmonary cells without affecting cell viability [63]. Similarly, Di Giorgio et al. studied the cyto- and genotoxic effects, as well as the inflammatory response and ROS production, of SWCNTs on the mouse macrophage cell line RAW 264.7 [64]. There, the authors reported that SWCNTs induced ROS release, cell ultrastructural damage, necrosis and chro-

tion by the extracellular matrix in bovine articular chondrocytes [62].

Chemical Society.

240 Cytotoxicity

mosomal aberrations, but did not cause an inflammatory response.

MWCNTs are defined as a nested coaxial array of SWCNTs, each nanotube being formed by a graphene sheet rolled into a cylinder of nanometer size diameter [65].

It has been postulated that MWCNTs can provide an innovative and promising alternative to conventional drug formulations for cancer therapy, as they can be conjugated with various bioactive molecules such as drugs, surfactants, diagnostic agents and antibodies in order to target receptors that are overexpressed in cancer cells [66–68].

The generation of carboxyl groups by oxidation on the surface of CNTs is one of the most used strategies for introducing hydrophilic moieties onto the CNT hydrophobic surface and in order to conquer a lack of solubility and to improve their biocompatibility [69–71]. Thus, Liu et al. have studied the effects of carboxylated c-MWCNTs on the human normal liver cell line L02 and found a reduction in the toxicity, when compared to pristine MWCNTs with a reduction of around 60% of cell viability at the highest concentration tested after 72 h and concluded that this effect is probably due to a reduced activation of the mitochondria mediated apoptotic pathway [72]. Moreover, as charged entities, c-MWCNTs bind to proteins in the bloodstream through noncovalent interactions to form a protein corona. De Paoli et al. have characterized the interactions of c-MWCNTs with common human proteins such as albumin, fibrinogen, gimmunoglobulins and histone H1 and found that the association of proteins to c-MWCNTs depends on the protein's charge, size and structural flexibility and that it affects the agglomeration state and charge of the CNTs [73].

As with SWCNTs, molecules can be covalently and noncovalently attached to the surface of MWCNTs [74]. The main disadvantage of noncovalent attachment is the lack of biomolecule specificity upon adsorption, which affects the CNTs dispersion stability by replacing the functional surface coating with proteins and molecules contained in all physiological fluids (cell culture media or blood) [65]. Heister et al. have compared five types of CNTs, varying in their dimensions and surface properties, for a multidimensional analysis of dispersion stability and their toxicity toward cancer cells (Figure 6), from which it was emphasized that the covalent link between PEG and oxidized MWCNTs leads to stable dispersion and biocompatibility in various biological environments [65].

It has been proposed that the metal impurities trapped inside the MWCNTs may be responsible for their toxicity that partially occurs through the generation of ROS [75]. Fe impurities trapped inside the MWCNTs may be partially responsible for neurotoxicity, as postulated by Meng et al., who investigated and compared the effects of two kinds of MWCNTs with different concentrations of Fe impurities in rat pheochromocytoma PC-12 cells [76]. They found that the exposure to Fe MWCNTs can reduce cell viability up to 80% after 72 h exposure and increase cytoskeletal disruption of undifferentiated PC-12 cells, diminish the ability to form mature neurites and then adversely influence the neuronal dopaminergic phenotype in NGF-treated cells.

Additionally, MWCNTs have been shown to affect the immune system. Pescatori et al. used a whole-genome expression approach to assess whether functionalized MWCNTs could stimulate

Figure 6. MTT cytotoxicity assay on WiDr human colon cancer cells after being incubated for 96 h with various samples of oxCNTs. No dose-dependent cytotoxicity is observed at this concentration as shown in the range dose-response curves for the five different types of CNTs, displaying (A). Cell viability percentage plot for Nanolab oxidated SWNTs with different surface functionalizations, where PEGylation results in a statistically significant enhancement in cell viability. The cells control correlates with 100% cell viability. Adapted with permission from Heister et al. [74]. Copyright 2010 American Chemical Society.

through apoptosis, triggered by the activation of caspase-3 and chromatin condensation

necrosis. Adapted with permission from Jin et al. [80]. Copyright 2008 American Chemical Society.

Figure 7. DNA-binding acridine orange staining of L929 mouse fibroblast cells. (A) Control cells with no TiO2 NPs show normal green nuclei with an organized cellular structure; (B) Cells cultured with 30 μg/mL of TiO2 NPs show weakly condensed chromatin; and (C) Cells cultured with 600 μg/mL of TiO2 NPs show fragmented chromatin, an indicator of

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Although the size of single TiO2 NPs reported by Jin et al. was of 5 nm [80], they were clustered in 20–30 nm aggregates, an effect that could enhance cytotoxicity. It was later shown that there is a correlation between the cytotoxicity of TiO2 NPs and their aggregate size, as larger aggregates (600 vs. 166 nm) elicited a stronger decrease in cell viability, as well as the expression of genes related to stress and inflammation [83]. In contrast, TiO2 NPs of 12 nm in diameter aggregated in 450 nm clusters and only at higher doses slightly decreased the viability of glomerular mesangial IP5 and epithelial proximal HK-2 cells, suggesting specific cell responses [84]. Additionally, although ROS was generated in the presence of TiO2 NPs, the cells were able to maintain their antioxidant potential, thereby showing no

Cellular uptake studies with 30 nm TiO2 NPs have been carried out in human amnion epithelial WISH cells using transmission electron microscopy (TEM), with images showing most of the particles localized either inside vesicles or freely in the cytoplasm [85]. In addition to the already mentioned cytotoxic response, WISH cells experience an oxidative response due to

Cytotoxicity data of TiO2 NWs are scarce, with only a handful of studies published. Magrez et al. observed that TiO2-based NWs of 5 μm in length and 75 nm in length had a negative impact on the cell proliferation and cell viability of H596 human lung tumor cells in a dosedependent manner and for concentrations up to 2 μg/mL [86]. NWs were observed to reside in the periphery of the nuclei, which were often enlarged and lobulated or fragmented. In another study, H2Ti3O7 NWs at a dose of 10 μg/mL induced the generation of cell debris in eight different cell lines, which the authors associated with an increase in autophagosome-like

Zinc is a biologically active element that plays a role in different processes, such as the immune system, cell metabolism, cell proliferation, enzymatic function and gene expression, among

ROS accumulation, as well as DNA double strand breaks and cell cycle arrest.

through ROS [82].

oxidative stress.

vacuoles in the cytosol [87].

3.2. Zinc oxide nanoparticles and nanowires

distinct molecular changes in immune cells, with transcriptomic changes analyzed in human immune cells THP1, a monocytic cell line, Jurkat cells and a T lymphocyte cell line [77]. They found a cell-specific action on monocytes for three types of MWCNTs, which specifically enhanced innate immunity activation mechanisms. The pathways activated are functionally relevant and critical for the development of an effective inflammatory response.
