3. Semiconductors

#### 3.1. Titanium dioxide nanoparticles and nanowires

A comprehensive review of the numerous biomedical applications of titanium dioxide (TiO2) throughout the years was published by Yin et al. [78]. In summary, mostly due to their low cost, strong optical absorption and high chemical stability, TiO2 NPs have shown great potential in applications such as photodynamic cancer therapy, drug delivery, cell imaging and biosensors, among others.

One of the initial cytotoxicity studies with TiO2 NPs was performed on human dermal microvascular endothelial cells [79]. There, it was shown that NPs with an average diameter of 70 nm at a dose of 50 μg/mL caused a minor pro-inflammatory response in the form of an increase in the levels of IL-8. Later, a study with mouse fibroblast L929 cells exposed to TiO2 NPs was conducted by Jin et al. [80]. For 3–600 μg/mL doses, cells appeared to shrink and became round in culture, with a dose-dependent reduction of cell metabolic activity, LDH release and ROS generation. Chromatin fragmentation was also reported, indicating possible DNA damage (Figure 7). It was also found that both human neural astrocyte-like U87 cells and human fibroblast HFF-1 cells exposed to 25 nm TiO2 NPs for 48 h had a decrease in cell survival for doses up to 100 μg/mL, with cell death reported as a combination of apoptosis and necrosis [81]. On the other hand, BEAS-2B cells underwent cell death

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

through apoptosis, triggered by the activation of caspase-3 and chromatin condensation through ROS [82].

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 oxidative stress.

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

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 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

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

A comprehensive review of the numerous biomedical applications of titanium dioxide (TiO2) throughout the years was published by Yin et al. [78]. In summary, mostly due to their low cost, strong optical absorption and high chemical stability, TiO2 NPs have shown great potential in applications such as photodynamic cancer therapy, drug delivery, cell imaging and

One of the initial cytotoxicity studies with TiO2 NPs was performed on human dermal microvascular endothelial cells [79]. There, it was shown that NPs with an average diameter of 70 nm at a dose of 50 μg/mL caused a minor pro-inflammatory response in the form of an increase in the levels of IL-8. Later, a study with mouse fibroblast L929 cells exposed to TiO2 NPs was conducted by Jin et al. [80]. For 3–600 μg/mL doses, cells appeared to shrink and became round in culture, with a dose-dependent reduction of cell metabolic activity, LDH release and ROS generation. Chromatin fragmentation was also reported, indicating possible DNA damage (Figure 7). It was also found that both human neural astrocyte-like U87 cells and human fibroblast HFF-1 cells exposed to 25 nm TiO2 NPs for 48 h had a decrease in cell survival for doses up to 100 μg/mL, with cell death reported as a combination of apoptosis and necrosis [81]. On the other hand, BEAS-2B cells underwent cell death

relevant and critical for the development of an effective inflammatory response.

3. Semiconductors

American Chemical Society.

242 Cytotoxicity

biosensors, among others.

3.1. Titanium dioxide nanoparticles and nanowires

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 others [88, 89]. Due to these biological functions of zinc, coupled with initial biocompatibility studies [90], ease of fabrication and relevant properties [91], zinc oxide (ZnO) nanostructures have been proposed as suitors for several biomedical applications, including cancer cell therapy [92, 93], drug delivery [94, 95] and imaging [96]. However, possible undesirable effects of the interactions between ZnO nanostructures and biological systems could arise and cause a toxicological response. Additionally, ZnO nanostructures are known to dissolve under acidic conditions. The phase-solubility diagram of ZnO [97] indicates that ZnO NPs will dissolve at a pH value below 6.7 at physiological temperature, and they will rapidly dissolve in the acidic pH of the lysosomes (pH 5.7) after their uptake [98]. Zinc oxide NPs can dissolve in an aqueous media to form hydrated Zn2+, which is enhanced in acidic pH as well as in the presence of biological components, such as amino acids and peptides [99]. A review on studies published between the years 2009 and 2011 on the toxicity of ZnO NPs to mammalian cells was reported by Vandebriel et al. [100]. They concluded that the induction of oxidative stress is the most important and the most likely mechanism underlying ZnO NP toxicity.

The liver, playing a major role in human metabolism, may be a target organ for NPs after they enter into the body. As such, it is an important toxicity evaluation method. Similarly, to previous results, Sharma et al. found that human liver HepG2 cells had a dose-dependent response to ZnO NPs at doses up to 20 μg/mL and for exposure times from 12 to 24 h [107]. Cell death was also shown as being through the apoptotic pathway, due to ROS generation, oxidative stress and mitochondrial and DNA damage. Taken together, all these results suggest that the cytotoxic response to ZnO NPs is dependent on the target cell tissue as well as on

Review of In Vitro Toxicity of Nanoparticles and Nanorods—Part 2

http://dx.doi.org/10.5772/intechopen.78616

245

Kao et al. observed the effects of ZnO NPs of <50 nm in the homeostasis of intracellular Zn2+ in human leukemia Jurkat cells and human lung carcinoma H1355 cells and found an increase in the concentration of cytosolic and mitochondrial Zn2+, most probably due to NP dissolution [108], as shown previously [86]. Caspase-3 activation, mitochondrial membrane depolarization and LDH release were also reported, which suggests an apoptotic death pathway due to mitochondrial dysfunction. In a more recent study, the intracellular concentration of Zn2+ of breast cancer MDA-MB-231 cells was also increased after treatment with ZnO NPs, leading to the generation of ROS,

Limited literature exists regarding the biocompatibility of ZnO NWs, but similar cytotoxicity effects as those of NPs were shown by Li et al. using HeLa cells and connective tissue L-929 cells [110]. Although the NWs used were rather large (200 μm in length and 1 μm in diameter), both cell lines seemed to maintain their viability for concentrations up to 10 μg/mL and exposure times of 24 h. Similar viability data were then reported for NWs of 10 μm in length and 327 nm in diameter at the same dose in human macrophages (Figure 8) [111]. There, too, it

Figure 8. Cytotoxicity of ZnO NWs on HMM human monocyte macrophages. Cell viability was assessed using the neutral red assay, with the red bars denoting doses of Zn in the form of ZnCl2 or ZnO NWs, respectively. Adapted with

permission from Müller et al. [111]. Copyright 2010 American Chemical Society.

damage to the cell membrane and mitochondria and culminating in apoptosis [109].

changes in NP dimensions.

Initial cytotoxicity studies in human T lymphocytes showed a significant decrease in cell viability only for concentrations higher than 5 mM using ZnO NPs of 13 nm in diameter [101]. It was then found that ZnO NPs preferentially kill cancerous human T lymphocytes compared to normal ones via ROS and apoptosis [92]. Similarly, NPs of the same size were tested against human lung BEAS-2B cells and RAW 264.7 macrophages, and a dose and timedependent cytotoxicity was found in both cases for doses up to 50 μg/mL and incubation times of 16 h [99]. Moreover, the ZnO NPs were reported to induce the generation of ROS, as well as the activation of the pro-inflammatory marker TNF-α and the pro-inflammatory pathway Jun kinase, as well as intracellular calcium release, a major oxidative stress response. Finally, ZnO NPs were found to reside in caveolae in the case of BEAS-2B cells, whereas in the RAW 264.7 cells, they resided inside lysosomes, with intracellular dissolution and release of Zn2+ shown in both cases. In a different study, ZnO NPs also impaired the survival of human neural astrocyte-like U87 cells in a dose-dependent manner [81].

The degree of cytotoxicity of ZnO NPs also depends on their size, as shown by Hanley et al. [102]. Using 4, 13 and 20 nm NPs, they determined an inverse relationship between nanoparticle size and cytotoxicity in terms of cell viability and ROS generation in immune cells. Among these, monocytes were the most sensitive to the ZnO NPs, whereas lymphocytes were the most resistant, as reported previously [101]. In contrast, glomerular mesangial IP5 cells showed a similar dose-dependent decrease in cell viability for ZnO NPs of both <100 nm and >1 μm, along with the generation of ROS [84]. The cell viability of neural stem cells appeared to be indifferent of NP size [103]. In a different work, a differential cytotoxic response was reported, when comparing the effects of 20–30 nm ZnO NPs in human myeloblastic leukemic HL60 cells and normal peripheral blood mononuclear cells (PBMCs) [104]. For concentrations up to 1000 μg/mL, PBMCs maintained a steadily high viable cell population, whereas a dose of 50 μg/mL was enough to bring the cell viability of HL60 cells down to 50%. DNA fragmentation analysis and annexin V staining confirmed that cell death was through the apoptosis pathway. The potent tumor suppressor that regulates the cell cycle and prevents DNA damage, p53 [105], is believed to be a molecular master switch toward apoptosis, and reports show that the p53 pathway was activated in BJ cells (skin fibroblasts) upon ZnO NPs treatment with a concomitant decrease in cell proliferation [106].

The liver, playing a major role in human metabolism, may be a target organ for NPs after they enter into the body. As such, it is an important toxicity evaluation method. Similarly, to previous results, Sharma et al. found that human liver HepG2 cells had a dose-dependent response to ZnO NPs at doses up to 20 μg/mL and for exposure times from 12 to 24 h [107]. Cell death was also shown as being through the apoptotic pathway, due to ROS generation, oxidative stress and mitochondrial and DNA damage. Taken together, all these results suggest that the cytotoxic response to ZnO NPs is dependent on the target cell tissue as well as on changes in NP dimensions.

others [88, 89]. Due to these biological functions of zinc, coupled with initial biocompatibility studies [90], ease of fabrication and relevant properties [91], zinc oxide (ZnO) nanostructures have been proposed as suitors for several biomedical applications, including cancer cell therapy [92, 93], drug delivery [94, 95] and imaging [96]. However, possible undesirable effects of the interactions between ZnO nanostructures and biological systems could arise and cause a toxicological response. Additionally, ZnO nanostructures are known to dissolve under acidic conditions. The phase-solubility diagram of ZnO [97] indicates that ZnO NPs will dissolve at a pH value below 6.7 at physiological temperature, and they will rapidly dissolve in the acidic pH of the lysosomes (pH 5.7) after their uptake [98]. Zinc oxide NPs can dissolve in an aqueous media to form hydrated Zn2+, which is enhanced in acidic pH as well as in the presence of biological components, such as amino acids and peptides [99]. A review on studies published between the years 2009 and 2011 on the toxicity of ZnO NPs to mammalian cells was reported by Vandebriel et al. [100]. They concluded that the induction of oxidative stress is the most

Initial cytotoxicity studies in human T lymphocytes showed a significant decrease in cell viability only for concentrations higher than 5 mM using ZnO NPs of 13 nm in diameter [101]. It was then found that ZnO NPs preferentially kill cancerous human T lymphocytes compared to normal ones via ROS and apoptosis [92]. Similarly, NPs of the same size were tested against human lung BEAS-2B cells and RAW 264.7 macrophages, and a dose and timedependent cytotoxicity was found in both cases for doses up to 50 μg/mL and incubation times of 16 h [99]. Moreover, the ZnO NPs were reported to induce the generation of ROS, as well as the activation of the pro-inflammatory marker TNF-α and the pro-inflammatory pathway Jun kinase, as well as intracellular calcium release, a major oxidative stress response. Finally, ZnO NPs were found to reside in caveolae in the case of BEAS-2B cells, whereas in the RAW 264.7 cells, they resided inside lysosomes, with intracellular dissolution and release of Zn2+ shown in both cases. In a different study, ZnO NPs also impaired the survival of human neural

The degree of cytotoxicity of ZnO NPs also depends on their size, as shown by Hanley et al. [102]. Using 4, 13 and 20 nm NPs, they determined an inverse relationship between nanoparticle size and cytotoxicity in terms of cell viability and ROS generation in immune cells. Among these, monocytes were the most sensitive to the ZnO NPs, whereas lymphocytes were the most resistant, as reported previously [101]. In contrast, glomerular mesangial IP5 cells showed a similar dose-dependent decrease in cell viability for ZnO NPs of both <100 nm and >1 μm, along with the generation of ROS [84]. The cell viability of neural stem cells appeared to be indifferent of NP size [103]. In a different work, a differential cytotoxic response was reported, when comparing the effects of 20–30 nm ZnO NPs in human myeloblastic leukemic HL60 cells and normal peripheral blood mononuclear cells (PBMCs) [104]. For concentrations up to 1000 μg/mL, PBMCs maintained a steadily high viable cell population, whereas a dose of 50 μg/mL was enough to bring the cell viability of HL60 cells down to 50%. DNA fragmentation analysis and annexin V staining confirmed that cell death was through the apoptosis pathway. The potent tumor suppressor that regulates the cell cycle and prevents DNA damage, p53 [105], is believed to be a molecular master switch toward apoptosis, and reports show that the p53 pathway was activated in BJ cells (skin fibroblasts) upon ZnO NPs treatment with a concomitant decrease in cell

important and the most likely mechanism underlying ZnO NP toxicity.

astrocyte-like U87 cells in a dose-dependent manner [81].

proliferation [106].

244 Cytotoxicity

Kao et al. observed the effects of ZnO NPs of <50 nm in the homeostasis of intracellular Zn2+ in human leukemia Jurkat cells and human lung carcinoma H1355 cells and found an increase in the concentration of cytosolic and mitochondrial Zn2+, most probably due to NP dissolution [108], as shown previously [86]. Caspase-3 activation, mitochondrial membrane depolarization and LDH release were also reported, which suggests an apoptotic death pathway due to mitochondrial dysfunction. In a more recent study, the intracellular concentration of Zn2+ of breast cancer MDA-MB-231 cells was also increased after treatment with ZnO NPs, leading to the generation of ROS, damage to the cell membrane and mitochondria and culminating in apoptosis [109].

Limited literature exists regarding the biocompatibility of ZnO NWs, but similar cytotoxicity effects as those of NPs were shown by Li et al. using HeLa cells and connective tissue L-929 cells [110]. Although the NWs used were rather large (200 μm in length and 1 μm in diameter), both cell lines seemed to maintain their viability for concentrations up to 10 μg/mL and exposure times of 24 h. Similar viability data were then reported for NWs of 10 μm in length and 327 nm in diameter at the same dose in human macrophages (Figure 8) [111]. There, too, it

Figure 8. Cytotoxicity of ZnO NWs on HMM human monocyte macrophages. Cell viability was assessed using the neutral red assay, with the red bars denoting doses of Zn in the form of ZnCl2 or ZnO NWs, respectively. Adapted with permission from Müller et al. [111]. Copyright 2010 American Chemical Society.

was found that an intracellular increase of Zn2+ precedes cell death, indicating the intracellular dissolution of the ZnO NWs after uptake [112–120].

### 3.3. Silicon nanoparticles

Coated silicon nanoparticles (Si NPs) have attracted both a great deal of concern and attention, especially in biomedical applications such as disease diagnosis, tumor cell tracking, imaging, drug delivery and gene therapy [121, 122]. They have been widely studied for such applications because of their active surface state and high suspension ability [121, 123, 124]. However, there are some recent reports that limit the use of Si NPs because of potential side effects on the cells, when using them in such a scale and in high concentrations [125].

One of the first studies on Si NPs focused on their cellular uptake [123]. Si NPs were coated with a fluorone dyes called Rhodamine 6G isothiocyanate (RITC), the fluorescence signal of which indicated uptake. NPs of 50 nm diameter at a dose of 80 μg/mL were accumulated in the cytoplasm of HeLa cells after 4 h of incubation at 37C, while the uptake was reduced by 80% at 4C. A year later, Lin et al. focused on the toxicity effect of the size, concentration and exposure time of Si NPs on human lung cancer cells (calveolar carcinoma-derived cells) [126]. The cell viability decreased significantly as a function of both nanoparticle dosage (10–100 μg/mL) and exposure time (24, 48 and 72 h). However, the cytotoxicity of two different sizes of Si NPs (15 and 46 nm) did not show a significant difference.

A different study compared the cytotoxicity of a variety of sizes of Si NPs (19, 43, 68 and 498 nm) at 100 μg/mL [127]. After 4 h of incubation with human liver HepG2 cells, it was noticed that the cytotoxicity of Si NPs is size-dependent (i.e., the smaller size the higher cytotoxicity. The live cells were counted by a cell-counting kit (CCK-8). Further, Sahu et al. proved that Si NPs (10–20 nm) are much more toxic than micro-sized ones (0.5–10 μm) for a concentration range of 5–500 μg/mL, after exposing them to human lung epithelial (L-132) and human monocytes (THP-1) for 24 h [128]. The cellular uptake efficiency and pathway of different sized NPs has also been confirmed to be size-dependent, with smaller particles (55 nm) being internalized faster than larger ones (307 nm) [129]. The largest NPs (307 nm) internalized through clathrin-coated pits, whereas medium ones (167 nm) internalized through clathrin-coated vesicles and the smallest (55 nm) were internalized through an energy independent pathway. Despite differences in their internalization pathway, all three sizes showed a high-level of biocompatibility.

viability, when compared to A549 and HepG2 cells [132]. On the other hand, positively charged (NH2-coated Si NPs) displayed higher cytotoxicity than negatively charged ones (COOH-coated NPs) in human adenocarcinoma Caco-2 and rat alveolar macrophage NR8383 cells [133]. However, the opposite has also been suggested for HaCaT keratinocyte cells [134]. A summary of the viability dependences on the Si NPs' size, concentration and cell type is

Figure 9. Cytotoxicity of SiO2 particle is size, concentration and cell-dependent in (a) L-132 cells and (b) THP-1 cells. Results were mean SEM of three independent experiments each carried out in triplicate, in comparison to untreated

Review of In Vitro Toxicity of Nanoparticles and Nanorods—Part 2

http://dx.doi.org/10.5772/intechopen.78616

247

controls. Adapted with permission from Sahu et al. [128]. Copyright 2016 Hindawi Publishing Corporation.

Si NWs show several advantages over Si NPs. For instance, they tend to not agglomerate in solution compared to Si NPs [135] and they enhance the drug-loading capacity due to their high surface area [136, 137]. However, it has been indicated that Si NWs have more toxic effects to macrophages cells at lower concentrations compared to Si NPs due to the large surface area, which increase the interaction and induce the cell death [138]. Naturally, the concentration of Si NWs plays a role on cell viability. Si NWs of 2 μm long, 55 nm diameter were co-cultured with HeLa and Hep-2 cells at different concentrations [138]. While no toxicity was found on either cell line for concentrations below 190 μg/ml, the cells died and released 75% of their contents into the supernatant at high concentrations (1900 μg/ml) after 72 h of incubation. Zhang et al. used amino-modified (APTES), folate-functionalized Si NWs to study cell interactions [139]. The NWs lengths were between 2.5 and 8.0 μm, with a concentration of

shown in Figure 9 [128].

3.4. Silicon NWs

In a similar approach as the one of Lin et al., the cytotoxic effects of increasing concentrations of Si NPs (0, 25, 50, 100 and 200 μg/mL) on HepG2 cells were analyzed in terms of ROS level, mitochondrial membrane potential and apoptotic rate. All three tests showed that the level of toxicity of the NPs increases while increasing the concentration from 25 to 100 μg/ml. Additionally, it was shown that the expressions of the apoptotic genes cytC and Caspase-3 were up-regulated with increasing NP concentrations. Additionally, the downregulation of the antiapoptotic Bcl-2 gene and upregulation of the genes p53 and BAX have also been reported [129, 130].

Other approaches have focused on cell-dependent cytotoxicity and surface charge [131]. Kim et al. found that NIH/3T3 fibroblasts appear to be more susceptible to Si NPs in terms of cell

Review of In Vitro Toxicity of Nanoparticles and Nanorods—Part 2 http://dx.doi.org/10.5772/intechopen.78616 247

Figure 9. Cytotoxicity of SiO2 particle is size, concentration and cell-dependent in (a) L-132 cells and (b) THP-1 cells. Results were mean SEM of three independent experiments each carried out in triplicate, in comparison to untreated controls. Adapted with permission from Sahu et al. [128]. Copyright 2016 Hindawi Publishing Corporation.

viability, when compared to A549 and HepG2 cells [132]. On the other hand, positively charged (NH2-coated Si NPs) displayed higher cytotoxicity than negatively charged ones (COOH-coated NPs) in human adenocarcinoma Caco-2 and rat alveolar macrophage NR8383 cells [133]. However, the opposite has also been suggested for HaCaT keratinocyte cells [134]. A summary of the viability dependences on the Si NPs' size, concentration and cell type is shown in Figure 9 [128].

#### 3.4. Silicon NWs

was found that an intracellular increase of Zn2+ precedes cell death, indicating the intracellular

Coated silicon nanoparticles (Si NPs) have attracted both a great deal of concern and attention, especially in biomedical applications such as disease diagnosis, tumor cell tracking, imaging, drug delivery and gene therapy [121, 122]. They have been widely studied for such applications because of their active surface state and high suspension ability [121, 123, 124]. However, there are some recent reports that limit the use of Si NPs because of potential side effects on the

One of the first studies on Si NPs focused on their cellular uptake [123]. Si NPs were coated with a fluorone dyes called Rhodamine 6G isothiocyanate (RITC), the fluorescence signal of which indicated uptake. NPs of 50 nm diameter at a dose of 80 μg/mL were accumulated in the cytoplasm of HeLa cells after 4 h of incubation at 37C, while the uptake was reduced by 80% at 4C. A year later, Lin et al. focused on the toxicity effect of the size, concentration and exposure time of Si NPs on human lung cancer cells (calveolar carcinoma-derived cells) [126]. The cell viability decreased significantly as a function of both nanoparticle dosage (10–100 μg/mL) and exposure time (24, 48 and 72 h). However, the cytotoxicity of two different sizes of Si NPs (15 and

A different study compared the cytotoxicity of a variety of sizes of Si NPs (19, 43, 68 and 498 nm) at 100 μg/mL [127]. After 4 h of incubation with human liver HepG2 cells, it was noticed that the cytotoxicity of Si NPs is size-dependent (i.e., the smaller size the higher cytotoxicity. The live cells were counted by a cell-counting kit (CCK-8). Further, Sahu et al. proved that Si NPs (10–20 nm) are much more toxic than micro-sized ones (0.5–10 μm) for a concentration range of 5–500 μg/mL, after exposing them to human lung epithelial (L-132) and human monocytes (THP-1) for 24 h [128]. The cellular uptake efficiency and pathway of different sized NPs has also been confirmed to be size-dependent, with smaller particles (55 nm) being internalized faster than larger ones (307 nm) [129]. The largest NPs (307 nm) internalized through clathrin-coated pits, whereas medium ones (167 nm) internalized through clathrin-coated vesicles and the smallest (55 nm) were internalized through an energy independent pathway. Despite differences in their internalization pathway, all three sizes

In a similar approach as the one of Lin et al., the cytotoxic effects of increasing concentrations of Si NPs (0, 25, 50, 100 and 200 μg/mL) on HepG2 cells were analyzed in terms of ROS level, mitochondrial membrane potential and apoptotic rate. All three tests showed that the level of toxicity of the NPs increases while increasing the concentration from 25 to 100 μg/ml. Additionally, it was shown that the expressions of the apoptotic genes cytC and Caspase-3 were up-regulated with increasing NP concentrations. Additionally, the downregulation of the antiapoptotic Bcl-2

Other approaches have focused on cell-dependent cytotoxicity and surface charge [131]. Kim et al. found that NIH/3T3 fibroblasts appear to be more susceptible to Si NPs in terms of cell

gene and upregulation of the genes p53 and BAX have also been reported [129, 130].

cells, when using them in such a scale and in high concentrations [125].

dissolution of the ZnO NWs after uptake [112–120].

46 nm) did not show a significant difference.

showed a high-level of biocompatibility.

3.3. Silicon nanoparticles

246 Cytotoxicity

Si NWs show several advantages over Si NPs. For instance, they tend to not agglomerate in solution compared to Si NPs [135] and they enhance the drug-loading capacity due to their high surface area [136, 137]. However, it has been indicated that Si NWs have more toxic effects to macrophages cells at lower concentrations compared to Si NPs due to the large surface area, which increase the interaction and induce the cell death [138]. Naturally, the concentration of Si NWs plays a role on cell viability. Si NWs of 2 μm long, 55 nm diameter were co-cultured with HeLa and Hep-2 cells at different concentrations [138]. While no toxicity was found on either cell line for concentrations below 190 μg/ml, the cells died and released 75% of their contents into the supernatant at high concentrations (1900 μg/ml) after 72 h of incubation. Zhang et al. used amino-modified (APTES), folate-functionalized Si NWs to study cell interactions [139]. The NWs lengths were between 2.5 and 8.0 μm, with a concentration of 100 μg/mL. It was found that the length of NWs affected the internalization, with NWs longer than 5 μm being more difficult to be internalized, due to geometrical restrictions.

4. Conclusion

Recent studies on the in-vitro cytotoxicity of carbon structure and semiconductors in biomedical applications were reviewed, taking into account nanoparticles and nanowires/nanorods.

Review of In Vitro Toxicity of Nanoparticles and Nanorods—Part 2

http://dx.doi.org/10.5772/intechopen.78616

249

Comparisons between the cytotoxicities of those different nanomaterials are generally difficult to make due to the vast range of methods, concentrations, dimensions, cell lines etc. For instance, the concentrations reported in the different studies were typically evaluated using either ICP or Cryogenic TEM. However, the concentration or dose of the nanomaterial plays a significant role in the cytotoxic response as well as the biomedical applications. Similarly, the reported toxicology of the nanomaterials depends on their interaction with the assay. For example, carbon nanostructures interact with the MTT-formazan crystals but not with XTT or INT reagents.

While the concentrations and exposure times are critical factors, the toxicity of these nanostructures is also material-dependent. These relations can be seen in Figure 10, which presents the average values reported for the cell viabilities (ignoring differences in concentrations, incubation times etc.), when exposed to the nanomaterials in the studies covered in Table 1. ZnO NPs

In addition, the particle size plays a major role in the cytotoxic properties of the nanostructure, whereby both the cellular uptake efficiency and pathway are affected, with smaller particles

The induction of ROS after dissolving the nanostructures in the lysosomes was shown to be the primary underlying cause of the toxicity in several cases, leading to cell death through the apoptotic pathway, due to ROS generation and mitochondrial damage. The acidic condition inside the lysosome increases the digestion of the particles, enhancing the release of ions that affect the viability of the cells. This is a particularly relevant issue in case of CdSe-core QDs,

Adding a coating to the nanostructure typically affected both the toxicity and the surface charge of the nanostructure, where cationic surfaces are more toxic than anionic. For instance, the toxicity of QDs was reduced by adding a BSA corona, and the release of Cd was prevented by the addition of polymeric and inorganic coatings. The type of the coatings was shown to

The cytotoxicity of the nanomaterial depends also on the nanostructure's shape. In this regard, several advantages have been reported for NWs over NPs. For instance, they enhance the drugloading capacity due to their large surface area. An interesting observation from Figure 10 is that NWs/NRs are, on average, less cytotoxic than NPs, with titanium dioxide being the only exception. However, one study has shown that the large surface area of Si NWs has a more toxic effect at lower concentrations compared to NPs. This was attributed to the increased interaction of the

A summary of the results of representative studies is provided in Table 1.

showed the highest toxicity, while the lowest has been reported for silicon.

which release Cd ions upon oxidation, leading to fast cell death.

nanomaterial with the cells due to the large surface area.

being internalized faster than the larger ones.

affect the cell viability differently.

#### 3.5. Quantum dots

Semiconductor quantum dots (QDs) are light-emitting particles that have broad excitation spectra, long fluorescence lifetimes compared to traditional fluorescent probes and are more resistant to photobleaching [140, 141]. Also, they can easily be conjugated to proteins [140], which makes them excellent choices for bioimaging [142–146] and other biomedical applications [141, 147, 148]. Tsoi et al. summarized the toxicity of QDs by two mechanisms: degradation with the release of free cadmium (Cd) and generation of ROS [149]. Each design of QD is a unique combination and has its own physicochemical properties that may influence its biological activity and toxicity. As a result, tremendous research efforts have been devoted to produce high quality QDs by optimizing synthetic procedures, as well as functionalizing their surface in order to enhance biocompatibility [142, 150].

An early study by Derfus et al. demonstrated that CdSe-core QDs oxidized and degraded, releasing Cd ions which induced cell death [151]. When CdSe QDs were exposed to a UV-light for 1, 2, 4 and 8 h and then incubated with hepatocytes, it showed a 6, 42, 83 and 97% decrease in the cells' viability, respectively [143]. Cd is a known carcinogen with potential damage to the renal, skeletal, pulmonary and reproductive systems [152]. Interestingly, Chen et al. showed that the cell viability of HEK293 cells treated with 37.5 nM of 5 nm CdTe QDs was not significantly altered, compared to the control (i.e., untreated cells) after 3 days of incubation [153]. However, high concentrations (300–600 nM) of QDs completely inhibited cell growth from the very beginning. The cytotoxicity of QDs has also been linked to the generation of ROS, which in turn damages cellular proteins, lipids and DNA [149]. The p53 gene was also shown to be inhibited by CdTe QDs, leading to apoptosis and cell death [150].

Tracking the QDs internalization pathways could help explain their toxicological properties. To this end, microscopy studies showed that QDs localize within cellular endosomes and lysosomes, exposing them to an acidic or oxidative microenvironment [149]. It was determined that the hypochlorous acid present in phagocytic cells oxidized polymer-encapsulated CdS and ZnS-capped CdSe QDs, releasing cadmium, zinc, sulfur and selenium into the cytoplasm. Some studies have suggested that the QDs toxicity might derive from multiple factors including the environment and the QDs physicochemical characteristics (such as size, shape and surface chemistry). A surface coating with a ZnS shell [149] or BSA corona [150] reduced the QDs toxicity. In addition, polymeric coatings (i.e., phospholipid-PEG) and inorganic coatings (e.g., Si) can prevent the release of Cd into the biological media [142]. In a different approach, Soenen et al. studied cell viability using Cd-free QDs (ZnSe/ZnS and InP/ZnS QDs) at concentrations ranging from 0 to 100 nM [154]. Cytotoxic effects were observed starting from 60 nM for ZnSe to 80 nM for InP QDs. Further, no increase in cytotoxicity was reported up to 7 days after the initial cell labeling compared to normal QDs due to the absence of Cd.
