**New Perspectives in Rehabilitation after Traumatic Brain Injury**

Editors

**Nada Andelic Cecilie Røe Eirik Helseth Emilie Isager Howe Marit Vindal Forslund Torgeir Hellstrøm**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Nada Andelic University of Oslo Norway

Emilie Isager Howe Oslo University Hospital Norway

Cecilie Røe Oslo University Hospital Norway

Marit Vindal Forslund Oslo University Hospital Norway

Eirik Helseth Oslo University Hospital Norway

Torgeir Hellstrøm Oslo University Hospital Norway

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Journal of Clinical Medicine* (ISSN 2077-0383) (available at: https://www.mdpi.com/journal/jcm/ special issues/Rehabilitation Traumatic Brain Injury).

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**2. Nonmagnetic metallic nanostructures**

#### **2.1. Gold nanoparticles**

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 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 properties, making nanostructures highly versatile for different applications [2, 3]. 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 the clinical use in order to ensure their safety and effectiveness. Currently, very few NP-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

210 Cytotoxicity

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

tion, activated signaling pathways, and cellular response of different kinds of NPs.

Here, we review relevant studies assessing the *in vitro* cytotoxicity of both nanoparticles (NPs) and nanowires (NWs)/nanorods (NRs) with the potential to be used in biomedical applications. Due to their prevalence within the applied nanomaterials in biomedicine, this review

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 NPs of different materials, whereas the latter is a more in depth review of the internaliza-

Gold (Au) NPs are some of the most heavily used nanostructures in biomedical applications, most notably in medical imaging and therapy. The absorbance and fluorescence of Au NPs are higher than that of bulk Au and they can be finely tuned from the visible spectrum to the near infrared by changing their size and morphology [26]. Au can also readily bind different kinds of functionalizing molecules, giving them great versatility [27]. These properties make Au nanostructures popular candidates for X-ray-based imaging and radiotherapy, as well as photothermal therapy, when coupled to their ability to transform absorbed light into heat [26–30].

Two of the first cytotoxicity studies with Au NPs were performed by Tkachenko et al. [31]. In their first approach, they found that NPs conjugated with bovine serum albumin (BSA) and different peptides could enter the cell cytoplasm via an energy-dependent, receptor-mediated endocytosis pathway, with a decrease in cell viability of only around 5%, using the lactate dehydrogenase (LDH) colorimetric assay. The following year, they tested the same principle in three different cell lines and found that the uptake of Au NPs, as well as their ultimate fate (endosomal or nuclear), was cell dependent [32]. Later, Goodman et al. tested the effects of cationic and anionic Au NPs in the metabolic activity of red blood COS-1 cells, and showed that the former are more cytotoxic, probably due to them being drawn by the cell membrane's negative charge and then taken up [33]. A different approach using human leukemic K562 cells was performed by Connor et al. using different surface modifiers and the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, concluding that, despite the Au fabrication precursor being cytotoxic, Au NPs are inherently not, even though they are internalized and engulfed in endocytic vesicles [34]. This last finding was also confirmed by Chan and colleagues, who reported a size-dependent, clathrin-mediated uptake of citric acid, and transferrin-coated NPs in HeLa cells (**Figure 2**) [35, 36]. Similarly, size-dependence cytotoxicity was subsequently reported by Pan et al. [37] They showed that fibroblasts, epithelial cells, macrophages, and melanoma cells incubated with small (1.4 nm) Au NPs for 2 days have an IC50 ranging from 30 to 56 μM, whereas the same cells can tolerate concentrations 60-fold higher, when changing the particle diameter to 15 nm. The same group then went on to confirm the size-dependent cytotoxicity of Au NPs coated with TPPMS, now extending their findings to elucidate a necrotic death pathway in HeLa cells, due to oxidative stress, intracellular formation of reactive oxygen species (ROS) and a compromised mitochondrial activity [38].

The cytotoxicity of Au NPs has also been tested in other relevant cell lines, such as the alveolar type II-like A549 and NCIH441 [39]. Using a combination of metabolic activity, cell proliferation, and release of LDH assays, it was determined that 9.5 nm Au NPs, though internalized and stored in endocytic vesicles, do not have an effect on either of the cell lines' metabolic activity. A 50% decrease in cell proliferation was detected for A549 cells after 24 hours of exposure, decreasing further as time progressed. A mild dose-dependent LDH release was also reported for 24 and 48 hours for Au NP concentrations up to 0.7 mM, although after 72 hours, the release was significantly higher for this concentration, at around 35 and 90% for A549 and NCHIH441 cells, respectively. Macrophages have also been subjected to cytotoxicity assessments using Au NPs [40]. The results in this case are particularly relevant, due to their shedding light on a potential immunological response. Au NPs of three different sizes (2–4, 5–7, and 20–40 nm) were tested in macrophage J774 A1 cells at two concentrations, 1 and 10 ppm. Whereas only the small and medium-sized NPs produced a slight decrease in cell proliferation for the lower concentration, NPs of all three sizes decreased cell proliferation to around 30–40% for the 10 ppm concentration. Au NPs were shown to be inside vesicles in the cytoplasm, as was reported for human leukemic [34], HeLa [35], and alveolar type II-like cells [39]. Additionally, it was reported that Au NPs upregulate the expression of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α). This data show that macrophages, being one of the principal immune effector cells, activate a pro-inflammatory response, when in the presence of Au NPs of either size.

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Cho et al. looked closely at the effects of different surface functional groups on the uptake and cell membrane adsorption of Au NPs by breast cancer SK-BR-3 cells [41]. They tested Au NPs of two sizes (15 and 45 nm) with three different surface groups: poly(ethylene glycol) (PEG), anti-HER2, and poly(allylamine hydrochloride) (PAH). The smaller NPs were found to be more readily internalized than the larger ones, in contrast with previous findings [35, 36]. The PAH-modified NPs showed the greatest amount of internalization, followed by anti-HER2 and then PEG NPs. Using inductively coupled plasma mass spectrometry (ICP-MS) and an etching method to remove the NPs adsorbed to the cell membrane [42], they were able to distinguish between internalized nanoparticles to those that remained attached to the cell membrane and found that PEG-modified NPs had the lowest adsorption rate, thereby internalizing most of the NPs that came in contact with the cells. PAH-modified NPs had a very

strong affinity to the cell membrane, probably due to their electrostatic interactions.

**2.2. Gold nanorods and nanowires**

A more recent study compared NPs of 10, 25, and 50 nm and found that larger NPs are more readily taken up by NRK cells [43]. It was also reported that Au NPs enter the cells through endocytosis, ultimately accumulating in lysosomes and impairing their degradation capacity through alkalinization: Au NPs cause the dissociation of the V1 protein from the acidificationregulator complex H+(V)-ATPase, down-regulating its activity. The impairment of lysosomal function thus reduced the turnover of autophagosomes, carriers of intracellular content to their final degradation in lysosomes, leading to their accumulation in the cell cytoplasm.

Gold NRs and NWs, unlike NPs, possess a transverse and a dominant longitudinal plasmon [44]. This intense absorption band is near the infrared region, which biological tissue hardly absorbs, thus making these Au nanostructures attractive in the biomedical field. Early

**Figure 2.** Size-dependent uptake of Au NPs of different sizes in HeLa cells at different positions from B-F, as observed by TEM. While (A) Quantify the number of each Au NPs per vesicle. Adapted with permission from Chithrani et al. [35]. Copyright 2006 American Chemical Society.

The cytotoxicity of Au NPs has also been tested in other relevant cell lines, such as the alveolar type II-like A549 and NCIH441 [39]. Using a combination of metabolic activity, cell proliferation, and release of LDH assays, it was determined that 9.5 nm Au NPs, though internalized and stored in endocytic vesicles, do not have an effect on either of the cell lines' metabolic activity. A 50% decrease in cell proliferation was detected for A549 cells after 24 hours of exposure, decreasing further as time progressed. A mild dose-dependent LDH release was also reported for 24 and 48 hours for Au NP concentrations up to 0.7 mM, although after 72 hours, the release was significantly higher for this concentration, at around 35 and 90% for A549 and NCHIH441 cells, respectively. Macrophages have also been subjected to cytotoxicity assessments using Au NPs [40]. The results in this case are particularly relevant, due to their shedding light on a potential immunological response. Au NPs of three different sizes (2–4, 5–7, and 20–40 nm) were tested in macrophage J774 A1 cells at two concentrations, 1 and 10 ppm. Whereas only the small and medium-sized NPs produced a slight decrease in cell proliferation for the lower concentration, NPs of all three sizes decreased cell proliferation to around 30–40% for the 10 ppm concentration. Au NPs were shown to be inside vesicles in the cytoplasm, as was reported for human leukemic [34], HeLa [35], and alveolar type II-like cells [39]. Additionally, it was reported that Au NPs upregulate the expression of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α). This data show that macrophages, being one of the principal immune effector cells, activate a pro-inflammatory response, when in the presence of Au NPs of either size.

Cho et al. looked closely at the effects of different surface functional groups on the uptake and cell membrane adsorption of Au NPs by breast cancer SK-BR-3 cells [41]. They tested Au NPs of two sizes (15 and 45 nm) with three different surface groups: poly(ethylene glycol) (PEG), anti-HER2, and poly(allylamine hydrochloride) (PAH). The smaller NPs were found to be more readily internalized than the larger ones, in contrast with previous findings [35, 36]. The PAH-modified NPs showed the greatest amount of internalization, followed by anti-HER2 and then PEG NPs. Using inductively coupled plasma mass spectrometry (ICP-MS) and an etching method to remove the NPs adsorbed to the cell membrane [42], they were able to distinguish between internalized nanoparticles to those that remained attached to the cell membrane and found that PEG-modified NPs had the lowest adsorption rate, thereby internalizing most of the NPs that came in contact with the cells. PAH-modified NPs had a very strong affinity to the cell membrane, probably due to their electrostatic interactions.

A more recent study compared NPs of 10, 25, and 50 nm and found that larger NPs are more readily taken up by NRK cells [43]. It was also reported that Au NPs enter the cells through endocytosis, ultimately accumulating in lysosomes and impairing their degradation capacity through alkalinization: Au NPs cause the dissociation of the V1 protein from the acidificationregulator complex H+(V)-ATPase, down-regulating its activity. The impairment of lysosomal function thus reduced the turnover of autophagosomes, carriers of intracellular content to their final degradation in lysosomes, leading to their accumulation in the cell cytoplasm.

#### **2.2. Gold nanorods and nanowires**

endocytosis pathway, with a decrease in cell viability of only around 5%, using the lactate dehydrogenase (LDH) colorimetric assay. The following year, they tested the same principle in three different cell lines and found that the uptake of Au NPs, as well as their ultimate fate (endosomal or nuclear), was cell dependent [32]. Later, Goodman et al. tested the effects of cationic and anionic Au NPs in the metabolic activity of red blood COS-1 cells, and showed that the former are more cytotoxic, probably due to them being drawn by the cell membrane's negative charge and then taken up [33]. A different approach using human leukemic K562 cells was performed by Connor et al. using different surface modifiers and the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay, concluding that, despite the Au fabrication precursor being cytotoxic, Au NPs are inherently not, even though they are internalized and engulfed in endocytic vesicles [34]. This last finding was also confirmed by Chan and colleagues, who reported a size-dependent, clathrin-mediated uptake of citric acid, and transferrin-coated NPs in HeLa cells (**Figure 2**) [35, 36]. Similarly, size-dependence cytotoxicity was subsequently reported by Pan et al. [37] They showed that fibroblasts, epithelial cells, macrophages, and melanoma cells incubated with small (1.4 nm) Au NPs for 2 days have an IC50 ranging from 30 to 56 μM, whereas the same cells can tolerate concentrations 60-fold higher, when changing the particle diameter to 15 nm. The same group then went on to confirm the size-dependent cytotoxicity of Au NPs coated with TPPMS, now extending their findings to elucidate a necrotic death pathway in HeLa cells, due to oxidative stress, intracellular formation of reactive oxygen species (ROS) and a compromised mitochondrial activity [38].

**Figure 2.** Size-dependent uptake of Au NPs of different sizes in HeLa cells at different positions from B-F, as observed by TEM. While (A) Quantify the number of each Au NPs per vesicle. Adapted with permission from Chithrani et al. [35].

Copyright 2006 American Chemical Society.

212 Cytotoxicity

Gold NRs and NWs, unlike NPs, possess a transverse and a dominant longitudinal plasmon [44]. This intense absorption band is near the infrared region, which biological tissue hardly absorbs, thus making these Au nanostructures attractive in the biomedical field. Early cytotoxicity data showed that HeLa cells incubated with PEG-stabilized NRs at concentrations up to 0.5 mM survived with more than 90% viability after 24 hours, as per MTT assay [45]. Takahashi et al. observed similar viability effects under the same conditions, but using NRs modified with phosphatidylcholine [46]. Chithrani et al. reported that an increased uptake was observed for NRs in terms of the number of particles per cell with NRs with smaller aspect ratios coated with citric acid ligands [35] and transferrin [36]. Similarly, NRs of varying surface charges provided by layers of polyelectrolyte coatings seem to maintain a cell viability of around 90%, with an increased cellular uptake observed with NRs with a positive surface charge [47]. These results were later confirmed using NRs coated with the polyelectrolytes polyacrylic acid (PAA) and PAH, with human colon cancer HT-29 cells showing 90% viability after incubation with 0.4 nM Au NRs with either coating [48]. Cell growth was also reported not to be impaired, when compared with control cells, although higher internalization numbers were found for PAH-coated NRs compared to PAA ones. This is in agreement with previous findings [47]: positively charged particles (PAH-coated) are more readily taken up, when compared to negatively charged ones (PAA-coated), possibly due to the cell membrane's negative charge attracting the positively charged particles, leading to a higher membrane adsorption, as was observed with Au NPs [33, 41].

of the generation of ROS on particle size and the evaluation of the inflammatory response [53–55]. In all three studies, a dose-dependent decrease in mitochondrial metabolic activity and an increase in LDH leakage were reported for Ag NPs of 15 nm in rat liver BRL 3A cells, C18-4 germline stem cells, and macrophages for doses up to 75 μg/mL. Changes in cell morphology and uptake of NPs were also reported, with low levels of apoptosis. Interestingly, larger NPs (55 nm) induced a lesser cytotoxic response in macrophages, a result that can be attributed to the larger agglomerates not being easily internalized. Additionally, Ag NPs significantly impacted ROS generation and the release of inflammatory mediators including TNF-α, MIP-2, and IL-1β in macrophages for doses starting at 5 μg/mL. In contrast, Yen et al. reported no upregulation of the pro-inflammatory genes TNF-α, IL-1, and IL-6, though the

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It was later confirmed by Miura et al. that the ROS-related genes ho-1 and mt-2A are upregulated in HeLa cells [56], further cementing its role in the cytotoxic response. On the other hand, Autrup and co-workers showed that polyvinylpyrrolidone (PVP)-coated Ag NPs of 70 nm in diameter seemed to elicit both an apoptotic and necrotic response in THP-1 monocytes [57] and human alveolar A549 cells [58]. Although necrosis was markedly higher, the effect could

Other parameters, such as genotoxicity, have also been studied. Using human lung fibroblast IMR-90 cells and glioblastoma U251 cells as test models, AshaRani et al. concluded that starch-coated Ag NPs (6–20 nm in size) are taken up and reside inside the mitochondria and nucleus and, on top of generating ROS and reducing the metabolic activity and cell viability (**Figure 3**), they also reduced the ATP content of the cells and induced DNA damage and chromosomal aberrations in a dose-dependent manner. The latter resulted in cell cycle arrest

damage [59]. On the other hand, RAW 264.7 macrophage cells exposed to increase concentrations of 70 nm Ag NPs showed a significant increase in TNF-α, protein, and gene levels. The secretion of nitric oxide, a second messenger in the inflammatory response, as well as

**Figure 3.** Cytotoxicity of Ag NPs on U251 glioblastoma and IMR-90 fibroblast cells. Different concentrations of NPs with sizes ranging from 6 to 20 nm in diameter were cocultured with the cells for incubation times up to 72 hours. Adapted

with permission from AshaRani et al. [59]. Copyright 2009 American Chemical Society.

/M phase with no significant cell death observed, possibly due to the repair of DNA

be a progression from an early apoptotic stage to a late apoptotic/necrotic one.

doses tested were considerably higher [40].

in the G2

More recent studies have continued the analysis of the toxicological properties of Au NRs with a higher aspect ratio, or Au NWs, as they provide enhanced properties such as absorption and scattering, due to their increase in length [49]. One of the first approaches aimed to compare the cytotoxic effects of Au NRs with Au NWs with an aspect ratio 10 times larger [50]. Both instances of particles were coated either with tannic acid (TA) or carboxylated PEG (PEG-COOH) and the cytotoxicity to human keratinocyte cells was evaluated using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) (MTS) assay, an indicator of mitochondrial metabolic activity similar to the MTT assay. Following the same trend as previous cytotoxic data with surface-modified Au NRs, both TA and PEG-COOH-coated Au NRs showed a cell viability of up to 90% after 24 hours of incubation and for concentrations as high as 100 μg/mL; however, in the case of Au NWs, TA decreased the cell viability to around 70% at the 50 μg/mL concentration, whereas PEG-COOH-Au NWs maintained the viability above the 90% mark. These results indicate that, as Au NRs, Au NWs show very low cytotoxic potential, though specific surface coatings may elicit a toxic response. The authors also reported an increased uptake for Au NWs, when compared with NR independent of the surface coating. However, the values are reported in mass of Au per cell, which could be explained by the amount of material, due to the increase in size, and not be a clear representation of the amount of particles internalized.

#### **2.3. Silver nanoparticles and nanowires**

Silver (Ag) NPs have proven themselves greatly useful for their antimicrobial activity [51] and they are widely used in therapeutics and as a treatment for burns [52]. Naturally, a plethora of toxicity assessment studies has been carried out in order to understand the potential side effects of these nanostructures. The first cytotoxicity studies observed the effects of NPs on metabolic activity and membrane damage through LDH leakage, as well as the dependence of the generation of ROS on particle size and the evaluation of the inflammatory response [53–55]. In all three studies, a dose-dependent decrease in mitochondrial metabolic activity and an increase in LDH leakage were reported for Ag NPs of 15 nm in rat liver BRL 3A cells, C18-4 germline stem cells, and macrophages for doses up to 75 μg/mL. Changes in cell morphology and uptake of NPs were also reported, with low levels of apoptosis. Interestingly, larger NPs (55 nm) induced a lesser cytotoxic response in macrophages, a result that can be attributed to the larger agglomerates not being easily internalized. Additionally, Ag NPs significantly impacted ROS generation and the release of inflammatory mediators including TNF-α, MIP-2, and IL-1β in macrophages for doses starting at 5 μg/mL. In contrast, Yen et al. reported no upregulation of the pro-inflammatory genes TNF-α, IL-1, and IL-6, though the doses tested were considerably higher [40].

cytotoxicity data showed that HeLa cells incubated with PEG-stabilized NRs at concentrations up to 0.5 mM survived with more than 90% viability after 24 hours, as per MTT assay [45]. Takahashi et al. observed similar viability effects under the same conditions, but using NRs modified with phosphatidylcholine [46]. Chithrani et al. reported that an increased uptake was observed for NRs in terms of the number of particles per cell with NRs with smaller aspect ratios coated with citric acid ligands [35] and transferrin [36]. Similarly, NRs of varying surface charges provided by layers of polyelectrolyte coatings seem to maintain a cell viability of around 90%, with an increased cellular uptake observed with NRs with a positive surface charge [47]. These results were later confirmed using NRs coated with the polyelectrolytes polyacrylic acid (PAA) and PAH, with human colon cancer HT-29 cells showing 90% viability after incubation with 0.4 nM Au NRs with either coating [48]. Cell growth was also reported not to be impaired, when compared with control cells, although higher internalization numbers were found for PAH-coated NRs compared to PAA ones. This is in agreement with previous findings [47]: positively charged particles (PAH-coated) are more readily taken up, when compared to negatively charged ones (PAA-coated), possibly due to the cell membrane's negative charge attracting the positively charged particles, leading to a

More recent studies have continued the analysis of the toxicological properties of Au NRs with a higher aspect ratio, or Au NWs, as they provide enhanced properties such as absorption and scattering, due to their increase in length [49]. One of the first approaches aimed to compare the cytotoxic effects of Au NRs with Au NWs with an aspect ratio 10 times larger [50]. Both instances of particles were coated either with tannic acid (TA) or carboxylated PEG (PEG-COOH) and the cytotoxicity to human keratinocyte cells was evaluated using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) (MTS) assay, an indicator of mitochondrial metabolic activity similar to the MTT assay. Following the same trend as previous cytotoxic data with surface-modified Au NRs, both TA and PEG-COOH-coated Au NRs showed a cell viability of up to 90% after 24 hours of incubation and for concentrations as high as 100 μg/mL; however, in the case of Au NWs, TA decreased the cell viability to around 70% at the 50 μg/mL concentration, whereas PEG-COOH-Au NWs maintained the viability above the 90% mark. These results indicate that, as Au NRs, Au NWs show very low cytotoxic potential, though specific surface coatings may elicit a toxic response. The authors also reported an increased uptake for Au NWs, when compared with NR independent of the surface coating. However, the values are reported in mass of Au per cell, which could be explained by the amount of material, due to the increase in size, and not be a clear representation of the amount of

Silver (Ag) NPs have proven themselves greatly useful for their antimicrobial activity [51] and they are widely used in therapeutics and as a treatment for burns [52]. Naturally, a plethora of toxicity assessment studies has been carried out in order to understand the potential side effects of these nanostructures. The first cytotoxicity studies observed the effects of NPs on metabolic activity and membrane damage through LDH leakage, as well as the dependence

higher membrane adsorption, as was observed with Au NPs [33, 41].

particles internalized.

214 Cytotoxicity

**2.3. Silver nanoparticles and nanowires**

It was later confirmed by Miura et al. that the ROS-related genes ho-1 and mt-2A are upregulated in HeLa cells [56], further cementing its role in the cytotoxic response. On the other hand, Autrup and co-workers showed that polyvinylpyrrolidone (PVP)-coated Ag NPs of 70 nm in diameter seemed to elicit both an apoptotic and necrotic response in THP-1 monocytes [57] and human alveolar A549 cells [58]. Although necrosis was markedly higher, the effect could be a progression from an early apoptotic stage to a late apoptotic/necrotic one.

Other parameters, such as genotoxicity, have also been studied. Using human lung fibroblast IMR-90 cells and glioblastoma U251 cells as test models, AshaRani et al. concluded that starch-coated Ag NPs (6–20 nm in size) are taken up and reside inside the mitochondria and nucleus and, on top of generating ROS and reducing the metabolic activity and cell viability (**Figure 3**), they also reduced the ATP content of the cells and induced DNA damage and chromosomal aberrations in a dose-dependent manner. The latter resulted in cell cycle arrest in the G2 /M phase with no significant cell death observed, possibly due to the repair of DNA damage [59]. On the other hand, RAW 264.7 macrophage cells exposed to increase concentrations of 70 nm Ag NPs showed a significant increase in TNF-α, protein, and gene levels. The secretion of nitric oxide, a second messenger in the inflammatory response, as well as

**Figure 3.** Cytotoxicity of Ag NPs on U251 glioblastoma and IMR-90 fibroblast cells. Different concentrations of NPs with sizes ranging from 6 to 20 nm in diameter were cocultured with the cells for incubation times up to 72 hours. Adapted with permission from AshaRani et al. [59]. Copyright 2009 American Chemical Society.

the gene expression of matrix metalloproteinases (MMPs) MMP-3, MMP-11, and MMP-19, which play a key role in extracellular matrix degradation and can be activated through ROS were also reported [60]. Further, another parameter that has been observed is the effect of Ag NPs of different sizes on the differentiation of embryonic stem cells [61]. Differentiation into cardiomyocytes was inhibited in a dose-dependent manner, with Ag NPs of 20 nm having a stronger effect compared to larger ones.

as high as 1 mg/mL, uncoated, 10–15 nm NPs reduced the viability to around 70% at a concentration of 250 μg/mL [75]. Similar results were observed with pullulan-coated and uncoated NPs within the same size range [76]. There, it was also shown that bare NPs significantly reduce cell attachment and disrupt the distribution of actin filaments and microtubules, while also being taken up at a higher rate compared to the coated ones. Uncoated NPs were also reported to have cytotoxic effects only at higher doses (100–250 μg/mL) in terms of cell viability and LDH leakage in rat liver BRL 3A cells [53]. In agreement with these findings, hydroxy-tetramethylammonium-coated SPIONs at higher concentrations (23 mM) did not induce a reduction inviability of kidney COS-7 cells, though the time of incubation tested

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Ma et al. studied the uptake of 30 nm aminosilane-coated NPs by human lung cancer SPC-A1 and human lung WI-38 cells and found that the intracellular Fe content was 15 times higher for the cancerous cells compared to normal counterparts [78]. As with other NPs, they are likely endocytosed through phagocytosis and found within endosomes and lysosomes. Human monocytes-macrophages were also found to endocytose SPIONs and retained them inside lysosomes, remaining highly viable with no apparent activation of pro-inflammatory

Using bare 20–30 nm magnetite NPs, Karlsson et al. tested other parameters of cytotoxicity in the human alveolar A549 cells, such as DNA damage and intracellular ROS [80]. No DNA

DNA lesion was found at this dose. In contrast, another study showed that uncoated SPIONs elicited a significant level of apoptosis on mouse fibroblasts (L929), whereas PVA-coated ones did not show a loss of cell viability, apoptosis, necrosis, or cell cycle arrest for up to 72 hours of incubation and concentrations up to 200 mM [81]. However, an increase in the concentration to 400 mM did induce apoptosis and cell cycle arrest, possibly due to DNA damage through oxidative stress. Naqvi et al. obtained similar results for Tween 80-coated NPs in macrophage J774 A1 cells: >95% cell viability for low concentrations (25–200 μg/mL) and low incubations times, with a decrease to 55–65% for higher concentrations (300–500 μg/mL) associated to an

In contrast to previous findings, both citric acid and dextran-coated NPs were found to produce a dose-dependent cytotoxicity in human umbilical vein endothelial cells (HUVECs) [83]. Concentrations as low as 0.1 nM decreased the cell viability for both NPs to around 80% after 24 hours and increasing the value to 20 nM would decrease the cell viability to less than 15%. Additionally, as shown by Soenen et al. [84], actin filaments and microtubules appeared disrupted, thinner, and less organized and vinculin adhesion points were diminished. Further, NPs also reduced the migration and vasculogenesis capabilities of HUVECs. Similar results regarding cell attachment and cytoskeleton morphology were also reported in a multipara-

With an aim to understand the differences in cytotoxicity between the charges provided by different coatings on SPIONs, a study showed that when different functional groups were added in order to provide either a positive or negative charge on SPIONs, cell viability and cell membrane integrity remained above 85% up to 24 hours for doses as high as 1000 ppm

, although a slight oxidative

cytokines for up to 14 days following the incubation with 0.4 mg/mL SPIONs [79].

damage or intracellular ROS were found for doses up to 40 μg/cm2

apoptotic death pathway through ROS generation [82].

metric study with NPs with different coatings on various cell lines [84].

was only of 4 hours [77].

In recent studies, it has been proposed that the intracellular release of Ag ions from the NPs is one of the causes of their cytotoxicity. Singh et al. showed that, after being taken up through scavenger receptor-mediated phagocytosis in macrophages, intracellular dissolution of Ag NPs had a 50 times faster rate than in water, at around 5% of the total dose being dissolved [62]. It was suggested that Ag ions are a cytotoxic response initiator in human lung BEAS-2B cells [63].

Ag NW cytotoxicity, on the other hand, has not been extensively studied. In a comparative study of Ag NWs with a diameter of around 100 nm and lengths of 3, 5, 10, 14 and 28 μm, it was found that only the wires with a length of 28 μm could elicit a significant decrease in cell proliferation and membrane instability in THP-1 cells [64]. Using light microscopy and back-scattered electron imaging, it was also proven that NWs of 14 and 28 μm are not properly internalized, resulting in a frustrated phagocytosis, or an inability to engulf its target, which is in turn an initiator of the inflammatory response. In a different approach, red blood cells exposed to Ag NWs of 2 μm in length and a diameter of 40 nm were confirmed to suffer structural changes, aggregation, and hemolysis in a dose-dependent manner [65].


**3. Obstacles to globalization, from drop in the growth to political ones**

### **3.1 Drops in the growth due to financial crises or emerging conflicts**

After the protracted upward development of globalization described in Section 1, world trade fell sharply in 2009 (**Table 8**).

Figures represented in **Table 8** clearly show the drop in both growth and hopes caused by the bankrupt of both Fannie Mae and Freddie Mac.

The global financial crisis of 2008 was followed by an extremely fluctuating and sometimes stagnant trend in international trade (**Figure 2**).

In these sudden (or gradually postponed) storms, both businessmen and politicians ask themselves *what's around the corner*, while the latter ones—in short or mid-terms, also due to different visions of the world (*Veltanschauung*), competences, and vested interests—succeed in finding some solutions in near every case, normally damaging State budget by the increase of expenses to be "solved" by increasing debt or printing currency (or both).

The problem referred here is a well-known one, whose most important consequences are connected to i) the spatial extension and ii) the time span of the crisis.

While the latter can be traced back to the abovementioned political "solution," the former one is strictly connected to such factors as follows:

Factors 1–3 are self-explaining ones, and in a sense the fourth too, about which we limit here to recall i) the dramatic effects of *Black Friday* on the world economy, ii) the large but not-so-dramatic effects of Freddie Mac & Co. in 2009, and iii) the effects of crises in some Latin America countries, repeated ones but regularly absorbed up to now.

*Source: https://www.statista.com/statistics/264682/worldwide-export-volume-in-the-trade-since-1950/ (accessed 6 May 2022).*

**Table 8.**

*Global export value of trade in goods 2008–2009 (billion dollars).*

#### **Figure 2.**

*Global export value of trade in goods 2010–2020 (billion dollars). Source: Authors' elaboration based on data from https://www.statista.com/statistics/264682/worldwide-export-volume-in-the-trade-since-1950/ (accessed 6 may 2022).*

Two more notations seem anyway of some interest here:

Financial crises are anyway not alone in causing (heavy) disturbances to world economy, and globalization in particular. One more category storms includes all the more or less important mismatches occurring along years in different continents, *sudden overturning of regimes*, *revolutions*, *famines*, *vars*.

### *Our Globalization Era among Success, Obstacles and Doubts DOI: http://dx.doi.org/10.5772/intechopen.105545*

To recall here only the most dramatic of them, we could rapidly mention the French Revolution (1789), the crumbling down of the Chinese Empire (1911), the Soviet Revolution in Russia (1917), the pulling out of the Shah in Imperial Persia (1979), not to mentions the so many local military rebellions in Africa, South America, and elsewhere.

Mention apart is deserved, to better clear our topic, by the World War I, originated by apparently silly causes (the assassination of an Austrian Archiduke), but as a matter of fact signing the dramatic end of the unbelievable more than 40 years of expansion and growth of the Western World which on due course since the Franco-German War of 1871.

Treating these two kinds of problems, scholars turn by turn qualify these disturbances as *unexpected*/ *unimaginable*/*unpredictable* and so on. In parallel, they resume the old double couple of environmental "challenges and threats" for firms must strategically answer coping with their own "strengths and weaknesses." Be it of this kind, or even a more sophisticated one by the help of econometric models, this interpretation can be radically criticized on the bases of the systematic approach to the problem, suggested by a seminal contribution of 1933 by Ragnar Frisch (Nobel Prize in Economics 1969) [42].

This approach, which gave origin to a set a studies in particular in non-Anglo-Saxon world [43, 44]4 , distinguishes two basic kinds of forces operating within economic dynamics, moving it currently or by exception, in regular v. erratica a way.

So, squeezing some thousands of pages in a few lines, we could describe Frisch interpretation as such the following:

Obvious to mention among most important impulse variables as the last 100 years are concerned, the World War I and II, the Sino-Japanese war of 1931 ff., the Italian war against Ethiopia 1934–1935 and—after some 20 years of peace 1944 (Bretton Woods) to Vietnam war (1964 ff.)—the set on international wars and disturbances, in addition to abovementioned ones, also the two *oil crisis* (1973 and 1980), the crumbling away of the Berlin Wall, and the disintegration of U.R.S.S. (1991) to give space at the end to the series of further wars in Iraki, Serbia, Lybia, and elsewhere.

Generally speaking, the political, sociocultural, economic, and industrial consequences of those impulse variables are well known ones to the reader, with general overturning—in worst cases—of boundaries, population distribution, public finance, industrial equilibria, but, at the same time, a fast development of technology, and so many advantages for the winner, if any. One more feature anyway to be mentioned physical and financial destructions apart—is the common and general increase of

4 See in particular Demaria, *cit*., *I propagatori*, 51–751, *Entelechiani e antientelechiani* (*Impulse variables*), 755–1279.

prices, industrial, and consumer ones, which change the equlibria of families, firms, and in case the State in some cases in a dramatic way.

Propagation variables, each of them normally working with better or worse an equilibrium, are on their turn differently shocked by the aforementioned impulses, anyway under an immediate disorder under the pressure of the impulse ones. We conclude this flash leaving on one side the analysis of the consequences once more to the reader, and on the other listing the most important propagation variables:

A. natural propagation forces:

	- institutions,
	- international relations,
	- money, banking, and finance;
	- type and size of firms,
	- competition structures,
	- distribution of income and wealth (according to Pareto theories) [43].