Reactive Oxygen Species-Related Nanoparticle Toxicity in the Biomedical Field

The unique physicochemical characteristics of nanoparticles have recently gained increasing attention in a diverse set of applications, particularly in the biomedical field. However, concerns about the potential toxicological effects of nanoparticles remain, as they have a higher tendency to generate excessive amounts of reactive oxygen species (ROS). Due to the strong oxidation potential, the excess ROS induced by nanoparticles can result in the damage of biomolecules and organelle structures and lead to protein oxidative carbonylation, lipid peroxidation, DNA/RNA breakage, and membrane structure destruction, which further cause necrosis, apoptosis, or even mutagenesis. This review aims to give a summary of the mechanisms and responsible for ROS generation by nanoparticles at the cellular level and provide insights into the mechanics of ROS-mediated biotoxicity. We summarize the literature on nanoparticle toxicity and suggest strategies to optimize nanoparticles for biomedical applications.


Introduction
Nanoparticles (NPs) are a class of novel synthetic particles with dimensions < 100 nm. Depending on their shape and size, the distinct physical and chemical characteristics give NPs different functions. NPs are widely used in many consumer products, including textiles, cosmetics, water purification, and food packaging [1,2]. They are also used in the engineering of photocatalysts, energy, and optoelectronics [3][4][5][6].
Despite the potential for positive applications of NPs in various fields, an increasing number of studies have indicated their adverse effects on organisms [16,17] and cells following NP exposure [18,19]. The toxic potential of NPs is dependent on their size and shape, which determined their propensity to induce the generation of reactive oxygen species (ROS) [20,21]. The excess generation ROS may induce an array of physiopathologic outcomes, including genotoxicity, apoptosis, necrosis, inflammation, fibrosis, metaplasia, hypertrophy, and carcinogenesis [18,22,23]. The toxicity of NPs has also been shown to enhance the expression of proinflammatory cytokines and activate inflammatory cells, such as macrophages, which further increase the generation of ROS [23,24]. The increased generation of ROS following exposure to NPs has been also shown to induce the modulation of cellular functions, with fatal results in some cases [17,23,25]. In this review, we discuss the main mechanisms underlying the ROS bursts induced by NPs, analyze the primary reasons for the cytotoxicity of NPs, and summarize the potential pathogenic effects of NPs. Our present review provides overwhelming evidence that the over-production of ROS is the major cause of the biotoxicity of NPs. Therefore, novel research should aim to reduce the cytotoxicity of NPs by designing NPs which induce low ROS production.

The Application of NPs in the Biomedical Field
NPs have been used in a variety of medical applications, and several novel NPs exhibit properties which are promising for their use in novel biomedical materials. As summarized in Table 1, Nano-C60 can be used as an anticancer agent, which inhibits cancer cell proliferation, both in vivo and in vitro [26]. ZnO NPs have been used as fillers in orthopedic and dental implants [38]. TiO 2 can be used as antibacterial agents, in air and water purification, and for dental prostheses [52-54]. Davaeifar et al. reported that a phycocyanin-ZnO nanorod could protect the cell by decreasing endogenous ROS generation [68]. Pacurari et al. pointed out that SWCNTs could be applied as a clinical diagnostic agent and as bioengineering materials [88]. Beyond that, numerous NPs can be used as antimicrobial agents, which kill bacteria by inducing ROS bursts ( Table 1).

The Mechanisms of Increased ROS Induced by NPs in Cells
ROS are chemically reactive particles that contain oxygen, including hydrogen peroxide (H 2 O 2 ), reactive superoxide anion radicals (O 2-), and hydroxyl radicals (•OH) [92,93]. ROS are predominantly generated in organelles such as the endoplasmic reticulum (ER), in peroxisomes, and most notably in the mitochondria [94]. During oxidative phosphorylation, oxygen is used for the synthesis of water by the addition of electrons through the mitochondrial electron transport chain (ETC). Some of these electrons are accepted by molecular oxygen to form O 2-, which can further transform H 2 O 2 and •OH [93].
In a physiological context, ROS are produced as a natural response to the normal metabolism of oxygen [95] and serve a vital role in various cellular signaling pathways [96,97]. Dröge and Holmstrom et al. reported that ROS could activate numerous signaling cascades, including the epidermal growth factor (EGF) receptor, the mitogen-activated protein kinase (MAPK) cascades, the transcription factor activator protein-1 (AP-1), and the nuclear factor-KB (NF-κB), and further participated in the process of mammalian growth, proliferation, and differentiation [98,99]. Further studies showed that ROS also regulated wound repair [100], survival after hypoxia [101], intracellular pH homeostasis [102], and innate immunity [103].
Nevertheless, following exposure to NPs, the intracellular generation of ROS may sharply increase by inducing ROS bursts in cells [20] (Table 1 105,106]. The dissociated metal ion (i.e., Ag + ) also causes cellular enzyme deactivation, membrane structure disruption [31,107], disturbed electron-shuttling process [108], depleted redox potential levels, reduced mitochondrial membrane potentials (MMP) [109], and further enhances the accumulation of intracellular ROS. NPs have been also reported to promote the intracellular ROS accumulation by disturbing the electron transfer process [32,110], increasing the NADP + /NADPH ratio [30], and interfering mitochondrial function [18]. NPs further interfere with the expression of oxidative stress-related genes, such as soxS, soxR, oxyR, and ahpC [58]; antioxidant genes, like sod1 and gpx 1 [111,112]; and the NADPH productionrelated gene met9 [30]. The instability in the expression of oxidative and antioxidant genes caused by NPs accelerates intracellular ROS accumulation.
Interestingly, increased ROS production has been strongly associated with particular sizes and shapes of NPs [113,114]. For example, TiO 2 NPs contributed to intracellular ROS generation, which led to nucleic acid and protein damage [10]. Liao et al. found that 10 nm TiO 2 NPs had higher genotoxicity than other sizes tested and therefore could induce more ROS generation [115]. In another case, Se NPs promoted the production of ROS in cells, and the yield of intracellular ROS was highly associated with the diameter of Se NPs. In this case, a diameter of 81 nm induced more ROS production than other sizes tested [113]. Cho et al. further showed that the shape of NPs strongly affected their capacity to induce ROS production. Day flower-mimicking metallic nanoparticles (D-NP) lead to a significantly higher production of ROS than night flower-mimicking metallic nanoparticles (N-NP), resulting in an enhanced cell killing effect [114] (Fig. 1).
NPs can induce intracellular ROS bursts at a very low concentration (showed in Table 1), for example, Nano-C60 at 1 μg/mL can significantly increase cell apoptosis

ROS Results in Lipid Peroxidate and Membrane Structure Damage
Lipids, especially unsaturated fatty acids, are important intracellular macromolecules, which play key roles in the structure and functioning of the cell membrane. NPs are strongly attracted to the cell membrane, where they can generate ROS and lead to outer membrane lipid peroxidation. The altered fatty acid content of the cell membrane may result in increased cell permeability, which results in the uncontrolled transport of NPs from the extracellular environment into the cytoplasm, where cellular damage may progress further [76,122]. Intracellular NPs induce the next round of ROS bursts. Overburdened ROS lead to the rupturing of the membranes of organelles, the leakage of the organelles' contents [52,123], the inactivation of cell receptors [124], the release of lactate dehydrogenase (LDH), and further irreversible cell damage [125].

ROS Attacks Proteins and Results in Functional Inactivation
ROS attacks the hydrophobic residues of amino acids, contributing to the breakage of peptide bonds and interfering with the function of these proteins [126][127][128]. Carbonylation is another feature of proteins subjected to oxidative damage [129]. Carbonylated proteins form aggregates that are chemically irreversible and cannot be degraded via proteasomes, leading to the permanent loss of function in these proteins [130,131]. Gurunathan et al. [132] showed that PtNPs could enhance the generation of ROS and increase carbonylated protein levels, which inhibited osteosarcoma proliferation and contributed to apoptosis. In one case, combustion and frictionderived nanoparticles (CFDNPs) had accumulated in the brain of young adults with Alzheimer's disease, which likely promoted ROS generation, resulting in protein misfolding, aggregation, and fibrillation [133]. Furthermore, Pelgrift et al. showed that Mg NPs may inhibit gene transcription or damage proteins directly [10].
The increased production of ROS is the main cause of gene miscoding, aneuploidy, polyploidy, and the activation of mutagenesis in cells exposed to NPs [145][146][147][148]. Among the nucleotide pools, guanine is the most vulnerable and is easily oxidized to 8-oxoG by ROS [149]. The increased level of 8-oxo-dG in DNA results in the mismatch of DNA bases [150]. Similarly, the incorporation of A:8-oxoG causes an increased rate of G:C > T:A deleterious transversion mutations [151,152]. The ratio of G:C > T:A transversion to G: C > A:T transition mutation has also been used as an index to quantify the oxidative DNA damage [153].
The generation of ROS induced by NPs resulted in the accumulation of DNA damage, which drives the development of mutagenicity [154], oncogenesis [155], multidrug resistance [156,157], aging, and immune escape [158]. Jin et al. showed that the overproduction of ROS dramatically increased mutagenesis of DNA-binding transcriptional regulator genes, which resulted in an expedited antibiotic efflux [159], which in turn promotes the multiple-antibiotic resistance of bacteria [34]. Giannoni et al. reported that mitochondrial DNA mutations occurred with increasing intracellular ROS and further damaged the activity of ETC complex I and resulted in mitochondrial dysfunction [160,161].
DNA damage induced by NPs has been shown to inhibit amino acid synthesis, replication [162], and cause the aberrant accumulation of p53 [163] and Rab51 proteins [82,142]. DNA damage may also delay or fully arrest the cell [164]. Cells with damaged DNA lose the capacity for growth and proliferation [165] and may eventually result in cell death [166] (Fig. 3).

Increased Production of ROS Induces Cell Damage and Disease Occurrence
NP cytotoxicity is associated with oxidative stress, endogenous ROS production, and the depletion of the intracellular antioxidant pools. The increased oxidative stress leads to oxidative damage to biomacromolecules, which further affects the normal functioning of the cell and contributes to the occurrence and development of various diseases [167].
NPs induce membrane damage and enhance the transport of NPs into the cytoplasm. NPs concentrate in lysosomes, mitochondria, and the nucleus, which results in catastrophic consequences for the cell [168,169]. It has been reported that NPs can reduce adenosine triphosphate (ATP) generation [89], deplete glutathione, induce protein mistranslation [170], rupture lysosomes [171], and inhibit the ribosomal subunit from binding transfer RNA (tRNA). These cellular events indicate a collapse of the fundamental biological process in the cell and lead to a significant decrease in cell viability [47]. Singh and Scherz-Shouval et al. reported that NPs could disturb cytoskeletal functions by inducing ROS generation and activate the process of autophagic and apoptosis in cells [89].
NPs enter the body via different routes, for instance through the skin, lungs, or intestinal tract (Fig. 4a) and can have a wide variety of toxicological effects and induce biological responses such as inflammation and immune responses [172][173][174]. In one case, exposure of cells to silica NPs caused macrophages to secrete a large amounts of interleukin-1β (IL-1β), which ultimately resulted in cell death [175]. Gao and colleagues reported that pulmonary inflammation was considerably higher in mice after exposure to carbon nanotubes, which could activate alveolar macrophages and induce a strong inflammatory response [176]. In another study, guinea pigs exposed to ZnO NPs suffered pulmonary damage, which leads to a decrease in total lung capacity and vital capacity [177][178][179].
ZnO NPs also induced severe injuries in the alveolar epithelial barrier and caused inflammation in the human lungs [180]. In another case, NPs absorbed into the intestines caused the inflammation and degradation of the intestinal mucosa [181]. Shubayev et al. noted that Mg NPs enhanced the migration of macrophages to the nervous system by degrading the blood-brain and bloodnerve barriers in an MMP-dependent manner [182]. Furthermore, mice which inhaled carbon nanotubes exhibited immunosuppression and repressed antibody response in naive spleen cells [183]. Finally, Cd NPs caused a severe decrease in blood monocyte viability, ultimately resulting in immunodeficiency [184].
In addition to the above pathologies, the highly variable level of ROS has been identified as the main cause of the development of numerous human diseases. Tretyakova and Liou et al. showed that oxidized DNA tends to form DNA-protein conjugates, which accumulate in the heart and brain and contribute to the occurrence of cancer, aging-related diseases, and chronic   [185,186]. Andersen [187] concluded that diabetes, as well as cardiovascular and neurodegenerative diseases, were highly related to the imbalance of ROS. Additionally, Pérez-Rosés et al. showed that increased ROS promoted Alzheimer's and Parkinson's disease development [188].
It has been further reported that NPs promote the apoptosis of breast cancer cells [35] and destroy malignant tissues and pathogens by promoting the generation of ROS [189,190]. However, ROS has also been found to induce the proliferation of both normal and cancerous cells, stimulating mutations, and initiating carcinogenesis in normal cells and multidrug resistance in cancerous cells [191,192]. Handy et al. found that fish exposed to carbon nanotubes exhibited granulomas in their lungs and tumors in their livers with extended exposure times [193]. Some NPs have caused multiple organ failure, primarily affecting the heart, lung, kidneys, and liver. TiO 2 NPs have been shown to promote reduced body weight, spleen lesions, blood clotting in the respiratory system, necrosis and fibrosis in liver cells, and in alveolar septal incrassation [194,195]. In one study, NPs also prevented stem cell differentiation, which aggravated organ damage [196]. Further research has also reported that NPs decreased sperm quality [197] and that exposure of sperm to carbon NPs influenced their ability to fertilize eggs and impaired the development of the embryos in purple sea urchins [198]. Mounting evidence shows the toxicological effects of NPs on microorganisms, algae, nematode, plants, animals, and humans specifically [22,199,200] (Fig. 4b).

The New Type of NPs with Fewer or No Cytotoxicity
NPs possess a range of biomedical properties that make them valuable (e.g., as antibacterial and anticancer agents [26-28]). Their main mode of action is their ability to increase the production of ROS in cells; however, this property also makes these particles toxic, by causing gene mutation, apoptosis, and even carcinogenesis [45,49,58]. Consequently, there is an urgent need to develop new NPs which retain their required properties without leading to excessive ROS production. Recent studies have reported on novel types of NPs which could remove intracellular ROS. These types fall into two classes: (1) NPs which can scavenge ROS [77] and (2) NPs which are coated with additional materials to decrease their cytotoxicity [87].
Panikkanvalappil and colleagues showed that Pt NPs inhibit the double-strand breakage of DNA by degrading ROS [201]. In another case, Mn 3 O 4 NPs modulated cellular redox resulting in the protection of biomacromolecules against oxidative stress [77]. Furthermore, the CeO 2 NP is a novel agent that protects cells and tissues against oxidative damage with its free radical-scavenging capacity [79,202].
H 2 O 2 is the main by-product of NP-cell interactions. H 2 O 2 destroys important biomolecules including proteins, lipids, and nucleic acids. However, when cells were treated with specialized MNPs coated with mercaptopropionic acid (MPA-NPs) or aminated silica (SiO 2 -MNPs), such damage was not observed [203,204]. Similarly, GO coated with polyvinylpyrrolidone (PVP) has fewer toxic effects on dendritic cell (DCs), T-lymphocytes, and macrophages than without this coating. PVP-GO has been shown to reduce the apoptosis of T-lymphocytes and even increase the activity of macrophages [205]. Pt-coated AuNRs (PtAuNRs) retain the efficacy of traditional gold nanorods (AuNR) and can trigger cell death of desired cells while scavenging the ROS, thereby protecting healthy, untreated cells from the indirect death induced by ROS production [87].

Conclusions and Outlook
NPs that possess unique physicochemical properties (e.g., ultra-small size, large surface area to mass ratio, and high reactivity) make them highly desirable in different applications. Engineered NPs for commercial purposes have been rapidly increasing. For that reason, the biosafety of NPs has gained more attention in the public. In this review, we summarized the mechanisms and responsible for ROS formation by NPs at the cellular level as well as recent advances of ROS-related NP toxicity in the biomedical field and highlighted the emerging field of cell-friendly NPs. The generation of ROS induced by NPs associated with their size, morphology, surface area, and component. In addition, ROS has bio-multifunctional in cell biology and biomedicine as well as the key mediator of cellular signaling, including cell apoptosis, viability, and differentiation.
However, to improve the biosafety of NPs and accelerate their use in the biomedical field, some bottlenecks need to be overcome and much work is still required. First, it is expected that high-throughput methods (HTMs) are designed to efficiently detect the biotoxicity of NPs in vitro and in vivo. HTMs could save time and resources, combine multiple parameters on a single system, and minimize methodological or systematic errors. It also would offer a deep understanding of the relationship between NP properties and cell responses, which could help us identify the optimal condition. Second, the molecular and cellular mechanisms related to the biotoxicity of NP-induced ROS are still unclear. There is a demand to further explore the mechanisms associated with the formation of ROS by NPs, which would provide more information to modify the chemicophysico features of NPs to control the ROS generation. This could help researchers develop novel strategies to reduce the hazards of engineered NPs for accelerating their clinical and commercial translation in the biomedical filed.
Finally, due to their structural characteristics, NPs may enter the body freely via multiple routes, and the accumulation of NPs in the body can induce inflammation and immune responses, which result in cell injury or death, organ dysfunction, and ultimately stimulate the occurrence of numerous diseases, such as Alzheimer's, Parkinson's, liver inflammation, and dysembryoplasia. These issues have become more pressing with the widespread use of NPs.