Graphene Toxicities

#### **Chapter 8**

## Graphene Oxide Nanotoxicity: A Comprehensive Analysis

*Mohammad Mahdi Sepahi and Marzieh Azizi*

#### **Abstract**

Graphene oxide (GO) has emerged as a promising nanomaterial with physicochemical properties that make it a candidate for a wide range of applications. However, the potential toxicity of GO is a significant threat that must be addressed before GO's safe use in biomedical and environmental applications can fully be realized. Numerous studies have demonstrated that GO has the ability to induce oxidative stress, inflammation, genotoxicity, and cytotoxicity in cell types and animal models. Importantly, the toxicity of GO is influenced by its size, morphology, charge, and surface functional groups. The current review summarizes recent research findings on the toxicity of GO by focusing on its cellular uptake, biodistribution, and biological effects. It provides an overview of the current understanding of GO nanotoxicity and highlights the need for additional research to assess its safety in various applications. By addressing these concerns and developing appropriate safety measures, we can fully exploit the potential of GO while ensuring its safe utilization in biomedical and environmental settings. Thus, the comprehensive evaluation of GO's potential risks is crucial for its full exploitation and utilization.

**Keywords:** biodistribution, cellular uptake, genotoxicity, graphene oxide, inflammation, nanotoxicity, oxidative stress

#### **1. Introduction**

Graphene oxide (GO) has garnered significant attention as a highly promising nanomaterial due to its exceptional physicochemical properties, including its large surface area, high mechanical strength, excellent electrical conductivity, and remarkable optical properties [1–4]. These characteristics make GO an attractive candidate for a wide range of applications in the fields of biomedicine, electronics, energy storage, and environmental remediation among others [5, 6]. However, numerous in vitro and in vivo studies using different cell types and animal models have demonstrated that GO can induce various adverse effects, including oxidative stress, inflammation, genotoxicity, and cytotoxicity, among other toxicities [2, 7, 8]. These toxic effects have raised concerns about the safe use of GO in biomedical and environmental applications highlighting the need to investigate its potential toxicity (**Figure 1**).

The mechanisms underlying the toxic effects of GO involve its interactions with cellular components such as proteins, lipids, and nucleic acids. These interactions can disrupt cellular functions and cell signaling pathways, leading to cellular damage and other adverse biological effects [9–13]. The toxicity of GO is influenced by

**Figure 1.** *Shape and size-dependent nanotoxicity of GO.*

its physicochemical properties, including size, morphology, charge, and surface functional groups [14–16]. Ou et al., for example, reviewed the toxicity of graphene family nanomaterials (GFNs) and found that the nanotoxicity of the material was influenced by, among other factors, the size, surface structure, functionalization, charge, impurities, aggregations, and corona effect. Different mechanisms have been identified to explain GFN toxicity, including physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. Toll-like receptors (TLRs), transforming growth factor β (TGF-β), and tumor necrosis factoralpha (TNF-α) dependent pathways are involved in the signaling pathway network, with oxidative stress playing a crucial role in these pathways [17]. Understanding the relationship between these properties and the toxicity of GO is crucial for designing safer nanomaterials and minimizing the risks of their use.

Thus, in order to assess the toxicity of GO comprehensively, it is essential to evaluate its cellular uptake, its biodistribution through the various biological matrices, and the accompanying biological effects. The understanding of how GO is internalized by cells, and how it gets distributed within the body leading to the specific biological responses that it elicits can provide valuable insights into its potential hazards and, therefore, guide the development of appropriate mitigation measures [18]. Moreover, the full analysis of the current knowledge of GO nanotoxicity can help identify knowledge gaps and highlight areas where further research is required in assessing its safety in various applications.

By addressing these concerns and developing effective strategies to minimize the adverse effects associated with GO, we can exploit its immense potential while ensuring its safe applications. The current analysis, therefore, aims to summarize recent research findings on the toxicity of GO and underscore the need for additional research to assess its safe use in different fields. The physicochemical properties of GO are important in understanding its nanotoxicity because they determine the interactions between the nanomaterials and living organisms. The combined effect of size and shape in nanotoxicity studies is paramount, influencing cellular uptake, biodistribution, clearance, and

#### *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

overall interactions with living organisms [19]. The size and shape of GO nanoparticles are linked to their reactivity and cellular uptake in biological systems. The potential toxicity of GO nanoparticles can affect the nervous tissues and other crucial body systems [20] and understanding these factors, therefore, is essential for designing safer and clinically relevant GO-based nanosystems. Furthermore, the different shapes of nanomaterials, such as sheets [10], flakes, and nanoribbons, can influence the speed and extent of their internalization and interactions with biological structures. GO nanosheets, for example, have a higher potential for membrane penetration than the other GO morphologies, leading to increased cytotoxicity. In contrast, GO nanoribbons have been implicated in inducing greater genotoxic effects due to their ability to cause DNA damage [9, 12]. Additionally, GO sheets with irregular shapes are cleared more slowly from the body, resulting in prolonged exposure and increased toxicity over time. Conversely, GO nanoparticles with a more spherical shape are cleared more quickly from the body, reducing their potential toxicity over time [21]. These findings emphasize the critical role of the shape of the GO nanoparticles in controlling the toxicity and safety of GO-based materials. Consequently, the size of GO materials is another critical determinant of its cytotoxicity [18, 22].

Surface functionalization of GO has been widely studied in its performance in biomedical applications, such as drug delivery and diagnostics. This technique allows tailoring the properties of nanomaterials to improve their interactions with biological systems for specific applications [23, 24]. Surface functionalization involves modifying the surface properties of nanomaterials by attaching various molecules to alter their stability, solubility, and interactions with biological systems [25, 26]. Different functional groups can influence the toxicity of nanomaterials by affecting cellular uptake, biodistribution, and biocompatibility [27]. However, surface functionalization can also affect particle agglomeration and enhance stability [28], which can impact their behavior and toxicity in biological systems. Also, functionalization of GO nanoparticles can alter the physicochemical properties of GO, influencing its toxicity [16]. For instance, functionalization with hydrophilic groups can reduce aggregation and enhance biocompatibility, while functionalization with targeting ligands can improve the specificity of GO-based drug delivery systems [29]. By carefully selecting appropriate surface functionalization strategies, researchers can mitigate potential nanotoxicity risks associated with GO.

In the realm of biomedical applications, surface functionalization emerges as a promising strategy in enabling targeted delivery of therapeutic agents using GO nanoparticles. However, the selection of functional groups and their stability is a crucial consideration in ensuring both the safety and effectiveness of targeted applications. This multifaceted approach to utilizing GO, in conjunction with a comprehensive understanding of its concentration-dependent effects on cell viability, paves the way for innovative and tailored applications with potentially far-reaching benefits.

The resulting surface GO nanoparticles control their interactions with biological systems and it plays a critical role in determining the toxicity of GO in cells and tissues by impacting their stability, aggregation, and interactions with biological systems [12, 30–32] as well as their cellular uptake pathways (**Figure 2**). Positively charged sheets are taken up through phagocytosis and clathrin-mediated endocytosis, while negatively charged sheets cross the membrane through phagocytosis and sulfate-receptor-mediated endocytosis [33]. Also, the charge of nanoparticles controls their affinity for cell membranes, leading to variations in cellular uptake and toxicity [34]. Furthermore, the surface charge of nanoparticles can lead to an imbalance in the

**Figure 2.**

*Key points regarding the effect of GO surface functional group on nanotoxicity.*

redox state of cells, which, in turn, can cause malfunctions in the internal metabolism of cells and in pathologies at the organismal level [8].

Whichever the case, the surface chemistry of GO has a significant influence on its nanotoxicity, affecting various aspects of their behavior and interactions with biological systems. Understanding the role of surface chemistry in GO nanotoxicity is therefore essential for assessing potential risks and developing strategies to mitigate toxicity.

### **2. Biodistribution**

#### **2.1 Effects of GO size and shape on biodistribution**

The effects of particle size on biodistribution have different aspects, impacting circulation time, organ accumulation, and the kinetics of clearance. Smaller nanoparticles tend to have a longer circulation time within the body and can accumulate in specific organs or tissues, magnifying their toxic potential. On the other hand, larger nanoparticles are cleared more rapidly from the body, reducing their overall toxicity. For example, nanomaterials smaller than 20 nm tend to accumulate in the kidney, while those in the range of 20–100 nm preferentially deposit in the liver [35].

Nonetheless, different shapes, such as spheres, rods, or sheets, can exhibit varying degrees of cellular uptake, distribution, and toxicity. GO sheets with irregular shapes are cleared more slowly from the body compared to those with more regular shapes. Prolonging the exposure period through slower rates of clearance intensifies the toxicities of the particles over time [36]. Conversely, GO nanoparticles with a more spherical shape are cleared more rapidly from the body, reducing their potential toxicity over time. **Figure 3** shows the clearance and biodistribution of GO in the different study.

*Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

#### **Figure 3.**

*Clearance and biodistribution of GO. (A) Proposed pathway for the photo-Fenton reaction, which contributes to the degradation of GO, potentially leading to the formation of oxidized polycyclic aromatic hydrocarbons [18]. (B) The blood circulation curve of GO is depicted, highlighting its behavior in the bloodstream. (C) The number of black spots observed in liver sections at different time points post-injection (PI) indicates the accumulation of GO in the liver. (D) The time-dependent distribution of GO in different organs, including the LI: liver; SP: spleen; K: kidney; H: heart; LU: lungs; ST: stomach; I: intestine; SK: skin; M: muscle; BO: bone; BR: brain; and T: thyroid, is presented. (E) The signal of GO in the liver and spleen is shown at different time points PI. (F) The presence of GO in urine and feces is demonstrated at different time points PI [18].*

#### **2.2 Functional group effects on biodistribution**

Functionalization of GO nanoparticles strongly influences their biodistribution. For example, intravenous administration of dextran-functionalized GO led to extensive fecal excretion [37, 38], whereas PEGylated GO showed lesser feces excretion suggesting that the choice of functional groups can impact the biodistribution and clearance pathways of GO nanoparticles [39]. This also indicated that dextranfunctionalized GO might be excreted through the gastrointestinal system, while PEGylated GO may be cleared differently [40]. **Table 1** provides the clearance and biodistribution of 10–30 nm GO with 1 nm thickness and the corresponding tracking methods and the influencing factors.


#### **Table 1.**

*Clearance and biodistribution of 10–30 nm GO with 1 nm thickness and the corresponding tracking methods and the influencing factors [18].*

In a study by Yang and colleagues, PEGylated GO biodistribution was tracked over time. The pharmacokinetics analysis revealed a two-compartment model: the central compartment with fast material exchange (e.g., blood, heart, and liver) and the peripheral compartment with slow material exchange (e.g., muscle, bone, and fat). The blood circulation of GO was found to consist of a rapidly distributed component and a slower elimination component. The half-lives of the blood circulation in the first and second phases were 0.39 ± 0.097 and 6.97 ± 0.62 hour, respectively [18]. At 1-hour post-injection, GO was distributed across different organs of the mice. Strong signals were observed in the bone marrow, which may be due to macrophage uptake of GO into the bone marrow. The radiation signal was mainly concentrated in the mononuclear phagocyte system (MPS), such as the liver and spleen. This experience evidenced the influence of circulation time of GO nanoparticles in various body compartments. They were found to distribute across different organs, including the bone marrow, liver, and spleen. It showed that the surface chemistry of GO nanoparticles influences their interactions with macrophages in the MPS, affecting their accumulation in these organs [18].

Long-term biodistribution studies found that GO was significantly present in the liver, indicating accumulation, which was attributed to enzymatic and photo-Fenton degradation. However, a small amount of GO remained in the liver even after 20 days, raising concerns about potential long-term toxicity [48]. GO also accumulated in the kidneys and intestines, suggesting its excretion through urine and feces [18].

#### **2.3 Organ toxicity**

The biodistribution of GO in various organs, such as the liver, lungs, and spleen, depends on the route of administration and dosage. Higher doses of GO can lead to organ toxicity, characterized by inflammation, oxidative stress, and histopathological changes. These findings spotlight the importance of dose and route of administration in organ toxicity GO [39, 49].

Animal studies have been conducted to assess the in vivo toxicity of GO. Intravenous injection of GO of various sizes in mice resulted in acute and chronic damage to the lung and kidney. This highlights the potential adverse effects of GO on vital organs and the importance of considering toxicity in animal models. Similar in vivo studies have shown that blood exposure to GO can cause accidental death in mammals, including non-human primates and mice, due to acute anaphylactic reactions. The severity of GO's toxic effects on living cells and organs varies based on factors such as administration route, dose, synthesis method, and physicochemical properties. Smaller GO nanoplatelets have shown lower toxicity and potential as drug carriers while enhancing the anticancer effect of chemotherapeutic drugs. This emphasizes the need for further studies to fully understand the potential toxicity of GO and its impact on various aspects, including behavior, reproduction, development, and genotoxicity [49].

#### **2.4 Clearance**

The size and shape of GO nanoparticles also play a crucial role in their clearance from the body, impacting the resident contact time with tissues and long-term potential toxicity. Smaller and thinner 2D GO sheets exhibit distinct clearance patterns in the body. Well-dispersed and smaller GO sheets have the capability to pass through the lung capillaries and get excreted through the glomerular filtration barrier (GFB)

#### *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

in the kidney, a process facilitated by their structural features, such as thinness, flexibility, and orientation in flow [50]. The urinary excretion of GO sheets following systemic circulation administration has been reported in several studies [37, 39, 51, 52]. In an animal study using a direct bladder puncture method, about 50–60% of GO was excreted in the urine 2 hours after injection [53, 54] and no significant changes in kidney function or structural damage to the glomerular and tubular regions were found after injection of well-dispersed GO nanosheets. GO's ability to pass through the kidney's filtration barrier is remarkable, considering that most GO nanosheets are larger than the pore size in the kidneys. Nevertheless, they can traverse these pores through rolling, crumpling, or folding, facilitated by their mechanical flexibility. This process allows thin graphene with high mechanical flexibility to be easily discharged from the body (**Figure 4**) [18].

It is noteworthy that the feces excretion of GO has also been observed, although to a lesser extent. The extent of feces elimination has been linked to specific functionalization moieties and routes of administration. For instance, intravenous administration of dextran-functionalized GO [37, 38] resulted in extensive feces excretion, while PEGylated GO showed it to a lesser extent [39]. Oral administration of GO similarly led to extensive feces elimination [50].

Although most GO nanosheets are excreted in the urine, a small but significant part remains isolated in the spleen at approximately 1.6% ID/g at 60 days after intravenous injection [51]. GO accumulates in the marginal zone macrophages of the mouse spleen [43]. Over time, the quantity of GO in the spleen decreases, but a portion remains inaccessible for removal due to slow degradation, a process taking over 270 days [50]. The spleen-residing GO gradually accumulates defects, resulting in structural changes and a transition from the initial sp2-hybridized carbon structure to mainly tetrahedral sp3 amorphous carbon [18, 43]. The physiological environment of the spleen is essential for the degradation process of GO, as GO incubated in vitro without exposure to light at 37°C did not exhibit similar structural changes [50].

This extensive exploration of GO's distribution and elimination in the body emphasizes the complex relationship between its size, shape, and to a lesser extent, mode of administration and surface functionalization, and the body's clearance processes. Smaller and thinner as well as more regular-shaped GO sheets exhibit unique excretion pathways and faster clearance, while larger, thicker sheets with more irregular morphology tend to accumulate in specific body organs, especially the spleen, leading to a prolonged clearance process and simultaneous toxic damage.

#### **Figure 4.**

*Morphological reconfiguration of GO nanosheets during entrance to the glomerular filtration barrier that enables the thin and flexible GO nanosheets to cross the GFB and be excreted in the urine [18].*

#### **3. Penetration**

The size of GO also plays a crucial role in its interaction with living organisms. Smaller nanoparticles exhibit a larger surface area-to-volume ratio, enhancing their reactivity and cellular uptake over those of the larger particles.

#### **3.1 Influence of size and shapes**

This is because the smaller nanoparticles experience less steric hindrance and have an increased propensity to penetrate biological barriers, which can result in greater interaction and higher toxicity levels. Also, smaller particles possess a larger surface area-to-volume ratio, enhancing their potential for interactions with biomolecules [31]. It has been reported that both sizes of GO (a ∼500 nm protein-coated commercial GO and GO sheets of 1 μm particle range) crossed over into the lysosomes for excretion and that the smaller nanoparticles induce more pronounced cellular stress responses and oxidative damage exacerbating their cytotoxic effects compared to their larger ones [8]. In a study on mouse mesenchymal progenitor C2C12 cells, a ∼500 nm proteincoated commercial GO was internalized through clathrin-mediated endocytosis, while larger GO sheets of 1 μm particle range were taken up through phagocytosis. Enhanced cellular uptake can result in higher toxicity, as internalized nanoparticles interact with cellular components and induce adverse biological responses. On the other hand, different shapes of GO materials possess varying degrees of surface area, surface charge, and functional groups, all of which influence their binding affinity and interactions with biomolecules [7, 19, 31]. Rod-shaped nanoparticles, for instance, have been observed to exhibit greater cellular internalization relative to spherical nanoparticles, although some studies have yielded contradictory conclusions [55]. This effect, which is influenced by shape, underscores the importance of different shapes of GO in determining the safety and nanotoxicity of the material. Moreover, studies involving GO nanoflakes have shown their uptake by both cancerous and non-cancerous cell lines through cellular uptake pathways tracked by label-free Raman imaging [56].

Mendes et al., illustrated during a 12-hour incubation period, human macrophages (differentiated THP-1 cells) were exposed to nano-graphene oxide (NGO) at different concentrations. The data from the study did not show a clear difference in internalization between the concentrations or sizes of NGO flakes. Regardless of the conditions, the NGO was observed as large collections of flakes rather than individual flakes inside the cells. Notably, at a concentration of 100 μg/mL, the cells were able to internalize agglomerations of NGO with diameters of up to 4 μm. The study also conducted a detailed transmission electron microscopy (TEM) analysis to differentiate the NGO flakes from other intracellular structures, such as cellular membranes and endosome membranes. The TEM analysis confirmed the ability to distinguish NGO flakes from other internal cellular structures (**Figure 5**).

It illustrates a key concept that the surface area-to-volume ratio of small nanoparticles enhances their reactivity and propensity for cellular uptake. Furthermore, the shape of nanomaterials significantly influences their interactions with cells and tissues.

#### **3.2 Influence of surface chemistry**

The charge of GO nanoparticles also has a strong impact on their cellular uptake mechanisms and, in turn, their potential toxic effects [19, 33, 43, 55]. The electrostatic interactions between the charge of GO and cell membranes are essential determinants *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

#### **Figure 5.**

*Differentiated THP-1 cells that treated by NGO flakes for 12 hours. The NGO flakes had an average diameter of 453, 277, and 46 nm and were used at concentrations of 10 μg/mL (a–c) and 100 μg/mL (d–f). The red-shaded areas indicate the internalized NGO flakes or clusters. Additionally, the red arrows in (c) and (e) point to the NGO flakes and clusters that are in contact with the cellular membrane.*

of the cellular uptake of GO nanoparticles. This means that the surface charge of GO nanoparticles directly affects their ability to internalize into cells and the specific cellular endocytosis pathways they utilize. In particular, positively charged GO nanoparticles exhibit a strong electrostatic attraction to negatively charged cell membranes, making them more readily absorbed by cells compared to negatively charged and neutral GO nanoparticles. This difference in cellular uptake mechanisms can have profound implications for the potential nanotoxicity of GO [31]. Studies have shown that negatively charged GO nanoparticles have a lower rate of endocytosis and do not typically utilize the clathrin-mediated endocytosis pathway, which is a common mechanism for cellular uptake. In contrast, positively charged GO nanoparticles internalize more rapidly via the clathrin-mediated pathway. This difference in uptake mechanisms can have implications for the intracellular fate of GO nanoparticles and the potential biological responses they trigger [57]. For example, research by Yang et al. in 2017 confirmed that positively charged GO nanoparticles were internalized more efficiently compared to negatively charged or neutral GO nanoparticles in human lung epithelial cells (A549). Once internalized, GO nanoparticles interface with intracellular components and trigger complex inflammatory responses. This observation highlights the importance of the surface charge of GO in influencing its cellular uptake, which can subsequently impact its nanotoxicity [58]. The extent of cellular uptake and internalization is, however, tied to the physicochemical characteristics of GO, underlining the significance of these properties in shaping the toxic outcomes [31].

#### **4. Protein interaction**

#### **4.1 Effect of size and shape on GO protein interactions**

Recent research has illuminated the profound influence of the GO shape on its cellular interactions. For instance, GO micropatterns with different shapes, including triangles

and squares, impact differently the cell migration parameters, such as distance, speed, and directionality, underlining the critical role of shape-dependent effects of GO in living organisms [33]. However, the impact of shape and size on GO nanotoxicity extends beyond cellular interactions and encompasses its influence on protein interactions. The size and shape of GO nanomaterials play a critical role in governing their interactions with biomolecules, particularly proteins and DNA. GO size and shape play a pivotal role in dictating its interactions with subcellular components in living organisms, leading to the induction of functional perturbations in cells and tissues. The shape and size-dependent GO nanotoxicity is, therefore, a critical aspect to be considered when examining the potential hazards associated with GO nanomaterials. In this context, the size of GO nanoparticles assumes significance, as smaller nanoparticles exhibit an enlarged surface area-to-volume ratio, intensifying their reactivity in biological systems. Consequently, the size of GO nanoparticles emerges as an important factor in shaping their interactions with biomolecules. The shape of GO materials also affects their surface area, which, in turn, has a direct bearing on their proteins and other bio-molecular pharmacokinetics [59]. GO nanoflakes, characterized by a higher surface area, have been found to adsorb a greater amount of proteins compared to GO sheets with a lower surface area, significantly enhancing their biological activities and toxicity over those of the nanosheets [33]. Moreover, the shape of nanomaterials significantly determines their interactions with cells and tissues. Nanomaterials characterized by sharp edges or high aspect ratios have been associated with more pronounced damage to cells and tissues [22, 34]. Thus, comprehending the influence of nanomaterial shape on cellular interactions is vital for designing safer and more clinically relevant nanosystems. This highlights the significance of shape-dependent interactions with cells, irrespective of their physiological state.

#### **4.2 Effect of surface chemistry on GO protein interactions**

The functional groups and the resulting surface charge of GO nanoparticles strongly influence GO's interactions with proteins and, in turn, the extent of its cytotoxicity. The charge of GO nanoparticles can lead to damage of cells through its effects on cellular proteins and nucleic acids. Positively charged nanoparticles have been shown to disrupt cell membranes, induce oxidative stress, and trigger inflammatory responses. In contrast, negatively charged nanoparticles interfere with cellular processes and induce genotoxic effects. Overall, positively charged GO nanoparticles are often found to be more cytotoxic compared to negatively charged or neutral GO nanoparticles [60]. This was illustrated in a study by Peng et al. in 2018; the effect of charge on GO's protein adsorption was investigated using human serum albumin (HSA). The study revealed that positively charged GO nanoparticles adsorbed more HSA compared to negatively charged or neutral GO nanoparticles. This observation was attributed to the electrostatic attraction between positively charged GO and negatively charged HSA, highlighting the role of charge in influencing protein interactions with GO [61].

The charge of GO nanoparticles not only affects their interactions with proteins but also their compatibility with blood components. Again, positively charged nanoparticles tend to interact more with blood proteins, leading to the formation of protein coronas [62]. These protein coronas can significantly impact the behavior of nanoparticles, including their circulation time and potential for clearance from the bloodstream. These interactions play a crucial role in influencing the overall toxicity of GO nanoparticles. A study by Zhang et al. in 2018 investigated the effect of charge on GO's hemolytic activity using red blood cells (RBCs) [63]. The study found that positively charged GO nanoparticles were more hemolytic compared to negatively charged or

#### *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

neutral GO nanoparticles. This observation suggested a greater electrostatic interaction between positively charged GO and negatively charged RBC membranes [63].

In addition to charge, the surface functional groups of GO nanoparticles also play a vital role in modulating the cellular proteins' interactions with GO. These functional groups can affect protein adsorption, leading to the formation of a protein corona. The composition and structure of the protein corona have a significant impact on the biological effect, fate, and toxicity of GO nanoparticles [62]. This aspect provides opportunities to control protein corona formation and mitigate potential adverse effects, making it an important consideration in the design and evaluation of GO-based nanomaterials. Furthermore, the surface functional groups alter the surface charge, hydrophobicity, and other properties of GO nanoparticles. These modifications affect interactions with various blood components and different organs inducing such changes as in circulation time, rate and extent of accumulation in specific tissues, and clearance from the body, which bear direct implications for the overall toxicity profile of GO nanoparticles [18, 62].

The intricate reciprocity between the surface chemistry of GO and its potential to induce oxidative stress, specifically reactive oxygen species (ROS) generation, is a multifaceted phenomenon with significant implications for nanotoxicology and cellular responses. GO nanoparticles have been widely recognized for their propensity to cause oxidative stress, which is linked to genotoxicity and cytotoxicity [60, 64]. While several studies have elucidated the role of surface chemistry in modulating these outcomes, the impact of GO's surface area and functionalization on ROS generation is an intriguing facet that merits further exploration. Notably, a study conducted with SH-SY5Y cells revealed that GO with a surface area of 750 m2 /g exhibited an unexpectedly lower potential to induce oxidative stress through lipid peroxidation, as evidenced by significantly decreased malondialdehyde (MDA) levels [65]. For the 150 m<sup>2</sup> /g graphene, no significant increase in MDA was observed at any concentration or time compared to the control group (p > 0.05). However, a significant decrease in MDA levels was observed at a concentration of 400 μg/mL after 24 and 48 hours compared to the cell control group (p ≤ 0.01, p ≤ 0.001) [65]. **Figure 6** shows the impact of two different types of graphene on MDA levels in SH-SY5Y cells [25, 65].

This unexpected finding challenges traditional notions about the pro-oxidative potential of GO and highlights the importance of GO surface area considerations. Furthermore, the observed increase in cellular glutathione (GSH) levels following exposure to GO, a known antioxidant marker, hints at a potential antioxidant role for certain GO formulations (**Figure 7**) [26, 65].

**Figure 6.**

*The impact of two different types of graphene, (A) 150 m2 /g and (B) 750 m2 /g, on MDA levels in SH-SY5Y cells [65].*

#### **Figure 7.**

*The impact of two types of graphene, (A) 150 m2 /g and (B) 750 m2 /g, on total GSH production was evaluated. Statistical analysis showed that \*p < 0.5, \*\*p* ≤ *0.01, \*\*\*p 0.001, and \*\*\*\*p* ≤ *0.0001 for the observed effects [65].*

However, it is essential to note that the pro- or anti-oxidant behavior of GO is not solely dictated by surface area, but also by surface charge and functionalization. A study by Liu et al. demonstrated that single-layered GO (SLGO) induced lower levels of ROS production compared to multi-layered GO (MLGO), highlighting the consequence of the GO layer structure and oxidation levels [66]. Additionally, pristine graphene, characterized by lower surface oxygen content, was found to exhibit reduced cytotoxicity, and magnitude of the role of surface chemistry in modulating toxicological outcomes [11].

Furthermore, the generation of ROS by GO coatings has been recognized as an effective mechanism for preventing bacterial colonization on various surfaces, demonstrating the potential utility of ROS generation in GO applications. Researches demonstrated that the toxicity of graphene nanomaterials (GNMs) to bacteria is linked to surface functionalization, but the exact mechanisms involved are not fully understood. One study investigated the toxic mechanisms of differently functionalized GNMs on bacteria, focusing on physical interaction, oxidative damage, and cell autolysis. Three basic functionalizations of GNMs, including carboxylation (G-COOH), hydroxylation (G-OH), and amination (G-NH2), were examined. G-COOH (66% viability vs. CT group) and G-OH (54%) graphene exhibited higher toxicity to *Escherichia coli* (*E. coli*) than G-NH2 (96%) within 3 hours at a concentration of 50 mg/L. The three materials displayed distinct physical interaction modes with bacterial cells. G-COOH and G-OH made contact with the cell membrane via their sharp edges, causing more damage than G-NH2, which covered the bacteria by attaching along the basal plane. The three GNMs demonstrated similar radical generation capacities, indicating that direct radical generation is not the mechanism causing the toxicity. Instead, the GNMs can oxidize the cellular antioxidant glutathione (GSH), leading to oxidative damage. The oxidative capacity followed the order: G-COOH > G-OH > G-NH2, which correlated with the antibacterial activity. Cell autolysis, the degradation of the cell wall component peptidoglycan, was found to be a new mechanism inducing bacterial death. G-COOH and G-OH caused more cell autolysis than G-NH2, which partially accounts for the different toxicity of the three GNMs [26].

#### **5. Cytotoxicity and genotoxicity**

The cytotoxicity of GO nanoparticles is intricately influenced by their size and shape, both of which play effective roles in determining the extent of cellular damage *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

and the specific mechanisms underlying it. This complex relationship is crucial for understanding the potential hazards associated with GO and its applications. Graphene family nanomaterials (GFNs) can enter cells through various means, which can lead to the generation of ROS, increase in lactate dehydrogenase (LDH) and MDA, and release of Ca2+ (**Figure 8**). As a result, GFNs can cause different types of cell damage, including harm to the cell membrane, inflammation, DNA damage, mitochondrial disorders, and cell death through apoptosis or necrosis [17].

#### **5.1 Effect of GO size and shape on its cytotoxicity**

The higher surface area graphene samples, linked to smaller-size graphene nanoparticles, induces greater cell damage, illuminating the correlation between surface area and cytotoxicity [65]. Smaller GO particles can readily penetrate cellular membranes and interact with various cellular components to trigger a cascade of events, including oxidative stress, inflammation, and ultimately cell death. These particles induce these responses, in part, through the generation of intracellular reactive oxygen species (ROS) and the activation of the mitochondrial pathway, ultimately leading to apoptosis [67]. The larger GO particles, on the other hand, tend to exert their cytotoxic effects through physical damage to cells and the induction of mechanical stress. They may not

**Figure 8.** *GFNs cytotoxicity mechanism [17].*

penetrate cells as easily as their smaller counterparts, but their sheer size and presence can disrupt cellular structures and induce mechanical stress, leading to cell damage and death [67]. The size of NGO flakes, for example, was found to impact cell viability, with larger flakes showing more pronounced reductions in cell viability compared to smaller flakes. Moreover, the size of NGO flakes was found to impact cell viability, with larger flakes showing more pronounced reductions in cell viability compared to smaller flakes [68]. Elsewhere, GO's lateral size has been identified as a significant parameter affecting its cytotoxic effects. Larger lateral sizes have been associated with stronger cytotoxic impacts on liver cells, emphasizing the importance of size in nanotoxicity [32]. Several studies have shown that GO nanosheets possess a higher potential for membrane penetration, leading to increased cytotoxicity and nanomaterials with sharp edges or high aspect ratios have been associated with greater damage to cells and tissues [22, 34].

#### **5.2 Effect of GO size and shape on its genotoxicity**

Investigating the genotoxicity and cytotoxicity of GO in the context of its physicochemical properties is of paramount importance to comprehensively assess its safety and potential impacts on cellular health and genetic material. GO's physicochemical attributes can exert a significant influence on its genotoxic and cytotoxic effects.

Recent investigations have delved into the impact of GO nanoparticle size and surface area on genetic materials. Smaller nanoparticles have displayed heightened interactions with cellular components and genetic material, culminating in escalated genotoxic effects [65]. This phenomenon is further corroborated by research that examined the neurotoxicity of graphene with varying surface areas in a dopaminergic neuron model [65]. While both sizes of graphene demonstrated an intriguing trend of increased cell viability as concentration decreased, an unexpected twist unfolded concerning the surface area aspect, which is a function of the GO particle size as well as shape. These findings collectively shed light on the knotty relationship between nanoparticle characteristics and their biological consequences.

Furthermore, studies have revealed that the shape of nanomaterials plays a crucial role in influencing proliferation, differentiation, and metabolism in both mammalian and microbial cells [31]. Domenech et al., discussed that graphene-based materials (GBM), like other nanomaterials, can induce genotoxicity through primary or secondary mechanisms (**Figure 9**). The primary genotoxicity arises from the direct interaction of GBMs with DNA or its associated histones or by GBMs interfering with DNA replication and/or repair processes, such as by triggering lipid peroxidation or the production of ROS. On the other hand, secondary genotoxicity occurs when GBMs interact with inflammatory cells, leading to downstream effects in the target cells. It is important to note that standard in vitro assays typically focus on non-inflammatory cell monocultures, limiting their ability to detect genotoxic effects caused by secondary mechanisms. In contrast, in vivo experiments conducted on whole organisms provide a broader perspective and can capture genotoxic effects induced by both primary and secondary mechanisms [69].

#### **5.3 Effect of GO surface chemistry on its cytotoxicity and genotoxicity**

The surface chemistry of GO, characterized by the presence of functional groups, emerges as a key player in its cytotoxic and genotoxic potential. The oxygencontaining functional groups, such as hydroxyl, carboxyl, and epoxy groups, interact with cellular components and genetic material, leading to genotoxic effects [70].

*Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

#### **Figure 9.**

*Genotoxicity mechanism of graphene-based materials, and the methods used to identify these effects both within cells and living organisms [69].*

Furthermore, the presence of these functional groups significantly influences the cellular uptake and internalization of GO, thereby modulating its cytotoxicity. Studies reveal that non-covalent conjugates based on GO and an alkylating agent demonstrated significant cytotoxicity toward specific cell lines [34].

The electronic charge of GO nanoparticles plays a prominent role in determining their genotoxic and cytotoxic effects. Positively charged nanoparticles are associated with higher cellular uptake and internalization, potentially leading to amplified genotoxicity and cytotoxicity. In contrast, negatively charged or neutral nanoparticles exhibit different interactions with cellular components and genetic material, resulting in varying levels of genotoxic and cytotoxic effects [12]. The zeta potential distribution of various GO derivatives, such as LA-PEG-GO, PEG-GO, and PEI-GO, influences their genotoxicity and cytotoxicity profiles (**Table 2**). Notably, the surface charge of GO derivatives has a profound effect on cell damage, and GO derivatives with lower positive electronic charges appear to mitigate their toxic effect on cells [12].

Furthermore, pristine graphene, a closely related material to GO, has been shown to induce cytotoxicity in various cell types. It can deplete mitochondrial


#### **Table 2.**

*Zeta potential distribution of GO, lactobionic acid-polyethylene glycol (LA-PEG)-GO, PEG-GO, and polyethylenimine (PEI)-GO [12].*

membrane potential, increase intracellular ROS generation, and trigger apoptosis through the activation of the mitochondrial pathway [71]. Additionally, GO has been found to generate ROS, leading to inflammation and apoptosis in alveolar macrophages and alveolar epithelial cells, further accentuating its influence on cytotoxicity [72].

The aggregation state of GO nanoparticles holds significance in determining their genotoxic and cytotoxic potential. Aggregated nanoparticles may experience reduced cellular uptake and internalization, which can lead to lower genotoxicity and cytotoxicity compared to dispersed nanoparticles. The formation of stable colloid dispersions, especially in hydrophilic graphene nanomaterials, is critical for avoiding aggregation and enhancing cellular internalization and removal [67].

In addition, exposure to higher concentrations of GO has been shown to result in genotoxic effects, encompassing DNA damage, chromosomal aberrations, and alterations in gene expression. The genotoxic potential of GO has been evaluated using techniques such as the comet assay and micronucleus assay, which have become standard methods for assessing the genotoxic effects of GO. Intriguingly, GO nanosheets have been demonstrated to have the capacity to intercalate into DNA molecules, hinting at their ability to interact with genomic DNA. These findings reinforce the importance of studying genotoxicity not just in isolation but in concert with cytotoxicity, as GO nanoparticles have demonstrated dose-dependent DNA damage, alterations in gene replication, and changes in gene expression patterns [58].

This complex relationship between GO's physicochemical properties and its genotoxic and cytotoxic effects highlights the multifaceted nature of evaluating the safety and potential applications of GO in various biological contexts. It emphasizes the need for thorough characterization, the modulation of surface properties, and an acute awareness of dose-dependent effects of the genotoxicity and cytotoxicity of GO in diverse cell types and biological systems. It underlines the need for further research and vigilance concerning the safety aspects of graphene nanoparticles.

In vitro studies have demonstrated that the concentration of GO plays a pivotal role in determining its cytotoxicity and impact on cell viability [33, 57, 60, 73]. One such study by Zhang et al. exemplified the concentration-dependent cytotoxicity of GO. Utilizing purified natural graphite as a starting material, the authors synthesized GO with irregular morphology and small-sized sheet structures. The cytotoxicity of GO was assessed using the Cell Counting Kit-8 (CCK8) assay on MDA-MB-231 human breast cancer cells, with GO concentrations ranging from 100 to 500 μg/mL. The results unequivocally revealed dose-dependent cytotoxicity, emphasizing that

#### *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

higher concentrations of GO correlate with reduced cell viability [74]. This outcome serves as a fundamental illustration of the direct relationship between GO concentration and its impact on cell survival.

Ribeiro et al., illustrated the nuanced interaction between GO surface chemistry and cell viability by investigating the interaction of GO with human breast cancer cells. The study explored the effects of 24, 48, and 72-hour exposure to GO, GO modified with DAB-AM-16 (GOD), and PAMAM dendrimers (GOP) at a concentration of 2.4 μg/cm2 . Intriguingly, the results demonstrated that at this particular concentration, there was no detectable toxicity in the cells during the specified exposure times. However, when higher concentrations of GO were employed, low cytotoxic effects became evident, revealing the sensitivity of cell viability to GO concentration [74]. Moreover, the research revealed that the affinity of GO, GOD, and GOP particles to cellular membranes induced morphological alterations and modifications in the organization of microfilaments and microtubules. However, when cells were treated with 24 μg/cm2 of GO, GOD, or GOP for 24 hours and then allowed to recover for an additional 24-hour period in normal medium, the nanoparticles persisted in the cytoplasm of some cells, without causing significant alterations in cellular morphology. This observation aligned with the results of the cell proliferation experiment, which indicated that the cells remained viable for up to 72 hours (**Figure 10**). These findings accentuate the dynamic nature of GO-cell interactions and the significance of concentration-dependent effects.

#### **Figure 10.**

*The immunofluorescence reaction of GOX, GOXP, and GOXD treated and untreated MCF-7 cells at a concentration of 2.4 μg/cm2 over a 48-hour period. The scale bar is 200 μm. The nuclei were stained with DAPI (blue), actin was stained with Alexa 633-phalloidin (red), and microtubules were evidenced by a monoclonal antibody against α and β tubulin and anti-mouse FITC-antibody (green). The figure also includes zoomed-in inserts of selected areas (white rectangles) [74].*

#### **6. Inflammation and other immune response**

#### **6.1 Inflammation response**

In the realm of size and surface area, the dimensions of GO nanoparticles emerge as decisive determinants in modulating their interaction with immune cells, which, in turn, has a fundamental bearing on the ensuing inflammatory response. Smaller GO nanoparticles, owing to their larger surface area per unit mass, exhibit an enhanced proclivity for interaction with immune cells, which translates into a heightened activation of inflammatory pathways and the subsequent release of pro-inflammatory cytokines. The increased contact area with immune cells substantiates their proinflammatory potential, marking a notable contribution to the overall inflammation induced by GO [75].

#### **6.2 Immune responses**

The immune response elicited by GO exposure has become a crucial element of its biocompatibility and its possible biomedical applications. GO elicits dosedependent immune responses characterized by the activation of immune cells and the subsequent release of pro-inflammatory cytokines. Higher doses of GO have been correlated with more pronounced immune reactions and notable alterations in immune components. These alterations extend to changes in the populations of different hepatic macrophage types and platelet infiltration in vital organs such as the liver and spleen. The uptake of GO nanomaterials by macrophages within these organs plays a central role in promoting the secretion of pro-inflammatory cytokines and has been robustly associated with inflammation [8, 75, 76]. Studies have pointed to the secretion of pro-inflammatory cytokines, including interleukin-6 (IL-6), IL-12, tumor necrosis factor-β (TNF-β), and interferon-gamma (IFN-γ), as a significant outcome of GO nanomaterial uptake by macrophages, thus magnitude the intricate link between GO exposure and the immune response. Moreover, the dose-dependent cytotoxicity of GO nanomaterials has been widely observed, underscoring the importance of balancing the immune response and cytotoxicity in determining the overall impact of GO [75]. Strikingly, the interplay between GO exposure, immune response, and inflammation has illuminated a novel avenue for mitigating inflammation through the modulation of autophagy. GO nanomaterials have been shown to disrupt lysosomal function, thus impeding autophagy flux. Paradoxically, this disruption of autophagy has been linked to a decrease in the levels of pro-inflammatory cytokines such as IL-6, IL-8, TLR4, and CXCL2, while concomitantly enhancing the levels of the anti-inflammatory cytokine IL-10. This results in the amelioration of conditions such as colitis [77]. In addition to these intriguing insights, GO nanostructures have exhibited multifaceted properties, including the ability to suppress the migration and metastasis of cancer cells and to inhibit prostaglandin-mediated inflammatory responses. These findings not only highlight the extensive potential of GO in biomedical applications but also emphasize the significant impact of dose-dependent immune responses on shaping its safety and effectiveness profiles [75].

Overall, the intricate balance between GO exposure and the immune response holds significant implications for the safe and efficacious utilization of GO in diverse biomedical applications. The collaboration between GO, the immune system, and inflammation unveils a dynamic and multifaceted relationship that warrants further exploration and offers new avenues for the development of strategies aimed at

optimizing the potential of GO in various medical and therapeutic contexts. These findings collectively spotlight the importance of fine-tuning GO dosages and its effects on the immune system to harness its full potential while mitigating potential immune-mediated side effects.

#### **6.3 Role of GO physicochemical properties on inflammatory and immune responses**

Inflammation is a key indicator of the nanotoxicity exhibited by GO. It serves as a multifaceted orchestrator of cellular and tissue responses, setting in motion the intricate cascade of events leading to the production of pro-inflammatory cytokines and recruitment of immune cells. Several studies have elucidated the role of GO's physicochemical properties, including its charge, in mediating the magnitude and duration of the ensuing inflammatory responses [34]. It is imperative to explore this connection to harness a comprehensive understanding that would empower the development of strategies aimed at mitigating the inflammatory consequences of GO and improving its biocompatibility. The presence of oxygen-containing functional groups on the GO surface, such as hydroxyl, carboxyl, and epoxy groups, governs its interaction with immune cells and the subsequent induction of pro-inflammatory cascade responses. These GO functional groups engage immune cell receptors, initiating intricate signaling pathways that culminate in the production of pro-inflammatory cytokines. The density and distribution of these functional groups on the GO surface critically influence its pro-inflammatory potential [75]. ROS generation, incited by GO exposure, adds another layer of complexity to the inflammation response. GO's surface chemistry and size wield profound influence over its capacity to induce ROS generation, and ROS, which in turn, activate inflammatory pathways and amplify the release of pro-inflammatory cytokines. This intricate relationship between GO's physicochemical attributes and ROS generation reinforces the dynamic and multifaceted nature of GO-induced inflammation [76]. Moreover, the interplay between GO nanoparticles and proteins in biological systems serves as an additional regulator of inflammatory reactions. The binding of proteins to the GO surface can profoundly change their structure and function, leading to immune responses and inflammation. Furthermore, the interaction between GO and immune-related proteins or receptors can stimulate inflammatory signaling pathways, introducing another level of intricacy to the reciprocal impact between GO's physical and chemical properties and inflammation [61].

#### **7. Dose-dependent nanotoxicity of GO**

#### **7.1 Effect of concentration**

The dose-dependent nanotoxicity of GO represent a key aspect of its safety profile and efficacy in various biomedical, electronics, energy storage, and environmental remediation applications. It has been consistently demonstrated that GO elicits a dose-dependent response, where higher concentrations correlate with increased cytotoxicity and adverse effects, both in in vitro and in vivo settings. In in vitro experiments, the impact of GO on cells has been investigated, revealing the dosedependent nature of its nanotoxicity. High concentrations of GO have been associated with the inducement of oxidative stress, DNA damage, and cell death [64].

Doses of GO exceeding 50 μg/mL have been shown to induce cytotoxicity in human fibroblast cells, leading to decreased cell adhesion and promoting cell apoptosis [78]. Furthermore, GO's impact on immune cells has been demonstrated, with dendritic cells experiencing decreased functional activity and immune proteasome unit down-regulation in response to GO exposure [11]. Commercial pristine graphene dispersed in 1% Pluronic F108 at concentrations up to 20 mg/mL interacted with murine macrophage-like RAW 264.7 cells. The thickness of graphene dispersed in Pluronic ranged from 2–3 to 500–1000 nm. The cells underwent apoptosis in a dosedependent manner, which was associated with a decrease in mitochondrial potential and an increase in ROS levels. Autophagy was induced in murine macrophage-like RAW 264.7 cells when incubated with approximately 100 μg/mL of GO, and Tolllike receptors associated with inflammatory responses were expressed. Dendritic cells treated with up to 25 μg/mL of GO showed a decrease in functional activity. Specifically, antigen inhibition and down-regulation of intracellular levels of an immune proteasome unit responsible for antigen processing in these cells have been reported [11]. Similarly, the cytotoxicity of GO-PEG nanoparticles was concentration-dependent in the regulation of human myeloid suppressor cells (MDSC) [73]. Nonetheless, doses of up to 100 μg/mL of both GO sizes GO (a ∼500 nm proteincoated commercial GO and GO sheets of 1 μm particle range) did not inhibit cell proliferation [19, 33].

Similarly, in vivo studies have reinforced the significance of dose-dependent nanotoxicity of GO. Exposure to elevated concentrations of GO has been shown to result in dose-dependent lung inflammation and organ damage [49, 79]. Elsewhere, the effects of NGO sheets on the viability and motility of spermatozoa were investigated in vivo. **Figure 11**. shows the dose-dependent effects, indicating that the viability and motility of spermatozoa decreased from approximately 75% to 40% as the concentration of NGO sheets increased from 0 to 2000 μg/mL. At concentrations of ≤20 μg/mL, there was only a slight reduction (~10%) in viability and motility, suggesting minimal

#### **Figure 11.**

*The viability, motility, and progressive motility of spermatozoa in male Balb/C mice after approximately ~8 weeks of weekly injections of NGO sheets at different concentrations ranging from ~2 to 2000 μg/mL. The control samples were untreated mice spermatozoa. The study used at least three mice per group (N = 3), and significant results are indicated with asterisks (\*) for p-values less than 0.05 [80].*

toxicity. However, significant in vivo toxicity was observed at injected concentrations ≥200 μg/mL, with a reduction of approximately 45% in sperm viability and motility at 2000 μg/mL or ~4 mg/kg of body weight [80].

However, it is essential to note that not all cell types and concentrations exhibit high cytotoxicity when exposed to GO. For example, GO nanoparticles of different sizes demonstrated low cytotoxicity on mouse neural stem cells (mNSCs) at a concentration of 20 μg/mL [81].

Nevertheless, these findings underline the importance of understanding the dose-response relationship to establish safe exposure limits and to design appropriate dosing strategies when considering GO for biomedical applications [57, 60, 73]. These observations are crucial in understanding the potential systemic effects of GO and are instrumental in determining safe exposure limits in clinical applications [49, 79]. The establishment of appropriate dosing strategies and safe exposure limits will ultimately facilitate the development of GO-based biomedical interventions with a high degree of precision and effectiveness.

#### **7.2 Time-dependent nanotoxicity**

While GO exhibits dose-dependent toxicity, time-dependent factors play a substantial role in understanding the persistence of GO within biological systems and its potential for long-term biocompatibility. Time-dependent nanotoxicity of GO provide crucial insights into its fate and potential long-term effects within biological systems. One noteworthy observation is the gradual degradation of GO within living organisms. In vivo studies have revealed dynamic changes in GO behavior over time, shedding light on its biodegradability, biodistribution, and clearance mechanisms. Research has shown that GO can be broken down and metabolized over time, as evidenced by the disappearance of the Raman signal associated with GO in the spleen after 9 months [43]. The temporal aspects of GO's toxicity are, therefore, linked to its clearance mechanisms. Studies have indicated that GO particles can be rapidly excreted through the urinary system, with evidence of GO presence in urine within just 2-hours post-injection. This signifies that GO has efficient excretion pathways, contributing to its time-dependent behavior within the body [53]. The interaction between time-dependent nanotoxicity and GO's physiological fate is of great significance in evaluating its safety profile and potential biomedical applications. The dynamic degradation and clearance mechanisms offer insights into how GO may be processed by the body, which is critical in determining the long-term effects of GO exposure.

Siqueira et al., discuss the potential environmental impact of residual traces of GO and rGO in aquatic environments and their effects on zebrafish hepatocytes. The study found that GO and rGO nanosheets were internalized by the fish cells, and exposure for 72 hours caused harmful effects such as higher ROS production and stopping cell replication. Both GO and rGO were found to be toxic after 72 hours, but with different mechanisms of toxicity [82]. Akhavan et al., investigated the timedependent biodistribution of NGO sheets in Balb/C mice after intravenous injection. The results showed high uptake of NGO sheets by residual organs like the liver and spleen, indicating effective filtering of the blood circulation and the kidneys also exhibited high uptake, suggesting regular renal clearance. Additionally, the thyroid and testis showed significant uptake, indicating potential effects on hormone secretion and spermatozoa performance (**Figure 12**) [80].

**Figure 12.**

*Time-dependent biodistribution of fluorescent-labeled NGO sheets in male mice treated with NGO at an injected concentration of 2000 μg/mL. The measurements were taken at various post-injection times, starting from the first weekly injection. Error bars were included in the figure, representing the standard deviations calculated from three mice per group (N = 3) [80].*

Overall, the time-dependent nanotoxicity of GO serve as a valuable aspect in the comprehensive evaluation of its safety, efficacy, and translational potential. These findings highlight the dynamic nature of GO's interaction with biological tissues and its potential to undergo transformation within the body [43]. However, these findings point to the need for further exploration of the kinetics and dynamics of GO within living organisms, facilitating the development of safer and more effective applications in biomedicine.

#### **7.3 Systemic effects**

The widespread impact of GO exposure on the body has attracted significant interest because of its implications for the safe use of GO in medical and related applications. Significant systemic effects have been linked to high doses of GO, making it a crucial factor in assessing its safety. Research has identified various systemic factors affected by GO, emphasizing the importance of careful dose selection and careful monitoring in different applications [19, 42, 74, 75, 83]. Hematological parameters have surfaced as a key area of impact, with findings suggesting that GO nanoparticles contribute to a decrease in red blood cell count, hemoglobin concentration, and mean corpuscular volume in a dose-dependent manner [83]. Such alterations in hematological parameters underline the systemic effects of GO and their potential consequences for biological systems. Furthermore, GO's influence extends to biochemical parameters, encompassing cholesterol, triglyceride, glucose, protein, albumin, creatinine, and bilirubin levels. These changes, when elucidated,

#### *Graphene Oxide Nanotoxicity: A Comprehensive Analysis DOI: http://dx.doi.org/10.5772/intechopen.114205*

could provide further insights into the systemic ramifications of GO exposure although specific changes in these parameters following GO treatment still remain inconclusive [83].

Liver enzymes, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), have also been scrutinized in the context of GO exposure, albeit without specified alterations [83]. The impact on liver enzymes hints at potential liver damage induced by GO, emphasizing the importance of assessing hepatic function. Mendonca et al., demonstrated that the intravenous administration of rGO at a single dose of 7 mg/kg resulted in minor indications of toxicity, not only in the blood, but also in the liver, and the kidney after 7 days, with no evidence of an ongoing inflammatory process. These effects were temporary and did not cause permanent harm. Despite a previous study showing disruption of the blood-brain barrier and the presence of rGO in the hippocampus, no noticeable changes were observed in the response of hippocampal neurons or astrocytes. Additionally, the behavior of both control and rGO-treated animals in their cages was similar, with no observable clinical signs of toxicity. The data suggested that systemic GO administration posed no significant health risk for rats under these experimental conditions. However, it was evident that the interactions between graphene-based materials and biological systems depend on the experimental design and the physicochemical properties of the nanomaterial. Therefore, comprehensive toxicological investigations are therefore necessary for understanding the biological effects and addressing safety concerns before the practical application of graphene-based materials in clinical settings. This study provided a foundation for further toxicological examinations of rGO following long-term in vivo exposure and it encouraged research into the mechanisms underlying the interaction between rGO and biological systems [84, 85].

Renal dysfunction has emerged as another facet of GO-induced systemic effects, with elevated levels of blood urea nitrogen (BUN) and creatinine observed in animal models post-GO exposure [86]. These markers of renal function underscore the potential nephrotoxicity associated with GO exposure.

Nonetheless, it is essential to note that the systemic effects of GO are complexly dependent on factors such as dose, exposure duration, and the specific biological system under investigation [19, 42, 74, 75, 83]. Thus, while certain studies have reported substantial systemic alterations, others have found GO nanoparticles to be relatively benign, with no significant impact on lung, liver, kidney, and blood parameters in rats. Notably, in these studies, GO nanoparticles were retained in the body, primarily as agglomerates, without adverse effects on systemic parameters. Presence of nanoparticles was detected in the body tissues of all groups treated with GO. The largest solid aggregates, which could be up to 10 mm in diameter, were found near the injection site in the stomach serous membrane, between the connective tissues of the abdominal skin, muscles, and peritoneum. There were also many smaller, spherical-shaped aggregates with diameters around 2 mm that were lodged among the mesentery. The mesentery in the GO was almost black due to the high concentration of nanoparticles. Smaller aggregates were also observed in abdominal lipid tissue near the injection site and mesentery (**Figure 13**). This suggested the possibility of GO application in drug delivery systems without causing systemic harm [86].

These contrasting findings emphasize the necessity for in-depth research into the dose-dependent systemic effects of GO, clarifying the conditions under which it may

#### **Figure 13.**

*Systemic effects of GO nanoparticles after multiple intraperitoneal injections into rats taken by digital camera. Solid aggregates were found in injected body regions (a, b, c) and mesentery (d). Average-sized dots of GO nanoparticle aggregates were also localized in abdominal lipid tissue (e). Black arrows were used to indicate the presence of the GO nanoparticles. The macroscopic structure of the kidney (f), lungs (g), heart (h), spleen (i), and liver (j) was also examined. No pathological features were found [86].*

be safely utilized in various biomedical applications. Moreover, these investigations highlight the paramount importance of establishing precise dose thresholds and safety protocols to ensure the effective and secure application of GO-based materials in medicine and other fields.

#### **8. Conclusion**

Over the past few years, graphene-based nanomaterials have become popular for a variety of technological and biomedical applications, including biosensing, drug delivery, tissue engineering, and diagnosis. However, it is essential to establish the potential risks and safety levels of using graphene materials in biomedical applications. Currently, there is limited information available about the in vitro and in vivo toxicity of graphene and GO, and more studies are needed. The toxicity of graphene and GO depends on various factors, including the surface, size, number of layers, cell type, administration route, dose, time of exposure, and synthesis methods. Reactive oxygen species generation in target cells is the most important cytotoxicity mechanism of graphene. Small and hydrophilic graphene nanomaterials tend to form a stable colloid dispersion and are more likely to be internalized and removed/excreted from the application site.

Understanding the complex relationship between these properties and graphene and GO's potential toxicity is essential for the safe and effective use of graphene and GO in biomedicine. By considering these properties and their effects on oxidative stress, inflammation, genotoxicity, and cytotoxicity, researchers can develop strategies to minimize the risks associated with graphene-based materials.

Graphene-based materials are promising candidates for biomedical applications due to their unique properties and lack of metallic impurities. However, toxicological studies must consider the purity of the sample and the non-molecular behavior of graphenebased nanomaterials. With further research and advancements, graphene and GO hold great promise in revolutionizing biomedicine while ensuring patient safety.

### **Author details**

Mohammad Mahdi Sepahi1,2\* and Marzieh Azizi3,4

1 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran

2 Department of Chemistry "Sapienza" University of Rome, Rome, Italy

3 Laboratory of Bioanalysis, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

4 Department of Chemistry, University of Oslo, Oslo, Norway

\*Address all correspondence to: m.m.sepahi@ut.ac.ir

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Chemistry of Graphene - Synthesis, Reactivity, Applications, and Toxicities* presents an in-depth exploration of new trends, concepts, and phenomena in the general field of graphene research to introduce graphene science in a multidimensional manner providing a definitive foundation and provoking new ideas and critical insight into the emerging areas of research for the professional researchers and other practitioners. The development of graphene science, which has been rapidly expanding since the year 2024, still attracts a lot of attention with expanding new opportunities and potential in the global market. The current book opens with an in-depth survey of novel methods that have characterized graphene production research over the last three decades. It lends itself to the shifting trends toward the scalability of graphene technologies, the diversified emerging graphene-related materials, processes, and opportunities for fashioning novel scalable applications. The book continues to explore the prospects of graphene in electrochemical applications and wraps up with a survey of the potential harmful effects of graphene and its implications on graphene applications in medicine, environment, and related fields. The work represents an in-depth effort that can serve to introduce early career scholars and practitioners to graphene research but also remains adequately engaging to provoke new thought lines that stimulate the development of new research topics for expert professionals.

*Jung Huang, Nanotechnology and Nanomaterials Series Editor*

Published in London, UK © 2024 IntechOpen © fotoliaxrender / Fotolia

Chemistry of Graphene - Synthesis, Reactivity, Applications and Toxicities

IntechOpen Series

Nanotechnology and Nanomaterials, Volume 4

Chemistry of Graphene

Synthesis, Reactivity,

Applications and Toxicities

*Edited by Enos W. Wambu*