## 3 Graphene Oxide
### Toxicity and Biocompatibility  
The toxicity of graphene oxide (GO) has been studied by several researchers. In a study undertaken by [PERSON] et al. [12] evaluating the toxicity of GO on A549 human lung cells, it was shown that GO does not enter the A549 cell and causes no evident cytotoxicity. However, based on the applied dose, it can lead to oxidative stress in cells and some loss of cell viability at high concentrations. [PERSON] et al. [13] showed that GO has a moderate level of toxicity to human bone marrow neuroblastoma cells and human epithelial carcinoma cells. When studied with zebrafish (Danio rerio) embryos, a 20% inhibition in cell growth was observed. Additionally, when a high dosage of 50 mg/L was used, this caused a delay in the hatching of the zebrafish embryos, but it did not increase apoptosis significantly. [PERSON] et al. [14] synthesized GO-chitosan nanohybrid films for wider biomedical applications and characterized the film's structure, antibacterial activity, and cytotoxicity. The results of the antibacterial tests showed that GO-chitosan hybrids had greater antibacterial activity compared to that of pure GO. Additionally, the attachment of graphene sheets to GO decreased the level of cytotoxicity and, in some cases, led to increased cell proliferation. In another study, [PERSON] et al. [15] evaluated the cytotoxicity of GO and graphene sheets with different sizes and oxygen levels on human red blood cells and adherent skin fibroblasts using WST-8 viability and hemolysis assays. The results of the WST-8 assay and hemolysis data proved that compact graphene sheets were more toxic to mammalian fibroblasts than the less dense graphene oxide. Additionally, the graphene sheets produced more reactive oxygen species on the surface of the dermal fibroblasts. They concluded that the toxicity of graphene and graphene oxide depended on the exposure environment, such as the level of aggregation and the type of cell interaction (i.e., cell suspension versus adherent cells). Exposure of reduced graphene oxide (rGO) particles with a diameter between 100 and 110 nm and a thickness between 3 and 5 nm to human PC12 neuronal cells increased the activation of caspase-3, the release of lactate dehydrogenase, and the generation of reactive oxygen species. However, rGO had a moderate effect on protein levels during contact with human HepG2 hepatoma cells [16]. According to the literature, the toxicity of GO and rGO is strongly dependent on their concentration, and increases with the amount of GO or rGO. For instance, a GO concentration greater than 50 mg/mL showed toxicity to human fibroblasts, decreased the amount of cell adhesion, and promoted cell apoptosis [17]. [PERSON] et al. [15] also investigated the blood compatibility of GO and graphene sheets on human red blood cells and adherent skin fibroblasts. The observed level of hemolysis for the GO samples was strongly dependent on the extent of exfoliation of the injured dermal tissue and particle size. Smaller-sized GO particles had a higher hemolytic activity compared to larger sized GO particles. Their results demonstrated that the size, shape, and density of the graphene particles, oxygen content, and electrostatic charge on the surface of the particles strongly affected the toxicological and biological responses by red blood cells. They also found that covering GO with chitosan removed any hemolytic activity. [PERSON] et al. [18] investigated the biocompatibility and distribution of GO in mice. The results showed that GO did accumulate in the lungs over an extended period of time. GO also contributed to a slower blood circulation time, with 5.3 + 1.2 h half-life compared to other carbon nanomaterials. When the mice were exposed to 1 mg kg\({}^{-1}\) body weight of GO for 14 days, no pathological changes were observed in the examined organs. GO exhibited good biocompatibility with red blood cells.