**3. Evidence regarding the biocompatibility and toxicity of graphene platforms**

The available literature indicates that research on GQDs has grown widely in relation to their uses, and that is why we now know their biomedical applications include the elimination of bacteria, the administration of drugs, the development of nanocarriers, cancer therapy, and tissue engineering [35–37, 68]. The therapeutic applications of nanomaterials remain quite limited, and there is no safe and effective formulation yet that can be administered in humans [69–71]. While QDs produce a series of morphological and functional alterations that lead to tumor cell death, what happens to healthy cells is unknown [72]. Therefore, the toxicological profile of each nanomaterial is needed to make decisions regarding potential risks vs. benefits. However, what is known about the biocompatibility of GQDs and what evidence is there of the toxicity of drug delivery platforms?

GQDs and their derivatives have variable toxicity in biological systems ranging from prokaryotic to eukaryotic, depending on the dose and the functional groups with which they are coated [34]. They have also been evaluated in a series of human cell lines. For example, studies carried out on leukocytes showed that there was

significant uptake of GQDs in monocytic and granulocytic cells, suggesting that phagocytic cells can incorporate GQDs. The toxicity observed in this study was relatively low (10%) after a 36-hour exposure period at concentrations of 500 μg/mL [73]. In another study using GQDs functionalized with NH2, COOH, and CO∙N(CH3)2 it was observed that A549 and C6 cells showed a slight increase in their proliferation at concentrations of 200 μg/mL, but no death due to apoptosis [74]. GQDs have also produced toxic effects on mesenchymal stem cell self-renewal and differentiation [75]. Several studies have pointed to the toxic effects of graphene derivatives [76–81]. These functionalized QDs can produce a variety of toxic effects at the cellular level and in vivo due to the series of impurities produced during the oxidation process. The same happens in the coating process with other molecules [82]. However, when GQDs are coated with polyethylene glycol at concentrations of 320 μg/mL, they do not affect the viability and differentiation capacity of neural stem/progenitor cells (NSPCs) [83]. Also, reduced toxicity, absence of ROS production, absence of apoptosis, and lack of morphological changes have been observed in HeLa and A549 tumor cells under concentrations of 100 μg/mL [84, 85].

The cellular and nuclear effects that GQDs produce are due to their high permeability in biological membranes. It is known that the uptake and localization of GQDs are highly dependent on size, shape, coating, and pH, among other factors. Previous studies have shown that GQDs use membrane lipid rafts for their transport across the cell membrane. This process is better, the smaller the QDs are [86]. However, protein-coated GQDs enter mainly by phagocytosis and with smaller coatings by clathrin-mediated endocytosis [87, 88]. GQDs with amide groups enter the cell through energy-dependent mechanisms by endocytosis, mediated by caveolae and phagocytosis [89]. Within the cell, GQDs are distributed in different organelles producing a variety of cellular effects. They are later distributed through endosomal trafficking and reach lysosomes, mitochondria, and the nucleus, and can produce autophagy, apoptosis, and DNA damage [90–92]. At the nuclear level, the NPC Kap2 and Nup98 genes can participate in the uptake of GQDs and can produce morphological and functional alterations associated with genotoxicity, including oxidative stress and DNA damage [93, 94].

There are many reports in the literature regarding the toxic effects of both GQDs and their derivatives in a variety of human cell lines and it is impossible to mention them all in this chapter. What is evident is the ease with which they penetrate cells, position themselves and participate in strategic cellular processes, thus potentially affecting cell functionality and leading to cell death. However, of the studies reviewed so far, most were done in tumor cell lines where physiological processes are altered and there are specific survival and adaptation mechanisms. To date, there are no studies carried out on cell lines from healthy tissue, so we cannot rule out the fact that GQDs could produce morphological and functional modifications associated with toxicity in healthy cells.

What effects do they produce in higher organisms and experimental animals? What is known about the processes of absorption, distribution, metabolism, and excretion (ADME) of GQDs? The information so far is limited. Previous studies in nematodes have shown that nitrogen-bound GQDs (N-GQDs) produce degeneration of dopaminergic and glutamatergic neurons at concentrations of 100 μg/mL [95]. A series of studies on the biocompatibility and biodistribution of GQDs in adult and embryonic zebrafish have been reported and provide important information on embryos' developmental delays, pigmentation inhibition, pericardial edema, and delayed hatching among other things. In adults, GQDs showed high biocompatibility
