In vitro toxicity assessment of graphene quantum dots using a 3D HepG2 model

In the present study, two types of graphene quantum dots (GQDs) were investigated: green-emitting (G-GQDs) and blue-emitting (B-GQDs). Physicochemical characterisation was performed using transmission electron microscopy (TEM), zeta potential, and hydrodynamic radius measurements to evaluate the morphology, particle size, aggregation behaviour, and colloidal stability of the GQDs in both water and cell culture medium. G-GQDs exhibited superior colloidal stability and more uniform dispersion than B-GQDs, whereas both types showed reduced aggregation and surface charge in cell culture medium due to protein corona formation. Toxicological characterisation was performed using an in vitro human hepatocellular carcinoma (HepG2) 3D spheroid model, with GQDs exposures up to 250 µg/mL (100 µg/cm2). Cytotoxicity was measured using the CellTiter-Glo luminometric assay, while genotoxicity was evaluated by the comet assay and flow cytometric analysis of γH2AX and phosphorylated histone H3 (p-H3) after 24 h of exposure. Both GQDs induced dose-dependent cytotoxic effects in HepG2 spheroids. At non-cytotoxic concentrations, a dose-dependent increase in DNA damage was observed, as determined by the comet assay. However, no evidence of DNA double-strand breaks (γH2AX) or elevated p-H3 levels was detected, suggesting the absence of clastogenic and aneugenic activity. The observed DNA single-strand breaks may be partly attributed to reactive oxygen species induction. These results indicate that, although GQDs induced cytotoxicity and single-strand DNA damage, no clear evidence of more severe genotoxic effects was observed under the tested conditions. Further studies are warranted to elucidate underlying mechanisms and comprehensively assess the safety profile of GQDs for biomedical applications.