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

Graphene quantum dots (GQDs) are nano-sized fragments of graphene sheets, with great potential for applications in optics, electrochemistry, and biomedicine, including drug delivery. In this study, two types of GQDs were investigated: green-emitting (G-GQD) and blue-emitting (B-GQD) and their genotoxic potential was examined on the protein level. Flow cytometric analysis of γH2AX, a marker of DNA double-strand breaks, and phosphorylated histone H3 (p-H3), a marker of aneugenic activity, was performed in HepG2 spheroids after 24 hours of exposure to G-GQDs and B-GQDs. HepG2 spheroids were exposed to graded concentrations of B- and G-GQD (12.5, 25, 50, and 100 μg/mL) for 24 hours, respectively. Spheroids were dissociated into single-cell suspensions, washed twice with 1x PBS and fixed in 4% PFA. For flow cytometric analysis, cells were labelled with anti-H2AX pS139 antibody and anti-histone H3 pS128 antibody. REA-APC and REA-PE controls were applied to exclude non-specific antibody binding. For each sample, 10,000 events were acquired using a MACSQuant Analyzer 10 flow cytometer and MACSQuantify™ software (Miltenyi Biotech, Germany). Raw data were exported from the MACSQuantify software and analysed with FlowJo V10 software (Becton Dickinson, New Jersey, USA).