Genotoxic effects of graphene quantum dots in an advanced in vitro human hepatic 3D model

Graphene quantum dots (GQDs) are nanoscale carbon-based materials characterized by a tunable bandgap and unique physicochemical features. Typically composed of only a few atomic layers and measuring under 10 nm in lateral dimension, they show excellent biocompatibility and low toxicity, making them attractive for biomedical uses. Their strong photoluminescent properties support applications in optical and electrical sensing, bioimaging, cancer therapy, and the development of high‑performance nanocomposites. In this study, two types of GQDs—green‑emitting (G‑GQD) and blue‑emitting (B‑GQD)—were examined, differing primarily in their optical properties, particularly the colour of emitted light dictated by their bandgap. Potential genotoxicity was evaluated using a 3D human hepatocellular carcinoma (HepG2) spheroid model. DNA damage induction was assessed with the comet assay, after 24‑hour exposure to 12.5, 25, 50, and 100 µg/mL of B‑ and G‑GQDs. Single-cell suspensions from spheroids were obtained using a combination of mechanical disruption and enzymatic digestion (Štempar et al., 2019). Following 24-hour exposure to B- and G-GQD at concentrations of 12.5, 25, 50, and 100 µg/mL (corresponding to 5, 10, 20, and 40 µg/cm2) and 30 μg/mL BaP as the positive control, spheroids were collected and treated with a mixture of collagenase and TrypLE, diluted in serum-free medium (1:20:9), for 10 minutes. Spheroids were then mechanically dissociated into a single-cell suspension by pipetting. The subsequent steps followed the standard monolayer cell culture protocol. Briefly, 30 μL of the cell suspension was mixed with 70 μL of 1% low-melting-point (LMP) agarose and applied to fully frosted slides pre-coated with a layer of 1% normal-melting-point (NMP) agarose. The slides were lysed in a solution containing 0.1 M EDTA, 2.5 M NaOH (pH 10), 0.01 M Tris, and 1% Triton X-100 for 1 hour at 4°C. DNA was unwound and electrophoresed in an alkaline solution (300 mM NaOH, 1 mM EDTA, pH 13) for 20 minutes at 25 V and 300 mA (0.5–1 V/cm). The slides were then neutralised using 0.4 M Tris buffer (pH 7.5), and the gels were stained with GelRed. Images were captured and analysed using an Eclipse 800 fluorescence microscope (Nikon, Japan) equipped with a Basler camera and the Comet IV image analysis software (Perceptive Instruments, UK). Three independent experiments were conducted, with 50 randomly selected nuclei analysed per experimental condition. Results were expressed as the percentage of tail DNA. References: Štampar M, Tomc J, Filipič M, Žegura B. Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing. Arch Toxicol 2019;93:3321–33. https://doi.org/10.1007/s00204-019-02576-