Nanozymes, nanomaterials with intrinsic enzyme-like properties, are becoming increasingly important in biomedical applications. Due to their unique bioactivity, it is essential to thoroughly characterise them and assess potential health risks prior to use. In this master’s thesis, we investigated the (geno)toxic effects of three different metal oxide-based nanozymes: γ-Fe₂O₃ (N4), Zn₀.₇Fe₂.₃O₄ (N8), and Mn₀.₄Fe₂.₆O₄ (N9). As a test model, we used the human hepatocellular carcinoma cell line HepG2, cultured in a three-dimensional (3D) format - spheroids. The spheroids were exposed to the nanozymes for 24 and 96 hours to evaluate short-term and long-term effects. To assess cytotoxicity, we used a luminescence-based assay that quantifies ATP levels. All tested nanozymes exhibited cytotoxic effects on HepG2 spheroids, with IC₅₀ values inthe reange of 50 - 85 µg/mL (24 h) and approximately 30 µg/mL (96 h). Subsequently, we used non-cytotoxic concentrations to evaluate oxidative stress and DNA damage. We employed the dichlorodihydrofluorescein diacetate (DCFH-DA) assay to measure reactive oxygen species (ROS), the malondialdehyde (MDA) assay for lipid peroxidation, and the comet assay to assess DNA damage. The results showed that nanozymes N4 and N9 slightly increased ROS levels but did not induce lipid peroxidation. Nevertheless, all nanozymes caused DNA damage. This was observed after 24 hours of exposure to N8 and N9 (25 µg/mL), while after 96 hours, all tested nanozymes induced DNA damage even at a concentration of 5 µg/mL. Our findings indicate that nanozymes can induce DNA damage even at low concentrations, highlighting the need for further studies to elucidate their mechanisms of action and to comprehensively evaluate their safety for biomedical applications.
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