DNA Damage Induction After Exposure to Ferrite Nanoparticles for biomedical applications (MxFe3-xO4, M = Fe, Zn, Mn) in an Advanced 3D Human Hepatic in vitro Model

Nanosized spinel-type ferrites have gained recognition as a unique class of engineered nanomaterials with promising applications, but their safety profiles remain insufficiently explored. Although iron (Fe), zinc (Zn), and manganese (Mn) are biologically relevant elements, the use of Zn- and Mn-containing ferrite nanoparticles in biomedical contexts demands careful (geno)toxicity evaluation. In this study, three ferrite nanoparticles – γFe2O3 (FeNPs), Zn0.7Fe2.3O4 (ZnNPs), and Mn0.4Fe2.6O4 (MnNPs) – synthesised through a microwave-assisted polyol route, functionalized with citric acid to improve colloidal stability, were evaluated for their potential (geno)toxic effects in an advanced in vitro 3D cell model, HepG2 spheroids. DNA damage induction was studied using the alkaline comet assay. After 24 and 96 hours of exposure, spheroids were dissociated into a single-cell suspension by collagenase treatment. Each spheroid was incubated in 0.25% trypsin–EDTA for 3 minutes and then gently dissociated into a single-cell suspension using cut pipette tips. After this the comet assay was conducted under conditions described in the Supplement material (Table 1). The cell suspension was combined with 1% low-melting-point (LMP) agarose and layered onto fully frosted slides pre-coated with 1% normal-melting-point (NMP) agarose. Slides were lysed, the nucleoids unwound and electrophoresis preformed, followed by neutralisation of the gells. Table 1: Comet assay conditions. Phase Buffer composition Conditions Cell lysis 0.1 M EDTA, 2.5 M NaOH, 0.01 M Tris, 1% Triton X-100  pH 10, 4 °C, 60 min Unwinding 300 mM NaOH, 1 mM EDTA pH 13, 4 °C, 20 min Electrophoresis 300 mM NaOH, 1 mM EDTA 25 V and 300 mA (0.5–1 V/cm), 4 °C, 20 min Neutralization 0.4 M Tris pH 7.5, 4 °C, 15 min For comet scoring, gells were stained using the GelRed nucleic acid stain (Biotium, USA) according to the manufacturer's instructions. Analysis and scoring were performed using a fluorescent microscope and Comet Assay IV software (Instem, Philadelphia, USA). A positive (30 and 5 µM benzo[a]pyrene; BaP for 24 and 96 hours, respectively) control was included. Each experiment was conducted independently three times, analysing 50 nuclei per experimental point from a poled sample of 3-4 spheroids. The datasets represent the exported analysis performed in Comet Assay IV.