P18-90 The Role of Dynamic 3D Models in Evaluating MXenes Toxicity: A Focus on Pulmonary and Hepatic Cells

The SAFARI project aims to develop sustainable and safe hybrid formulations of MXenes and Graphene (2D materials) by leveraging their unique physicochemical properties for applications in electronics, energy storage, and biomedicine. However, their increasing use raises concerns about potential health risks, particularly oral, inhalation and dermal exposure [1] . Despite their broad applications, studies on their health effects remain limited. This study investigates MXene-induced pulmonary toxicity and hepatotoxicity, focusing on occupational safety and public health. We compared traditional 2D cell cultures with 3D spheroid models (3DA549 and 3D-HepG2) [2] , and advanced dynamic liver cultures (3DDHepG2) [3] . Additionally, dynamic HepG2 cell growth was characterized over 21 days before MXene exposure. Valproic acid validated the model as an in vitro system mimicking in vivo assay. Furthermore, to validate the lung and liver models, transcriptomic analysis was performed to compare gene expression profiles across 2D, 3D, and 3DD models using the Human Atlas. To assess toxicity, a dose-response study evaluated cell viability after 24 hours of exposure to MXene concentrations (0.39–100 µg/mL) in lung and liver cell models. In monoculture cells, no significant effects on viability or dose-response relationships were observed. However, notable differences emerged in 3D models. 3D lung spheroids showed statistically significant effects at concentrations up to 25 µg/mL, while 3D-HepG2 constructs exhibited toxicity at all tested concentrations. The IC 50 was also evaluated in 3DD-HepG2 model. Surprisingly, when IC 50 values were normalized to biomass (dose per unit weight), they were higher in 3D spheroids than in 3DD spheroids. These results suggest that dynamic spheroids may be more compact and metabolically active, potentially making them more susceptible to treatment. These findings indicate that the lack of a dose-dependent response in 2D models may result from uniform exposure, which does not accurately replicate the physiological conditions. In contrast, 3D models, with their cellular heterogeneity, provide a more realistic, concentration-dependent response. Among these, 3D dynamic models offer significant advantages such as improved cell differentiation and better nanoparticle interaction assessment, making them more representative of human lung and liver tissue than 2D or 3D models. The dynamic HepG2 spheroid model holds promise for human toxicological assessments, with applications in chronic toxicity studies, biomarker identification, and the implementation of the 3Rs principle (replacement, reduction, and refinement) in animal testing.