Integrated human-relevant NAMs for biomedical evaluation of selenium nanoparticles linking epithelial barrier translocation to neuronal effects

New Approach Methodologies (NAMs) are increasingly needed to evaluate the safety and efficacy of nanomaterials in biomedicine while generating human-relevant evidence and reducing reliance on animal studies. For therapeutic nanoformulations, single in vitro assays often fail to capture the sequence of events that determines both functional performance and potential adverse outcomes, including interaction with exposure interfaces, epithelial transport, and response at the target tissue. Here, we present an integrated human-relevant NAMs workflow to evaluate selenium nanoparticles (SeNPs) stabilised with polyvinylpyrrolidone (PVP) or polysorbate 20 (Tween). These SeNPs were used as a relevant nanomedical case study, as such systems are increasingly being designed with antioxidant, neuroprotective, and drug-delivery properties. The workflow combined three complementary in vitro models: a tri-culture alveolar barrier at a semi-air-liquid interface, a mucus-competent intestinal barrier with continuous transepithelial electrical resistance monitoring, and differentiated dopaminergic-like SH-SY5Y neurons, representing two major therapeutic administration interfaces and a disease-relevant downstream neuronal target. Across models, SeNPs showed coating- and cell-type-dependent cytotoxicity, preserved barrier integrity at non-cytotoxic doses, and exhibited measurable basolateral transport. Neuronal uptake was confirmed, and both SeNP formulations attenuated L-dopa-induced oxidative stress without eliciting measurable pro-inflammatory cytokine release at effective concentrations. By linking two administration-relevant epithelial barrier models with a disease-relevant neuronal target, this study demonstrates how integrated NAMs can support early de-risking, formulation prioritisation, and more informed preclinical development of nanomedical systems.