Caco-2-derived spheroids and organ-on-a-chip for food safety risk assessment: mechanisms of intestinal inflammation induced by 20 nm polystyrene nanoplastics

Microplastics released from food packaging have become an emerging food safety concern and may contribute to intestinal inflammation. This study employed Caco-2-derived spheroids combined with self-developed microfluidic organ-on-a-chip technology to systematically investigate the effects of 20 nm polystyrene nanoplastics (PS-NPs) on oxidative stress and functional homeostasis in the three-dimensional (3D) intestinal epithelial microenvironment. We detected oxidative stress markers, antioxidant capacity, inflammatory factors, and epithelial function indicators; evaluated Villin and ZO-1 expression by immunofluorescence staining; performed metabolomic analysis on chip effluents; conducted styrene-based computational target prediction and exploratory molecular docking, identifying ALOX5 as a computational candidate; and independently analyzed a public IBD single-cell RNA-sequencing dataset using in silico ALOX5 perturbation for exploratory analysis. The results demonstrated that 40 μg/mL and 400 μg/mL PS-NPs could induce oxidative, inflammatory, and epithelial-associated alterations. This study is the first to integrate Caco-2-derived spheroids and organ-on-a-chip for nanoplastic toxicity evaluation, which provides a novel experimental platform and theoretical basis for investigating potential mechanisms underlying nanoplastic toxicity and risk assessment of food-related nanoplastics.