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A major bottleneck in evaluating the environmental health implications of micro-nanoplastics (MNPs) is the inadequacy of analytical techniques for their precise quantification within complex environmental and biological matrices. Additionally, there is a conspicuous paucity of studies addressing environmentally relevant, photo-aged MNPs. In this study, the effects of UV aging on toxicity and bioavailability were investigated utilizing inductively coupled plasma mass spectrometry (ICP-MS)-traceable 25 nm gold-core polystyrene shell nanoplastics (AuPS25 NPs) and a triculture small intestinal epithelium (SIE) model coupled with simulated digestions to mimic physiological bio-transformations post-ingestion. Employing dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS), the physicochemical and morphological alterations of AuPS25 NPs as a function of UV exposure time were investigated, revealing significant photo-oxidation within 14 days. Toxicological evaluations demonstrated that, contrasting with un-aged AuPS25 NPs, the digesta from UV-aged AuPS25 NPs at oral concentrations of 4 and 40 µg/mL weakened barrier integrity by ~15% and ~18% and heightened cytotoxicity by ~4.3% and ~5.4%, respectively. Although the NP translocation rates were similar for both aged and un-aged PS NPs, the uptake by SIE of aged AuPS25 NPs was significantly higher, reaching 72.2% at 4 µg/mL and 59.2% at 40 µg/mL. In contrast, less than 0.5% of the un-aged PS NPs at both 4 µg/mL and 40 µg/mL were taken up by SIE. These findings highlight the imperative to integrate environmentally aged MNPs into toxicological assessments, as they facilitate “real-world” MNPs. Finally, the use of ICP-MS-traceable core–shell MNPs enables the identification and quantification of PS MNPs in cell lysates and biological media via ICP-MS, showcasing the use of such a tracer MNP approach in cellular uptake and in vivo biokinetic studies. Full article