Quantifying Cellular Nanoplastic Accumulation Using Raman Microscopy

Abstract Micro- and nanoplastic contamination poses emerging health risks through ingestion of contaminated food and water, thereby placing the intestinal barrier as the primary interface for nanoparticle translocation following ingestion. In this context, Caco-2 epithelial cells provide a well-established in vitro model of the small intestine barrier, yet label-free quantification of nanoparticle uptake at the single-cell level remains a significant methodological challenge in nanotoxicology. Here, we report a confocal micro-Raman spectroscopy strategy for per-cell quantification of polystyrene nanoparticle (150 nm diameter) accumulation in Caco-2 intestinal epithelial cells. Our approach combines ratiometric analysis, referencing the polystyrene marker band at ∼1002 cm–1 to the cellular amide I band at ∼1660 cm–1, with control-derived thresholding to distinguish cell-associated nanoparticles from endogenous phenylalanine background, followed by single-cell masking to compute quantitative uptake descriptors. Application to cells exposed to 16 and 25 μg/mL polystyrene for up to 24 h revealed concentration-dependent uptake dynamics. Whereas qualitative spatial occupancy suggests greater nanoplastic accumulation at the lower dose, a rigorous quantification─difficult to achieve with conventional fluorescence-based approaches─uncovers the true dynamics at both concentrations, reconciling apparently paradoxical trends with the complementary transcriptional response and cell viability assays presented herein. These findings demonstrate that the ratiometric Raman mapping framework proposed here provides a chemically specific and robust approach for studying nanoparticle–cell interactions relevant to nanoplastic risk assessment, suggesting that spatial organization of cell-associated nanoparticles as well as relative uptake burden may act as critical determinants of cellular bioactivity.