Quantifying Cellular Uptake of Nanoplastics In Vitro: A Workflow for Fluorescently Labeled Nanosized Polystyrene...

The accumulation of micro- and nanoplastics (MNPs) in the environment increasingly entails human exposure to this class of highly diverse polymer particles. To evaluate potential risks to human health, hazard assessments commonly involve in vitro testing using human-derived cell lines to establish dose-response relationships for MNPs. However, the reliable quantification of actual particle uptake in cells is particularly challenging for nanoscale materials (particles less than 1 μm in size). We present a workflow for the characterization and quantification of the in vitro uptake of fluorescently labeled polystyrene (PS) nanoparticles in human-derived A549 lung epithelial cells as a relevant model for inhalation exposure. This study employs well-characterized PS nanobeads with a mean diameter of 180 nm. The semiquantitative characterization of uptake by confocal fluorescence microscopy and flow cytometry is complemented by two orthogonal quantification methods based on automated fluorescence imaging microscopy and online pyrolysis gas chromatography mass spectrometry (Py-GC-MS), the latter also rendering unlabeled MNPs accessible. Applying the developed workflow, we confirm that PS nanoplastics are taken up in significant amounts by A549 cells. Analysis by flow cytometry revealed that almost all cells take up particles. Applying an automated high-throughput fluorescence microscopy platform, we determined a dose-dependent uptake resulting in an average accumulation of 1100 ± 450 particles per cell (mean ± SD) when incubated for 24 h with the highest tested dose of 31 μg·cm^-2. These findings were confirmed by Py-GC-MS, a method used for MNP quantification in human cells in vitro for the first time, yielding on average 3.2 ± 0.6 pg PS (950 ± 200 particles) per cell. The workflow described in this study facilitates characterization and quantification of cellular MNP uptake in vitro, allowing for the calculation of average particle counts and polymer mass contents of nanosized plastic particles per cell.