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Quantifying Cellular Uptake of Nanoplastics In Vitro: A Workflow for Fluorescently Labeled Nanosized Polystyrene Particles.
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Scientists developed a reliable way to measure exactly how many nanoplastic particles get absorbed into human cells, using lung cells as a test case since we breathe in these tiny plastic bits from the air. Using two different measurement methods that confirmed each other, they found that lung cells readily soak up plastic nanoparticles, nearly every cell took some in, accumulating around 1,000 particles each after just one day of exposure. This matters because having a trustworthy measurement tool is a crucial first step for scientists to figure out whether the nanoplastics we're constantly exposed to actually pose health risks.
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 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 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. These findings were confirmed by Py-GC-MS, a method used for MNP quantification in human cells 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 allowing for the calculation of average particle counts and polymer mass contents of nanosized plastic particles per cell.
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