Differential cascading cellular and subcellular toxicity induced by two sizes of nanoplastics

Nanoplastics (NPs) can be potentially accumulated by living organisms, but how they interact with cells at the cellular or subcellular level in the physiological environment is still largely unknown. In this study, time-resolved flow cytometry coupled with confocal imaging as well as other biomolecular approaches were used to investigate the cellular and subcellular responses to amine-modified polystyrene NPs of two different sizes (100 nm and 1000 nm). We first demonstrated that the two sizes of NPs displayed contrasting cytotoxicity to embryonic zebrafish fibroblast cell lines ZF4. Using the fluorescent-labeled NPs, the differentially internalized patterns between the two-sized NPs in a time-resolved manner were observed. Confocal images showed that the two sizes of NPs were deposited in lysosomes but could escape through lysosomal rupture, as evidenced by the induction of lysosomal acidification (for 1000 nm) and alkalization (for 100 nm) as well as permeabilization. Subsequent deposition of 100-NPs in the cytosol induced loss of mitochondrial membrane potential and significant reactive oxygen species production, and finally stimulated the activation of caspases, disrupted the mitochondrial mitophagy, leading to irreversible cell death. In contrast, 1000-NPs toxicity in ZF4 cells did not involve lysosomal permeabilization and loss of mitochondrial membrane potential. Lysosomal deposition of such larger sized nanoplastics mainly induced lysosome acidification, activated the autophagy as well as disrupted the integrity of cell membrane, but at the same time provoked the activation of caspases and finally triggered the apoptosis. Our study demonstrated a complicated relationship among lysosome damage, autophagy activation, and apoptosis, leading to contrasting toxicity of NPs of different sizes.