Time- and dose-dependent toxicity profiles and molecular correspondence of nanoplastics in human embryonic stem cells (H9)

Nanoplastics (NPs) pose a potential risk to the human reproductive system, but their toxicity to human embryonic stem cells (hESCs) remains poorly understood. This study evaluated the in vitro toxicity of polystyrene (PS), polyethylene (PE), and polyvinyl chloride (PVC) nanoparticles using H9 hESCs. All three polymers reduced cell viability in a concentration- and time-dependent manner. PE showed the most significant decrease in IC₅₀, from 172.6 μg/mL at 24 h to 37.93 μg/mL at 48 h, and further to 11.25 μg/mL at 72 h. Flow cytometry and confocal imaging revealed that PS could be effectively internalized, with uptake increasing from 2.5% at 5 μg/mL to 77.65% at 100 μg/mL after 24 h, and approaching 84% after 72 h. All NPs increased intracellular reactive oxygen species with PS-induced responses being the strongest, reaching 14.6-fold and 22.8-fold higher than the control group at concentrations of 50 and 100 μg/mL, respectively. Western blot analysis showed that caspase-3 remained largely unchanged, while Oct-4 levels increased and p-p38 levels decreased, especially under PVC exposure. Pharmacological inhibition of p38 MAPK only resulted in a slight increase in Oct-4 levels, indicating that the weakened p38 signaling pathway plays a limited role in PVC-related molecular responses. RT-qPCR further showed that Nanog was significantly upregulated at concentrations of 50 and 100 μg/mL under PVC exposure, and Sox2 was significantly upregulated at a concentration of 100 μg/mL. These findings reveal the potential mechanism by which nanoparticles induce hESC toxicity and provide a scientific basis for assessing their developmental risk.