Multi-endpoint neurotoxicity assessment of nanosilver with different particle sizes in human neuronal cell model

QIU Jing-ru; LI Shuang-xing; JI Ying-lu; YANG Yan-wei; ZHANG Di; GENG Xing-chao; QU Zhe

doi:10.20251/j.cnki.1003-3734.2025.22.010

Abstract：Objective:To evaluate the neurotoxic effects of silver nanoparticles(Ag-NPs)of two different particle sizes using neuronal cells differentiated from human induced pluripotent stem cells(hiPSC)with multiple endpoints.The reason to conduct this study is that in recent years,metal nanoparticles have shown a great deal of potential in pharmaceutical research,but they also pose potential toxicity to human nervous system.Methods:hiPSC-derived neuronal cells were cultured in vitro.After 72 h of administration,CCK-8 assay was used to detect cell viability of neurons,ROS levels were detected using DCFH-DA to evaluate neuronal oxidative stress injury,and yH2AX levels were detected to evaluate neuronal DNA damage.After 24 and 72 h of administration,levels of cytokine(TNF-α,IL-1β,and IL-6)were detected using enzyme-linked immunosorbent assay(ELISA),and the impact on neurite outgrowth was detected using high-content cell imaging.Results:40 and 70 nm Ag-NPs were cytotoxic to neurons.40 nm Ag-NPs showed a significant reduction effect(P＜0.001)on cell viability from the dose of 12.5 μg·mnL-1 and its LD50 was 21.82 μg·mL-1,70 nm Ag-NPs showed a significant reduction effect(P＜0.05)on cell viability from the dose of 25 μg·mL-1 and its LD50 was 51.13 μg·mL-1.From the dose of 2.5 μg·mL-1,40 and 70 nm Ag-NPs induced a dose-related increase in neuronal ROS levels and DNA damage levels from the dose of 2.5 μg·mL-1.40 nm Ag-NPs had an inhibitory effect on neurite fiber width and area from the dose of 2.5μg·mL-1,70 nm Ag-NPs had an inhibitory effect on neurite count,length,width,branch points,and other parameters from the dose of 10 μg·mL-1.The level of neuronal cytokine release was not significantly altered after the administration of Ag-NPs.Conclusion:In this study,we constructed a human neuronal cell model with multi-endpoints for evaluating the neurotoxicity of Ag-NPs in vitro,effectively assessed the cytotoxicity,oxidative stress damage,DNA damage and inhibition of neurite outgrowth induced by two particle sizes of Ag-NPs,and provided a reference for the safe use of Ag-NPs.