Size-dependent Neurotoxicity Induced By Silica Particles

With excellent physicochemical properties,nanomaterials have been widely used in food,cosmetics,energy,biomedicine and other fields.However,their safety has attracted public attention.Nanomaterials can enter human body via skin contact,inhalation or gastrointestinal absorption,and then can cross or bypass the blood brain barrier to enter central nervous system(CNS)and cause neurotoxicity.As CNS plays a leading role in the regulation of human physiological activity,it's crucial to evaluate the neurotoxicity of nanomaterials.Untill now,the researches on neurotoxicity of nanomaterials are still insufficient to establish the structure-activity relationship between nanomaterials' physicochemical properties and neurotoxicity.In this study,silica particles were used as the research object,and three different in vitro neurotoxicity model cells including N9 microglia,bEnd.3 brain-derived microendothelial cells and HT22 neurons were used to evaluate the neurotoxicity of silica particles.The size effects on neurotoxicity of silica particles will be explored together with the mechanisms behind.First,a series of testing methods including transmission electron microscopy(TEM),fourier infrared spectrum(FTIR),zeta potential and particle size analysis were used to characterize the physicochemical properties of commercialized silica particles(50,100,300 nm)involving morphologies,surface functional groups,hydrodynamic size and dispersion.DCF assay was conducted to detect their potential to produce abiotic reactive oxygen species(ROS).Secondly,the toxicity profiles of different-sized silica in N9,bEnd.3 and HT22 cells were detected,which showed that silica particles had no signiticant toxicity in bEnd.3 and HT22 cells,but caused obvious size-dependent toxicity in N9 cells,with 50 nm silica particles exhibiting the largest toxicity.In-house synthesized silica using St?ber method also induced similar size-dependent toxicity in N9 cells.Further studies showed that the toxicity of silica particles in N9 cells was related to their content of silanols.Silica with smaller size had more silanols per area,therefore caused higher toxicity.The cytotoxicity of silica particles decreased significantly after their silonals were gradually removed by heat treatment.Finally,the mechanisms of silica NP-induced neurotoxicity were explored.Silica particles induced the generation of mitochondrial ROS in N9 cells,and 50 nm silica induced the largest amount of mitochondrial ROS.Additionally,the content of glutathione,an intracellular antioxidant substance,decreased accordingly.Besides,N9 cells showed cell swelling,cell membrane bubbling,release of pro-inflammatory cytokine IL-1?,and expression of N-terminal GSDMD protein(N-GSDMD),and all of which proved that silica particles induced pyroptosis in N9 cells.The results of this study are helpful to establish an evaluation system for the determination of engineered nanomaterial(ENM)-induced neurotoxicity.They can also provide a theory basis for the safety evaluation and design of ENMs,and further benefit its application in the research of nanomedicine and clinical treatment.