Silver Nanoparticles Cause Cytotoxicity Via Ag~+heavy Metal Toxicity And Lysosomal Membrane Permeabilization-induced Apoptosis

ObjectiveSilver Nanoparticles(AgNPs)have great potential for appliction in medicine due to its antibacterial properties and unique physical and chemical properties.However,the cytotoxicity of AgNPs and underlying mechanisms remain unclear,which largely limit the wide use of AgNPs.In this study,we synthetised and characterized AgNPs for investigating cytotoxicity of AgNPs.In addition,the metabolism of AgNPs after entering fibroblasts,the heavy metal toxicity and the effects on lysosomal membrane permeabilization of AgNPs were determined.Methods1.The AgNPs were synthesized with ethanol as the reducing agent and PVP as the stabilizer.The absorption spectrum of AgNPs was characterized by Uv-Vis.The particle size and size distribution were measured using TEM and DLS.Dialysis method was employed to detect the solution stability of AgNPs.2.The cytotoxicity of AgNPs in human skin fibroblasts was detected by CCK 8 assay in vitro.To evaluate the toxicity effect in mice,a dose of 48?g/g.bw AgNPs was subcutaneous injection in mice back and the several organs(liver,kidney,spleen,heart)functions were determined.3.Lyso-tracker Red probe was employed to observe the distribution of AgNPs after entering into cells.Western Blot for metallothionein content,ICP-MS for the Ag~+released from AgNPs and flow cytometry technology for the apoptosis of cells in different time points were used.BAL,as Ag~+chelating agent,was utilized to evaluate the role of heavy metal toxicity induced by Ag~+in cell apoptosis.4.Detection of the cathepsin B activity was used for evaluating the lysosomal membrane permeabilization(LMP)that caused by AgNPs.After Chelating Ag~+with BAL or inhibiting LMP with 3-O-MeSM/UTI,respectively or together,the metallothionein,CB activity and apoptosis rate of fibroblasts were measured for studying the relationship between Ag~+and LMP.Results1.The size distribution of AgNPs is 22.69±4.15nm(Mean±SD)and the AgNPs is very stable in DMEM with a ratio of 1.46±0.03%transferring into Ag~+after 48 hours infusing.2.Comparing to Ag~+,AgNPs show a very low cytotoxicity to fibroblasts(24?g/ml vs3?g/ml),but it is still cytotoxic to fibroblasts with a dose and time dependent manner.The AgNPs mainly transfer into liver and spleen after a single-dose of AgNPs48?g/ml subcutaneous injection in mice back.In addition,a mild liver damage is found after 48 hours.3.AgNPs release a large amount of Ag~+into the cytoplasm after into lysosomes,which causes heavy metal toxicity.Forthermore,the generation of reactive oxygen species and cell apoptosis(49.7±3.57%)are induced by heavy metal toxicity after 48 hours.The rate of apoptosis reduces to 26.7±3.06%after treatment with BAL chelating Ag~+.Our results suggest that the heavy metal toxicity of Ag~+is not the only cytotoxicity mechanism for AgNPs.4.The increased activity of cathepsin B induced by AgNPs is presented in this study,which indicates the increasing of lysosomal membrane permeabilization.Meanwhile,an increased apoptosis rate has been obsevred.Both lysosome membrane protector and lysosomal enzymes inhibitor partially decrease apoptosis rate that induced by AgNPs,but the metallothionein level has no change.In addition,the Ag~+chelating agent does not reduce cathepsin B activity.The combination of Ag~+chelating agent and inhibition of lysosomal membrane permeability significantly reduces the metallothionein level and the activity of cathepsin B in fibroblasts,resulting in a very low apoptosis rate.Conclusion1.Comparing to Ag~+,the necrosis through the damage of cell membrane induced by AgNPs is significantly reduced(cell necrosis rate<0.5%),but AgNPs can cause cell apoptosis.2.Subcutaneous use of AgNPs(48?g/g.bw)can cause liver founction damage.3.AgNPs degradation is promoted by the acidic lysosomal environment after internalizing into cells and release Ag~+into cytoplasm,which resulting in ROS production and inducing cell apoptosis.This heavy metal toxicity is one of mechanisms for AgNPs cytotoxicity.4.AgNPs induce lysosomal membrane permeabilization after entering into lysosomes and release cathepsins into the cytoplasm,which result in a cathepsin dependent apoptosis.This is another cytotoxic possibility for AgNPs cytotoxicity,called independent cytotoxicity mechanism of Ag~+.5.The combination of Ag~+chelating agent and inhibiting lysosomal membrane permeability can fully inhibit fibroblast apoptosis caused by AgNPs.