Effects Of Low Concentrations Of Silver Nanoparticles On Cell Proliferation In Skin Fibroblasts And Neuroendocrine Cells

BackgroundSilver nanoparticles have been widely used in textiles,cosmetics and medical aspects.Silver nanoparticles could enter human body through skin contact,respiratory tract,digestive tract and other ways that increase the risk of the body exposed to silver nanoparticles.Antimicrobial dressing containing silver nanoparticles have been widely used in the clinical application,but its potential risk is still controversial.ObjectiveTo investigate the effects of low concentrations of silver nanoparticles on cell proliferation and cellular dospotion in cells,skin fibroblasts and neuroendocrine cells were used.References of its security applications in the process of skin wound healing were expected.Methods1.Skin fibroblasts were cultured by explant method from back and tail of a newborn rat.Neuroendocrine cells were also cultured.2.MTT assay was used to detect the cell viablity of skin fibroblasts and neuroendocrine cells by different concentrations gradient of silver nanoparticles and differernt times.3.Select different concentrations of silver nanoparticles solution to incubate skin fibroblasts and neuroendocrine cells for differernt times respectively,then observed the change of cell morphology and the cellular deposition of silver nanoparticles under electron microscope observation.4.The final concentrations of silver nanoparticles solution at 20 ng/ml,200 ng/ml,2000 ng/ml were selected to incubate skin fibroblasts for 48 h.Then DNA were extracted to complete agarose gel electrophoresis for detecting the DNA ladder.5.The final concentrations of silver nanoparticles solution at 200 ng/ml,2000 ng/ml were selected to incubate neuroendocrine cells for 4 h,24 h,48 h respectively.Then proteins were extracted to complete western blot for detecting the express of cytochrome c.6.Control groups were setted in all of the experiments above,which were not added silver nanoparticles.Results1.Several cells near the skin tissues could be visible 3-5 days after tissues were explanted,the cell number was increased in the 7th day and the cells could be covered with flask wall in the 10 th day.The skin fibroblasts from back and tail of the newborn rat were found to express vimentin by immunofluorescence staining.The shapes of most neuroendocrine cells were round which keeping semi adherent state.2.MTT assay results showed that the cell viability of skin fibroblasts was not affected when the concentrations of silver nanoparticles were less than 200 ng/ml;The cell showed concentration-dependent cytotoxicity when the concentrations of silver nanoparticles in 200-10000 ng/ml;The neuroendocrine cells were showed concentrationdependent and time-dependent cytotoxicity after incubated by silver nanoparticles.3.The electron microscopy results found that silver nanoparticles went into the cytoplasm when the skin fibroblasts were incubated by silver nanoparticles at 2000 ng/ml;While only a small amount of silver nanoparticles were found in the cytoplasm when the concentration of silver nanoparticles at 20 ng/ml and 200 ng/ml;And when the deposition amount of silver nanoparticles into the cytoplasm were less,preferentially accumulated in lysosomes;With the concentrations and time of incubation prolonged,the deposition amount of silver nanoparticles in the cytoplasm became more,microfilament,microtubule and mitochondria and nucleus were also found silver nanoparticles.When the neuroendocrine cells were incubated with different concentrations of silver nanoparticles,the cell apoptosis and necrosis were observed under the electron microscope as well,except for the same deposition of silver nanoparticles within the cytoplasm.4.DNA ladders were not examined by DNA agarose gel electrophoresis.5.The expression level of cytochrome c after incubated neuroendocrine cells with silver nanoparticles at 2000 ng/ml for 48 h was significantly different compared to the control group(P<0.05).Conclusions1.The cell proliferation of skin fibroblasts and neuroendocrine cells were inhibited by silver nanoparticles.There is still a potential safety risk of silver nanoparticles in low concentrations.2.The cytotoxic mechanism of silver nanoparticles may be related to the apoptosis pathway induced by mitochondrial.