| In this study, human liver cell line HL-7702 was used as cell model to evaluate the cytotoxicity of silver nanopaticles coated with mPEG-SH. The distribution of silver nanopaticles in cells was observed by transmission electron microscopy, the cytotoxicity of silver nanopaticles was measured by MTT assay and LDH release assay, the lipid peroxidation effects were examined by biochemical method, the mitochondria membrane potential was detected by flow cytometry(FCM) after Rhodamin-123 staining, changes of cell cycle were observed by FCM after PI staining. The experimental results of the study were shown as follow:1. Distribution of Ag-NPs particle in cells observed by TEMThe study of electron microscope showed that 25μg/mL of Ag-NPs could enter into the cells after 24 h, high density small particles could be seen in the cytoplasm which were darker than in the nucleus, that may be the Ag-NPs through the cell. Electal microscope showed that nucleus was smooth and nuclear membrane was integrity, the nucleus could also be found a high density Ag-NPs. Mitochondrion swelled and mitochondrial crista shortened and endoplasmic reticulum dilated was dedected obviously.2. The cytotoxicity of HL-7702 induced by Ag-NPsMTT results showed that different concentrations (6.25, 12.50, 25.00, 50.00,100.00μg/mL) of Ag-NPs were exposured to the HL-7702 cell for 12,24,48,72h, cell survival rate decreased gradually with increasing of the concentration of Ag-NPs. Compared with the nomal control group, there was the difference significant (P < 0.05) with the dose-effect relationship, with the time extension of exposuring Ag-NPs, the cell survival rate was declining and showed time-effect relationship. Intra-cellular LDH of results show that different concentrations (6.25, 12.50, 25.00, 50.00, 100.00μg/mL) of Ag-NPs were exposured to the HL-7702 cell for 24 h, Intra-cellular LDH content decreased gradually with the concentration increasing, 12.50, 25.00, 50.00 and 100.00μg/mL dose group was lower significantly than the nomal control group (P < 0.05). The results suggested that Ag-NPs could cause cell membrane damage and have the inhibitory effect on HL-7702 cell proliferation.3. Lipid peroxidation of HL-7702 induced by Ag-NPsBiochemical methods were used to detected SOD, GSH-Px activity and MDA content. The results showed that different concentrations (6.25, 12.50, 25.00, 50.00, 100.00μg/mL) Ag-NPs exposured to the HL-7702 cell for 24h, SOD,GSH-Px activity decreased with the increasing of concentration of Ag-NPs, 25.00,50.00 and 100.00μg/mL dose group are compared with normal control group, and there was the significant difference (P < 0.05), MDA content increased gradually with the increase concentration of Ag-NPs, 12.50, 25.00, 50.00 and 100.00μg/mL dose group are compared with normal control group, and the difference was significant (P < 0.05). The results suggested that Ag-NPs could induce lipid peroxidation on HL-7702 cells.4. Changes of mitochondrial membrane potential in HL-7702 induced by Ag-NPsFCM with Rhodamin-123 staining method was used to detect the changes of mitochondrial membrane potential.After exposure to the different concentrations (6.25, 12.50, 25.00, 50.00, 100.00μg/mL) of Ag-NPs for 24 h in HL-7702 cells,mitochondrial membrane potential decreased with the increasing dose,and there were significant differences between exposure and normal control group (P < 0.05). The result showed that Ag-NPs could decrese mitochondrial membrane potential of HL-7702 cells.5. Effects of Cell cycle in HL-7702 induced by Ag-NPsFCM with PI staining method was performed to detect the cell cycle. After exposure to the different concentrations (6.25,12.50,25.00,50.00,100.00μg/mL)of Ag-NPs for 24 h in HL7702 cells, with the increasing of the concentration, the percentage of cells in G0/G1 phase decreased, 50.00 and 100.00μg/mL dose group were compared with normal control group, there was the significant difference (P < 0.05), the rate in S phase increased gradually with the increasing of dosage of Ag-NPs, while compared with control group, the difference was not statistically significant (P > 0.05), the percentage of cells in G2/M phase increase gradually, 100.00μg/mL dose group was compared with normal control group, there was the significant difference (P < 0.05). All suggested Ag-NPs could interfere the process of cell cycle and induce Ag-NPs phase arrest.In conclusion, Ag-NPs could enter into the cell, they were found to distribute throughout the cytoplasm and nucleus. Ag-NPs could induce the cell toxic effects on HL-7702 cells, including decreasing the activity of anti-oxidative enzyme(GSH-Px, SOD), increasing the concent of MDA, inducing lipid peroxidation, increasing mitochondrial membrane potential, impacting the progress of cell cycle. The research had important theoretical meaning in studying the toxic effects of Ag-NPs on liver and related mechanism,which would provide experimental basis for safety evaluation of Ag-NPs .
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