Abstract: Objective: To prepare the cell membrane nanovesicles (NVs) derived from the human embryonic kidney cells and the doxorubicin (DOX)-loaded nanovesicles(NVs-DOX),and to investigate the cellular uptake of NVs-DOX and the killing effect on the melanoma cells. Methods: The cell membrane was prepared from HEK293 cells with ultracentrifugation method and the cell membrane was treated with Liposome extruder to obtain the cell membrane NVs;DOX was encapsulated into the inner cavity of NVs by electric shock to get the NVs-DOX. The size of NVs-DOX was determined by nanoparticle size analyzer. The cells without vescicles were used as control group,and the cell membrane NVs were used as experiment group.The biocompatibilities of NVs on the NIH3T3 cells under different concentrations(5,10,20,50 and 100 mg·L^-1)were detected by MTT method. The free DOX (10 mg·L^-1) was sued as control group,and different concentrations(0.001,0.005,0.010,0.050,0.100,0.500 and 1.000 μmol·L^-1) of NVs-DOX were used as experiment groups.Fluorescent imaging and flow cytometry were used to analyze the fluorescence distribution of NVs-DOX in melanoma B16F10 cells.In cytotoxicity experiment,the survival rates of cells in various groups were detected by MTT method;Live/dead cell staining method was used to detect the death of B16F10 cells after treatment with NVs-DOX (1 μmol·L^-1). Results: The NVs with a mean size of 254.3 nm were prepared with cell membrance materials and NVs-DOX with a mean diameter of 289.6 nm were also obtained with electric shock.The MTT results indicated that the survival rates of cells in different concentrations of NVs groups were higher than 100%.The fluorescence imaging results showed that after 3 h of drug incubation,the fluorescence intensity in the cell nucleus in NVs-DOX group was slightly lower than that in small molecule DOX group.The flow cytometry results showed that the internalization rate in NVs-DOX group(91.07%) was slightly lower than that in free DOX group(95.47%). The cytoxicity experiment results showed that both NVs-DOX group and small molecule DOX group displayed concentration-dependent killing effects on the B16F10 cells.When the concentration of DOX was 0.1 μmol·L^-1,the survival rate of B16F10 cells was lower than 20%. There was no significant difference in the survival rate of B16F10 cells between NVs-DOX group and free DOX group(P>0.05).The Live/dead cell staining results showed that there was no significant difference in the ratio of dead cells to total cells in the melanoma B16F10 cells between NVs-DOX group and free DOX group(P<0.05). Conclusion: The cell membrane NVs derived from human embryonic kidney cells and new DOX-loaded drug NVs-DOX are successfully prepared. NVs-DOX have good cellular uptake in the B16F10 cells and significantly killing effect on the melanoma cells.

Key words: cell membrane, nanovesicle, doxorubicin, cellular uptake, killing effect