Cancer Cell Membrane Hybrid Liposome-loading Paclitaxel Targeted Treatment Of Melanoma

Objective: The anti-tumor drug paclitaxel(PTX)will be loaded with cancer cell membrane and HA-mediated liposome as the carrier,and PTX will be actively targeted and delivered to the melanoma lesion by injection to improve the bioavailability and enhance the anti-tumor efficacy of the drug.Method: The PTX liposome was prepared by the film dispersion method,the prescription was determined and characterized by the single factor experiment.HA was covalently grafted onto dioleoyl phosphatidylethanolamine(Dioleoyl Phosphoethanolamine,DOPE)molecules,and the synthetic phospholipid material(HA-DOPE)was inserted into the phospholipid membrane of liposome to form HA-modified liposomes(HA-LS).The B16F10 melanoma cell membrane was extracted and purified by differential centrifugation and fused with liposome to form cancer cell membrane-mediated liposomes(CCM/LS),and the HA-LS was hybridized with the cancer cell membrane to form cancer cell membrane and HA-mediated liposomes(HA-CCM/LS).Through the cell uptake experiment,the way how cancer cell membrane be hybridized with the liposome,the total protein concentration of the cancer cell membrane,and the amount of HA-DOPE are determined.Membrane proteins were characterized by SDS-PAGE and Western blotting techniques.The B16F10 melanoma cells were selected to evaluate the cytotoxicity of blank nanoparticles and drug-loaded nanoparticles.The uptake ability of B16F10 to nanoparticles was detected by flow cytometry and intracellular colocalization experiments.The safety of nanoparticle injection was investigated by in vitro hemolysis test.The nude mouse model of melanoma was constructed to investigate the distribution and anti-tumor effect of nanoparticles in tumor-bearing nude mice.Results: The preferred preparation method by single factor method is film dispersion method,the amount of cholesterol was 2 mg/m L,the hydration temperature was 40 °C,the ultrasonic power was 97.5 W,the ultrasonic time was 15 min,and the ratio of drug to lipid was 1:15.The prepared particle size was 149.77±4.35 nm,the PDI was 0.227±0.05,the encapsulation efficiency was 95.12±0.39 %,and the drug loading was 5.14±0.47 %.The synthesis of HA-DOPE was verified by FT-IR.After hybridization of cancer cell membrane,the size of liposome decreased to 131.57±1.21 nm,and the particle size distribution was more uniform.The drug loading and encapsulation efficiency were 4.66±0.12 % and 93.45±3.00 %,respectively.CCM/LS showed a double membrane structure under transmission electron microscopy.The modification of HA and cancer cell membrane will not affect the encapsulation efficiency and drug loading,but the hybridization of the cell membrane improves the particle size distribution of the nanoparticles and the stability of the preparation.SDS-PAGE and Western blotting techniques validated the retention of membrane proteins on hybrid nanoparticles.In vitro release experiments showed that drug released slower after modification with HA or / and CCM.In vitro cultured cell level studies showed that the blank nanoparticles had no obvious cytotoxicity to B16F10 cells,and the hybridization of cancer cell membranes and modification of hyaluronic acid in drug-loaded nanocarriers can enhance the ability of nano drug delivery system to kill B16F10 melanoma.Uptake of cells to Coumarin 6(C6)-labeled formulations indicated that HA or CCM modifications promoted cellular uptake,whereas CCM-modified liposomes showed significantly higher tumor cell internalization than HA modified group.In vitro hemolysis experiments showed that the hemolysis rate of each nano-formulation was less than 20 % at 2.5?100 ?g/m L,indicating good blood compatibility.The imaging experiments of small animals show that compared with unmodified LS,cancer cell membrane and/or HA-modified LS can be well enriched in tumors with good tumor targeting.CCM/LS and HA-CCM/LS had the best inhibitory effect on melanoma,and the most significant effect on tumor cell apoptosis in tumor tissues,followed by HALS group and LS group.This is consistent with the results of in vitro cultured tumor cytotoxicity experiments.Combined with the results of in vitro cultured tumor cell uptake and tissue distribution in vivo,it was confirmed that after modification by homologous cancer cell membrane,liposome has stronger ability to actively target tumor tissue,thereby exerting a better anti-tumor effect.Conclusion: The liposome hybridized with the homologous cancer cell membrane exhibits specific adhesion effect on tumor cells,and enhances the ability of the nanocarrier to actively target the tumor tissue in vivo,thereby exerting stronger tumor suppressing effect.However,HA and cancer cell membrane co-modified liposomes did not show stronger active targeting,and the reasons for further study are pending.