Construction Of Cascade Targeted Photothermal Nano-Drug Delivery System To Enhance The Efficacy Of Immunotherapy

Objective:In vivo and in vitro experiments such as cytology,pharmacology and molecular biology were used to study the therapeutic effects of photothermal therapy,PD-1 antibody therapy and photothermal therapy combined with PD-1 antibody in the treatment of mouse hepatocellular carcinoma.Cascaded double-targeted liposomes were constructed to study the effect of photothermal therapy on the expression of PD-L1 binding site,and to explore the mechanism of photothermal therapy combined with PD-1 antibody therapy.Methods:1.The model of H22 hepatocellular carcinoma balb/c bearing mice was constructed and divided into blank group,hyperthermia group and operation group.Flow cytometry and immunofluorescence were used to analyze the infiltration of CD4+T cells and CD8+T cells in mouse tumor tissues.2.The expression of PD-L1 and IFN-gamma in tumor cells and mouse tumor tissues in vitro were analyzed by immunoblotting,Q-PCR and immunohistochemistry after different treatments.3.Flow cytometry was used to analyze the expression of CD38 in tumor microenvironment of mice.4.To construct cascade dual-targeting tumor microenvironment-responsive thermosensitive liposome carriers and characterize cascade dual-targeting tumor microenvironment-responsive thermosensitive liposome carriers.5.The mice bearing H22 tumors were divided into groups,and the volume change,survival rate and organ toxicity of the tumors were recorded and analyzed.6.The 4T1 tumor-bearing mice were divided into groups,and the volume change,survival rate and lung metastasis were recorded and analyzed.Results:1.Flow cytometry and immunofluorescence experiments showed that compared with the blank control group and the operation group,the infiltration of CD4+T cells and CD8+T cells in the tumor microenvironment of mice in the hyperthermia group increased significantly.2.The results of immunoblot analysis,Q-PCR and immunohistochemistry showed that compared with the blank control group,the expression of PD-L1 in tumor cells in vitro increased significantly after hyperthermia stimulation.The expression of PD-L1 and IFN-gamma in mouse tumors increased significantly.3.Flow cytometry analysis showed that the expression of CD38 was decreased compared with other treatment groups.4.After the construction of the vector.H22 and 4T1 mice were treated.The experimental results showed that the therapeutic effect of the vector combined with PD-1 antibody was better than that of other treatment groups.Conclusion:Hyperthermia can stimulate the infiltration of immune cells in tumor microenvironment,increase the expression ievel of PD-L1 in tumor microenvironment,inhibit the expression of CD38 in tumor microenvironment,and improve the synergistic therapeutic effect with PD-1 antibody.