Study On Low-Temperature Photothermal Therapy Combined Immunotherapy For Breast Cancer Mediated By Engineering Gene Delivery Vectors

Objective:To synthesize an engineered nanoparticle delivery carrier loaded with photothermal agent and connected with small interfering RNA of heat shock protein to achieve low temperature photothermal therapy.At the same time,the nanoparticle was coated with tumor cell membrane with high expression of CD86,so as to realize the combination of photothermal therapy and immunotherapy while achieving targeting of tumor cells and activating the T cells of tumor microenvironment.And to evaluate the efficacy of synthetic nanomaterials in breast cancer treatment.Methods:DSPE-PEG-NH₂ was connected to the heat shock protein 90 small interfering RNA（HSP90-si RNA）,and the photothermal agent F0 was coated by its self-assembly property.Murine breast cancer 4T1 cells were transfected with lentivirus to express CD86 on their cell membranes.The expression and location of CD86 were verified by immunofluorescence assay.4T1 cell membrane with high CD86 expression was extracted and coated on the surface of nanoparticles.Verification of synthesized nanomaterials is performed through material characterization,which involves determining their morphology through transmission electron microscopy,as well as measuring their hydrated particle size and Zeta potential.The protein expression of nanomaterials was verified by protein electrophoresis,and the release of HSP90-si RNA was verified by nucleic acid electrophoresis.The nanomaterial was co-cultured with4T1 cells to verify its targeting,its biosafety was verified by CCK-8 cytotoxicity assay,its blood stability was confirmed by hemolysis assay,and its stability was evaluated by measuring its particle size changes in different solutions.Western blot was used to verify the inhibitory effect of the nanomaterials on heat shock proteins.Calcein staining and flow cytometry were used to verify the ability of the nanomaterials to kill tumor cells in vitro.Primary T cells were extracted from mice,and the activation of T cells by nanomaterials was verified by detection of LDH and detection of cytokines（TNF-αand IFN-γ）by ELISA.The mouse model of subcutaneous breast cancer was used.Nanomaterials were injected into caudal vein and NIR irradiation was given.The efficacy of the treatment was evaluated by monitoring the changes in tumor volume during the treatment,and the safety of the treatment was evaluated by the changes in body weight and H&E staining of the slices of important organs.Results:In this study,the engineered nano-carrier DT@FNPs was successfully prepared to mediate low temperature photothermal combined immunotherapy.Material characterization verified that the nanoparticle had good particle size,electric potential and other properties,retained tumor cell membrane proteins and transfected CD86proteins,and could release si RNA in a simulated tumor environment in vitro.Through CCK-8 cytotoxicity and hemolysis experiments,it was verified that DT@FNPs has good biological safety and blood stability,and it also has good stability in different solutions.In vitro targeting experiments verified that tumor cells have a good uptake effect on DT@FNPs.Western blot verified that DT@FNPs could effectively inhibit the expression of HSP90 in 4T1 cells.In vitro killing experiments,calcein staining and flow cytometry both proved that DT@FNPs had a good killing effect.Immunoactivation experiments have verified that DT@FNPs has a good effect on activating T cells,which can enhance the killing ability of T cells and the ability to secrete cytokines.In vivo experiments on mice have proved that DT@FNPs has a good therapeutic effect on 4T1 subcutaneous tumor of mice,and has little effect on body weight and important organs of mice,with good biological safety.Conclusion:In this study,targeted nanocarriers（DT@FNPs）with gene delivery function were successfully prepared.At the same time,low temperature photothermal therapy was combined with immunotherapy,which had good antitumor therapeutic effect in vivo and in vitro while taking into account safety and stability.