| Background:Photothermal therapy（PTT）is a promising anti-cancer therapy,which has significant effect on causing tumor cell apoptosis and necrosis,reducing tumor burden,and enhancing immune response.However,in the process of cancer occurrence and development,T cells undergo exhaustion and cause dysfunction,which greatly hinders the efficiency of cancer treatment.By triggering the Gal-9/TIM-3 signaling pathway,T cell exhaustion inhibits the expansion of Th1 and Th17,promotes Th1 cell apoptosis and induces peripheral immune tolerance,weakens the body’s anti-tumor immune response in the tumor microenvironment and leads to the occurrence of tumor immune escape.Therefore,Gal-9 has become a new potential target for immunotherapy.Objectives:This study aims to construct a new type of composite nano-drug FSGG/si Gal-9for the treatment of melanoma,which is capable of down-regulating the immune checkpoint Gal-9,possessing the effect of photothermal immunotherapy,and reversing T cell exhaustion and enhancing anti-tumor immunity,finally,inhibiting the growth of melanoma.Methods:1.Preparation of new composite nanocarrier FSGG:A new multifunctional composite nanocarrier Fe3O4@Si O2-Glu-GNR（FSGG）was made by a layer-by-layer synthesis method.The newly synthesized FSGG was detected by ultraviolet absorption spectroscopy,transmission electron microscope,and Zeta potential.2.Gel migration test was used to detect the drug-carrying ability of FSGG to si RNA.3.MTT method was used to detect the in vitro cytotoxicity and photothermal effect of FSGG on B16-F10 cells.4.The transfection efficiency of FSGG/si RNA to B16-F10 cells in vitro was detected by fluorescence microscope and flow cytometer.5.The in vitro silencing efficiency of FSGG/si Gal-9 was determined Q-PCR and Western-blot.6.The long-term toxicity of FSGG in vivo was evaluated by transdermal administration.7.The in vivo targeting of FSGG/si RNA was assessed by frozen sections.8.Mouse melanoma model was used to test,synergistic treatment by down-regulating immune checkpoints using FSGG/si Gal-9,in combination with photothermal effect.9.T lymphocytes from the spleen of mice in each experimental group were collected for following immune assays.The apoptosis of T cells was detected by flow cytometry;The expression of Gal-9 in DC was detected by flow cytometry and Q-PCR.The expression of immunosuppressive molecules PD-1,TIM-3,BTLA and cytokines IL-2,TNF-α,IFN-γon T cells were detected by Flow cytometry,ELISA kit and Q-PCR.10.The CD8+T cells in the tumor were separated by magnetic bead.The proliferation ability of T cells is detected by CCK-8,while the Cyto Tox 96?non-radioactive cytotoxicity test was used to detect its killing effect on tumor cells.Results:1.TEM results showed that the size distribution of FSGG nanoparticles was uniform,and GNR-MUA was uniformly adsorbed on the surface of FSG,with good dispersibility.Zeta potential analysis showed that the zeta potential of the material at each stage had obvious positive and negative alternating changes,indicating the composite nano material was successfully synthesized.UV-Vis absorption spectrum showed that FSGG had a significant red shift compared with GNR-MUA,indicating that the composite nanomaterials well retained the optical properties of GNR-MUA.2.The gel migration test showed that when the mass ratio of si RNA to FSGG is1:6（w/w）,si RNA can be effectively loaded.3.The MTT eassay showed that FSGG had lower cytotoxicity.After near-infrared laser irradiation,FSGG effectively inhibited the viability of tumor cells in vitro.4.Fluorescence microscope demonstrated that FSGG can effectively deliver Cy3-si GAPDH to the cytoplasm,whereas the magnetic field significantly enhanced the cell delivery efficiency of FSGG to si RNA.The flow cytometry further confirmed that the transfection efficiency achieved as high as 88.13%.5.Gal-9 gene expression in B16-F10 cells was significantly inhibited after FSGG/si Gal-9 transfection.The protein expression level was reduced by59.29±2.69%,and m RNA expression level was reduced by 76.15±5.77%.6.After local transdermal administration of FSGG,the percentages of CD4+T and CD8+T lymphocytes in the spleen of mice did not change significantly;the results of GOT,GPT,creatinine,and urea in the serum showed that FSGG treatment had no obvious toxicity to liver and kidney on mice.HE staining showed that no histopathological abnormalities occurred in mouse organs;and TEM results showed that there was no obvious accumulation of FSGG,FSG or GNR-MUA in liver and spleen.7.Frozen section results showed that after transdermal administration of FSGG/Cy3-si GAPDH,si RNA can be efficiently targeted and delivered to tumor tissues with the assistance of a magnetic field.8.The treatment of tumor-bearing mice showed that FSGG/si Gal-9 can synergistically inhibit the growth of mouse melanoma by down-regulating immune checkpoint Gal-9 combined with photothermal effect.9.Silencing Gal-9 gene resulted in repression of immunosuppressive receptors PD-1,TIM-3 and BTLA on the exhaustive T cells as well as upregulation of immune-related cytokines such IL-2,TNF-αand IFN-γ.Enhancement of the cytotoxic killing effect of CD8+T lymphocytes and proliferation of T cells.Conclusion:1.In this study,we successfully constructed a new type of tumor targeting nanomaterial FSGG.2.FSGG has good biocompatibility and excellent photothermal effect.It has no long-term toxicity to the body and is easy to remove.It achieves high-efficiency targeted transdermal delivery of si RNA,and effectively induces Gal-9 gene silencing in vivo and in vitro.3.FSGG/si Gal-9 reverses CD8+T cell depletion by inhibiting the immune checkpoint Gal-9,and the coordinated photothermal effect can effectively enhance the anti-tumor immune response of T cells and inhibit the growth of melanoma in mice. |
