The Gold Nanorod For Tumor-targeted Gene Silencing Of PD-L1 And Photothermal Therapy For Melanoma

Objective: The tumor-derived immunosuppressive molecule PD-L1 on the surface of tumor cells interacts with PD-1,which is immune suppressive receptors expressed on the surface of T cells.The interaction between PD-L1/PD-1 results in a phenomenon called T cell exhaustion,which leads to inhibition of T cell mediated immune response and,impairment of CD8+T cell–mediated killing response.In this study,we used a new nano-carrier which can deliver siRNA to tumor cells.FA-GNR-siPD-L1 can silence PD-L1 gene in tumor cells,so that we can block the PD-L1/ PD-1 signaling pathway,and eventually recover T cells activity.Besides,we also explored the combination of RNAi technology and gold nanorod-mediated PTT on the treatment of mouse melanoma.Methods: 1.The capacity of loading siRNA was tested by gel retardation assay.2.The cell toxicity of FA-GNR and MUA-GNR were determined by MTT test.3.The transfection efficiency was detected by the fluorescence microscopy and the flow cytometry.4.The gene silence efficiency of FA-GNR-siPD-L1 was determined by q-PCR and flow cytometry.5.The B16-BL6 cell after gene silencing of siPD-L1 was co-cultured with T cells which is derived from B16-BL6 melanoma-bearing mice.The m RNA expression of PD-1,TIM-3 and BTLA of T cells and the m RNA expression of IL-2,TNF-? and IFN-? were tested by q-PCR.The apoptosis of T cells was detected by flow cytometry.6.To establish a melanoma mouse model,B16-BL6 cells were injected into C57BL/6 mice,the tumor-bearing mice were treated with FA-GNR-siPD-L1 by intravenous injection and combined with PTT.Tumor size of every group was observed and evaluated.7.The lymphocyte of every group was collected after treatment.The change of T cells phenotype was measured by flow cytometry,and the apoptosis of T cells was tested by Annexin V/PI assay.The m RNA expression of IL-2,TNF-? and IFN-? were tested by q-PCR.8.CD8+ T cells were isolated from tumor bearing mice using magnetic beads.The cytotoxic capability of CD8+ T cells was determined by LDH assay.The ability of proliferation was tested with CCK8.Results: 1.The gel retardation experiments showed that when the weight ratio was 3.5:1,the FA-GNR can combine all siRNA.2.The cytotoxicity of FA-GNR was significantly reduced after the functionalize modification of gold nanorods.3.With the modification of FA,FA-GNR can deliver siRNA into cells,the transfection efficiency of siRNA by FA-GNR to tumor cells was high,while the best concentration for transfection was 20 ?g/ml.4.The q-PCR result showed that the rate of gene silencing of PD-L1 in B16-BL6 was above 80%,and flow cytometry result showed that the rate of protein silencing of PD-L1 in B16-BL6 reached 68%.5.The B16-BL6 cell that silenced by siPD-L1 inhibited the PD-L1/PD-1 signaling pathway,as evidenced by the decreased expression of inhibitor receptor of PD-1,TIM-3 and BTLA on the surface of T cells,as well as the suppressed apoptosis of T cells.6.The combination therapy of FA-GNR-siPD-L1 and PTT significantly suppressed the growth of tumor,and improved the capability of anti-tumor in tumor-bearing mice.7.After combination therapy,the exhausted phenotype of T cell in the treated group could be significantly recovered,whereas the secretion of cytokines was increased.8.The combination therapy can enhance the cytotoxic capability of CD8+ T cells in the treated tumor-bearing mice,and improved the proliferative capacity of T cells.Conclusion 1.The delivery system of FA-GNR-siPD-L1 can effectively deliver the siRNA into tumor cells,and significantly silence the expression of target gene in tumor cells.2.The combination of RNAi technology and gold nanorod-mediated PTT can effectively suppress the tumor growth in the mice bearing melanoma,recover T cells exhaustion and improve the capability of anti-tumor of T cells in tumorbearing mice.