Treatment Of Lung Cancer In Mouse Models Via The Synergic Therapy Of GNR-MUA-PEI-Man/siIDO Targetedly Silencing IDO Of DCs And Flt3-L Proliferating DCs In Vivo

Objectives:Dendritic cells(DCs)are currently known as the most potently professional antigen-presenting cells and mature DCs can effectively activate naive T cells,which is very important for the immune response of the body,but DCs can produce some important immune negative reg?latory molec?les such as Indoleamine 2,3-Oxygenase(IDO),which can prevent the tumor cells from the immune system monitoring and result in tumor invasion and metastasis and affect the clincal efficacy of DC vaccines finally.This study is to build a new type nanosystem GNR-MUA-PEI-Man/si IDO that can target DCs,deliver si RNAs and silence the target molecule IDO,and then to study its targeting DCs and silencing IDO efficiencies,and eventually to combine this new nanostructure and Flt3-L amplifying DCs to treat the lung cancer of mouse models in vivo.Methods:1.Synthesis of GNR-MUA-PEI-Man: Firstly,the gold nanorods(GNR-CTAB)for this study were synthesized by the classical seed growth method,and then were further modified to GNR-MUA-PEI-Man.The size and dispersion were observed by transmission electron microscopy.The localized surface plasmon resonance spectr?m was measured by UV spectroscopy.The surface mannose molec?les were certified by NMR.2.CCK8 was used to examine the cytotoxicity of gold nanorods before and after modification.3.GNR-MUA-PEI-Man combined with si RNA according to different weight ratio by the gel block experimental to observe combined effect.4.Flow cytometry was used to measure the transfection efficiency of GNR-MUA-PEI-Man/Cy3-si RNA into DCs and determine the optimalconcentration and time for transfection.5.Silencing efficiency of GNR-MUA-PEI-Man/si IDO on DC was detected by Q-PCR.6.GNR-MUA-PEI-Man/Cy3-si GAPDH was injected intravenously into vein and treated the lung cancer-bearing C57BL/6 mice,then,taking its liver,heart,lung,spleen,kidney made frozen sections,observe the role of body in vivo.7.GNR-MUA-PEI-Man/si IDO was injected intravenously into and treated the lung cancer-bearing C57BL/6 mice of which DCs were amplified by Flt3-L and the therapeutic effects were observed.8.After the above treatment,the mice were sacrificed and the spleens were collected for detecting the amplification effects of DCs by Flt3-L and the apoptosis of T cells and the production of Tregs.9.CD11c+DCs seperated from the spleens by magnetic beads were measured the maturity and the silencing IDO effects of GNR-MUA-PEI-Man/si IDO in vivo.10.CD4+T cells and CD8+T cells isolated from the spleens by magnetic beads as well were analysed the killing abilities and the the proliferation of CD4+T cells.Results:1.Before and after modification,the UV absorption peak of gold nanorods was redshift and that of GNR-MUA-PEI-Man was 810 nm.Under the observation of transmission electron microscope,the size was uniformand the dispersion was good.NMR detected mannoses on GNR-MUA-PEI-Man,indicating that the modification was successf?l.2.After the functionalization of gold nanorods,the cytotoxicity of GNR-MUAPEI-Man was significantly reduced,and its biocompatibility and biosafety were significantly improved.3.The gel block experiments showed that when the mass ratio of GNR-MUA-PEI-Man to si IDO is 2:1,si IDOs are just all combined by GNR-MUA-PEI-Man.4.Flow cytometry showed that when the mass ratio of GNR-MUA-PEI-Man to si IDO was 5:1,the transfecting DC efficiency was the highest about 69.3% after24 hour transfection.5.After GNR-MUA-PEI-Man/si IDO transfecting DCs for 48 hours in vitro,Q-PCR res?lts shows the silencing efficiency was 71.2%,and silencing IDO can significantly promote the maturity of DCs.6.The results of frozen sections showed that the fluorescent substance was mainly accumulated in the spleen and macrophages rich in DC and other macrophages after 48 hours of injection of GNR-MUA-PEI-Man / Cy3-si GAPDH tail vein.See obvious accumulation.7.I.v.administration of GNR-MUA-PEI-Man/si IDO thro?gh tail vein into C57BL/6 mice can effectively silence IDO expression on DCs in vivo.Synergetic therapy for lung cancer-bearing mice with the i.v.administration of GNR-MUA-PEI-Man/si IDO that targetedly silences IDO on DCs and Flt3-L that promotes and proliferates DCs can apparently inhibit the t?mor growth in vivo.8.In vivo after using the built GNR-MUA-PEI-Man/si IDO downreg?lating IDO on DCs in lung cancer-bearing mice,the apoptosis of T cells was reduced,the n?mber of Tregs reduced,the killing ability of cytotoxic T cell enhanced,and the proliferation ability of CD4+T cell increased.Conclusion:1.Gene nanocarrier of GNR-MUA-PEI-Man was successf?l synthesized,which can targetedly deliver si RNA into DCs and effectively silence target molec?les in vitro and in vivo.2.GNR-MUA-PEI-Man/si IDO combined with Flt3-L can treat lung cancer synergetically and effectively in vivo.