A Novel Anti-cancer Therapy Using Gold Nanorods Mediated Targeteded Gene Silencing Indolamine 2,3-dioxygenase Combined With Photothermal Effect On Lewis Lung Cancer

Objectives: IDO1 is an critical immune negative regulatory molecule.It plays a very important role in modulating tumor immune escape,through manipulating the ability of tumor invasion and metastasis,thus is a potential target for clinical cancer treatment.This study establishs a mouse model of lung cancer and constructs a nanocomplex GNR-MUA-PEI-FA(GMPF)/siIDO1 that can target lung cancer,delivers si RNAs and induces gene silencing to the target molecule IDO1.This study assessed the tumor-specific targeting and gene silencing effect of IDO1 in of Lewis lung cancer,as well as evaluated a novel anti-cancer therapy using GNR-MUAPEI-FA(GMPF)/siIDO1,through gene silence in combination with the photothermal therapy on Lewis lung cancer treatment.Methods: 1.Synthesis of GNR-MUA-PEI-FA(GMPF): 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-FA.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 1H-NMR.2.MTT was used to examine the cytotoxicity of gold nanorods before and after modification.3.The capacity of GNR-MUA-PEI-FA incooperating si RNA was determined according to different weight ratio,Release of si RNA from GMPF and si RNA stability in serum,and was detected by the gel block experiment.4.Flow cytometry was used to measure the transfection efficiency of GMPF/Cy3-si GAPDH into LLC cells,whereasthe optimal transfection concentration and the targeting of LLC cells were thus determined.5.Q-PCR and western blot(WB)are used to detect the silencing effect of GMPF/siIDO1 on LLC cells.6.To determine the in-vivo distribution of newly synthesized nono-carrier of si RNA,GMPF/Cy3-si GAPDH was injected intravenously through tail vein of lung cancer-bearing C57BL/6 mice.The organs(liver,heart,lungs,spleen,kidneys and tumors were collected.The frozen sections were made and the distribution of GMPF/cy3-si GAPDH was observed under fluorescence microscope..7.To evaluate the therapeutic effect,GMPF/siIDO1 was injected intravenously into and treated the lung cancer-bearing C57BL/6 mice,followed by photothermal therapy(PTT)under laser irradiation.The anti-tumor effects were assessed by tumor growth curve and tumor size that were meatured daily until end point of experiment.8.To determine the in-vivo gene silencing effect,the tumor tissues of each group were removed,and the silencing effect of IDO1 in tumor tissues was detected by Q-PCR and WB.In addition,the tumor tissue were made into paraffin sections,the IDO1 expression was measured by immunohistochemical;9.The apoptosis was detected by TUNEL,which evaluates the effects of GMPF/siIDO1 gene silencing combined with photothermal therapy in vivo.Results: 1.Characterization of GMPF: Before and after modification,the UV absorption peak of gold nanorods whowed redshift and that of GMPF was 790 nm.Under the observation of transmission electron microscope,the size was uniformand and the dispersion was evenly.The modification of mannoses on GMPF was successful as detected by 1H-NMR.2.Cytoxicity and biocompatibility: After the functionalization of gold nanorods,the cytotoxicity of GMPF was significantly reduced,and its biocompatibility and biosafety were significantly improved.3.Protection of si RNA by GMPF: The gel block experiments showed that when the mass ratio of GMPF to siIDO1 is 4:1,siIDO1 are just all combined by GMPF;the gel retardation experiments show that the GMPF/siIDO1 can release si RNA in the presence of SDS and stable in serum of 50% to 72 hours.4.si RNA delivery by GMPF: Flow cytometry showed that the optimum concentration of GMPF/cy3-si GAPDH transfected into LLC cells was 40?g/ml and the efficiency was 92.3%;In addition,flow cytometry was used to detect GMR-MUA-PEI(GMP)and GMPF transfection efficiency,the results proved that GMPF can targeted to LLC cells.5.In vitro Gene silencing by GMPF/siIDO1: Q-PCR and WB results showed that the silencing efficiency of GMPF/siIDO1 in vitro was 60% and 43%,respectively.6.In vivo distribution of GMPF/siIDO1: Frozen tissues showed the fluorescent substance mainly accumulated in the tumor,a small amount of accumulation in the organs of metabolism like liver and kidney tissues,24 h after administration of GMPF/cy3-si GAPDH.There is no other organs showing obvious accumulation of GMPF/cy3-si GAPDH.7.In vivo Gene silencing by GMPF/siIDO1: GMPF/siIDO1 can be effectively silence IDO1 in vitro by hydrodynamic injection into C57BL/6 mice.The results of Q-PCR and WB showed that the silencing effect in GMPF/siIDO1 and in GMPF/siIDO1+laser groups were 66% and 60%,respectively.8.Anti-tumor effects by GMPF/siIDO and PTT: The tumor growth was significantly arrested by the treatment with GMPF/siIDO1+laser in LLC lung cancre-bearing mice.9.Immunohistochemistry showed that the expression of IDO1 protein in tumor tissue and the GMPF/siIDO1+laser group decreased significantly.Apoptosis induced by GMPF/siIDO and PTT:The results of TUNEL assay showed that the percentage of apoptosis in GMPF/siIDO1+laser group was 45%.Conclusion: 1.The GMPF/siIDO1 nanocomposite was successfully constructed,which can effectively deliver si RNA to tumor cells in vitro and in vivo,as well as inducing targeted gene silencing of IDO1 in LLC cells.2.Treatment with GMPF/siIDO1 exerts synergistic effects of photothermal therapy and effectively inhibited tumor growth in lung cancer.