A Novel Functionalized Gold Nanorod For Tumor-targeted Gene Silencing Of BRAF And Photothermal Therapy For Melanoma

Objectives:To explore the effect of BRAF gene on cell biological function in silenced mouse melanoma cells(B16-BL6)by gold nanorods(GNR)-mediated RNAi technology.To study the effect of photothermal therapy PTT)on mouse melanoma cell apoptosis,as well as to study the gold nanorod-mediated RNAi technology combined with PTT on the treatment of mouse melanoma.Methods:1.The gold nanorods(GNR)were prepared by the seed synthesis method,and functionally modified.After modified,the size and dispersion,the surface plasma resonance effect and the surface molecular structure after functionalization were observed by TEM,UV Vis spectra and NMR respectively.2.The cell toxicity of before and after modification of GNR were determined by MTT test.3.The effect of loading siRNA were determined by gel retardation assay.4.The transfection efficiency were determined by the cell flow cytometry.5.The silence efficiency of BRAF gene/protein and the expression of MEK-ERK signal pathway related proteins in B16-BL6 cells/tumor-bearing mice were observed by qPCR and Western Blot.6.The changes of BRAF silencing and migration,proliferation were determin-ed by the Scratch assay/Transwell migration,MTT assay,flow cytometry respective-ly.7.The photothermal effects of GNR was determined by Thermometers.The photothermal effects of GNR on the cells and the effect of gold nanorod-mediated RNAi combined with PTT on the apoptosis of B16-BL6 cells were detected by MTT assay and flow cytometry.8.A C57BL/6 B16-BL6 tumor-bearing mouse model was established and treated with transdermal administration to observe the effect on tumor growth.Results:1.Compared with GNR before modification,after modification,the UV Vis absorption peak was redshifted to 790nm,the size and dispersion were no obvious changes.2.The cell cytotoxicity significantly declined.3.When the weight ratio was 3.5:1 just combining all siRNA.4.The results of flow cytometry showed that the transfection efficiency of GNR-FA to siRNA was significantly higher than that of GNR-PEI,and increased with the increase of concentration in safety range.Less affected by serum.5.The results of qPCR and Western blot showed that the rate of BRAF gene and protein silencing in vitro reached more than 80%and reached 40%in vivo.6.The migration ability of 24 hours cells decreased by 13.2%.7.After silencing,the proliferation and migration ability of cells were significantly decreased,the MEK-ERK signal pathway P-MEK,P-ERK protein down significantly.8.PTT at 4w/cm~2 and 5min in optical density was obvious.Flow cytometry was used to detect the apoptosis of B16-BL6 cells induced by RNAi combined with PTT.9.In vivo combination therapy group than other control group and treatment group significantly inhibited tumor growth rate,reduce tumor volume,reduce tumor weight.Conclusion:1.GNR-FA can safely and effectively target BRAF gene silencing in B16-BL6cells and inhibit its proliferation and differentiation by affecting MEK-ERK signaling pathway.Combined with PTT,GNR-FA can promote tumor cell apoptosis.2.GNR-FA mediated RNAi combined with PTT is more effective in inhibiting tumor growth in B16-BL6 tumor-bearing mice.