A New SiRNA Delivery System: Synthesis Of 1-methyl-?-caboline-3-RGDS And Studies On Inhibitions Of VEGF Expression And Tumor Growth

Use of siRNA in anti-angiogenic gene therapy is limited because its insufficient cellur delivery efficiency.In this study,we developed a new anti-tumor nano targeting carrier,which could effectively load and transfect siRNA into cells.The new siRNA loaded carrier exhibit anti-tumor effects in vivo and in vitro.Following studies are included:1.1-CH3-?-Carboline-3-Arg-Gly-Asp-Ser was synthesized.And with 1 H NMR,13C NMR,Mass spectra,IR spectrum,UV spectrum and HPLC,the structure andpurity were characterized.2.Thin film-dispersion method and orthogonal screening were taken to prepare thevehicle?CRS for short?,and by condensation,VEGF-siRNA was loaded to formVEGF-siRNA/CRS.Gel retardation assay and in vitro release trial confirmed thesuccessful loading and gradual releasing characteristic of the complex.Moreover,VEGF-siRNA/CRS could be observed in SEM and TEM as a globe shape of100-200 nm in particle size.And in 650 nm laser light,Tyndall effect wasobviously observed.3.Transfection could be effectively at 4 hours by confocal observation in vitro.Andanti-tumor bioassays were conducted on HeLa cells by CCK-8 method,whichwere cultured with different concentrations of a series of VEGF-siRNA/CRS,andcompared with VEGF-siRNA/CRS?CRS?VEGF-siRNA?DEPC water and VEGF-siRNA/lipo^TM2000 to study the cytotoxicity.4.Also,Gene silencing efficiency at protein level was conducted on HeLa cells to determine the down regulating of VEGF by ELISA.Results proved that VEGF-siRNA/CRS showed a higher gene silencing efficiency.5.Gene silencing efficiency at mRNA level was performed on HeLa cells.mRNA was amplified by RT-PCR and VEGF-siRNA/CRS showed a higher gene silencing efficiency in mRNA level.6.In vivo experiments were taken out in S180 tumor mouse.Fluorescence animal images were taken to verify the target characteristic of the carrier.Further,by acute toxicity trial and tumor growth inhibition experiment,it were concluded that the carrier exhibited lower toxicity and VEGF-siRNA/CRS distributed most in tumor site with good inhibiting on the growth of tumor compared with others.Summarily,A novel VEGF-siRNA delivery system:VEGF-siRNA/CRS was prepared and proved with enhanced anti-tumor activity and higher gene silencing efficiency.