| ObjectiveTo explore the effect of vatamin E-coupled nanoparticles for sustained delivery of siRNAs targeting hepatitis C virus and inhibit its replication.Methods1.HCV-replicable cell line,Huh7.5.1-HCV and Huh7.5.1-Core cell line were constructed and were analyzed with Western blotting and immunofluorescent staining assay.2.Specific siRNAs were designed to target the replicon of HCV virus gene and HCV core protein and transfected into Huh7.5.1-HCV and Huh7.5.1-Core respectively.The efficacy were examined by real-time PCR and western blot analysis for the expression of specific protein.3.Vatamin E-coupled nanoparticles were prepared by the thin-film hydration method.The optimal VE-DC/siRNA ratio for transfection were determined by incubating VE-DC carrying Cy3-labeled siRNA(VE-DC/siRNA-Cy3)in varied molar ratios.After transfection of Huh7.5.1 cells with fractionated molar ratios of VE-DC/siRNA-Cy3,the transfection efficiency was determined using flow cytometry.The particle size distribution,zeta potential,stability in serum and cytotoxicity of the VE-DC/siRNA were determined by laser particle size analyzer,TEM images,agarose gel electrophoresis and CCK8 assay,respectively.4.The uptake of VE-DC/siRNA by Huh7.5.1-HCV cells was detected using fluorescence microscopy.Specific VE-DC/siRNA were used to treat Huh7.5.1-HCV and Huh7.5.1-Core cell respectively and the suppressive effect of VE-DC/siRNA was validated by examining gene and protein expression with real-time PCR,western blotting and immunofluorescent staining assays.5.The Huh7.5.1 cells were co-transfected with pRL-PK and pGL3-5? UTR-luc or pGL3-Enhancer,and then treated with DC/siRNA or VE-DC/siRNA.The cells lysates were assayed for luciferase activity on a multiple function enzyme analyzer for the transfection efficiency.6.HCV core mouse model was constructed by hydrodynamic injection of pCore-3FLAG into BALB/c.The HCV core protein level of the liver tissue was determined by immunofluorescent staining and western blotting.7.VE-DC/siRNA(siRNA-core)was intravenously injected into HCV core mouse model.The suppressive effect and targeting ability of VE-DC/siRNA was analyzed by detecting HCV core expression level.The destructive effect of VE-DC/siRNA on HCV replication was evaluated by the expression of luciferase regulated by HCV 5? UTR which established by the hydrodynamic tail vein injection of plasmid pGL3-5? UTR-luc in BALB/c mice.8.The in vivo side effects of the VE-DC/siRNA were evaluated by histological changes in the tissues,blood count(CBC)and biochemical parameters.Results1.The particle size distribution and zeta potential of the VE-DC/siRNA was 125.5±9.0 nm and 39.1±4.8 mV.The VE-DC/siRNA had a spherical morphology and homogeneous distribution.These nanoparticles were not cytotoxic to hepatoma cells and well stability in serum.The highest transfection efficiency was observed at a molar ratio of 1:1 for VE-DC and siRNAs in the incubation solution.2.Huh7.5.1-HCV and Huh7.5.1-Core were constructed.A denser granular pattern of fluorescence was seen for 24 h in VE-DC/siRNA-Cy3-treated cells.The targeting gene and protein expression was significantly lower in VE-DC/siRNA-treated cells compared with control group.3.A liver specific HCV core model was contrstructed successfully.VE-DC was more effective for delivering siRNA into the liver and inhibiting targeting protein expression.4.The effect of VE-DC/siRNA to inhibit luciferase gene expression controlled by HCV 5? UTR was detected by in vivo image analysis.The relative luciferase activity was significantly lower in the VE-DC/siRNA group compared with controls.5.No innate immunity response and toxicity effects induced by VE-DC/siRNA were observed both in vitro and in vivo.ConclusionTherefore,delivery of siRNAs by Vitamin E-coupled nanoparticles could target liver and efficiently inhibit HCV replication.Gene delivery by Vitamin E-coupled nanoparticles would provide a new strategy for HCV therapy. |
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