A Vitamin A-modified Nanocarrier For Targeted TLR4 SiRNA Delivery To Hepatic Stellate Cells For Anti-fibrotic Treatment

Background and Object Liver fibrosis is characterized by excessive accumulation of extracellular matrix(ECM)protein,which occurs in most types of chronic liver disease,and is often accompanied by portal hypertension,liver failure,susceptibility to infection or HCC in the later stage.HSCs are located in the dise space and store a large amount of vitamin A(VA).Activated HSCs are the main cells involved in liver fibrosis,and the generation of a large number of ECM is considered to be the initial link of liver fibrosis.Therefore,inhibition of HSCs activation and proliferation may be an effective anti fibrosis therapy.To develop the polyethylene glycol and polyethyleneimine(PEG-PEI)modified by vitamin A(VA)for targeted TLR4 si RNA delivery to hepatic stellate cells(LX-2 cells).To evaluate the transfection efficiency,the level of TLR4/NF-kappa B signaling pathway and the ?-SMA expression in LX-2 cells treated with PEG-PEI-VA.At the cellular level in vitro,the TLR4 / NF-? B signaling pathway of HSCs can be targeted and regulated,so as to explore the possibility of a new strategy for the treatment of liver fibrosis.Method 1.Synthesis of vitamin a-polyethylene glycol-polyethyleneimine nano carrier.2.Determination of hydrated particle size and zeta potential of nanocomposites.3.Agarose gel retardation electrophoresis was used to evaluate the ability of nano carriers to load si RNA.4.The cytotoxicity of the nanodrug was evaluated by CCK8 assay.5.The transfection efficiency of the nanodrug was measured by flow cytometry and fluorescence microscopy.6.LX2 cells were stimulated by lipopolysaccharide(LPS)and transfected with different polyplexes(VA-PEG-PEI/si TLR4 or PEG-PEI/si TLR4).7.The m RNA level,protein levels of TLR4/NF-kappa B pathway,and ?-SMA expression in LX2 cells were respectively assessed by RT-PCRassay,Western Blot assay and immunofluorescence.Results LX2 cells incubated with VA-PEG-PEI /si TLR4 at a high concentration of 40 ug/m L still showed viabilities above 80 %,indicating low cytotoxicity of the nanodrug.The agarose gel retardation electrophoresis showed that nano carrier VA-PEG-PEI possessed good si RNA loading capacity.LX2 cells stimulated by LPS showed a obviously increased level of TLR4/NF-kappa B pathway,such an effect was significantly detracted by VA-PEG-PEI /si TLR4 treatment.Moreover,VA-PEG-PEI /si TLR4 effectively deactivated LX-2 cells as evidenced by the reduced level of fibrotic marker,i.e.,?-SMA.Conclusion VA-PEG-PEI /si TLR4 achieved a highly efficient si RNA transfection,and effectively downregulated the TLR4/NF-kappa B pathway and ?-SMA expression in LX-2 cells.Our in vitro investigation showes that VA-PEG-PEI /si TLR4 has great potential for treating liver fibrosis.