Preparation And Preliminary Evaluation Of MRI Visualized SiRNA Delivery System

Objective:To prepare a visualized lipid polycationic siRNA delivery system(LP-PEI₁₈₀₀-SPION)with dual functions of gene silencing and visual magnetic resonance imaging,and preliminarily evaluate its feasibility in the diagnosis and treatment of liver cancer.Method:Co-precipitation method was used to synthesize superparamagnetic iron oxide nanoparticles（SPION）.Polyethyleneimine（PEI MW 1800）was modified on the surface by thiourea reaction.X-ray diffractometer and infrared spectrometer were used to characterize SPION and PEI₁₈₀₀-SPION.Thermogravimetric analyzer was used to detect the wrapping rate of PEI on SPION surface.The lipid polycationic gene carrier LP-PEI₁₈₀₀-SPION was prepared by ethanol injection method,and characterized by a high-sensitivity Zeta potential and particle size analyzer and transmission electron microscopy.The best prescriptions for LP-PEI₁₈₀₀-SPION and siRNA were screened by agarose gel electrophoresis,cytotoxicity,and in vitro gene silencing experiments.The silencing effect of complex on green fluorescent protein was evaluated by fluorescence microscopy and flow cytometry.And the silencing effect of LP-PEI₁₈₀₀-SPION/siRNA on the surface specific expression and highly expressed protein GPC3 in hepatocellular carcinoma cells was investigated by western blot assay.Prussian blue staining was used to detect the uptake of SPION nanoparticles by the cells.The nuclear magnetic resonance imaging experiments in vitro was used to detect the imaging effect of LP-PEI₁₈₀₀-SPION delivery system.Results:The data showed,the LP-PEI₁₈₀₀-SPION/siRNA complex is spherical or ellipsoidal without obvious aggregation.The hydrodynamic size is 174.33±1.15 nm,and the Zeta potential is 18.06±1.72 mV.MTT assay showed the cell survival rate is more than 80%when the concentration of the drug was within the range of 1-50μg/mL.When the mass ratio of LP-PEI₁₈₀₀-SPION/siRNA is 20,the transfection efficiency of the composite is the highest,and the silencing efficiency of the target protein is the largest.Prussian blue staining experiment showed obvious blue-stained iron particles in HepG2 cells and SMMC-7721 cells.MRI image signals decreased with the increased LP-PEI₁₈₀₀-SPION/siRNA complexes concentration in vitro.Conclusion:The visualized polycationic lipid siRNA delivery vehicle(LP-PEI₁₈₀₀-SPION)was successfully prepared in this experiment,which provides a basis for further theranostic of liver cancer.