The Study on Inhibition of Retinal Neovascularization with Targeting VEGF siRNA Transfection by New Polymeric Liposomes
|School||Tianjin Medical University|
|Keywords||polymeric liposome gene transfection oxygen inducedretinopathy (OIR) neovascularization|
ObjectiveThe purpose of the present work was to formulate and evaluate polymeric liposomes(PL) nanoparticles as novel non-viral gene delivery. To explore its applicability and feasibility as a nonviral vector for gene transport. The membrane translocation and longer half-life features of the nanoparticles were investigated and characterized, respectively.Methods1. The PL was synthesized through carboxymethyl chitosan grafting with glycidyl octadecyl dimethylammonium chloride, then conjugated by Tat. Explore the optimal way to transfect plasmid into cultured cells; draft dissolution curve by supercentrifuge process; RPE cells were used for in vitro cytotoxicity evaluation. According to the optimal methods, compared transfection efficiency between PL and Lipo to RPE.2. Collectd the culture supernatant of hypoxic RPE to determine the changes of secreted VEGF by ELISA. The expression of VEGF protein and mRNA in cells were detected by immunohistochemistry and Real-time PCR.3. To construct the oxygen induced retinopathy (OIR) mouse(C57BL/6J)model on the basis of improved Smith’s methods. HE staining and fluorescein-dextran angiography of retinal vascular were performed to observe the morphologic alterations of retinal neovascularization. Frozen-section was used to show the membrane translocation of PL.Results1. We successfully synthesized the PL with Tat. The average diameter of the PLs was134nm with+39.64mV Zeta potential. The in vitro release behavior of PL loaded plasmid DNA (pDNA) was depicted in the cumulative percentage release. The results showed that about70%of total encapsulated pDNA was released within initial5days followed by a constant and sustained release for14days.2. The optical transfection ways include:mass ratio between PL and pDNA was2:1; incorporation time was30-40minutes without serum. According to these terms, the transfection efficiencies to RPE cells, Hela cells and RF/16were69%,52%and80% respectively.3. After transfection, GFP could be seen in PL and Lipo group through fluorescence microscope. VEGF expression decreased in both PL group and Lipogroup by ELISA test. The expression of VEGF mRNA and protein in PL group and Lipo group decreased apparently through immunohistochemistry stains and Real-time PCR. The differences were significant compared to model group respectively(P<0.05).4. In fluorescence angiograms, irregular neovascularization and fluoresce leakage were observed in OIR model. Flat-mounted retina showed GFP expressions just at first day post-intravitreal administration which could reach their peaks at sixth day, remaining till11th day in PL group. Fluorescein angiography of retinal vascular and HE staining were performed to observe the significantly improvements of retinal neovascularization in PL and Lipo group at P17. Moreover, inhibitory effects maintained at P22in PL group as HE staining and Western blot results showed. Frozen-section examination showed the property of membrane translocation:GFP expression could be seen in vitreous cavity just at first day post-intravitreal administration in PL and Lipo group, which could reach their peaks in external retina nearby RPE layer at P17, remaining at P22in PL group. At the genetic level, Real-time PCR detected the VEGFmRNA decreased obviously in PL and Lipo2000group compared with that in model group(P<0.01).Conclusions1. OQLCS/chol PLwere synthesized successfully with higher size stability and cellular uptake efficiency. OQLCS/chol showed many superior properties, including lower cytotoxic effect, high Zeta potential compared with Lipo2000. Plasmid loaded PL exhibited slow steady release profile over14days at37℃.2. The gene loaded polymeric liposome could efficiently transfect RPE, Hela and RF/16cells as Lipo2000did.3. The PL performed excellent ability of membrane translocation, and it was a kind of slow steady released gene vector.