Construction And Research On Amphiphilic Gene Carriers For Improving The Transfection Efficiency Of Endothelial Cells

Nowadays, the intervention therapy of cardiovascular and cerebrovascular diseases have always accompanied with neointimal proliferation and restenosis, which may endanger the patients’ lives. Owing to the inherent advantages of endothelial cells, the rapid endothelialization of vascular grafts is a radical approach to avoid a series of pathological processes. Gene therapy and surface modification as two effective strategies have been investigated for promoting endothelialization. However, the transfection efficiency of endothelial cells is relatively low, which decreases the endothelialization of vascular grafts. Therefore, improving the transfection efficiency of endothelial cells is very impotant for promoting endothelialization. Considering the molecular weight of carriers and targeting peptide playing an important role in delivering gene, several polycationic gene carriers with different molecular weight and targeting peptide modified carriers were prepared and used for investigating the influence on the transfection efficiency of endothelial cells.Chapter 2. In consideration of the transfection efficiency of endothelial cells being influenced by the polycationic carrier’s molecular weight, four kinds of polycationic gene carriers with different molecular weights were synthesized and used for the transfection of endothelial cells. Block copolymer of poly(2-hydroxyethyl methacrylate)-poly(L-lactide)(PHEMA-PLA) was synthesized for providing enough free hydroxyl groups as reaction sites, and then hydrophilic methoxy poly(ethylene glycol) ether(PEG) and poly(ethyleneimine)(PEI) chains bearing different molecular weight were randomly grafted onto PHEMA-PLA to prepare amphiphilic polycationic polymers PHEMA-PLA-PEG 2 kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 5 kDa-PEI 1.8 kDa, PHEMA-PLA-PEG 2 kDa-PEI 10 kDa, PHEMA-PLA-PEG 5 kDa-PEI 10 kDa. These four copolymers were assembled into G2I1.8, G5I1.8, G2I10 and G5I10 nanoparticles via dialysis method, respectively. Dynamic light scattering was used to investigate the hydrodynamic diameters and zeta potential values of these four nanoparticles and their complexes. EA.hy926 cells were used as model cells for investigating the cytotoxicity and transfection efficiency of endothelial cells mediated by these four gene complexes. The results indicated that the transfection efficiency of endothelial cells can be influenced by the molecular weight of polycationic carriers. The carriers with higher molecular weight presented a higher transfection efficiency value than the lower ones and the introduction of PEG in the gene carrier can improve the transfection efficiency of endothelial cells to some extent.Chapter 3. Herein, we developed a targeting REDV peptide functionalized polycationic gene carrier for delivery of pEGFP-ZNF580 plasmid with the aim to enhance the transfection capability of human endothelial cells. The amphiphilic block copolymer methoxy poly(ethylene glycol) ether-poly(L-lactide-co-ε-caprolactone)-poly(ethyleneimine)(mPEG-P(LA-co-CL)-PEI) was prepared by ring-opening polymerization and graft polymerization reaction. The polycationic gene carrier with REDV peptide(mPEG-P(LA-co-CL)-PEI-REDV) was prepared by the conjugation of Cys-Arg-Glu-Asp-Val-Trp(CREDVW) peptide with the mPEG-P(LA-co-CL)-PEI copolymer. mPEG-P(LA-co-CL)-PEI nanoparticles(NP) and mPEG-P(LA-co-CL)-PEI-REDV nanoparticles(REDV-NP) were formed by self-assembling of the corresponding polycationic polymers. Gel electrophoresis results showed that targeting REDV-NP could condense pEGFP-ZNF580 plasmids into stable complexes. MTT assay indicated that these targeted REDV-NP/pEGFP-ZNF580 complexes exhibit better cyto-compatibility than the non-targeted NP/pEGFP-ZNF580 complexes and the control PEI 1800 Da/pEGFP-ZNF580 complexes. In vitro transfection experiments of EA.hy926 endothelial cells showed that the transfection efficiency of the targeted REDV-NP/pEGFP-ZNF580 complexes were roughly consistent with that of PEI 25 kDa/pEGFP-ZNF580 complexes. More importantly, the scratch wound assay results demonstrated that the migration capability of EA.hy926 cells had been improved significantly by the expression of pEGFP-ZNF580 plasmid.Chapter 4. In this chapter, we synthesized the REDV peptide modified polycationic polymer as pEGFP-ZNF580 gene carrier for investigating the transfection efficiency in human endothelial cells. Amphiphilic block copolymer PHEMA-PCL was prepared by ring-opening polymerization of ε-caprolactone with PHEMA as macroinitiator. PHEMA-PCL-PEG-PEI polycationic polymer was synthesized by esterification of PEG with carboxylated-PHEMA-PCL and amidation reaction of PEI with carboxylated-PHEMA-PCL. Targeting polycationic copolymer PHEMA-PCL-PEG-PEI-REDV was prepared by the conjugation of PHEMA-PCL-PEG-PEI and CREDVW peptide. The targeting nanoparticles were prepared by self-assembling of PHEMA-PCL-PEG-PEI-REDV and used for the delivery of pEGFP-ZNF580 plasmid. This polycationic carrier effectively condensed pEGFP-ZNF580 plasmid to form REDV-targeted complexes. These REDV-targeted complexes exhibited low cytotoxicity but high transfection efficiency to EA.hy926 cells compared with the non-targeted complexes(PHEMA-PCL-PEG-PEI/pEGFP-ZNF580) determined by MTT assay and fluorescence images of endothelial cells. The western blot results showed that the relative protein level of endothelial cells transfected by REDV-targeted complexes was higher than that by non-targeted complexes.The results in chapter 2 showed that the molecular weight of polycationic gene carriers affected the transfection efficiency of endothelial cells significantly. And the transfection efficiency of endothelial cells can be improved by introduction of targeting gene carriers modified with peptide. We believe that the research may be a promising platform for enhancing the endothelialization of artificial vascular grafts, and can be potentially applied in the interventional therapy of cardiovascular diseases in future.