Targeting gene engineering should be considered as an effective method for promoting endothelialization of vascular grafts. Herein, we developed a targeting REDV peptide functionalized polycationic gene carrier for carrying the pEGFP-ZNF580 plasmid with the aim of enhancing the transfection and migration capability of human endothelial cells. This polycationic gene carrier with the REDV peptide (mPEG-P(LA-co-CL)-PEI-REDV) was prepared by the conjugation of the Cys-Arg-Glu-Asp-Val-Trp (CREDVW) peptide with the amphiphilic block copolymer methoxy poly(ethylene glycol) ether-poly(L-lactide-co-ε-caprolactone)-poly(ethyleneimine) (mPEG-P(LA-co-CL)-PEI). mPEG-P(LA-co-CL)-PEI nanoparticles (NP) and mPEG-P(LA-co-CL)-PEI-REDV nanoparticles (REDV-NP) were formed by the self-assembly of the corresponding polycationic polymers, and then their pEGFP-ZNF580 complexes were prepared via the electrostatic interaction with pEGFP-ZNF580 plasmids, respectively. Gel electrophoresis results show that the targeted REDV-NPs could compress pEGFP-ZNF580 plasmids into stable complexes and protect the plasmids against desoxyribonuclease degradation. MTT assay indicates 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 and western blot analysis of EA.hy926 endothelial cells show that the pEGFP-ZNF580 plasmid expression and the relative protein level transfected by targeted REDV-NP/pEGFP-ZNF580 complexes are roughly consistent with that transfected by PEI 25 kDa/pEGFP-ZNF580 complexes. More importantly, the scratch wound assay results demonstrate that the migration capability of EA.hy926 cells has been improved significantly by the expression of the pEGFP-ZNF580 plasmid. Our results indicate that the polycationic polymer with functional REDV peptides can be a potential candidate as a pEGFP-ZNF580 plasmid delivery carrier and may be used in the endothelialization of vascular grafts.
The lack of living endothelial cells (ECs) functional layer is one of the major reasons which account for thrombosis of synthetic vascular vessels. To overcome this obstacle, we employed ZNF580 gene complexed with biodegradable microparticles (MPs) to promote the rapid endothelialization by ECs. In order to realize the controlled release of ZNF580 gene from MPs/gene complexes, a series of amphiphilic triblock copolymers with different degradation rate , namely, methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-morpholine-2,5-dione)-graft-polyethyleneimine (mPEG-b-PMMD-g-PEI), methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-morpholine-2,5-dione-co-lactide)-graft-polyethyleneimine (mPEG-b-P(MMD-co-LA)-g-PEI) and methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-morpholine-2,5-dione-co-lactide-co-glycolide)-graft-polyethyleneimine (mPEG-b-P(MMD-co-LA-co-GA)-g-PEI), were synthesized. Then, MPs were formed by self-assembly. The hydrophobic cores of these MPs were composed of PMMD, P(MMD-LA) or P(MMD-co-LA-co-GA) segments, and provided crosslinking points for numbers of PEG and short PEI chains to form a high hydrophilic and positive charged corona/shell structure. Based on their positive charged surface, MPs can compact pEGFP-ZNF580 into MPs/pEGFP-ZNF580 complexes. The cell transfection result demonstrated that pEGFP-ZNF580 could be transported efficiently into ECs by these complexes. The result of western blot showed that the relative ZNF580 protein level can increase to 35.74%–46.11% by the overexpression of ZNF580 gene. Furthermore, the release of pEGFP-ZNF580 could be sustained at least 25 days due to the controllable degradation ability of the hydrophobic MPs' core. The MPs and MPs/pEGFP-ZNF580 complexes showed low cytotoxicity because of the introduction of PEG chains and low molecular weight PEI on the surface of these MPs. Notably, at the low concentration (20 μg/mL), the MPs and their complexes were non-cytotoxicity. The rapid endothelialization was promoted significantly by the expression of pEGFP-ZNF580.
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