Preparation Of Microparticles For Gene Carriers And Their Preliminary Application In Promoting Endothelial Cells’ Proliferation

In order to promote the proliferation and migration of human umbilical veinendothelial cells (HUVECs), biodegradable microparticles (MPs) were prepared in thethis dissertation. We employed ZNF580gene complexed with MPs to promote theproliferation of HUVECs.1. Amphiphilic triblock copolymers, methoxy-poly(ethylene glycol)-block-poly(3(S)-methyl-morpholine-2,5-dione-co-glycolide)-graft-polyethyleneimine(mPEG-b-P(MMD-co-GA)-g-PEI) were synthesized. MPs were obtained via self-assembly of these copolymers. The hydrophobic core composed of P(MMD-co-GA)segments provide crosslinking points for numbers of PEG and short PEI chains to forma high hydrophilic and positive charged corona/shell structure.Using these MPs, potential genes (ZNF580) for rapid endothelialization wereefficiently transported into HUVECs. Because of the introduction of hydrophilic PEGchains and low molecular weight PEI in the triblock copolymers, the cytotoxicity ofthese MPs and their complexes with pEGFP-ZNF580was decreased significantly. Thetransfection efficacy of MPs/pEGFP-ZNF580complexes was as high asLipofectamineTM2000reagent in vitro. The proliferation and migration of HUVECswere promoted greatly by the expression of pEGFP-ZNF580after60hours.2. In order to realize the controlled release of ZNF580gene from MPs/genecomplexes, 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(ethyleneglycol)-block-poly(3(S)-methyl-morpholine-2,5-dione-co-lactide)-graft-polyethyleneimine (mPEG-b-P(MMD-co-LA)-g-PEI) and methoxy-poly(ethyleneglycol)-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 cell transfection result demonstrated that pEGFP-ZNF580could betransported efficiently into HUVECs by these complexes. The result of western blotshowed that the relative ZNF580protein level can increase to35.74%-46.11%by theoverexpression of ZNF580gene. Furthermore, the release of pEGFP-ZNF580could be sustained at least25days due to the controllable degradation ability of the hydrophobicMPs’core.3. To realize the specific adhesion, two kinds of HUVECs targeted MPs wereprepared. Triblock copolymers of REDV-graft-polyethyleneimine-graft-poly(lactide-co-glycolide)-graft-polyethyleneimine-graft-REDV (REDV-g-PEI-g-P(LA-co-GA)-g-PEI-g-REDV) and REDV-graft-polyethyleneimine-graft-poly(lactide-co-glycolide-co-methyl-2,5-morpholinedione)-graft-polyethyleneimine-graft-REDV (REDV-g-PEI-g-P(LA-co-GA-co-MMD)-g-PEI-g-REDV) were synthesized firstly. After self-assembly,targeted MPs with hydrophobic core of P(LA-co-GA) or P(LA-co-GA-co-MMD)copolymers and hydrophilic PEI cationic shell were formed.Their functions were investigated in vitro. The results indicated that the release ofplasmids can be controlled by N/P molar ratios and the degradation of copolymers, andcould be maintained at least for28days in the present experiment condition. Moreover,at low concentration (40μg/mL), the MPs and their complexes not only exhibited non-cytotoxicity, but also promoted the cells proliferation. Furthermore, HUVECs andHUASMCs were co-cultured in the medium. Then the HUVECs targeted MPs/pEGFP-ZNF580complexes were added into the co-culture system. After24hours, theproliferation of HUVECs were found significantly, the proliferation of HUASMCswere inhibited obviously.