Block Copolymer Micelles As Gene Vectors In Promoting Endothelial Cells’ Proliferation

In this study, a kind of gene vectors based on polyethylenimine(PEI) were prepared to decrease the cytotoxicity and enhance the transfection efficiency of endothelial cells(ECs) in vitro. ZNF580 gene plasmid(plasmid fused to green fluorescence protein, p EGFP-ZNF580), which has the ability of enhancing the proliferation of vascular endothelial cells, was condensed with these vectors and transfected into EA. hy926 cells in order to enhance the proliferation and migration of ECs. The main results are summarized as follows.1. Complex micelles were prepared as low toxic gene vectors by self-assembly of two block copolymers in aqueous solution. The complex micelles consisted of a biodegradable poly(lactide-co-glycolide)(PLGA) core and a mixed methoxy-poly(ethylene glycol)(m PEG)/PEI shell. The ZNF580 gene plasmid, which has the ability of enhancing the proliferation of vascular endothelial cells(ECs), was encapsulated into these complex micelles. Using dynamic light scattering(DLS), the degradation behavior of the micelles was investigated in vitro. The hydrodynamic size and zeta potential of complex micelles and micelles/p DNA complexes were feasible to cellular uptake. MTT assay showed that the cytotoxicity of the complex micelles was very low when m PEG-b-PLGA/PEI-b-PLGA-b-PEI was 3/1. The micelles/p DNA complexes were found to be able to enhance the proliferation of ECs.2. In order to adjust the cytotoxicity and the transfection efficiencies of nonvrial vectors, complex micelles with a biodegradable poly(lactide-co-3(S)-methyl-morpholine-2,5-dione)(PLMD) core and a mixed methoxy-poly(ethylene glycol)(m PEG)/PEI shell were prepared by co-assembling of PEI-b-PLMD-b-PEI and m PEG-b-PLMD in aqueous solution. Then ZNF580 gene plasmid(p EGFP-ZNF580), which has the ability of enhancing the transfection of vascular ECs, was encapsulated into the complex micelles. The hydrodynamic size and zeta potential of complex micelles and micelles/p DNA complexes were feasible to cellular uptake and gene transfection. As expected, the transfection efficiency and cytotoxicity for ECs of these micelles/p DNA complexes could be tuned depending on changing the mass ratio of of m PEG to PEI in mixed m PEG/PEI shell. Moreover, the trend of ZNF580 protein expression of western blot analysis was consistent with the result of transfection efficiency. The results of wound healing assay showed that the migration capacity of cells treated by micelles/p DNA complexes increased. These results indicated that the co-assembled complex micelles could be a suitable gene vector with tunable gene transfection efficiency and cytotoxicity.3. In order to achieve efficient endocytosis and enhance transfection efficiency of gene vectors, micelles with ECs targeting peptides were prepared. Cys-Arg-Glu-Asp-Val-Trp(CREDVW) peptides were linked to star-shaped copolymer(PLMD-b-PEI)6 through PEG linker Maleimide-PEG3500-N-hydroxysuccinimide. Then micelles with a biodegradable PLMD core and PEI shell were prepared by assembling of(PLMD-b-PEI-b-PEG-CREDVW)6 in aqueous solution. CREDVW peptide could enable micelles with special recognition for ECs. ZNF580 gene plasmid, which has the ability of enhancing the transfection of ECs, was encapsulated into the micelles. MTT assay showed that the cytotoxicity of these targeting gene vectors was very low. In vitro transfection experiments and Western blot assay demonstrated enhanced transfection efficiency for EA.hy926 cells of these gene vectors, and the rapid migration of transfected ECs can be verified by wound healing assay. After 24 h, the migration area percentage of cells of two micelles/p DNA complexes linking peptide CREDVW reached 76.2%(PEI/PEG = 1/1) and 78.5%(PEI/PEG = 1/2), respectively.4. Amphiphilic block copolymer PEI-b-PLMD-b-PEI was synthesized and could self-assemble into micelles with the PLMD as the core and PEI as the hydrophilic shell. Low molecular weight PEI(Mw = 600) was cross-linked utilizing cross-linking reagents Dithiobis(succinimidylpropionate)(DSP) onto the surface of micelles. ZNF580 gene plasmid(p EGFP-ZNF580), which has the ability of enhancing the transfection of ECs, was encapsulated into the micelles. These micelles/p DNA complexes had low cytotoxicity and high transfection efficiency for EA.hy926 cells compared with micelles before cross-linking. With increasing the concentration of micelles/p DNA complexes, cell viability of micelles/p DNA complexes decreased from 78.3% to 59.8%(PEI/DSP = 2/1) and from 83.6% to 60.9%(PEI/DSP = 1/1), respectively. Also, Western blot assay and wound healing assay were consistent with the result of transfection efficiency, indicating enhanced ability of proliferation and migration for ECs of these cross-linked micelles.