Preparation And Characterization Of Charge-reversal Gene Delivery System Based On POSS And Functional Peptide

Due to the lack of the intact layer of endothelium,small-diameter vascular grafts often cause serious problems,such as thrombosis and intimal hyperplasia.Currently,gene therapy is considered as a fascinating strategy for relieving or solving these problems via accelerating the endothelialization of biomaterials.However,the development of this technology has been greatly restricted by lacking safe and efficient gene delivery systems.In this thesis,a type of p DNA delivery system with excellent biocompatibility and transfection efficiency was constructed.This highly efficient p DNA intracellular delivery could significantly improve the proliferation and migration of endothelial cells（ECs）and further induce angiogenesis in vivo.Besides,we also prepared a corresponding si RNA delivery system to transfect smooth muscle cells（SMCs）for inhibiting the ERK2 pathway,which could inhibit the proliferation and migration of SMCs.1.Preparation of multifunctional p DNA delivery system with charge-reversal moiety and progressively targeting peptide.Polyhedral oligomeric silsesquioxane was attractive on account of its stability,high biocompatibility and easy for modification.So,star-shaped cationic polymer was synthesized by grafting the integrated peptide TAT-G-NLS-GC with both cell-penetrating and nucleus localization function onto the eight arms of POSS.Afterward,the obtained star-shaped cationic polymer sequentially self-assembled with p EGFP-ZNF580（p ZNF580）and multifunctional anionic polymer to fabricate final p DNA delivery system through electrostatic interaction.The anionic polymer possessed charge-reversal property and ECs-targeting function.The zeta potential of this system was neutral,which could prolong blood circulation time in vivo.In addition,the outerlayer of the delivery system could target for ECs and then hydrolyze in acidic condition,which remarkably enhanced the cellular uptake and the subsequent endosomal escape processes.Therefore,highly efficient p DNA delivery was achieved.Experimental results confirmed that our designed system could accelerate the proliferation and migration of ECs and promote angiogenesis in vitro and in vivo.Therefore,we believed that this superior p DNA delivery system provided a potential platform for the therapy of cardiovascular diseases.2.Preparation of si RNA delivery system with charge-reversal function for preventing intimal hyperplasia via ERK2 silencing.Encouraged by the above advanced charge-reversal design,we grafted oligoarginine W-G-R₈-G-C onto the arms of POSS to synthesis star-shaped cationic polymer POSS-（C-G-R₈-G-W）₁₆.Then,cationic POSS-（C-G-R₈-G-W）₁₆ bound negatively charged ERK2-si RNA through electrostatic interaction to obtain stable binary si RNA complexes.The complexes exhibited enhanced capability of loading ERK2-si RNA and crossing the plasma membrane.Finally,cationic binary si RNA complexes were electrostatically coated by acid-labile anionic polymer polylysine-cis-acotonic anhydride（PLL-CA）to obtain the ternary ERK2-si RNA delivery system with core-shell structure.PLL-CA was synthesized by the amidation reaction between cis-acotonic anhydride（CA）and commercial polylysine（PLL）.Biocompatibility assay revealed that this system possessed well hemocompatibility and low cytotoxicity.Besides,cellular uptake and intracellular trafficking assay confirmed its outstanding performance in endosomal escape,which contributed to high ERK2-silencing efficiency.Furthermore,the inhibition of SMCs proliferation and migration was also observed in transwell assay.These above results could prove the superiority of the ternary delivery system.Importantly,it also provided a new technologic strategy for gene delivery.