Peptide Modification And Gene Engineering For Improving Hemocompatibility And Endothelialization Of Vascular Materials

The continuous development of artificial vascular materials is of great significance for the treatment of cardiovascular diseases.However,small-caliber artificial vascular grafts（<6 mm）and coronary stents still encounter worldwide problems such as thrombosis,intimal hyperplasia,restenosis,and even occlusion in clinical use.An effective solution is to improve the surface hemocompatibility and accelerate endothelialization after graft implantation.To this end,using surface modification and gene engineering technologies as the basic starting point,we designed and constructed multifunctional artificial vascular grafts surface,and then evaluate their endothelialization ability by in vitro and in vivo experiments.In this thesis,surface modification with biofunctional molecules,gene transfection to promote vascular endothelial cells（ECs）growth,and enzyme-responsive technologies were utilized to improve the hemocompatibility of biomaterials and promote ECs adhesion,migration,and proliferation on materials surface.Thus,the rapid endothelization and vascularization ability could be improved,and finally,the long-term patency of small-caliber artificial vascular grafts and stents is expected to achieve.1.We explored the possibility of immobilized bioactive peptides being used to improve the anticoagulation and anti-hyperplasia of biomaterial surface.The anticoagulant peptides(ACH₁₁)with sequence LTFPRIVFVLG were immobilized onto stent surface using hydrophilic polyethylene glycol（PEG）as the linker.The results showed that PEG-ACH₁₁ grafted surface could not only exhibit anti-FXa activation,anti-fibrinogen（Fg）degeneration,and anti-platelet adhesion/aggregation,but also effectively prevent granulation formation and fibrous encapsulation after in vivo subcutaneous implantation.This ensures good hemocompatibility and histocompatibility of the vascular stent after in vivo implantation.2.Anticoagulant peptide ACH₁₁ and EC-targeting adhesive peptide（Cys-Ala-Gly,CAG）were co-immobilized onto small-caliber artificial vascular grafts to improve the surface hemocompatibility and endothelialization ability.The artificial vascular grafts（PLCL/Gt）were fabricated by blend-electrospinning of poly（L-lactic acid-co-ε-caprolactone）（PLCL）and gelatin（Gt）.The results showed that the co-modified artificial vascular grafts not only possessed good hemocompatibility,but also effectively promoted EC-selective adhesion and proliferation on the material surface,while inhibited the SMC adhesion.Besides,the modified artificial vascular grafts confirmed high patency and rapid endothelialization after 6 weeks of rabbit carotid artery implantation.This surface modification strategy provides new ideas for in situ rapid healing of vascular endothelium of artificial vascular grafts and stents.3.An agmatine-grafted bioreducible polycation poly（disulfide-L-lysine）-g-agmatine（denoted as SSL-AG）,was designed and utilized to transfer ZNF580 plasmid into vascular ECs.The guanidine moiety has attractive cellular membrane-penetration and nuclear-localization capacity,and the disulfide bond rapidly degrades in the cytoplasmic reduction environment.The resulting complexes（SSL-AG/p ZNF580）not only possessed reductive degradation and low cytotoxicity,but also exhibited high cellular uptake,rapid endosome/lysosome escape,and high-efficiency nucleus accumulation ability.Also,the gene complexes could effectively improve the transfection efficiency in ECs,and thereby promote the proliferation,migration,and vascularization of ECs.This provides an effective strategy for gene transfection technology in promoting the proliferation of vascular ECs and rapid endothelialization of biomaterials surface.4.A matrix-metalloproteinase（MMP）-responsive EC-selective gene delivery surface was constructed for in situ smart release of genes from the biomaterial surface under EC-microenvironment.An enzyme-cleavable peptide（MCP）linker was firstly designed and prepared,which contained the MMP-susceptible sequence,the N terminal-modified biotin to link the gene complexes,and C terminal-modified cysteine for the fixation on the material surface.The scaffolds material selected commonly used polycarbonate urethane（PCU）.The biotin-modified ZNF580 gene complexes（NPs）were subsequently anchored to the PCU surface modified by MCP through the action of biotin-avidin,and the MMP-responsive gene delivery surface was constructed.Further,the glycine（G）sequences with different repeat numbers（Gm,m=0,4,and 8）were integrated with MCP peptides as a flexible spacer arm for reducing the steric hindrance of the surface,and further exerting enzymatic degradation function well.The experimental results confirmed the MMP-responsive EC-selective gene delivery surface（PCU-REDV/MCP（Gm）@NPs）achieved the smart responsive release of gene complexes and subsequent efficient cellular uptake by ECs.Finally,ECs were effectively transfected on this surface and achieved high expression of specific protein and m RNA,leading to enhanced migration/proliferation and neovascularization on the material surface.In conclusion,surface modification with biofunctional molecules,vascular ECs transfection,and enzyme-responsive technologies were utilized to design and construct multifunctional artificial vascular grafts surface,and then evaluate their endothelialization ability by in vitro and in vivo experiments.The results showed that anticoagulant peptide immobilized using hydrophilic polymer and EC-targeting adhesive peptide significantly improved the hemocompatibility and surface endothelialization of vascular materials;Using enzyme-responsive peptide as a linker to temporarily immobilize gene complexes onto materials surface successfully achieved the smart responsive release of gene complexes and subsequent efficient gene transfection by ECs,finally leading to rapid endothelialization.