| Gene therapy refers to the introduction of exogenous functional genes into target cells to compensate or correct gene defects and abnormalities,so as to achieve the purpose of treating diseases,which has become a promising biomedical technology for clinical application.However,genetic molecules cannot be used alone due to their sensitivity to the nuclease in serum and low cellular uptake.Therefore,gene vector with excellent performance is the key to the success of gene therapy.Cationic polymers have become one of the hotspots of nucleic acid carrier research due to the advantages of easy binding/loading with nucleic acid and diverse structure composition.Among them,hydroxyl-rich cationic polymers have significant advantages of low cytotoxicity.Meanwhile,it is necessary to further optimize the structural design of hydroxyl-rich cationic carrier materials to achieve high transfection efficiency and multifunctionalization,and explore the therapeutic effectiveness in multi-type disease models.In the chapter 2,a monomer with epoxy groups and disulfide bonds was prepared.Topamycin,one clinical aminoglycoside drug,reacted with the monomer to prepare polyaminoglycoside containing disulfide bond(SS-HPT)through amino-epoxy ring-opening reaction.The hydroxyl-rich structure endowed SS-HPT with high cytocompatibility in cardiomyocytes(H9c2),and disulfide bond also improved the transfection performance of reporter plasmids such as luciferase.For D5 R protein that regulates cardiac function,two functional nucleic acids(Drd5 plasmid,which encodes the protein,and Drd5 si RNA,a small interfering RNA that down-regulates the protein expression)were designed.SS-HPT delivered two kinds of nucleic acids efficiently in cardiomyocytes and regulated the function of cells by regulating D5 R protein expression.In the mouse model of cardiac hypertrophy,SS-HPT could effectively transport/enrich Drd5 plasmid in the heart and increase the content of D5 R protein.In chapter 3 of this paper,tumor targeted ligand phenyl boric acid(PBA)was introduced into SS-HPT structure by the ring-opening reaction between epoxy group and aminobenzenoborate acid to prepare SS-HPT containing PBA groups(SS-HPT-P).The surface of lung tumor cells(A549)overexpressed sialic acid,and PBA ligand endowed SS-HPT-P with higher phagocytosis efficiency in A549 cells,as well as higher transfection efficiency of reporter and therapeutic plasmids.SS-HPT-P delivered the p Cas9-surv(one CRISPR-Cas9 plasmid that targets and knockouts the survivin gene),which exhibited gene editing performance in A549 cell line and reduced survivin protein content,thereby inhibiting tumor cell migration and proliferation.SS-HPT-P/p Cas9-surv showed obvious advantages in the accumulation rate and amount at tumor site,and could effectively inhibit tumor by downregulation of survivin protein.In Chapter 4,topamycin reacted with epoxide monomer to prepare polyaminoglycoside(HPT).The photosensitizer(Rose Bengal,RB)and bacterium/cell-affinity PBA conjugated onto HPT to prepare HPT-RP,and HPT-RP self-assembled in water to form nanoparticles.HPT-RP realized high anti-bacterial ability owing to the receptor-mediated photodynamic therapy.The nanoparticle morphology and PBA conjugation endowed HPT-RP with high gene transfection.In a rat infected-skin-defect model,HPT-RP exhibited the remarkable performance of anti-infective/epidermal growth factor plasmid(p EGF)-delivery and accelerated healing.In order to meet the clinical transformation needs of nucleic acid transport system,a carrier material with simple preparation method and easy to optimize the large-scale preparation process was developed.In Chapter 5,based on the previous research on three kinds of polyaminoglycoside carriers with different structures/functions for three kinds of diseases,the quaternary ammonium chitosan(QCS)was prepared by the amine-epoxy ring-opening reaction of 2,3-glycidyl trimethyl ammonium chloride(GTA)and chitosan hydrochloride.The quaternary ammonium groups introduced by the amine-epoxy ring-opening reaction could improve the performance of QCS condensation with nucleic acid.Some primary amine groups of chitosan were modified into secondary amine groups,so that QCS owned good proton buffer/lysosomal escape capacity.Compared with polyethylene imine(PEI,the "international gold standard"),QCS had similar transfection performance,but lower cytotoxicity and hemolysis ratio.In mouse tumor model,QCS could effectively inhibit tumor by delivering large dose of p53 gene intravenously,and the performance was significantly better than PEI which could only deliver low dose of p53.In the rat skin defect model,QCS effectively transported EGF and accelerated skin wound healing.In summary,a series of hydroxyl-rich cationic polymer carriers were designed through the amino-epoxy ring-opening reaction of a variety of molecules containing epoxy groups and polyamine components(amino glycoside/polysaccharide),to meet the needs of nucleic acid delivery for a variety of diseases.The high performance of the four hydroxyl-rich cationic polymer carriers was verified by in vitro experiments.And the biosafety and effectiveness of the four gene delivery systems were verified by in vivo experiments in animal models of different diseases. |
