Lipid-Coated Gold Nanoparticles For Targeting Delivery The Gene Expression Plasmids To Repair Cell Junction In Vascular Endothelial Cells

Vascular endothelial cell junctions are junctions between adjacent vascular endothelial cell membranes.Vascular endothelial cell junctions and endothelial cells regulate the flow of substances into and out of blood vessels and maintain the barrier function of blood vessels.Damage to vascular endothelial cell junctions will lead to loss of vascular barrier function,which will cause a large amount of water and macromolecular proteins to enter tissues and cause tissue edema and even organ failure,which seriously threatens human life and health.The adhesion junction and tight junction between vascular endothelial cells are the key structures of vascular endothelial cell junction,and the two junctions work together to regulate the entry and exit of substances.The key proteins of adhesion junction and tight junction are VE-Cadherin and Claudin-5,respectively.The two proteins interconnect between adjacent vascular endothelial cells,closing the gap between vascular endothelial cells.When inflammation breaks out in the body,pro-inflammatory factors and chemokines will reduce the expression of VE-Cadherin and Claudin-5,destroy the structure of VE-Cadherin and Claudin-5,and then lead to the destruction of vascular endothelial cell junctions,resulting in increased vascular permeability.Studies have shown that increasing the expression of VE-Cadherin and Claudin-5 can effectively reduce vascular permeability and reduce the damage of inflammation to blood vessels.Gene therapy is a method to treat certain diseases by silencing disease-causing genes,increasing the expression of therapeutic genes,or correcting the diseasing-causing genes by introducing target genes into target cells through gene carriers.Therefore,directly up-regulating the expression of VE-Cadherin or Claudin-5 through gene therapy is a potential new strategy to repair vascular endothelial cell junction damage.In this thesis,by using the ability of gene therapy to precisely regulate the expression of target proteins at the gene level,and combining with the advantages of two non-viral vectors,gene therapy approaches based on cationic liposomes and AuNPs were designed and developed for the treatment of vascular endothelial cell junction damaged.The main body of this thesis is divided into three parts:1.Lipid-coated gold nanoparticles were designed to deliver the VE-Cadherin overexpression plasmids into vascular endothelial cells to increase the expression of the VE-Cadherin.AuNPs combined with cationic liposomes can not only improve delivery efficiency but also protect the VE-Cadherin overexpression plasmids from nuclease degradation.Modification of Ang-1 mimic peptide HHHRHSF on the liposome surface gives the complex the ability to target vascular endothelial cells.By Western blot assay,it is proved that the complex HLAV successfully transported the VE-Cadherin overexpression plasmids into vascular endothelial cells and increase the expression level of the VE-Cadherin protein.Transwell experiments demonstrated that complex HLAV could reduce vascular permeability.2.Lipid-coated gold nanoparticles were designed to deliver Claudin-5 overexpression plasmids targeting vascular endothelial cells,which could increase the expression of Claudin-5 and reconstruct the tight junctions between vascular endothelial cells.REDV peptide,which was binding to integrin α4β1 on endothelial cells,was modified laterally on the liposome and made the liposome target endothelial cells as well.By Western blot assay,it is demonstrated that complex RLAC can deliver Claudin-5 overexpressed plasmid into cells and increase the expression level of the Claudin-5 protein.Transwell results demonstrated that complex RLAC can reduce vascular permeability and repair tight junction damage.3.Lipid-coated gold nanoparticles were designed to deliver Claudin-5 overexpression plasmids targeting vascular endothelial cells,which could increase the expression of Claudin-5 and repair the tight junctions between vascular endothelial cells.Ang-1 mimic peptide HHHRHSF was modified in liposome surfaces,and the liposome complex was given the ability to target vascular endothelial cells by its specific binding to the Tie-2 receptor on the vascular endothelial cell membrane.The experimental results show that the HLAC complex has good stability and a high encapsulation rate.In this thesis,three complexes HLAV,RLAC,and HLAC were designed and synthesized to deliver the VE-Cadherin overexpression plasmids or Claudin-5 overexpression plasmids into vascular endothelial cells for expression.VE-Cadherin overexpression plasmids and Claudin-5overexpression plasmids in the endothelial cells significantly increased the expression level of VE-Cadherin protein or Claudin-5 protein.The vascular permeability was reduced after using HLAV and RLAC,which effectively repaired vascular endothelial cell junction injury.