Study Of Novel Polycationic Gene Vector Which Was Synthesized From Branched PEI 1800 Da And Bisformaldehyd Imidazole/2,6-pyridinedicarbox Aldhyd In Treatment Of Peripheral Ischemic Disease

Peripheral ischemic vascular disease is caused by inadequate blood supplement and will finally result in local tissue necrosis to ischemic site.It’s a chronic disease caused by congenital or acquireddiseasewith a high rate of mortality and morbidity.Traditional medical method and invasive surgery are the most common methods to remit ischemic vascular disease,however,neither of them can’t cure this disease,further more they carries a high risk of side reactions and repeated surgery operations,which will cause pain and huge cost to patients.Nowadays,Gene therapy has become a hot topic in the treatment of peripheral ischemic disease,which can not only compensate for the shortcomings of traditional methods,but also offers a solution to the controlled and specific delivery of genetic materials(DNA and RNA)to targeted cells.Vectors play an important role in gene thrapy.With high transfection efficiency,viral vectors can successfully deliver gene into cells,but some serious adverse events have occurred such as oncogenicity and immunogenicity,etc.Non-viral vectors are preferred for its low toxicity and ability to condense pDNA,but its low transfection efficiency hindered its application in clinical and pre-clinical research.Looking for high efficiency,low-toxicity vector has become a key factor in gene delivery.In this study,our laboratory has designed and synthesized twonovel p H-sensitive cationic biodegradable vectorswithpolythylenimine 1800 Da as the basic unit and safety-known drugmetabolite bisformaldehyd imidazole(IM)and 2,6-pyridinedicarboxaldhyd(PDA),these two novel biodegradable polyethyleneimine derivatives were named as IMPEI and PDAPEI.Based on these two polymers(IMPEI,PDAPEI),we investigated particle size and zeta potential of novel biodegradable polyethyleneimine derivatives-pDNA nanoparticles,and estimated cytotoxicity on HUVEC by CCK-8.Using plasmid with VEGFA sequence and GFP sequence,we also checked transfection efficiency of these two polymers.We successfully established peripheral ischemia animal model on C57/BL6 J mice to evaluate the therapeutic effect of IMPEI/PDAPEI-pDNA complex system on ischemic disease with plasmid with VEGFA sequence.Results:(1)Agarose gel electrophoresis indicated that these two polymers(IMPEI、PDAPEI)can successfully condense the entire pDNA at the w/w ratio of 0.2:1 or above.(2)Particle size and zeta potential of polyplexes were 160-220 nm and 20-30 m V respectively,TEM images of polyplexes formed of IMPEI/PDAPEI-pDNA at W/W ratio of 40:1indicated that polyplexes had a spherical shape,which were conductive to cellular uptake and form stable dispersion systems.(3)Cytotoxicity experiment results suggested that viability of HUVEC cells treated with IMPEI/PDAPEI were above 80%,which showed that these two polymers had lower cytotoxicity than commercial transfection agent PEI 25KDa at the same concentration and same polymer to pDNA W/W ratio.(4)Transfection images and FACS analysis of pDNA,PEI 25 KDa and polyplexes formed of IMPEI/PDAPEI-pDNA at various polymer to gene ratios(W/W)indicated that these two polymers had high transfection efficiency in HUVEC cells,and the transfection efficiency increased with the increasing trend of polymer to pDNA W/W ratio.(5)We successfully establishedperipheral ischemia animal model on C57/BL6J mice to evaluate the therapeutic effect of IMPEI/PDAPEI-pDNA complex system on ischemic disease with plasmid with VEGFA sequence.The results were ideal including gene expression of VEGFA by VEGFA concentration in mouse peripheral ischemic model at 7 and 14 days after administration and double immunofluorescent staining for CD31 and Brd U.As a result,VEGFA gene therapy via non-virus vector of cationic biodegradable carriers(IMPEI and PDAPEI)shows potential utility in treating ischemic disease.