| The incidence of lower limb ischemia increased gradually,seriously endangering human health.Our previous studies have confirmed gene-based therapies in patients with critical limb ischemia did not gain ideal long-term outcome,which might be single proangiogenic function of past gene therapy and compromised gene activity and duration caused by complex ischemic micro-environment.Cytochrome P450 epoxygenase-2J2(CYP2J2)and its downstream metabolite epoxyeicosatrienoic acids(EETs)have been demonstrated to possess various biological functions,including promotion of angiogenesis,anti-inflammatory effect,and inhibition of endothelial cell apoptosis.We speculate that gene therapy with CYP2J2 can multi-functionally and effectively stimulates angiogenesis in critical limb ischemia.We have designed and synthesized ROS-responsive nanospheres 3s-PLGA-po-PEG as a gene delivery vector.On this basis,3s-PLGA-po-PEG encapsulating CYP2J2 gene will be prepared to exert various biological functions of CYP2J2-EETs,and for targeted delivery,improved micro-environment and prolonged effect time of CYP2J2 gene by virtue of ROS-responsiveness and sustained-release capacity of 3s-PLGA-po-PEG.The purpose is elucidating that this nano-gene delivery system can play multiple roles of"functional diversity,improvement of ischemic microenvironment,and nano-long circulation",it provides a new target and strategy for gene treatment of ischemic diseases of the lower extremities.Part Ⅰ:We innovatively synthesized a novel three-arm star block copolymer(3s-PLGA-po-PEG)with peroxalate ester(PO)as an H2O2-responsive linkage through the esterification reaction of oxalyl chloride and hydroxyl groups,and we designed a CYP2J2 DNA delivery system using 3s-PLGA-po-PEG nanoparticles as a gene vector.Our results demonstrated good stability and slow-release ability of nanoparticle/pDNA complexes(NP/pDNA).Intracellular ROS scavenging ability of nanoparticle/pDNA complexes was tested utilizing ROS probe DCFH-DA,and our findings indicated the good reactive oxygen response of 3s-PLGA-po-PEG nanoparticles.The cytotoxicity assay demonstrated that nanoparticle/pDNA complexes showed no signs of cytotoxicity and could significantly stimulate the proliferation of HAECs.In addition,Visualization of cellular uptake and gene transfection by laser confocal microscopy also validated the good lysosome escape ability and high gene efficiency of NP/pDNA in vitro.Part Ⅱ:We verified the pro-angiogenesis effect of nanoparticle/pDNA complexes through in vitro and in vitro experiments.The results of in vitro study showed that nanoparticle/pDNA complexes effectively promoted HAECs migration and tube formation.The murine hind-limb ischemia model was used to evaluate the pro-angiogenic ability of NP/pDNA.Our finding revealed that 3s-PLGA-po-PEG nanoparticles prolonged the drug retention time in lower extremity muscles,and the results of in vivo study confirmed the high gene delivery and expression efficiency of nanoparticle/pDNA complexes in vivo.In brief,nanoparticle/pDNA complexes exhibited improved therapeutic outcomes by significantly increasing hindlimb blood reperfusion and resulted in rapid limb salvage in the mice tested.Additionally,NP/pDNA could significantly improve the pathological microenvironment of high oxidative stress and inflammatory infiltration in the ischemic gastrocnemius muscle.Part Ⅲ:We performed a preliminary investigation into the specific molecular mechanism of CYP2J2-EETs to promote angiogenesis.The results revealed that reductions in the PHD2 levels allow persistence of HIF-1α,with resulting increases in HIF-1-mediated VEGF expression,which is a major mechanism of CYP2J2/EETs induced angiogenesis.In addition,with strong H2O2 scavenging activity of 3s-PLGA-po-PEG,blank nanoparticles and nanoparticle/pDNA complexes could reverse the reduction in HIF-1α accumulation by modulating PHD2 activity. |
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