| Acute myocardial infarction(AMI)is a common clinical acute cardiovascular disease with a high fatality rate.It is the most important treatment strategies to restore blood flow timely through thrombolysis or percutaneous coronary intervention.However,the recovery of coronary blood flow will induce myocardial ischemia/reperfusion injury(MI/RI),and the therapeutic effect of reperfusion is subsequently reduced.Cyclosporine A(CsA)is an immunosuppressant.It has been found that CsA can play a role in the treatment of myocardial ischemia reperfusion injury by inhibiting the opening of mitochondrial permeability transition pore(m PTP).But CsA displays poor solubility and is difficult to be effectively distributed in ischemic myocardial tissue.The glycoproteins on the platelet membrane can specifically bind to von willebrand factor(v WF)which is highly expressed on the surface of injured blood vessels in ischemic myocardium.Thus,platelet membrane-wrapped nanoparticles can target to ischemic myocardium.Besides,the level of reactive oxygen species(ROS)in ischemic/reperfusion myocardial tissue is higher than that in other normal tissues.So,when CsA is encapsulated by ROS-cleavable material,CsA can be specifically released in ischemic myocardial tissue.Objective:A ischemic myocardial targeting core-shell structured nanoparticle(CsA@PPTK)was prepared by using platelet membrane as a shell and ROS-cleavable poly(5,5-dimethyl-4,6-dithio-propanediol azelate)(PTK)as a core to encapsulate CsA.CsA@PPTK can release CsA to the ischemic myocardial tissue and to improve the therapeutic effect of CsA on MI/RI.Methods:1.The 5,5-dimethyl-4,6-dithio-azelaic acid(TK)was synthesized from acetone and3-mercaptopropionic acid under the catalysis of trifluoroacetic acid.The poly(5,5-dimethyl-4,6-dithio-propylene glycol azelate)(PTK)was synthesized by using TK and 1,3-propanediol at high temperature and vacuum under the catalysis of scandium trifluoromethanesulfonate.2.Nanoparticle core CsA@PTK was prepared by emulsification solvent evaporation method,and the formulation to prepare CsA@PTK was optimized.The water bath ultrasound and co-extrusion method was used to prepare platelet membrane-coated nanoparticles CsA@PPTK.The CsA@PPTK was characterized by TEM,HPLC,laser particle size analyzer,SDS-PAGE and CLSM.3.The ROS response characteristics of CsA@PPTK was studied by HPLC.4.The effect of CsA@PPTK on the stability of erythrocyte membrane was observed through erythrocyte hemolysis experiment.5.The effects of blank nanoparticles(@PTK),free CsA,CsA@PTK and CsA@PPTK on the viability of normal H9c2 cell and hypoxia and reoxygenation(H/R)injured H9c2 cell was investigated by MTT method.6.The effect of CsA@PPTK on the m PTP opening in H/R injured H9c2 cell was studied by flow cytometry.The effect of CsA@PPTK on the mitochondrial membrane potential in H/R injured H9c2 cell was observed by fluorescence spectrophotometer.The effect of CsA@PPTK on ROS in H/R injured H9c2 cell and in mitochondria of H/R injured H9c2 cell was observed by CLSM.The uptake of CsA@PPTK by normal H9c2cell and H/R injured H9c2 cell was studied by HPLC.7.The specific binding of Cy7.5@PPTK with endothelium injured aortic vessels was observed by fluorescence microscopy,and the distribution of coumarin 6@PPTK in MI/RI mice was studied by using an in vivo imager.8.Myocardial ischemia reperfusion injury(MI/RI)model in mice was established.The effect of CsA@PPTK on the left ventricular ejection fraction(LVEF)and left ventricular fraction(FS)of MI/RI mice was studied by using VEVO 2100 ultrasound system.The effect of CsA@PPTK on the myocardial fibrosis in MI/RI mice was observed by Masson staining of mouse heart sections.The effect of CsA@PPTK on morphology of the myocardial tissue in MI/RI mice was studied by H&E staining.The effect of CsA@PPTK on the expression of connexin-43(Cx43)and matrix metalloproteinase-9(MMP-9)in the myocardial tissue in MI/RI mice was investigated by immunofluorescence staining of mice heart sections.Results:1.The synthesized TK and PTK were confirmed as the target products by using~1HNMR and MS analysis.2.The drug loading of CsA@PPTK was 4.97%.The average particle size and average zeta potential of CsA@PPTK was 170 nm and-17.6 m V,respectively.3.With the increase of sodium hypochlorite concentration,the release rate of CsA from CSA@PTK and CSA@PPTK was significantly accelerated.This indicated that the release of CsA from CSA@PTK and CSA@PPTK was ROS sensitive.In different concentration of sodium hypochlorite,the release rate of CsA from CsA@PPTK was almost the same as CsA@PTK,indicating that the platelet membrane coated on the surface of the nanoparticles did not affect the ROS triggered CsA release from CsA@PPTK.4.CsA@PPTK did not cause hemolysis of red blood cells.5.@PTK increased the viability of normal H9c2 cell and H/R injured H9c2 cell at concentrations of 0.5 mg/m L and 1 mg/m L.Free CsA,CsA@PTK and CsA@PPTK increased the viability of H/R injured H9c2 cell.When the concentration of CsA was 15μg/m L,the viability of H/R injured H9c2 cell in CsA@PTK and CsA@PPTK treated group was the same as that in free CsA treated group.When the concentration of CsA was30μg/m L,the viability of H/R injured H9c2 cell in CsA@PTK and CsA@PPTK treated group was greater than that in free CsA treated group.6.Free CsA,CsA@PTK and CsA@PPTK significantly reduced the opening of m PTP in H/R injured H9c2 cell.Free CsA,CsA@PTK and CsA@PPTK restored the mitochondrial membrane potential of H/R injured H9c2 cell.Besides,@PPTK,CsA@PTK and CsA@PPTK obviously reduced the level of ROS in H/R injured H9c2 cell,and CsA@PPTK scavenged much more ROS in H/R injured H9c2 cell than@PPTK and CsA@PTK.Free CsA,@PPTK,CsA@PTK and CsA@PPTK also significantly reduced mitochondrial ROS in H/R injured H9c2 cells,and CsA@PPTK scavenged much more mitochondrial ROS in H/R injured H9c2 cell than CsA@PTK.7.The uptake of CsA@PTK and CsA@PPTK by H/R injured H9c2 cell was higher than that by normal H9c2 cells.Compared with CsA@PTK,much more CsA@PPTK was uptaken by H/R injured H9c2 cell.8.CsA@PPTK could specifically bind with endothelium injured vascular.CsA@PPTK could target to ischemic myocardium of MI/RI mice.Free CsA,@PPTK,CsA@PTK and CsA@PPTK increased the LVEF and FS of MI/RI mice at 28th days after drug injection,and the LVEF and FS in CsA@PPTK(2.5 mg/kg)treated group was higher than that in free CsA and CsA@PTK treated group.In addition,Free CsA(2.5 mg/kg)and CsA@PPTK(1 mg/kg,2.5 mg/kg)also increased LVEF and FS of MI/RI mice at 70th days after drug administration,and the LVEF and FS in CsA@PPTK(2.5 mg/kg)treated group was higher than that in free CsA treated group at 70th days after drug administration.Furthermore,CsA@PPTK markedly reduced the expression of MMP-9 and increased the expression of Cx43 in the ischemic myocardium.CsA@PPTK also reduced the fibrosis of the ischemic myocardium.Conclusions:CsA@PPTK exhibited ROS triggered drug release characteristic,and CsA@PPTK could target to ischemic myocardium of MI/RI mice.CsA@PPTK markedly reduced the expression of MMP-9 and fibrosis of the ischemic myocardium,and increased the expression of Cx43 in the ischemic myocardium.Subsequently,LVEF and FS of MI/RI mice were increased,and the heart injury induced by MI/RI in mice was significantly repaired. |
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