| After acute myocardial infarction,thrombolysis or percutaneous coronary intervention for early recovery of myocardial perfusion are the most effective ways to reduce myocardial infarct size and improve clinical prognosis.However,the reperfusion of ischemic myocardium can cause myocardial ischemia reperfusion injury?MI/RI?,which reduces the effect of myocardial reperfusion.In recent years,cyclosporine A?CsA?,an immunosuppressive agents used in organ transplantation,was found to be able to effectively reduce the injury of reperfusion.The study indicated that CsA inhibited the opening of mitochondrial permeability transition pore?mPTP?.However,the systemic immunosuppressive effect of CsA limited its appliaction in prevention and treatment of MI/RI.Therefore,how to deliver CsA to the mitochondria of ischemic cardiomyocytes is the key point to improve the preventive and therapeutic effect of CsA on MI/RI.The enhanced permeability and retention?EPR?effect in ischemic myocardium is the same as that in tumor tissues.Thus,the nanoparticles loaded with CsA can target the myocardial tissue of ischemia reperfusion through the EPR effect.In addition,the affinity of polypeptide SS31 to the mitochondrial membrane is 5000 times more than that to other organelle membrane.Besides,the accumulation of SS31 in mitochondria does not depend on the mitochondrial transmembrane potential.Thus,SS31 is an ideal targeting ligand for mitochondria-targeted drug delivery system.Objective:Byusing polypeptide SS31 as mitochondriatargeting ligand and poly?lactic-co-glycolic acid??PLGA?as drug carrier,a mitochondria-targeted PLGA nanoparticles was prepared to specifically deliver CsA to mitochondria of ischemic myocardium to enhance the protective and therapeutic effect of CsA on MI/RI.Methods:1.CsA loaded PLGA nanoparticles modified with SS31?CsA@PLGA-PEG-SS31?were prepared by nanoprecipitation method.The prescription composition and preparation process of CsA@PLGA-PEG-SS31 were optimized.The nanoparticle was characterized by using high performance liquid chromatography?HPLC?,laser particle size analyzer and transmission electron microscope.2.HPLC was used to study the release profile of CsA@PLGA-PEG-SS31 in vitro.3.Erythrocyte hemolysis experiment was used to observe the effect of blank nanoparticle@PLGA-PEG and@PLGA-PEG-SS31 on the stability of erythrocyte membrane.4.The cytotoxicity of CsA,@PLGA-PEG,@PLGA-PEG-SS31 and CsA@PLGA-PEG-SS31 on H9c2 cardiomyocyte was studied by MTT method.5.The hypoxia reoxygenation injury model in H9c2 cardiomyocytes was optimized.The effects of CsA@PLGA-PEG-SS31 on the viability of H/R injury H9c2 cardiomyocyte were studied by using MTT assay and LDH Kit.The uptake of CsA@PLGA-PEG-SS31on H/R injured H9c2 cardiomyocyte was studied by using HPLC and confocal laser scanningmicroscope?CLSM?.Thedrugdistributiondeliveredby CsA@PLGA-PEG-SS31 on H/R injured H9c2 cardiomyocyte and its effects on mitochondrial reactive oxygen species?ROS?were studied by using CLSM.The effects of CsA@PLGA-PEG-SS31 on ROS and mitochondrial membrane potential of H/R injured H9c2 cardiomyocyte were determined by using fluorescence spectrophotometer.The effect of CsA@PLGA-PEG-SS31 on the opening of mPTP in H/R injured H9c2cardiomyocyte was determined by using flow cytometry.The effect of CsA@PLGA-PEG-SS31 on the activity of Caspase 3 in H/R injured H9c2 cardiomyocyte was determined by using Caspase 3 activity detection kit.6.MI/RI model in rats was established.The effect of CsA@PLGA-PEG-SS31 on myocardial infarction area of ischemia/reperfusion?I/R?rats was observed by using2,3,5-triphenyte-trazoliumchloride?TTC?and evans blue staining.The effects of CsA@PLGA-PEG-SS31 on cardiac function of MI/RI in rats was monitored by Lab chat7.0 biological signal acquisition and processing software.The effects of CsA@PLGA-PEG-SS31 on myocardial enzymatic profile and troponin I in MI/RI rats were also determined by using automatic biochemistry analyzer and enzyme-linked immunosorbent assay,respectively.The effects of CsA@PLGA-PEG-SS31 on histomorphology of cardiac tissue in MI/RI rats were studied by using H&E staining and TUNEL staining.The effect of CsA@PLGA-PEG-SS31 on mitochondrial morphology of myocardial tissue after MI/RI was observed by using transmission electron microscopy.The drug distribution delivered by mitochondria-targeted nanoparticles in various organs of MI/RI rats was observed by using fluorescence microscope.The drug distribution delivered by mitochondria-targeted nanoparticles in MI/RI rats heart was observed by using small animal living imager.Results:1.The optimized prescription composition and preparation process was as follows:water phase was deionized water,the ratio of aqueous to oil phase was 10:4?v/v?,and the ratio of CsA to PLGA-PEG-SS31 was 1:4?g/g?.The drug loading of CsA@PLGA-PEG-SS31 was 3.5%.The particle size of CsA@PLGA-PEG-SS31 was 54nm and the zeta potential was+14.5 mV.2.There was no significant change in particle size of CsA@PLGA-PEG-SS31 in 30days in water.In the release medium contained 40%ethanol,CsA@PLGA-PEG-SS31released 57.2%of the loaded CsA within 3 h and released all the loaded CsA within 2days.3.The hemolysis rate of blank nanoparticle@PLGA-PEG and@PLGA-PEG-SS31to erythrocyte was less than 5%.This indicated that the blank nanoparticles did not cause erythrocyte hemolysis,and nanoparticles were suitable for cell and animal experiment.4.CsA and CsA nanoparticles did not exhibit obvious cytotoxicity on H9c2cardiomyocyte when the dosage of CsA was less than 30?g/L.The survival rate of H9c2cardiomyocyte was 68.8%when hypoxia for 3 h and reoxygenation for 4 h?H3/R4 model?were performed,which met the next step experimental requirement.5.In the H3/R4 model,when the CsA concentration was 15-30?g/mL,CsA@PLGA-PEG-SS31 markedly reduced the release of LDH from H/R injured H9c2cardiomyocyte and showed significant protective effects on H/R injured H9c2cardiomyocyte.The uptaken of CsA@PLGA-PEG-SS31 by H/R injured H9c2cardiomyocyte was mainly through macropinocytosis and reached the maximum platform within 2 h.CsA@PLGA-PEG-SS31 significantly increased the distribution of CsA in mitochondria,and subsequently reduced the production of reactive oxygen species in mitochondria of H/R injured H9c2 cardiomyocyte.CsA@PLGA-PEG-SS31 markedly prevented mitochondrial membrane potential collapse,and obviously reduced the opening of mPTP in H/R injured H9c2 cardiomyocyte.Meanwhile,CsA@PLGA-PEG-SS31significantly lowered the activity of Caspase 3 in H/R injured H9c2 cardiomyocyte.6.The MI/RI model in rat was successfully established by observing the changes of electrocardiogram and left ventricular pressure.CsA@PLGA-PEG-SS31 significantly reduced infarct size after MI/RI.CsA@PLGA-PEG-SS31 promoted heart function recovery through increasing the±dp/dtmax,heart rate and left ventricular end-systolic pressure after MI/RI in rats.CsA@PLGA-PEG-SS31 significantly decreased the concentrations of CK-MB,LDH,AST and troponin I in the rats blood after MI/RI.Besides,CsA@PLGA-PEG-SS31 significantly reduced the swelling and lysis of mitochondria,andattenuatedapoptosisofcardiomyocytesafterMI/RI.CsA@PLGA-PEG-SS31 effectively accumulated in ischemic myocardium in MI/RI rats and delivered drug to ischemic myocardial tissue,subsequently reduced the accumulation of CsA in other organs and tissues.Conclusion:ThesmallparticlesizeCsA@PLGA-PEG-SS31wasprepared.CsA@PLGA-PEG-SS31 effectively accumulated in ischemic myocardium and specifically delivered CsA to mitochondria to recover the function of mitochondria in ischemic cardiomyocyte,consequently enhanced the protective and therapeutic effect of CsA on MI/RI in rats. |
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