Study On The Releasing Of Exo-Cur From Decellularized Extracellular Matrix Hydrogel For The Myocardial Infarction Treatment

Objective:The decellularized extracellular matrix(dECM)hydrogel was prepared to encapsulate Exo-Cur,which has the biological function of anti-myocardial fibrosis.The injectable composite hydrogels loaded with Exo-Cur was obtained,and its effects of anti-myocardial fibrosis after myocardial infarction was investigated.Methods:(1)Bone Mesenchymal Stem Cells(BMSCs)were isolated by adherent screening,and characterized by flow cytometry.Exosomes were isolated from BMSC supernatant by ultracentrifugation,and then curcumin was introduced into them by electroporation.The morphology,size,protein markers and biological activity of exosomes and Exo-Cur were detected by transmission electron microscope,nanoparticle tracking analysis,Western blot and fluorescent staining.The absorbance at 420 nm was measured by microplate reader to calculate encapsulation rate,solubility rate,and remainder rate.The dECM was prepared by acellular porcine left ventricle,and the degree of decellularization was detected by HE staining,DAPI staining and dsDNA determination,the main components of dECM hydrogel were characterized by Sirius red staining,Orcein red staining and Alcian blue staining;pore size and structure of dECM hydrogel were characterized by scanning electron microscopy.The rheology and turbidity gel kinetics of dECM hydrogel between different concentrations were measured,and NIH3T3 cells were cultured on its surface.Proliferation of cells was investigated by CCK8 method,and the hydrogel concentration with the best performance was screened for subsequent research.Exo-Cur was mixed into dECM hydrogels to investigate the sustained release of curcumin at different time points by absorbance determination.(2)The inhibiting fibrosis function of Exo-Cur in vitro was investigated by α-SMA fluorescence staining,EdU staining,Transwell experiment,Scratch experiment,CCK8 assay and Western blot.Finally,the composite hydrogel was injected into the infarction region and peripheral region,and echocardiography,Masson staining,Col Ⅰ,Col Ⅲ,α-SMA and CD31 fluorescence staining were used to evaluate its effect on heart repair in mice.Results:(1)Flow cytometry showed that CD29 and CD44 were positive on the surface of BMSCs.while CD29 and CD117 were negative;Compared with exosomes,Exo-Cur had no changes in morphology,size distribution,zeta potential,and surface marker expression;Compared with free curcumin.the solubility and stability of ExoCur were increased by 23.25 times and 36%:The biocompatibility experiment showed that the dECM hydrogel of 8 mg/mL to 16 mg/mL was suitable for cell adhesion growth.but the 12 mg/mL dECM hydrogel had better mechanics and turbidity gel dynamics.and the hydrogel could slowly release Exo-Cur to 28 days.(2)The results of in vitro experiments show that Exo-Cur can effectively inhibit the transformation of fibroblasts into myofibroblasts,as well as the proliferation and migration of myofibroblasts.Experiments in vivo have confirmed that,compared with the Control group,the dECM/Exo-Cur group can effectively increase ejection fraction,reduce infarction area.prevent myocardial fibrosis and promote angiogenesis.Conclusion:The use of exosome encapsulation can effectively improve the solubility,stability and bioavailability of curcumin.The dECM hydrogel has good mechanical properties and biocompatibility,and can deliver Exo-Cur.The composite hydrogel can effectively inhibit myocardial fibrosis,promote angiogenesis,reduce infarct area and improve cardiac function after MI in mice.