Protective Effect Of Redox-responsive Covalent Organic Framework Nanocarrier Loaded With Matrine On Myocardial Ischemia-reperfusion Injury In Mice

Purpose:To investigate whether the transient intervention of biodegradable redox-sensitive fluorescent covalent organic framework（TPE-ss COF）nanocarriers in the early stage of myocardial ischemia-reperfusion can achieve early myocardial cell apoptosis,improve cardiac function,and have a good therapeutic effect on the long-term prognosis of myocardial ischemia-reperfusion injury.Methods:（1）After the yellow powdered TPE-ss COF material was obtained by Schiff base reaction,the TPE-ss COF material was characterized and analyzed by transmission electron microscopy and thermogravimetric analysis.At the same time,matrine was loaded into TPE-ss COF material to verify the loading rate of the material.（2）In the cell experiment,human cardiomyocytes（AC16）were used and H₂O₂（hydrogen peroxide）injury model was used.The following experimental groups were set:Control group,H₂O₂ group,TPE-ss COF group,Matrine group and TPE-ss COF@Matrine group.In the early stage,the biosafety and biocompatibility of the materials were determined by cell endocytosis and MTT experiments,and the dosage of each group of materials and drugs was provided for subsequent experiments.Subsequently,reverse transcription-polymerase chain reaction（RT-PCR）was used to detect the effects of Matrine and TPE-ss COF@Matrine on the expression of myocardial cell-related m RNA levels in the early stage of myocardial ischemia-reperfusion.The level of reactive oxygen species（ROS）in cells was detected by flow cytometry,which proved that Matrine and TPE-ss COF@Matrine had a protective effect on H₂O₂-induced cardiomyocytes.（3）The results of small animal imaging showed that after loading aggregation-induced emission luminophores and redox-sensitive disulfide bonds into the COF skeleton to develop redox-sensitive and emission TPE-ss covalent organic framework（COF）nanocarriers,TPE-ss COF-loaded matrine can be effectively loaded and delivered,and accumulated in the heart,which can prolong the distribution and metabolic time of Matrine in the body,thus playing a therapeutic and protective role in MIR damage.（4）In animal experiments,70 male Kunming mice were used,8 were used as normal group,and 62 were subjected to MIR surgery.Among them,42 were injected with TPE-ss COF,Matrine,TPE-ss COF@Matrine at the moment of loosening the ligature.After48 hours,48 surviving mice were randomly divided into normal group,MIR group,TPE-ss COF group,Matrine group,TPE-ss COF@Matrine group and Matrine gavage group.The MIR group was injected with normal saline.Materials and drugs were injected through the tail vein within 4 weeks after injection,no other intervention,normal diet.Matrine gavage group was given normal gavage for 4 weeks.After 4 weeks,the changes of cardiac function and cardiac morphology were observed in each group.The pathological changes of myocardial tissue were observed by hematoxylin and eosin（HE）and Masson staining.The levels of N terminal pro B type natriuretic peptide（NT-pro BNP）,GSH（Glutathione）and cardiac troponin I（c Tn I）in serum were measured by enzyme-linked immunosorbent assay（ELISA）after 4 weeks.TUNEL（Td T-mediated d UTP Nick-End Labeling）staining was used to verify the protective effect of materials and drugs on cardiomyocyte apoptosis at 24 h after myocardial ischemia-reperfusion.By comparing the efficacy of Matrine group and TPE-ss COF@Matrine group and Matrine gavage group,it is proved that single transient treatment of reperfusion can produce good long-term prognosis,and TPE-ss COF@Matrine has better protective effect than Matrine.Its protective effect may be related to reducing myocardial cell apoptosis and protecting myocardial cells during the peri-perfusion period.Results:（1）The COF carrier has a large specific surface area（15.61±0.23%）and a high loading rate（92.3±3.1%）,which can efficiently deliver drugs in vivo.Moreover,the COF carrier has AIE structural fluorescence properties,which is conducive to real-time tracking.In addition,the COF carrier has the characteristics of controlling drug release and prolonging the duration of action and minimizing toxicity.After the synthesis of GSH-responsive covalent organic framework nanocarrier AIE-COF material,the drug is effectively encapsulated byπ-πstacking.Matrine was delivered to the myocardial tissue and released at the inflammatory site of myocardial ischemia,reducing the ROS level at the inflammatory site and responding to the overexpressed glutathione after reperfusion.（2）Based on cell endocytosis and MTT experiments,it has good biosafety and biocompatibility,which provides a biological basis for the next use of TPE-ss COF and TPE-ss COF@Matrine in cell and animal experiments.The results of RT-PCR showed that Matrine and TPE-ss COF@Matrine could protect H₂O₂-induced cardiomyocyte injury,and its protective effect may be related to reducing the level of early inflammatory factors in cardiomyocytes,while TPE-ss COF alone could not play a protective role.At the same time,flow cytometry results showed that Matrine and TPE-ss COF@Matrine could inhibit their ROS levels in early cardiomyocyte apoptosis and protect cardiomyocytes.（3）The results of small animal imaging showed that after loading aggregation-induced emission luminophores and redox-sensitive disulfide bonds into the COF skeleton to develop redox-sensitive and emission TPE-ss covalent organic framework（COF）nanocarriers,TPE-ss COF-loaded matrine can be effectively loaded and delivered,and accumulated in the heart,which can prolong the distribution and metabolic time of Matrine in the body,thus playing a therapeutic and protective role in MIR damage.（4）The results of animal experiments showed that when Matrine and TPE-ss COF@Matrine were injected into the MIR model by tail vein at the moment of reperfusion after 30 minutes of ischemia,MIR-induced cardiomyocyte injury could be significantly reduced.In addition,compared with the MIR group,after administration of TPE-ss COF@Matrine at the early stage of reperfusion,cardiomyocyte apoptosis was significantly reduced and cardiac function was significantly improved.At the same time,TTC results showed that TPE-ss COF@Matrine significantly reduced the myocardial infarction area of MIR mice,confirming the cardioprotective effect of TPE-ss COF@Matrine on MIR injury,demonstrating the great potential of using COF as an intelligent drug carrier for the peri-reperfusion treatment of ischemic heart disease,and realizing the long-term treatment of MIR injury.Conclusions:The biodegradable redox-sensitive fluorescent covalent organic framework nanocarrier has a protective effect on myocardial ischemia-reperfusion injury in mice through a single transient intervention during the peri-perfusion period.The protective mechanism may be related to the rescue of non-apoptotic cardiomyocytes and the reduction of inflammatory response during the peri-perfusion period.