An Antioxidant System Through Conjugating Superoxide Dismutase Onto Metal-organic Framework For Cardiac Repair

BackgroundMyocardial infarction(MI),a dominant origin of death worldwide,involves myocardial injury and cardiomyocyte(CM)death caused by acute occlusion or severe stenosis of coronary arteries,resulting in local persistent ischemia and hypoxia.During the occurrence of AMI,lots of reactive oxygen species(ROS)are produced because of persistent hypoxia and lack of ATP in CMs.This leads to activation of the apoptosis cascade reaction,CM death,and degradation and remodeling of cardiac extracellular matrix(ECM).Excessive ROS and necrotic CMs activate the immune system and produce a severe inflammatory response,which forms a vicious circle by promoting further elevation of ROS.It is known that the inflammatory process is the basis of the development and complications of MI.Appropriate inflammation is conducive to the repair of MI,while excessive inflammation leads to the secondary aggravation of myocardial injury.This accelerates the occurrence of fibrotic microenvironment/left ventricular(LV)remodeling.Then,over time,cardiac function deteriorates progressively,eventually leading to congestive heart failure.Early ROS clearance can reduce the death of CMs,lessen the generation of inflammatory factors and the degradation of ECM,and lower subsequent injury.The main antioxidant defense system in the body includes superoxide dismutase(SOD),which can catalyze O2·-into H2O2 and oxygen.However,many problems greatly restrict the application of natural SOD in therapy,such as high synthesis cost and non-specific targeting.And the major obstruction to the widespread application is the easy inactivation of enzymes when exposed to high temperatures and/or nonaqueous media during machining,which can intermingle the special and intricate interactionsof covalent and noncovalent that disrupt the stability of their tertiary structures under artificial conditions.Several strategies have been used to overcome the vulnerability of enzymes,such as immobilization.Immobilizing enzymes on the surfaces or inside of porous materials often permits easy separation of enzymes from their reaction products,which is a major advantage compared to dissolved enzymes reactions.Actually,biotechnology of immobilized enzymes has been applied in vivo and vitro research.The immobilization of enzymes by porous silicon has been a mature technology,while metal-organic framework(MOF)seems a better choice.Representing a recent species of support nanomaterial for enzymes,MOFs possess easy optimized chemical functionality and pore dimensions of a framework for the immobilization of specific enzymes under biocompatible synthesis conditions(H2O,room temperature),which has a precision that can’t be achieved by other porous materials and extensive scope for development.Zirconium(Zr)-based compounds are widely used in biomedical applications due to their good biocompatibility and also exhibiting good mechanical properties.Moreover,there have been many studies using Zr-based MOF(ZrMOF)-based NMs for in vivo and in vitro experiments,all of which have demonstrated the good biocompatibility of ZrMOF.However,to date,there is no application of ZrMOF in the cardiovascular field.Therefore,this study uses SOD as a raw material and reduces the size to produce a larger surface area to improve the efficiency of the catalytic reaction.By using a well-organized crystalline Zr-based MOF(ZrMOF)as a precursor,we found that the immobilized enzyme becomes uniformly dispersed and more stable.Through local myocardial injection,these stabilized immobilized enzymes were for the first time applied to study the repair of myocardial injury after MI.Part 1 Synthesis and characterization of SOD-ZrMOFPurpose:A novel immobilized enzyme with nanoscale size and antioxidant activity,SOD-ZrMOF,was prepared,and its characterization and antioxidant activity level should be examined.Methods:1 ZrMOF was synthesized by hydrothermal method,and SOD-ZrMOF was constructed by chemical bonding.2 The morphology of SOD-ZrMOF was characterized by SEM,TEM and DLS.3 The distribution characteristics of SOD and ZrMOF in three-dimensional space were studied by CLSM.4 The relative activities of SOD and SOD-ZrMOF were determined using SOD detection kit.Results:1 SEM show that SOD-ZrMOF presents a uniform and regular octahedral structure,and the statistical results show that its average diameter is 96 nm,which is highly similar to the SEM pertormance of blank ZrMOF;the TEM image of SOD-ZrMOF also shows similar morphological features;The average hydrodynamic particle size of SOD-ZrMOF measured by DLS experiments is about 154 nm,which is slightly larger than that of ZrMOF(133 nm).2 The CLSM results show that the green fluorescence of FITC and the red fluorescence of rhodamine 6G gradually increase from the top to the middle,and then gradually weaken from the middle to the bottom,and the strongest and weakest levels of the two fluorescences are consistent,namely SOD and ZrMOF have the same distribution characteristics in three-dimensional space.3 There was no statistical difference in the enzymatic activity of free SOD and SOD-ZrMOF.Conclusion:In this study,a novel immobilized enzyme SOD-ZrMOF was synthesized by relatively simple methods in the way of chemical bond connection.A series of in vitro research results prove that SOD-ZrMOF is a nano-scale with uniform regular octahedral structure and retaine a high level of SOD activity.Part 2 Protective effect and mechanisms of SOD-ZrMOF on hypoxic cardiomyocytes in vitroPurpose:The cytotoxicity of SOD-ZrMOF,the scavenging ability of ROS,and the protective effect on hypoxic CMs and related mechanisms were verified at cellular level.Methods:1 The cytotoxicity of ZrMOF and SOD-ZrMOF on H9c2 cells was detected by CCK-8.2 CCK-8 was used to detect whether ZrMOF and SOD-ZrMOF had protective effect on H9c2 cells under hypoxia,and to determine the optimal concentration of SOD-ZrMOF to treat H9c2 cells.3 DCFH-DA probe was used to detect whether ZrMOF and SOD-ZrMOF could scavenge ROS generated in H9c2 cells under hypoxia.4 JC-1 probe to detect the effect of SOD-ZrMOF on the depolarization rate of mitochondrial membrane potential in H9c2 cells under hypoxic conditions.5 Live/dead cell staining and apoptosis assay were used to verify whether SOD-ZrMOF could inhibit the death of H9c2 induced by hypoxia.6 Western blot was used to detect whether SOD-ZrMOF could inhibit the activation of NF-κB/HIF-1α pathway in H9c2 cells under hypoxia.Results:1 ZrMOF and SOD-ZrMOF are less toxic to H9c2 cells.2 ZrMOF has no protective effect on the cell viability of H9c2 cells under hypoxia,while SOD-ZrMOF can significantly reduce the damage of hypoxia on the cell viability of H9c2 cells.3 SOD-ZrMOF has no effect on the ROS level of H9c2 cells under normoxia;hypoxia could lead to a significant increase in the ROS level of H9c2 cells;ZrMOF has no effect on the ROS level of H9c2 cells under hypoxia,while SOD-ZrMOF could significantly decrease the ROS level of H9c2 cells under hypoxic conditions.4 SOD-ZrMOF has no effect on the mitochondrial membrane potential level of H9c2 cells under normoxia conditions;hypoxia could lead to a significant imbalance in the mitochondrial membrane potential of H9c2 cells;SOD-ZrMOF significantly inhibited the increase of mitochondrial membrane potential depolarization rate in H9c2 cells under hypoxia.5 The apoptosis and necrosis of H9c2 cells are significantly increased under hypoxia;SOD-ZrMOF has no adverse effect on the survival of H9c2 cells under normoxia,but significantly inhibites the apoptosis and necrosis of H9c2 cells under hypoxia.6 SOD-ZrMOF significantly inhibites the activation of NF-κB/HIF-1αpathway in H9c2 cells under hypoxia.Conclusion:In studies at the cellular level,SOD-ZrMOF showes good biocompatibility and could protect the mitochondrial function of CMs by scavenging excess ROS produced under hypoxia,and further protect CMs from hypoxia-induced apoptosis and necrosis.The inhibitory effect of SOD-ZrMOF on NF-κB/HIF-1α pathway may be the key mechanism of its treatment of hypoxia-induced cell damage.Part 3 Short-term efficacy and mechanisms of SOD-ZrMOF in myocardial infarction model micePurpose:The biocompatibility,protective effect and related mechanisms of SOD-ZrMOF against acute myocardial tissue injury caused by MI will be verified in vivo.Methods:1 Intramyocardial injection of SOD-ZrMOF was given to healthy mice,and 72 h later,serum and organs were collected for biochemical detection and HE staining to evaluate its acute toxicity in vivo.2 Inject the same amount of FITC-labeled SOD and SOD-ZrMOF into the myocardium of mouse,and select multiple time points to observe the fluorescence signal intensity on the heart with the help of fluorescence imaging technology to evaluate whether SOD-ZrMOF has better stability.3 The MI model mice were given myocardial injection of SOD-ZrMOF or PBS immediately after the ligation of LAD,and the Sham group was constructed atthe same time.After 24 hours,serum was collected,and ELISA was used to detect the levels of inflammatory factors in the mice in each group;The myocardial tissue of each group of mice was collected to detect the protein expression of NF-κB/HIF-la pathway by Western blot;Paraffin sections of myocardial tissue were made,and then the expression level of HIF-la protein was verified by immunofluorescence,and the the level of apoptosis was detected by TUNEL.Results:1 The results of blood biochemistry and HE staining show that SOD-ZrMOF has no adverse effect on heart,liver function,kidney function,morphological structure and inflammatory cell infiltration level of each organ in mice.2 Fluorescence imaging results show that SOD is highly unstable in vivo,and the immobilization strategy can indeed significantly enhance the stability of SOD and prolong its action time in vivo.3 Compared with the Sham group,the levels of inflammatory factors and the protein expression of NF-κB/HIF-la pathway in the model mice 1 day after MI significantly increase,and the apoptosis of CMs significantly increases.In contrast,SOD-ZrMOF significantly inhibits inflammatory response,CM apoptosis and the activation of the NF-κB/HIF-1α pathway in the acute phase of MI.Conclusion:In short-term in vivo studies,SOD-ZrMOF shows good biocompatibility and better stability than SOD.Interestingly,SOD-ZrMOF can inhibit apoptosis,inflammation and the activation of NF-κB/HIF-1α pathway to alleviate acute myocardial tissue damage caused by MI.Part 4 Study on the long-term efficacy of SOD-ZrMOF on myocardial infarction model micePurpose:The protective effect and related mechanism of SOD-ZrMOF on myocardial tissue damage over a long-term caused by MI will be verified in vivo.Methods:1 Immediately after the MI model mice were constructed,SOD-ZrMOF or PBS were injected into the myocardium,and the Sham group was constructed at the same time,and the cardiac function changes of the mice in each group were dynamically observed by means of small animal ultrasound at multiple time points.2 After 4 weeks,the body weight of the mice in each group was measured,and each organ was taken out,then the heart weight and the length of the tibia were measured to evaluate the cardiac remodeling.3 Paraffin sections of myocardial tissue were made,and the myocardial infarction area,left ventricular wall thickness,the proportion of viable myocardium in the infarcted area and the deposition of collagen fibers were evaluated by HE staining,Masson staining and Sirius red staining.4 The vascular density of the injured myocardial tissue of each group of mice was assessed by immunofluorescence.5 The liver,spleen,lung,kidney and non-MI tissues of heart were taken to make paraffin sections,and the long-term toxicity of SOD-ZrMOF to mice in each group was evaluated by HE staining.Results:1 Compared with the Sham group,the cardiac function of the model mice at different time points after MI decreased progressively over time,and SOD-ZrMOF significantly inhibited the decreasing trend.2 Compared with the Sham group,the HW/BW and HW/TI ratios of the mice in the MI group significantly increased,and SOD-ZrMOF significantly suppressed the trend.3 Compared with the Sham group,the MI area and collagen deposition level of the model mice 4 weeks after MI significantly increased,the thickness of the left ventricular wall significantly thinner,and the proportion of viable cells in the MI area significantly reduced,while SOD-ZrMOF significantly suppressed the above adverse effects.Conclusion:Long-term in vivo studies show SOD-ZrMOF is safe for long-term in vivo studies by intramyocardial injection.SOD-ZrMOF can effectively reduce myocardial infarction size,increase the vascular density of damaged myocardial tissue,dynamically improve cardiac function,and inhibit the progression of pathological ventricular remodeling in MI model mice.