Biomimetic Design Of Mitochondria-Targeted Hybrid Nanozymes As Superoxide Scavengers For Myocardial Ischemia Reperfusion Injury Protection

Ischemia-elicited tissue damage is the central cause of major cardiovascular disorders,such as myocardial infarction(MI)and ischemia-reperfusion(IR)injury.An early restriction of blood supply to cardiac tissue and the following imbalance in metabolic supply and demand within ischemic tissue are key drivers of these diseases.Therapeutic strategies for ischemia-elicited diseases aim to restore the blood flow through reperfusion and/or regeneration of functional vasculature networks,whereas the early reperfusion is commonly associated with an exacerbation of tissue damage/injury and a profound inflammatory response,namely IR injury.The injury mechanism is complicated,but it is well-characterized that the initiating factor of the IR injury is a burst of reactive oxygen species(ROS)from the mitochondrial respiratory chain upon reperfusion.Thus,protecting ischemic tissue from mitochondrial ROS-induced injury is necessary for the alleviation of ischemia-elicited diseases.Herein,we utilized ferritin nanocages(FTn),a human-derived self-assembled protein nanocarrier,as the enzyme scaffold for nanozyme synthesis.This artificial cascade nanozyme possesses superoxide dismutase(SOD)-and catalase(CAT)-like activities and also targets ischemic tissues.The protective advantages of the hybrid nanozyme for cardiac ischemia-reperfusion injury via systemic delivery and localized release from adhesive hydrogels were investigated,respectively.Nanozymes are nanomaterials with intrinsic enzyme-like characteristics.Some nanozymes have been shown to possess antioxidant enzyme-like activity which can be used as a potential free radical scavenger.Triphenylphosphonium(TPP),a highly positive lipophilic cation,was used as a mitochondrial targeting ligand due to its preferential accumulation into the mitochondria.Thus,it was proposed to construct mitochondria-targeted nanozymes to scavenge mitochondrial ROS by conjugating TPP with nanozymes.We prepared Mn O₂ nanoparticles in the hollow cavity of FTn,which showed superoxide dismutase(SOD)-and catalase-like activity.To explore the mitochondria-targeted ability,we thus conjugated TPP onto the surface of FTn-based nanozyme(Mito-Fenozyme).Our results showed that,compared to Fenozyme only,Mito-Fenozyme can escape from lysosome and target mitochondria to alleviate mitochondrial ROS.The mitochondrial protective effects of Mito-Fenozyme were demonstrated in vitro in H9C2 cells by increasing mt DNA copy number and ATP generation.In addition,we found the expression of TIM-2,a receptor for FTn in mouse,was increased in cardiac tissues subjected to IR surgery.Tail-vein injection of the Cy5-FTn-TPP particles resulted in elevated signal intensity of IR heart compared to sham heart.To further evaluate the protective effects of Mito-Fenozyme in vivo,we systemically administered Mito-Fenozyme in a cardiac IR mouse model.The echocardiographic analysis showed the recovery of heart function after Mito-Fenozyme treatment.Histological assessment of infarct wall thickness and scar area also demonstrated the protective effect of Mito-Fenozyme in the IR model.Biomimetic fabrication of a mussel-inspired,catechol-functionalized hydrogel has been demonstrated to provide a strong tissue adhesiveness.We next sought to explore whether mitochondria-targeted nanozyme was able to use for the treatment of cardiac IR injury via local administration.The mitochondria-targeted nanozyme was incorporated into tissue adhesive hyaluronan-catecholamine(HA-CA)hydrogels(Mitofegel)to construct cardiac patches.After local transplantation into the heart injury site,the Mitofegel showed long-lasting retention on the heart due to their tissue adhesive ability.We found that hydrogels supported the slow release and penetration of Mito-Fenozyme into cardiac tissue.TTC and Evans Blue double-staining were performed to check the area at risk,which illustrated the significant decrease of infarct size after Mitofegel treatment.Echocardiographic images and histological staining also revealed that,compared to control groups,the improvement of heart function was observed after Mitofegel treatment.The results demonstrated that FTn is an ideal general nanocarrier for vasculature regeneration and mitochondrial ROS scavenger,catered to various ischemia-elicited diseases.