Transplantation of active mitochondria condensed in liquid–liquid phase-separated hydrogels ameliorates myocardial ischemia-reperfusion injury

Abstract Mitochondrial dysfunction is a key driver of myocardial ischemia-reperfusion injury (MIRI), which exacerbates cardiac damage following the restoration of blood flow. Although single-phase hydrogel-based mitochondria transplantation therapy (MTT) holds promise for restoring cellular energy metabolism, its effectiveness is limited by extracellular calcium-induced mitochondrial degradation. In this study, we design a thermo-sensitive hydrogel utilizing liquid-liquid phase separation (LLPS), composed of gelatin and polyethylene glycol (PEG), to condense freshly isolated mitochondria. Compared to conventional single-phase hydrogels, the LLPS hydrogel significantly increases the sol-gel transition temperature above physiological levels, allowing it to remain injectable at body temperature while enabling rapid mitochondrial release following transplantation. More importantly, the LLPS structure enhances mitochondrial packing density and protects the activity of condensed mitochondria against calcium toxicity through spatial confinement and gelatin-mediated calcium chelation. In vitro, LLPS hydrogel-condensed mitochondria exhibit improved mitochondrial membrane potential and ATP production ability compared to those delivered via single-phase gelatin hydrogel. In vivo, mitochondria released from the LLPS hydrogel are effectively internalized by cardiomyocytes, resulting in improved cardiac function and reduced tissue damage after MIRI. These findings suggest that LLPS-based hydrogels offer a promising strategy to enhance MTT efficacy for MIRI and other mitochondria-related disorders.