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Mitochondrial dysfunction is the main cause of myocardial ischemia-reperfusion injury and limits cardiac repair after blood flow recovery. Although mitochondrial transplantation may help restore cellular energy metabolism, its therapeutic benefits are reduced due to extracellular calcium ion-induced mitochondrial damage. Here, we present a thermosensitive phase-separated hydrogel composed of gelatin and PEG, which can concentrate, protect, and deliver freshly isolated mitochondria. Compared to traditional single-phase hydrogels, this system remains injectable at physiological temperatures and can achieve rapid mitochondrial release after transplantation. Moreover, the phase-separated structure improves mitochondrial aggregation and maintains their activity through spatial confinement and the chelation effect of gelatin on calcium ions. In vitro, concentrated mitochondria show better membrane potential and ATP production capacity. In vivo, the transplanted mitochondria are efficiently internalized by cardiomyocytes, thereby improving cardiac function after myocardial ischemia-reperfusion and reducing tissue damage. These findings indicate that the phase-separated hydrogel is a promising platform for mitochondrial transplantation.
Cardiovascular diseases remain the leading cause of death worldwide, with ischemic cardiomyopathy being the most severe and life-threatening clinical manifestation. Although drug thrombolysis and percutaneous coronary intervention are necessary measures to save ischemic myocardium, the sudden reperfusion of blood after ischemia triggers a series of damage processes, which may lead to further necrosis. During myocardial ischemia-reperfusion injury, pathological Ca²⁺ influx causes calcium overload and increased production of reactive oxygen species, subsequently inducing the opening of the mitochondrial permeability transition pore, ultimately leading to mitochondrial damage. Given that mitochondria are the main sites of oxidative phosphorylation and ATP synthesis, mitochondrial dysfunction leads to a decrease in cellular energy metabolism levels. This damage impairs cellular energy, disrupts excitation-contraction coupling, and promotes adverse cardiac remodeling, ultimately leading to the occurrence and progression of heart failure.
Current standard drug treatments for MIRI mainly include angiotensin-converting enzyme inhibitors, beta-blockers, angiotensin II receptor antagonists, and statins. These drugs mainly reduce adverse ventricular remodeling by alleviating cardiac load, myocardial oxygen demand, and overall myocardial energy demand, thereby exerting cardioprotective effects. Although coronary artery reperfusion strategies and clinical management have been continuously optimized, the mortality and morbidity of I/R-induced heart failure remain high. One fundamental limitation of these therapies is that they cannot address the potential energy deficit in the failing myocardium, making it essentially incurable. Mitochondrial transplantation therapy has recently emerged as an innovative approach to restore the biological energy function of damaged tissues. In this strategy, active exogenous mitochondria are introduced into the damaged tissue, internalized by recipient cells, and integrated into the endogenous mitochondrial network. Transplanted mitochondria have been proven to restore ATP production, regulate mitochondrial dynamics, enhance mitochondrial autophagy and autophagy, and regulate the metabolism and inflammatory state of recipient cells. These benefits have been verified in preclinical models of myocardial infarction and pediatric patients with refractory cardiogenic shock. Additionally, developing strategies to protect mitochondria during transplantation has become a key research focus. Recently, some studies have explored delivery systems based on single-phase hydrogels to improve the survival rate of transplanted mitochondria during transplantation, including thermosensitive F127 hydrogel, alginate-based hydrogel, and HA-MC hydrogel.
However, despite the broad potential of MTT, there are still several translational barriers. A key limitation is the vulnerability of isolated mitochondria in the extracellular space, especially under conditions simulating the in vivo environment. The extracellular fluid contains approximately 1.8 mmol/L Ca²⁺, a concentration far exceeding the physiological intracellular range. This high calcium concentration induces mitochondrial swelling, loss of membrane potential, and enzyme inactivation, significantly reducing the vitality and therapeutic efficacy of transplanted mitochondria. Even when mitochondria are encapsulated in monomolecular hydrogel, their uniform distribution and the large pore size of the hydrogel allow for unrestricted Ca²⁺ exchange, which still may lead to mitochondrial damage. Therefore, maintaining mitochondrial activity during delivery and ensuring their functional integration into the recipient cells remains a major challenge.
To address this issue, we developed a mitochondrial condensation system within an online thermosensitive hydrogel sub-volume using liquid-liquid phase separation. By simply mixing polyethylene glycol, gelatin, and freshly isolated mitochondria, we induced LLPS to form a multi-volume hydrogel, in which mitochondria were concentrated in the spherical polyethylene glycol phase. The continuous phase, gelatin, endowed the LLPS hydrogel with thermosensitivity. Additionally, we found that LLPS significantly altered the sol-gel transition behavior of the hydrogel, specifically raising the sol-gel transition temperature to above body temperature (37°C). Notably, the mitochondrial condensates within the PEG microdomains increased mitochondrial packing density and enhanced mitochondrial activity through confinement effects and mitochondrial crowding effects. Moreover, the continuous gelatin phase provided a protective microenvironment through the chelation of Ca²⁺ with gelatin carboxyl groups, preventing Ca²⁺ from diffusing into the mitochondrial condensates within the PEG microdomains. This spatial buffering capacity of Ca²⁺ alleviated Ca²⁺-induced mitochondrial damage, as evidenced by maintaining mitochondrial membrane potential and ATP production capacity. Furthermore, both cell and animal studies demonstrated that compared to mitochondrial transplantation based on monomolecular hydrogels, LLPS hydrogels containing concentrated mitochondria delivered more functionally active mitochondria to ischemic myocardium and enhanced MTT efficacy. More active mitochondria upregulated mitochondrial fusion protein MFN1, enhanced aerobic respiration, and inhibited anaerobic pathways, thereby significantly improving the overall energy metabolism of myocardial cells to meet the increased energy demands after MIRI. Therefore, by maintaining mitochondrial activity, enhancing biological activity, and achieving controlled delivery to ischemic myocardium, Mito/LLPS hydrogels effectively overcome the key limitations of traditional MTT strategies based on monomolecular hydrogels. This strategy represents an important advancement in the effective clinical translation of mitochondrial transplantation for the treatment of MIRI.
Original link: https://doi.org/10.1038/s41467-026-71765-6
