Injectable Polyleucine Hydrogel for Local Amino Acid Supplementation Drives Muscle Regeneration through mTOR-Associated Metabolic Reprogramming

Background Skeletal muscle regeneration following volumetric muscle loss (VML) is hindered by impaired regenerative capacity and excessive fibrosis, which are associated with aberrant metabolism in the microenvironment of damaged tissue. Although dietary amino acids effectively promote muscle protein synthesis and myogenesis, their in situ supplementation to correct metabolic dysregulation remains a substantial challenge. Therefore, this study aimed to identify critical amino acid metabolites that drive myogenesis and to develop a local supplementation strategy that promotes muscle regeneration through metabolic reprogramming. Methods Targeted metabolomics was performed on differentiating C2C12 myoblasts, and L-leucine was identified as a key promoter of myogenic differentiation. An injectable poly(L-leucine) hydrogel (PEG-Leugel) was formed by self-assembly of the polyethylene glycol–polyleucine copolymer (PEG-Leu), which was synthesized by ring-opening polymerization. Its mechanical properties, degradability, and biocompatibility were systematically evaluated. The effects of PEG-Leugel on myoblast differentiation, mitochondrial metabolism, and mammalian target of rapamycin (mTOR) signalling were assessed in C2C12 cells. The hydrogel was further loaded with interleukin-4 (IL-4), and its effects on macrophages were evaluated. Therapeutic efficacy was further assessed in a VML rat model using ultrasonography, gait analysis, histological staining, biochemical assays, and transcriptomic analysis. Results PEG-Leugel exhibited shear-thinning behaviour, injectability, a porous microstructure, and sustained degradation. In vitro, PEG-Leugel promoted myoblast differentiation by increasing mitochondrial metabolism and the energy supply through activation of the mTOR signalling pathway. Furthermore, PEG-Leugel also served as a carrier for the loading and subsequent sustained release of therapeutic agents such as IL-4, thereby promoting M2 macrophage polarization. In a rat VML model, PEG-Leugel reduced the defect area and fibrosis by restoring metabolic enzyme activity and inhibiting protein degradation. These effects were augmented by IL-4-modulated pro-reparative immune responses. Conclusions The injectable leucine-based polypeptide hydrogel system effectively promoted muscle regeneration through the in situ regulation of bioenergetic metabolism. These findings provide proof-of-concept that local amino acid supplementation via a biodegradable polypeptide hydrogel can reprogram the bioenergetic metabolism of the injured muscle microenvironment and promote functional regeneration in a VML model.