**Background:** Chronic diabetic wounds affect millions worldwide and are characterized by persistent hyperglycemia-induced oxidative stress, microvascular injury, impaired angiogenesis, and prolonged inflammation. Mitochondrial fission has been strongly linked to hyperglycemia-induced ROS formation and vascular damage. Adipose-derived mesenchymal stem cell exosomes (ADSC-Exos) have therapeutic potential but suffer from rapid in vivo clearance. Metformin provides microvascular protection and glucose control. This study aimed to develop a self-healing conductive hydrogel co-loaded with ADSC-Exos and metformin and investigate its effects on chronic diabetic wound healing, focusing on mitochondrial dynamics.
**Methods:** A self-healing conductive hydrogel (PEG/Ag/CNT-M+E) was synthesized via Ag–S coordination between 4-Arm-PEG-Thiol and Ag⁺, with hydroxyl-modified multiwalled carbon nanotubes (CNTs) for conductivity, and loaded with metformin (1 mg/mL) and ADSC-Exos (0.1 mg/mL). The hydrogel was characterized for morphology (SEM), self-healing (rheology), injectability, tissue adhesion (lap-shear test), swelling, degradation, conductivity, hemocompatibility, and drug/exosome release. ADSC-Exos were characterized by nanoparticle tracking analysis (50–150 nm), electron microscopy, and Western blot (CD9, CD63, TSG101). In vivo efficacy was tested in a streptozotocin-induced diabetic mouse model with full-thickness 1-cm diameter skin wounds. Five groups were compared: Tegaderm control, PEG/Ag/CNT, PEG/Ag/CNT-M, PEG/Ag/CNT-E, and PEG/Ag/CNT-M+E (n=5/group). Wound closure was monitored on days 0, 3, 7, 10, and 14. Histological analyses included H&E, Masson staining, immunofluorescence (Ki67, CD31, αSMA, Ve-cadherin, Icam, Vcam), immunohistochemistry (IL-6, TNF-α), and electron microscopy for microvascular and mitochondrial morphology. In vitro studies used HUVECs in high-glucose (33 mM) medium. ROS and mtROS were measured by flow cytometry and immunofluorescence. Mitochondrial morphology was assessed by MitoFluor staining and Drp1 colocalization. Mitochondrial fission/fusion proteins (Drp1, Fis1, Mff, Mfn1, Mfn2) were analyzed by Western blot. The mitochondrial fission inhibitor mdivi-1 and activator fccp were used to confirm mechanism. Cell migration (Transwell), tube formation, and F-actin staining were performed.
**Key Results:** The PEG/Ag/CNT-M+E hydrogel exhibited a uniform macroporous structure, self-healing at 774% critical strain, injectability, tissue adhesion of ~8 kPa, conductivity of 3.04–3.65×10⁻⁴ S/m, hemolysis ratio <5%, and sustained release of metformin (90.2%±8.23% at day 12) and exosomes (>80% at day 12). In vivo, the PEG/Ag/CNT-M+E group showed significantly smaller wound length by day 3 (p<0.01) and nearly complete wound closure by day 10 (p<0.001). H&E and Masson staining revealed complete epithelization, well-organized collagen fibers, and hair follicle structures. Electron microscopy showed reversed vascular edema, lumen stenosis, and basement membrane thickening. Ki67 immunofluorescence was strongest in the dual-loaded group (p<0.001). CD31/αSMA staining showed the highest density of mature vessels. Ve-cadherin expression was restored, while Icam and Vcam were significantly decreased (p<0.001). IL-6 and TNF-α were markedly reduced on day 3. In vitro, the hydrogel promoted HUVEC proliferation, migration, and tube formation. ROS and mtROS were significantly reduced by the dual-loaded hydrogel and mdivi-1, while fccp partially counteracted this effect. Mitochondrial morphology was preserved (filamentous vs. fragmented). Drp1 mitochondrial translocation was reduced. Western blot showed upregulation of fusion proteins Mfn1/Mfn2 and downregulation of fission proteins Drp1, Fis1, and Mff. F-actin homeostasis was maintained. Fccp counteracted the protective effects on migration, tube formation, and Ve-cadherin/Icam/Vcam expression.
**Clinical Implications:** This study presents a promising therapeutic strategy for chronic diabetic wounds using a dual-loaded hydrogel that addresses multiple pathological features simultaneously. The sustained release system overcomes the limitation of rapid exosome clearance. The mechanistic link to mitochondrial fission provides a new therapeutic target. The hydrogel's self-healing, injectable, and conductive properties make it clinically adaptable for irregular wound shapes. However, translation requires further validation in larger animal models and human trials, assessment of long-term safety, and manufacturing scalability.