THERMORESPONSIVE POLYMERIC HYDROGELS FOR MITOCHONDRIA TRANSPLANTATION

Mitochondrial dysfunction underlies a wide spectrum of debilitating conditions, including neurodegenerative and metabolic disorders as well as traumatic injuries such as spinal cord injury (SCI). Mitochondrial transplantation (MT) offers a promising therapeutic strategy by replacing damaged mitochondria with functional ones. However, clinical translation has been hindered by difficulties in maintaining mitochondrial viability outside cells and achieving targeted delivery into injured tissue. This dissertation focused on hyaluronic acid (HA)-based hydrogels as protective and tunable delivery platforms for MT. To tailor gelation behavior and enhance thermoresponsive performance, two HA copolymer systems were evaluated: hyaluronic acid-methylcellulose (HA-MC) and hyaluronic acid-poly(N-isopropylacrylamide) (HA-PNIPAAm). HA-MC blends were evaluated as thermoresponsive semi-gels with unique biocompatible physicochemical properties. These semi-gels exhibited reversible sol-gel transitions and an interpenetrating polymeric network that lowered the lower critical solution temperature (LCST) of MC and enhanced water retention. They enabled sustained release of mitochondria while preserving mitochondrial viability and oxidative phosphorylation capacity, as confirmed by oxygen consumption rate (OCR) and respiratory control ratio (RCR) analyses. While promising, HA-MC’s LCST was found to remain relatively higher than physiological temperature, limiting its practical applications. To address HA-MC limitations, PNIPAAm grafted Hyaluronic Acid (HA-PNIPAAm) copolymers were synthesized and systematically characterized using spectroscopic and thermal analyses, which confirmed successful grafting and phase transition dynamics. Mitochondria-loaded HA-PNIPAAm semi-gels preserved mitochondrial viability ex-vivo and exhibited a biphasic release profile, with an initial burst followed by a slower dissolution-driven phase. Tetramethylrhodamine ethyl ester (TMRE) and Seahorse assays demonstrated that encapsulated mitochondria maintained significantly higher membrane potential (ΔΨm) and Oxygen Consumption Rate (OCR) than free mitochondria at physiological conditions, indicating that the hydrogel microenvironment provides functional protection. In-vivo transplantation studies in a rat SCI models further showed that mitochondria delivered via HA-PNIPAAm semi-gels retained greater viability and respiratory activity compared to free mitochondria controls. Animals receiving encapsulated mitochondria exhibited restored ADP-stimulated and maximal respiration, effectively rescuing oxidative phosphorylation and reserve capacity in injured spinal cords. This work establishes thermoresponsive HA-PNIPAAm hydrogels as a robust and biofunctional delivery platform for mitochondrial transplantation. By combining structural tunability, protective encapsulation, controlled release kinetics, and validated therapeutic efficacy in vivo, these systems advance the feasibility of organelle-based therapies and highlight their potential for translation to SCI and other diseases driven by mitochondrial dysfunction.