(540a) Synthesis and Optimization of Hyaluronic Acid-Methyl Cellulose Thermogel for the Controlled Release of Viable Mitochondria

During the initial time periods following a traumatic spinal cord injury (SCI), significant loss of mitochondria by the cells in the vicinity of contusion site results in reduced energy producing ability, subsequently contributing to accelerated cell death. It is hypothesized that transplantation of healthy mitochondria to failing cells can provide protections to diseased and injured cells after trauma, which is demonstrated for injured human lung and heart cells. However, mitochondrial transplantation requires that mitochondria to first be isolated from other cells (esp. muscle tissue) and the viability be maintained outside cells (extracellular environment) until they are taken up by the intended cellular targets. Direct injection of mitochondria around the contusion site does not result in full efficacy due to injection induced secondary injury and aggregate (bolus) formation. Here we propose that rapid, controlled delivery of viable mitochondria using temperature sensitive hydrogel (thermogel) can be an effective novel biological intervention technique, where the gel can also act as a sink for mitochondrial substrate, supporting (stabilizing) and synergistic chemicals to maintain key cytosolic features (osmotic pressure, reducing environment, proteins and substrate) outside the cells required for mitochondrial viability.

Here, an injectable erodible thermogelling system was prepared from hyaluronic acid (HA) and methyl cellulose (MC) for the localized controlled delivery of viable mitochondria and optimized in terms of swelling, release and mitochondrial viability. Initially, transport properties (mobility) of Mitotracker Green FM tagged mitochondria isolated from adult female Sprague-Dawley rat brain was analyzed in polymer solution (0-12 wt%) in isolation buffers or PBS with/without substrate (5 mM pyruvate , 2.5 mM malate and 10 mM succinate) using fluorescence correlation spectroscopy. Mitochondrial transport was slowed significantly by increasing polymer condition, which demonstrate the necessity for an erodible polymeric delivery system. HA-MC themogelling blends were prepared in PBS and isolation buffers separately at 4 °C and incubated at 37 °C with variable polymer blend ratio and total polymer concentration (0.5-3 wt%). Polymer blends are liquid at 4 °C, which becomes gel at 37 °C with transition time of <15 min. Fluorescent labeled latex beads (200 and 500 nm dia.) were incorporated in the pre-gel mixture to study the release kinetics during gel erosion using fluorescent spectroscopy. Comparatively faster release was found in isolation buffers in comparison to that in PBS. By tuning the polymer ratio between HA and MC, erosion and release time can be varied from less than an hour to several days. To study and compare polymer erosion rates with release, gel was synthesized using fluorescent dye modified HA and MC. Mitochondria release in 1% HA-0.5% MC gel in isolation buffers and PBS was also studied, which shows no difference based on release medium for sustained release of viable mitochondria up to 3 h. Most importantly, no aggregation or swelling of mitochondria was observed after release from the gel. Finally, mitochondrial viability was assessed by measuring their oxygen consumption rate (OCR) using Seahorse Bioscience XF^e24 flux analyzer, which showed that bioenergetic integrity of isolated mitochondria was maintained at 37 °C and HA-MC thermogels can be ideal carriers for localized and controlled delivery of viable mitochondria.