(379c) Mechanical Characterization, Release and Degradation of Hyaluronic Acid-Methyl Cellulose Thermogels for Viable Mitochondria Replacement Therapy

Spinal cord injury (SCI) can result in severe physical impairments where one is often left with limited to no motor or sensory function below the injury level. There are two sequential stages of traumatic damage to the spinal cord: 1) the initial onset of irreversible damage from trauma and 2) the surrounding inflammatory responses that further damages tissue over time. Cell loss in the surrounding tissue is attributed to impaired mitochondria, and once mitochondria are no longer viable the cell death cascade is accelerated. It has been shown that delivery of isolated viable mitochondria around sites of SCI results in their being taken up by various cell types and renders improved bioenergetics of the injured spinal cord tissues (Gollihue et al., J. Neurotrauma, 2018, 35(15): 1800-1818). While this burgeoning method seems promising, there are two potential issues to address: 1) intra-parenchymal accumulation of mitochondrial bolus injections cause mechanical perturbation and 2) isolated mitochondria have a limited viability extracellularly at body temperature (37ËšC). Therefore, a less invasive method which can also maintain mitochondrial viability extracellularly for extended periods of time appears warranted for effective delivery of healthy mitochondria to injured spinal cord.

The present study focused on the mechanical compatibility of an inverse thermal-gelation hyaluronic acid – methylcellulose (HAMC) hydrogel that was prepared for delivering isolated mitochondria. Manually loading the gel solution into syringes through the flange-end of the barrel while gently stirring in mitochondria proved to be sufficient in producing a homogeneous, injection-ready gel and mitochondria mixture. Rheological analyses were performed to study the inverse thermal-gelation behavior and swelling pressure of the gel. The high tan δ, which indicates predominant liquid-like behavior, demonstrates the gel to be injection-feasible at 4 ̊C, whereas its low value (showing solid-like behavior) at basal body temperature (37 ̊C) demonstrates it becoming localization-feasible. The gel’s swelling pressure was determined to be 30 Pa, which is well below pre-trauma (360 Pa and 930 Pa in rodents and human, respectively) and post-trauma (1200 Pa and 1750 Pa in rodents and human, respectively) intraspinal pressures. Fluorescently-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. Polymer erosion rates were studied with fluorescent dye modified HA and MC. Cumulative release profiling showed the beads releasing simultaneously with polymer release, establishing degradation to be an effective means for mitochondria release. Notably, we have found that a 1% HA – 1% MC thermogel can be successfully injected intrathecally in naive rats and remains localized proximal to the injection site. In summary, the injectability, degradation, thermal gelation, and swelling properties of the 1-1 HAMC gel indicate it to be a viable candidate for localized, controlled method of mitochondrial delivery to normal and injured tissues.