Enhanced Mitochondrial Vability via Engineered Hydrogels for Intrathecal Spinal Cord Delivery

Mitochondrial dysfunction is pivotal to the neuropathological sequelae following traumatic spinal cord injury (SCI). We have documented that maintaining endogenous mitochondrial bioenergetics with acetyl-l-carnitine (ALC), an alternative mitochondrial biofuel, or reducing oxidative stress by replenishing endogenous antioxidant, glutathione (GSH) with N-acetylcysteine amide (NACA) results in increased long-term functional neuroprotection post-SCI. In the current proposal, we will test neuroprotective efficacy of novel combinational therapeutic strategy to administer ALC and/or NACA systemically after contusion SCI, followed by intrathecal transplantation of mitochondria via controlled-release from polymeric hydrogels (MitoTxp). We have reported that acute intraspinal MitoTxp isolated from rat soleus muscle significantly preserves bioenergetics function 48hr post-SCI, like ALC/NACA; but this did not translate into long-term functional recovery. Since direct intraspinal injections are limited in distribution, we are proposing to deliver mitochondria intrathecally at the site of injury; locally applied mitochondria are able to diffuse into the spinal cord parenchyma by uncertain mechanisms. We hypothesize that the combination of systemic injections of ALC and NACA, in conjunction with intrathecal MitoTxp will help preserve both grafted and host cellular bioenergetics additively to promote tissue sparing and functional recovery. Moreover, injectable polymer hyaluronic acid/methyl cellulose hydrogels fabricated to protect healthy mitochondria will be used for localized diffusion. Proposed studies will establish 1) optimum constituents for isolated mitochondria to remain viable for extended periods in polymeric hydrogels, 2) whether exogenous mitochondria transplanted via less invasive subdural route equally preserve integrity of bioenergetics compared to intraspinal route and 3) consequences of acute or delayed MitoTxp in combination with ALC and/or NACA on bioenergetics, oxidative stress, and functional neuroprotection at sub-acute stages of contusion SCI. Specific Aim 1a: Determine key features of cytosolic environment that aide in maintaining and improving mitochondrial viability and efficacy Specific Aim 1b: Generate a Biomimetic Hydrogel cytosol to serve as Recapitulate these within a polymer network to create a biomimetic cytosol Specific Aim 1c: Test the hypothesis that subdural MitoTxp will significantly maintain bioenergetics after SCI. Specific Aim 2: Test the hypothesis that mitochondrial-targeted pharmaceuticals in combination with MitoTxp ameliorate acute and sub-acute biochemical and cellular outcome measures compared to individual therapies