(394d) Antioxidant-Encapsulating Nanoparticles for the Treatment of Glutamate Excitotoxicity

Introduction: Despite the neurological advancements of the past century, neurological diseases still have no effective cures, and cost nearly $800 billion annually in the United States alone. Most neurological diseases, including stroke, traumatic brain injury, and Alzheimer’s, exhibit glutamate excitotoxicity that perpetuates injury. Glutamate is a major neurotransmitter used for neuronal communication but leads to neuronal death when in excess by over-activating glutamate receptors. The mechanism behind excitotoxicity involves an excessive influx of calcium and generation of reactive oxygen species (ROS), and subsequently leads to neuroinflammation in many cases. After death, neurons release their glutamate depots, promoting further neighboring neuronal death. Stopping the excitotoxic cascade is crucial for preventing additional damage and promoting recovery. However, the many obstacles to drug delivery to the brain, including systemic clearance, blood-brain barrier (BBB) transport, and brain parenchyma penetration, as well as the complex etiology of disease initiation and propagation complicate the study and effective treatment of excitotoxicity. The use of polymeric nanoparticles for encapsulating and delivering therapeutic cargo can help overcome several of these barriers. Still, discerning whether a therapeutic failed due to ineffective biochemistry as opposed to poor delivery is challenging in the complex in vivo environment. Testing therapeutics on simplified, yet representative models, provides a systematic approach towards evaluating drug efficacy. By utilizing ex vivo whole hemisphere brain slice models, we can assess therapeutic efficacy, nanoparticle biocompatibility, as well as probe for deeper understanding of excitotoxicity mechanisms in high-throughput fashion.

Materials and Methods: We developed an organotypic whole hemisphere brain slice model of excitotoxicity. In this model, we incubated slices in excitatory conditions and evaluated cytotoxicity via lactate dehydrogenase (LDH) release, mRNA profiles with real-time polymerase chain reaction (RT-PCR), and microglial and mitochondrial morphologies with immunohistochemistry. We next determined the therapeutic efficacy of antioxidant enzymes, namely superoxide dismutase (SOD) and catalase. Additionally, we formulated antioxidant-encapsulating poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-PEG) polymeric nanoparticles (AE-NPs) using the double emulsion method, and evaluated their therapeutic potential and enzymatic activity in degradative conditions.

Results and Conclusions: We obtained a characteristic profile of cell viability and disease environment for excitotoxicity-induced brain slices. After application of SOD, we observed decreased cell death and a return to healthy mitochondrial and microglial morphologies. This effect was not seen with catalase treatment. Additionally, we formulated nanoparticles with an antioxidant/PLGA core and a dense PEG coating to achieve a hydrophilic, bio-inert surface. The AE-NPs exhibited a near-neutral surface charge and sub-100 nm size, and extended enzymatic activity compared to un-encapsulated enzyme in vivo and in the presence of pronase in vitro. We further used ex vivo slices to identify PEG toxicity of our nanoparticles, and for optimization of a biocompatible formulation. Our work provides evidence of SOD efficacy and supports the use of NPs to better deliver therapeutics to combat excitotoxicity. Beyond promoting SOD-NPs against excitotoxicity, our findings encourage the systematic approach of screening therapeutics on ex vivo brain slice models to isolate, understand, and overcome neurological disease processes. The slice model retains the multi-cell type complexity of the brain while bypassing systemic and BBB obstacles. By evaluating efficacy and biocompatibility without confounding delivery issues, we can focus on individual therapeutic development bottlenecks to ultimately design an effective therapeutic against glutamate excitotoxicity.