Brain cells depend on the extracellular matrix (ECM) for structural and functional support as well as sequestration and transport of key ions and neurotransmitters. Structural and compositional changes to the ECM occur in development and in response to injury and disease. Probing ECM structure and composition in real-time in a dynamic living brain would enhance our understanding of the ECM changes that drive disease. In our work, we use organotypic whole-hemisphere (OWH) brain slices to study the interaction between brain parenchymal cells and the ECM. We have applied multiple-particle tracking (MPT), an imaging technique that tracks movement of nanoscale probes with sub-micron resolution, to OWH slices exposed to different stimuli, including oxygen-glucose deprivation (OGD) and mitochondrial dysfunction by rotenone (ROT) exposure.  Our MPT data confirmed that ECM microstructure changes in a time and stimuli-dependent manner and this was associated with changes in cellular composition and morphology. In this study, we measured changes in expression of ECM transcripts using Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) of RNA isolated from OWH brain slices exposed to OGD and ROT. After exposure to 30 minutes of OGD or treated with 50 nM ROT, OWH slices were preserved at 2h and 24h in RNALater buffer for RNA isolation. The 2h time point aligns with the end of the MPT experimental window. Healthy unexposed OWH slices were controls. We measured expression of genes associated with ECM composition and remodeling including tenascin-R, aggrecan, neurocan, MMP9, and TIMP1; markers associated with cellular activation including Ki67, Cd45, Cd11B, and GFAP; inflammation markers including IL-1β, IL-4, IL-6, IL-9, and IL-10; and cell death markers including iNOS, nNOS, TNF-α, and Casp-3. Our results provide a quantitative measure of ECM composition that can be integrated with our MPT and imaging data to better define microstructural dynamics in the stimuli-exposed brain.

Multiple Particle Tracking (MPT) is a powerful technique for studying microscopic particles, such as viruses and nanoparticles, by tracking individual displacement and movement. One application of MPT is to measure microstructural changes in the brain extracellular environment (ECM) in development, aging, and disease progression. MPT of nanoparticle probes generates thousands of trajectories, from which geometric features, diffusion coefficients, and viscosities can be extracted. The vast array of trajectories presents an opportunity for deep learning models to uncover meaningful insights. However, to enable MPT data to be trainable and predictable by deep learning models, we need to curate the data to be useable by these models. To enable this, I have created a database and developed a data architecture that would allow MPT data to be useable within deep learning models. Building upon this foundation, I am currently working on creating a Self-supervised deep learning model utilizing equivariant graph neural network, equivariant transformer, and Explainable AI methods. The current iteration of this model can predict a masked point of a trajectory with a 34% error rate. The goal is to reduce this error to 10% and, more importantly, to differentiate between healthy and pathological trajectories. To achieve this, we will use Saliency Maps, an Explainable AI method, to understand how the model distinguishes between these two datasets. This approach will provide insights into which part of the trajectory the model finds most relevant. My hypothesis is that the model can effectively learn to distinguish between healthy and pathological trajectories based on the trajectory properties with an error rate of 10%. I will verify my model by modifying the trained model’s output layer to explicitly classify trajectories as healthy or pathological. By fine-tuning this model, we will evaluate performance using error metric, which I will further validate using Saliency Map visualizations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that damages motor neurons, leading to severe disability within 1-3 years of diagnosis. Though its precise mechanism is unknown, chronic microglial activation has emerged as a hallmark of ALS pathophysiology. This results in persistent neuroinflammation and a positive feedback loop of cell death. Anti-inflammatory drugs could help restore microglia to a neuroprotective state. However, delivering these therapeutics across the blood-brain barrier and into disease-mediating cells presents a major challenge. Our prior work demonstrated that poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles can overcome barriers to the brain in models of neurodegeneration such as Huntington’s disease. PLGA-PEG nanoparticles further exhibit localization and uptake in microglial cell populations. In this study, we aimed to develop PLGA-PEG nanoparticles for targeted delivery of danirixin (DNX), an anti-inflammatory agent, in ALS. We formulated DNX-loaded PLGA-PEG nanoparticles (PLGA-PEG/DNX) with different mixed organic solvents via sequential nanoprecipitation. Nanoparticle characterizations included dynamic light scattering for size, dispersity, and surface charge determination. We quantified drug loading and release using liquid chromatography-mass spectroscopy. PLGA-PEG/DNX achieved physical properties for effective brain delivery, including a small hydrodynamic diameter (<100 nm) with narrow dispersity (<0.20) and near-neutral surface charge (-10-0 mV). We identified an optimal mixed organic solvent system for synthesizing PLGA-PEG/DNX with high drug loading (>30%) and encapsulation efficiency (>70%). We further show that DNX retains activity following PLGA-PEG encapsulation with suitable lyophilization stability for in vivo administration. Future work will evaluate dose response, therapeutic efficacy, and pharmacokinetic properties for PLGA-PEG/DNX in pre-clinical ALS models. Successful completion of this study could help advance nanoparticle-based therapies into ALS clinical trials.

Nanoparticles are drug delivery carriers on the nanometer-length scale, and are promising targeted drug delivery solutions due to their small size and tailorability. However, current materials used to produce nanoparticles are synthetic and typically lead to large amounts of chemical waste and high costs. To explore more sustainable technologies, the Nance and Roumeli labs established a novel bacterial cellulose nanoparticle (BCNP) platform. BCNPs are formulated with a bacteria that produces cellulose and no byproducts when cultured, allowing for less reagents required and non-toxic biodegradable wastes. To be comparable to synthetic nanoparticles as a drug delivery platform, BCNPs must load and release drugs and be biocompatible with mammalian cells. In this project, I explored the tunability of BCNPs through size modification, performed cytotoxicity studies on a microglial cell line, and carried out drug loading studies. I found that higher mixing speeds during BC culturing led to a smaller BCNP size and variable particle concentration. Through cytotoxicity analysis in cell culture, I showed BCNPs were not toxic. Ongoing studies are assessing BCNP cytotoxicity as a function of BCNP dose. To demonstrate drug loading, I am incorporating catalase, an enzyme with the ability to mitigate oxidative stress markers, into BCNPs to analyze their efficacy in an in vitro model of oxidative injury. These results show BCNPs have the potential to become a sustainable nanomedicine platform and provide an important step towards reducing the environmental impact of synthetic nanoparticles.

Therapeutic delivery to the brain is challenging due to restrictive barriers such as the blood-brain barrier and the brain-parenchymal barrier. Although nanoparticles help overcome these barriers and improve therapeutic uptake, many nanoparticles are developed from synthetic materials and generate significant harmful waste. Bacterial cellulose nanoparticles (BCNPs) offer a sustainable alternative to current synthetic carriers. As a new platform, evaluating cytotoxicity and localization is essential to determine BCNP biocompatibility and potential for targeted drug delivery. To produce BCNPs, a BC pellicle was grown with gram-negative bacteria in the presence of yeast and washed with sodium hydroxide and deionized water. The BC was chemically and mechanically dissolved via sonication with dimethylacetamide and lithium chloride. Then, the BC dissolution media was added dropwise into a Pluronic F127 surfactant solution at room temperature and incubated for 2 h under stirring conditions to produce BCNPs. After washing and filtration, BCNPs were ~100 nm in size, had a slight negative zeta-potential, and demonstrated a polydispersity index <0.3, all parameters necessary for brain-targeting drug delivery. BCNPs were labeled with varying concentrations of carbotrace 680, a fluorescent dye used to specifically label cellulose materials. Cytotoxicity of BCNPs was assessed using healthy 10-day-old postnatal rat brain slices cultured for 4 days in vitro. BCNPs were topically applied to the brain slices (n=3 per experimental condition) at doses of 97 µg/mL – 290 µg/mL and incubated for 24 h. Slices were stained with propidium iodide (PI) before fixation and 4’,6-diamidino-2-phenylindole after fixation and imaged on a confocal microscope to quantify PI+ cells and determine BCNP localization. BCNPs resulted in <20% cytotoxicity at the applied doses confirming BCNPs do not cause cell death. These results demonstrate BCNPs are biocompatible and a promising alternative to synthetic carriers for drug delivery to the brain.

Bacterial cellulose (BC) nanoparticles (BCNPs) are a promising sustainable nanomedicine platform for drug delivery and provides a scalable, eco-friendly alternative to synthetic counterparts. We aim to develop a small library of BCNPs with different chemical moieties to incorporate a broad range of active agents for drug delivery use. To produce BCNPs, a BC pellicle is grown in a kombucha media of tea, sugar, vinegar, and bacterial co-cultures. The pellicle is isolated and chemically and manually broken down using dimethylacetamide, lithium chloride, and an ultrasonicator probe to produce an organic BC dissolution. The BC dissolution is precipitated into an aqueous Pluronic F-127 (F127) surfactant solution under 650 rpm stirring conditions and incubated for 2 h to form nanoparticles ~100 nm, near neutral charge, and low polydispersity index (<0.3). In this study, we optimize the dissolution and nanoprecipitation processes using acetylated and methylated BC pellicles to form acetyl- and methyl-functionalized BCNPs. The functionalized BCNPs were characterized using Fourier transform infrared spectroscopy, nanoparticle tracking analysis, electron microscopy, and light scattering to assess physicochemical properties. Our results demonstrate that functionalized BCNPs can be formulated using similar formulation parameters to unmodified BCNPs. Ongoing work evaluates drug loading and encapsulation efficiencies in the functionalized BCNPs using curcumin as a model drug. Engineering BCNPs with different chemical moieties enables incorporation of a wider array of drugs, which can improve the utility of BCNPs as a sustainable alternative to current synthetic nanomedicines.

Hypoxic ischemic encephalopathy (HIE) is a neurological condition resulting from reduced blood and oxygen flow to the brain and is a leading cause of morbidity and mortality in neonates. Limited treatment options necessitate accessible and scalable interventions to improve outcomes in newborns impacted by HIE. Extracellular vesicles (EVs) have been previously shown to attenuate oxidative stress and inflammation in the brain. Further research suggests that EVs secreted by astrocytes, a brain cell type involved with the inflammatory and injury response, may elicit neurotrophic or neuroprotective properties. In this study, I isolated, characterized, and evaluated the therapeutic potential of astrocyte-derived EVs (AEVs) in an ex vivo model of hypoxic-ischemic (HI) brain injury. AEV characterization via protein assays and nanoparticle tracking analysis showed that we were able to produce AEV particles about 100 nm in size at concentrations up to 10^11 particles/mL. To assess their therapeutic efficacy, I administered AEVs at varying doses (5, 12.5, 25, and 50 µg) to neonatal rat brain slices exposed to oxygen-glucose deprivation (OGD), an ex vivo model for HI injury. Following 24h of exposure, I evaluated cell viability. Our results indicate that AEVs decrease cytotoxicity in a dose-dependent manner. To further elucidate AEVs’ mechanisms of action, we conjugate AEVs with quantum dots to track AEV localization and cell-type specific uptake in brain tissues. Understanding AEV interactions with neural cells provides insight into both the roles of AEVs and different brain cells in modulating inflammatory responses and promoting neuroprotection. By characterizing AEVs and their therapeutic potential, these findings contribute to the growing body of research on EV-based therapeutics and lay a foundation for developing reliable and scalable therapies with the potential to advance treatments for neurodevelopmental disorders and aid brain injury recovery.