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.

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.

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.

Tumor angiogenesis is characterized by unregulated blood vessel formation, impairing vascular networks and biological transport. It represents a critical stage in cancer progression, where malignant tumors metastasize and exploit the human body’s resources, which lie in vascular networks. However, the complex tumor microenvironment presents significant challenges in studying tumor angiogenesis and identifying its biomarkers. Towards addressing this concern, hydrogels—water-swollen, polymeric networks—can be used to recapitulate the tumor microenvironment, whose physiochemical properties can be precisely tuned to match that found in vivo. The DeForest Lab has developed methods and techniques in bioorthogonal chemistry and light-based subtractive manufacturing to tune such hydrogel materials with precise and unique 4D control, all at subcellular resolutions. In this project, I will exploit image-guided multiphoton lithography to engineer natively complex tumor vasculature patterns within photodegradable hydrogels. We will further embed tumor vascular spheroids within these hydrogels, providing a platform to model and assay tumor progression in vitro. This study has exciting implications for translational research and preclinical studies, particularly for disease modeling and therapeutic screening, as well as reducing ethical concerns regarding tissue and animal models in preclinical studies.

Biological processes rely on the intricate functions of proteins, which drive essential biochemical reactions. Given their critical role, various methods have been developed to regulate protein functions in biomaterials and in vitro. Enhancing the precision of gene editing is crucial for advancing applications in gene therapy and minimizing off-target effects. My project focuses on integrating photoactivatable proteins with prime editors, a modified version of the widely known gene editor CRISPR/Cas9, to improve spatial and temporal control over gene modifications. By utilizing genetic code expansion, non-canonical amino acids are incorporated into human cells to express photocaged prime editor proteins and altering host genomes. This system enables optical stimulation to precisely regulate protein activity. Through the deployment of well-characterized photolabile groups, we expect to be able to render protein activity controllable in a dose dependent way. A key application of this approach is the development of a photoactivatable prime editor system to induce precise gene edits. Traditional CRISPR/Cas9 methods lack spatiotemporal control over activation. To address this, the system is adapted for use in hydrogels, where two-photon patterning allows visualization of prime editor protein activation in three dimensions. Our study aims to demonstrate the feasibility of optically controlling gene editing with high specificity, offering a novel strategy for advancing cell lineage tracing and gene therapy applications.

Architectural and spatiotemporal aspects of epigenetic regulation and cell behavior are critical for maintaining overall health. Unintentional genetic mutations can create dynamic dysregulation in the epigenome and transcriptomes at the cellular level which is implicated in diseases ranging from fibrosis to cancer. However, our tools to probe and understand these behaviors are limited by a lack of spatiotemporal control. To address this, we propose installing four-dimensional control over the potent CRISPR inhibition transcriptional effectors to establish epigenetic control at cellular scale resolutions.  CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic modification system that relies on Cas9 proteins to splice and deactivate genes as controlled by a programmable guide RNA sequence. CRISPR inhibition relies on a deactivated Cas9 protein that does not directly alter the genetic material in order to sterically hinder transcription. Our work aims to formulate a CRISPR inhibitor system which can unbind from the target DNA with two photon activation via a photo-cageable noncanonical amino acid insertion. This would allow for four-dimensional spatiotemporal control over the system, thus increasing the level of control in epigenetic regulation. Currently, work is being done to test the CRISPR inhibition system in HEK 293 cells that have been lentivirally transduced with both a test sequence and the deactivated Cas9 protein. After testing is completed for this simpler system, we will move toward creating a system that incorporates the photocaged noncanonical lysine variant, giving us control over the CRISPR inhibition system with regards to both space and time.