Advancements in HIV prevention include pre-exposure prophylaxis strategies (PrEP), which are not as effective for women due to poor partitioning of antiretrovirals (ARVs) to the female reproductive tract. Integrating ARV-releasing reservoirs with intrauterine devices (IUDs) offers a strategy for local sustained delivery to overcome the partitioning issue. Our lab investigates reservoirs containing polymer-drug conjugates (drugamers), where the HIV integrase inhibitor raltegravir (RAL) is covalently attached to a polymer through a hydrolyzable linker. A previously characterized RAL-polymer exhibited release over 30 days, which is insufficient for the targeted 1-3 years of IUD-mediated delivery. To address this kinetic problem, the drugamer linker chemistry was modified from an ester to an acetal carbonate. Since the rate-determining step of the acetal carbonate linker hydrolysis does not depend on the acidic RAL hydroxyl (pKa = 6.6), it was hypothesized that this acetal carbonate linker will slow the RAL release rate as opposed to the ester linker. An acetal carbonate-linked monomer of RAL was synthesized and led to a 30-fold reduction in hydrolysis rate. The corresponding drugamer was then synthesized via RAFT polymerization and characterized via NMR. In hydrophilic media, RAL released from the novel polymer significantly slower than in the current lab polymer, showing potential for lengthened duration of action in in vivo models. Future work includes measuring release from RAL-polymer in a matrix device for future IUD incorporation, assessing potential polymer cytotoxicity, and evaluating release rates in mouse models. These findings lay the groundwork for the development of long-acting formulations for sustained HIV prevention.

Neurodegenerative disorders, including Alzheimer’s, are characterized by the accumulation of fibril aggregates—made up of amyloid β-sheet peptides—which were historically thought to disrupt cellular function and contribute to neuronal death. Recent studies have revealed that these plaques are relatively benign; they are preceded by toxic oligomers—peptides that adopt a rare α-sheet secondary structure. These oligomers form decades before the appearance of plaques and have been linked to the neurodegenerative symptoms associated with these diseases. As precursors to full aggregates, toxic oligomers serve as valuable therapeutic and diagnostic targets. Custom peptides designed to bind to α-sheet toxic oligomers can be deposited onto the surface of a well plate to form the basis of a diagnostic assay. Similar to a sandwich ELISA, this soluble oligomer binding assay (SOBA) utilizes a two-antibody system to selectively detect the presence and relative concentration of α-sheet oligomers. In an effort to improve assay repeatability, we attempt to optimize the antibody system used in SOBA experiments. To evaluate assay performance, we test a variety of incubated Aβ oligomer samples and brain homogenates from transgenic mouse models to assess SOBA sensitivity and specificity. In the future, we aim to extend SOBA repeatability studies beyond Alzheimer’s to other aggregation-related disorders, such as type two diabetes and Parkinson’s. By improving the repeatability of this assay, we can enhance early detection methods for Alzheimer’s and related disorders. These experiments serve to develop a standard method for the detection of toxic oligomers, which could pave the way for future neurodegenerative disorder treatments and diagnostic strategies.

Traumatic brain injury (TBI) is one of the leading causes of death in young adults. It is initiated by loss of endothelial junctions during deterioration of the blood-brain-barrier. Investigation into endothelial barriers has been enabled by high-resolution imaging via confocal microscopy. However, existing image analysis tools struggle to capture the complexity of EC morphology due to their reliance on rigid segmentation, limiting their ability to extract meaningful insights. My project focuses on developing a more effective method for analyzing ECs by implementing a pixel-based computational tool that provides a more nuanced analysis of EC junction integrity and morphology beyond traditional segmentation methods. Immunofluorescence (IF) images were obtained from an in vitro model of TBI mimicking 3D brain microvessels which were treated with kinase inhibitors (KIs) to determine which kinases promote or hinder recovery. If ECs remained damaged after treatment, it indicate the inhibited kinase was essential for recovery. If they improved, the kinase was likely disruptive. To identify these effects of KIs on EC based on IF images, I developed a pixel-based clustering tool that analyzes junction intensity without forcing explicit segmentation. By analyzing pixel intensities relative to cell nuclei, I generated profiles of each vessel that represent the tightness of EC junction. I used an existing AI-based tool, Cellpose, to define nucleus masks and wrote a customizable Python script to compute pixel distances and intensity variations, providing a detailed, unbiased view of EC behavior. Preliminary findings suggest that this method enhances the accuracy and efficiency of cellular analysis by eliminating segmentation bias and capturing subtle morphological changes. Future work involves integrating this tool with regression models to identify kinases that regulate EC junction integrity. This research has broad applications in vascular disease modeling and drug discovery, offering a new approach for studying cell behavior and developing targeted therapies. 

Pulmonary Arterial Hypertension (PAH) is a deadly vascular disease, affecting the blood vessels of the lungs, with no existing cure. PAH is characterized by pulmonary arterial smooth muscle cell (PASMC) hypertrophy and hyperplasia, which increases resistance to blood flow within the pulmonary arteries, leading to rapid symptom progression and eventual death from right heart failure. My mentor and I hypothesize that defects in PASMC differentiation and alignment may contribute to PAH. To test whether alignment and phenotypic responses differ in patients with PAH, we designed a micropatterned collagen scaffold atop a glass coverslip. Explanted PASMCs from patients with PAH or failed donors (controls) were cultured on alternating 10-µm wide x 10-µm deep microchannels or unpatterned constructs and alignment, protein expression, and cellular morphology were compared across conditions. I evaluated 3 PAH and 3 control subjects and have collected preliminary data for each condition (control versus PAH), with three technical replicates each. Through these preliminary studies, I have demonstrated success of my model with consistent alignment observed on patterned substrates. Excitingly, PASMCs from patients with PAH expressed significantly decreased levels of the contractile protein, Calponin, when compared with control cells, including after responding to cues that promote alignment and contractility. This suggests that PAH PASMCs remain in an inappropriately synthetic or proliferative state. Moving forward, I plan to evaluate additional micropatterns by varying dimensions of rectangular and sine waves designs using an ablation protocol with a 2-photon microscope laser. Subsequent evaluation will include immunofluorescent staining of contractile and other SMC markers as well as transcriptomic evaluation of cellular responses to micropatterning. This work will enhance understanding of whether SMC abnormalities contribute to disease initiation and progression in PAH and will contribute to the broader effort of developing more complex models of pulmonary vascular disease.

VLA-4 is an integrin expressed on immune cells that plays an important role in their extravasation into tissues during an immune response. In the autoimmune disease multiple sclerosis (MS), pathogenic T cells extravasate and attack nerve cells by using VLA-4 to bind to VCAM-1, a cell adhesion molecule on endothelial cells that line blood vessels. Current treatments for MS rely on antibodies to bind VLA-4 and block its interaction with VCAM-1, thus preventing a pathogenic immune response. However, antibodies are expensive to manufacture, and their binding cannot be easily regulated to control drug-induced side effects. Aptamers are single-stranded DNA or RNA molecules that fold into sequence-defined structures capable of binding to their targets with affinities and specificities comparable to antibodies. Being chemically synthesized, they are much cheaper to manufacture and offer minimal batch-to-batch differences. Unlike antibodies, their binding in vivo can be rapidly reversed using a reversal agent, which could alleviate the side effects of disease treatments. However, aptamers have limitations in vivo: degradation by nucleases in blood serum, and rapid clearance into urine through the glomerular filtration barrier. This project focuses on the development of a VLA-4 aptamer for treating MS. We found that the VLA-4 aptamer prevents soluble VCAM-1 from binding to VLA-4-expressing leukocytes by flow cytometry. We then showed that the aptamer blocks VLA-4/VCAM-1 mediated leukocyte adhesion in vitro. We are currently assessing aptamer blockade of leukocyte transendothelial migration. We are also designing modifications to improve the stability of the aptamer for in vivo uses. Successful development of the aptamer will lead to an alternative treatment modality for MS with a potentially improved safety profile.

Therapeutic targets for monoclonal antibodies (mAbs) are currently limited to membrane proteins which consist of up to 30% of total proteins encoded by the human genome. The other 70% of cytosolic protein targets remain inaccessible inside the cell. Thus, research into intracellular protein delivery is critical to unleash the full potential of protein therapeutics. The Gao lab recently developed a highly efficient technology that allows small proteins to be directly delivered into the cytoplasm with minimal damage to the cell, by cholesterol tag. To further this research, we developed a new version of the tag via the covalent linkage of Coomassie Blue dye with 2-hexyldecanoic acid, branched alkyl chains. This new tag could deliver mAbs, specifically immunoglobulin G (IgG), labeled with fluorescent dye.