Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Leveraging Biomaterials to Direct Immune Function in Cancer and Autoimmunity(2022) Tsai, Shannon Joanne; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Immune dysregulation and difficulties in directing immune function in cancer and autoimmune disease pose complex challenges for existing vaccines and immunotherapies. In cancer, tumor cells exploit processes to evade the immune system. Conversely, autoimmune diseases such as multiple sclerosis (MS) occur when immune cells incorrectly attack healthy host tissue and cells. To address the dichotomy of dysregulated immune responses that can arise, next generation vaccines and immunotherapies demand better control over the specificity and types of immune of responses generated within lymph nodes (LNs). This dissertation investigated two approaches to improve immune signal delivery for precision control over immune responses. In the first approach, self-assembling vaccine nanoparticles were engineered with tunable charge and cargo loading to efficiently deliver immune signals in specific combinations and doses without compromising function. These studies offer new insight into biomaterial design for therapeutic cancer vaccines and demonstrate that the physiochemical properties of biomaterials - particularly the interplay between charge, uptake, and affinity - play an important role in the immune signals that can promote T cell expansion against tumor antigens. In the second approach, a biomaterial-based platform is used to control immune signal delivery to LNs during autoimmunity. Direct injections of therapeutic vaccine carriers into the LNs of mice offer new insight into how the localized combination of myelin peptide (MOG) and rapamycin (Rapa) - an immunomodulatory signal, promote potent and selective immune tolerance. This body of work demonstrates that immune function is highly localized to the signals delivered to the LNs, requiring an idealized combination of both self-antigen and immunomodulatory signal to promote the proliferation, retention, and polarization of antigen specific T cells towards regulatory T cells that can selectively limit inflammatory T cell phenotypes and combat autoimmunity. Together, these two approaches offer new insight into how biomaterials can be rationally harnessed to direct immune function across cancer and autoimmune disease.Item Biomimetic Polymer Capsules: Novel Architecture and Properties(2021) Ahn, So Hyun; Raghavan, Srinivasa R.; Bentley, William E.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This study focuses on polymer capsules made from biocompatible, water-soluble polymers. Typically, the capsule core is a hydrogel in which proteins, nanoparticles, or biological cells can be encapsulated, while the capsule shell is permeable to small, but not large molecules. We explore two new designs or architectures for such capsules. One is a multi-compartment capsule (MCC) where a capsule has several distinct compartments inside it. A second design is a multilayer capsule, where concentric layers of different chemistries surround a core. These new designs mimic structures commonly found in nature such as a eukaryotic cell or an onion. Our goal is to exploit these novel capsule architectures to achieve new or improved properties. In our first study, we introduce a new kind of multilayer capsule, wherein a protective shell of covalently crosslinked polymer (acrylate) surrounds a core formed by physical crosslinking (alginate). Alginate capsules are widely used for cell-encapsulation, but they are quite weak. We show that a covalent acrylate shell can be added to these capsules in a single step under mild conditions. The shell protects the core from degradation while allowing the encapsulated cells to remain viable and functional. A variation of the synthesis technique yields capsules with two concentric shells (alginate, then acrylate) surrounding a liquid core. Next, we create MCCs in which microbes from two different kingdoms, i.e., bacteria (Pseudomonas aeruginosa) and fungi (Candida albicans), are placed next to each other in distinct inner compartments. This MCC platform holds advantages over traditional co-culture as it eliminates physical contact between the two microbes and allows for real-time monitoring of cell growth in 3D. Using this platform, we study the effects of both physical variables (e.g., pH) as well as chemical additives (e.g., surfactants) on the growth of the two populations. We also detect crosstalk between the bacteria and fungi, i.e., as the bacteria grow, they inhibit the formation of hyphal filaments by the fungi, which make the fungi less invasive. Lastly, we create MCCs with ‘smart’ inner compartments, which are sensitive to various stimuli. An analogy is drawn to different organelles in a cell, which have different constituents and unique functions. We select the chemistry or architecture of each inner compartment of the MCC such that their responses are distinct and orthogonal. For example, one compartment alone breaks apart when the MCC is contacted with an enzyme, while another gets degraded by the introduction of hydrogen peroxide (H2O2), and a third is disrupted by ultraviolet (UV) light. Another concept is shown where the degradation of one compartment induces the degradation of another. We believe these new designs will make the MCC platform more attractive for various biological applications.Item Formulation and Delivery of Enhanced Extracellular Vesicles for Wound Repair(2021) Born, Louis Joseph; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. Extracellular vesicles (EVs) have recently emerged as an alternative approach to address this issue. In particular, EVs derived from mesenchymal stem/stromal cells (MSCs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. However, despite their clearly established potential, EVs have limitations that limit clinical relevancy, including low potency and rapid clearance from the body. Additionally, the ability to sustainably deliver EVs may enhance their efficacy in wound healing. Here, we leveraged the capability of EVs to be engineered via producer cell modification to investigate the therapeutic potential of EVs from MSCs transfected to overexpress a well-established pro-angiogenic long non-coding RNA HOX transcript antisense RNA (HOTAIR). We established that HOTAIR was able to be successfully loaded into MSC EVs (HOTAIR-MSC EVs) and delivered to endothelial cells in vitro with increased functional angiogenic activity. HOTAIR-MSC EVs injected intradermally around excisional wounds also showed increased angiogenic activity in vivo in two different species of rodents and improved wound healing in diabetic mice. We further report biomaterial-enabled sustained release of EVs using injectable hydrogel nanoparticles containing a composite of thiolated hyaluronic acid, maleimide functionalized poly(ε-caprolactone), and polyethylene glycol tetraacryalte as well as 3D-printed hydrogel discs composed of gelatin methacrylate for topical application. EVs released from the formulation of both of these biomaterials retained angiogenic bioactivity. Nanoparticles containing HOTAIR-MSC EVs were injected intradermally around an excisional wound in diabetic mice and were able to increase angiogenesis and improve wound healing. EVs released from 3D-printed EV-loaded GelMa hydrogels retained bioactivity in an in vitro endothelial scratch assay. Overall, these data suggest increasing the content of lncRNA HOTAIR in MSC EVs as a promising wound healing therapeutic. Additionally, establishing a biomaterial-enabled sustained release therapeutic represents a promising translational product for clinical implementation.Item Therapeutic contact lenses for extended drug delivery(2021) Torres Luna, Cesar Eduardo; Wang, Nam Sun; Briber, Robert M; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)There is significant interest in hydrogel-based drug-eluting contact lenses as platforms for topical ocular drug delivery. These devices have shown to provide an increased residence time of drugs at the surface of the eye, leading to enhanced bioavailability (~ 50%) when compared to eye drops (1–5%). One major limitation of contact lenses for drug delivery is that most drugs are released in a few hours, which limits their application for extended delivery. In this dissertation, we develop novel drug-eluting contact lenses that are capable of achieving extended in vitro drug delivery. In our first study, we describe the application of drug-participating catanionic aggregates in poly-(2-hydroxy-ethyl-methacrylate) based contact lenses. Contact lenses embedded with catanionic aggregates can achieve extended delivery of at least 1-week for two anionic drugs. Release kinetics is significantly dependent on the drug’s octanol-water partition coefficient, the hydrocarbon chain length and concentration of the cationic surfactant. Next, we focus on the use of unsaturated fatty acids in commercial contact lenses to extend the release of three cationic drugs. We demonstrate that lenses loaded with oleic acid can extend drug release kinetics to over 1 month. An opposite effect is seen for two anionic drugs, where oleic acid significantly accelerates release kinetics. These studies confirm the dominating impact of coupling charge interactions between drug molecules and fatty acid carrier molecules in contact lenses to adjust drug delivery rates. Finally, we extend the application of fatty acids in contact lenses to evaluate the effect of hydrocarbon chain length, ionic strength, and pH on the release kinetics. It is shown that fatty acids with carbon chain lengths equal or greater than 12 are capable of extending drug release of two cationic drugs, which confirms the importance of hydrophobic interactions with the silicone domain of the gel matrix. By decreasing ionic strength (from 1665 to 167 mM) or increasing the pH of the release media (from 5.5 to 7.4), release kinetics is significantly extended. In summary, the use of fatty acids to control the release of oppositely charged drug molecules represents a versatile tool to modify contact lenses for drug delivery applications.Item Leveraging Biomaterial Properties to Reprogram Immune Function in Autoimmunity(2020) Gosselin, Emily A; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Autoimmune diseases occur when immune cells incorrectly identify and attack the body’s tissues as foreign. In Multiple Sclerosis (MS), the immune system targets myelin, the protective layer that insulates nerves. Current MS therapies reduce disease severity without treating the cause, requiring frequent treatments to slow disease progression. Further, existing therapies cannot differentiate between dysfunctional myelin-reactive inflammatory cells and normal lymphocytes, leaving patients vulnerable to infection. To overcome these limitations, this dissertation investigated biodegradable polymeric microparticles (MPs) co-loaded with myelin peptides and rapamycin, an immunomodulatory signal. Directly injecting these tolerogenic MPs into key immune tissues (e.g. lymph nodes, LNs), induces myelin-specific regulatory immune cells that selectively control myelin-specific inflammation. This work aimed to advance pre-clinical studies and motivate clinical research in two ways: investigating the systemic impact of intra-LN tolerogenic MPs in two MS models, and enhancing MP stability using Chemistry, Manufacturing, and Controls (CMC) considerations. This work showed that across both progressive and relapsing-remitting disease, one tolerogenic intra-LN treatment promoted long-lasting improvements in disease-induced paralysis. Tolerogenic MPs delivered prior to symptom onset promoted tolerance and protected against disease. Treatment at peak disease reversed paralysis and prevented relapse, while treatment during relapse limited disease progression. Strikingly, mice vaccinated against a foreign protein on the same day as intra-LN treatment generated protein-specific T cells and antibodies at similar levels to healthy vaccinated mice, while simultaneously exhibiting significantly reduced paralysis – highlighting the myelin-specific nature of this therapy. While the low dosage requirements of these studies allowed for on-demand preparation, clinical translation requires investigation into manufacturing, preservation, storage, and stability of this immunotherapy. Thus, this dissertation also tested the impact of lyophilization (freeze-drying) and excipients (stabilizing molecules) on MP stability after storage. Lyophilization with low concentrations of excipients significantly improved MP stability and formulation recovery after reconstitution. Storage for 5 months at room temperature did not negatively impact cargo loading, MP size, or biofunctionality. MP formulations with excipients could deactivate inflammatory signaling and restrict myelin-specific immune cell proliferation as well as formulations without excipients. Together, these studies motivate the development of intra-LN delivery of tolerogenic MPs as a potential MS immunotherapy for clinical translation.Item DEVELOPMENT AND OPTIMIZATION OF A P47-BASED PLASMODIUM VACCINE TO BLOCK MALARIA TRANSMISSION(2020) Yenkoidiok-Douti, Lampouguin; Barillas-Mury, Carolina; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Malaria is an infectious disease caused by Plasmodium parasites that are transmitted to hosts by infected Anopheles mosquitoes. Over the last two decades, the widespread deployment of effective interventions, such as drugs and insecticides, has resulted in significant reductions of malaria cases. However, without an effective vaccine, the recent emergence of drug-resistant parasites and insecticide-resistant mosquitoes are threats to this progress, motivating the need for newer tools to control and ultimately eliminate malaria. Recently, reducing Plasmodium transmission from humans to mosquitoes has become an actively pursued approach to eradicate malaria. One unique strategy to achieve this goal is through transmission-blocking vaccines (TBVs). TBVs generate antibodies in immunized individuals that are transferred to mosquitoes during a blood meal to block the Plasmodium life cycle. Recently, our laboratory discovered that the P. falciparum surface protein P47 (Pfs47) allows parasites to evade mosquito immune system. This makes Pfs47 critical for the parasite’s survival, and a valuable target for a TBV. The work in this dissertation reveals the potential of P47 as a TBV target in two models of malaria. In the first aim, the development, optimization, and efficacy of a P47 vaccine were investigated using Pfs47 as an antigen. Recombinant Pfs47 protein was expressed in Escherichia coli, and vaccine immunogenicity was assessed in mice. Antibodies targeting a key region of Pfs47 reduced Plasmodium density in mosquito. This result supports TBV as an effective approach to control the spread of malaria. Since delivering vaccines using traditional injection is challenging in developing countries, new technologies that improve vaccine accessibility are also needed. Thus, Pfs47 vaccine was loaded into microneedles, dissolvable micron-scale structures, and tested for function. In the second aim, the efficacy of a P47 vaccine was evaluated in a challenge model of malaria using the Plasmodium berghei mouse malaria antigen Pbs47. The key region in Pbs47 where antibody binding confers protection was mapped. This in vivo system provides preclinical evidence that a vaccine targeting Pfs47 could be effective in humans. Together, this thesis presents P47 as a new malaria vaccine target and introduces MNs as an effective platform to deliver vaccines in resource-poor settings.Item ENGINEERING THE LYMPH NODE MICROENVIRONMENT TO MODULATE ANTIGEN-SPECIFIC T CELL RESPONSE(2019) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccines and immunotherapies have provided enormous benefit to human health. However, the development of effective vaccines and immunotherapies for many diseases is hindered by challenges created by the complex pathologies of these targets. For example, in cancer the tumor microenvironment suppresses the function of tumor-specific T cells. In autoimmune diseases, lymphocytes specific for self-antigens attack self-tissue. New technologies providing more sophisticated control over immune response are needed to address these challenges. Lymph nodes (LNs) are tissues where adaptive immune responses develop. Therefore, local delivery of combinations of immune signals is a potential strategy to modulate antigen-specific T cell response for pro-immune or regulatory function. However, application of this idea is hindered since traditional administration routes provide little control over the kinetics, combinations and concentrations with which immune signals are delivered to LNs. Biomaterials have emerged as important tools to overcome these challenges as they provide unique capabilities, including co-delivery, targeting, and controlled release. The research presented here harnesses biomaterials to control immune signals present in LNs to modulate antigen-specific T cell response. In one area, intra-LN injection (i.LN) was used to deposit microparticles (MPs) encapsulating tumor-antigens, adjuvants and immunomodulators to promote tumor-specific central memory T cells. These cells display increased proliferative capacity and resistance to tumor-mediated immunosuppression. MPs encapsulating CpG, an inflammatory adjuvant, and a melanoma antigen potently expanded tumor-specific T cells. MPs delivering low doses of rapamycin – a regulatory immune signal – promoted tumor-specific central memory T cells when co-delivered with the melanoma vaccine. Another important aspect of T cell phenotype which can be modulated for therapeutic benefit is regulatory immune response to control autoimmunity. In this second area, biomaterial-based strategies were used to deliver regulatory immune signals to expand regulatory T cells (TREG) and promote immune tolerance. In one direction, liposomes were designed to deliver regulatory metabolic modulators to bias T cells. In a parallel direction, MPs encapsulating rapamycin and islet self-antigens were designed to promote tolerance in T1D. i.LN delivery of MPs expanded islet-specific TREG and inhibited disease in a mouse model of T1D. Together this work demonstrates potent and modular strategies to therapeutically modulate T cell response.Item DESIGN OF ENZYME CASCADE AND CRISPR-CAS9 NETWORKS FOR ENABLING BIOELECTRONIC COMMUNICATION(2017) Bhokisham, Narendranath; Bentley, William E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)There is an increasing emphasis on building closed loop systems in human health where real time monitoring and analysis is connected to feedback and treatment. Building such systems requires bridging the information loop across the different signal modalities of biology and electronics. In this work, I have created two different networks at biology-electronic interface to enable the communication from biology to electronics and vice versa. The first network is a multi-step enzyme cascade assembled on a microchip to enable conversion of biologic information into electro-chemical information. I first devised a modular construction approach using microbial transglutaminase (mTG) based conjugation chemistry where multiple enzyme components are assembled on an abiotic surface in a ‘plug and play’ fashion. Integration of bio-components with electronics requires a scaffold material for functionalization of the bio-electronic interface. To address this challenge, I engineered a self-assembling Tobacco Mosaic Virus-Virus Like Particle into a 3D scaffold displaying desired functional groups at the interface. Using the 3D scaffolds and mTG mediated conjugation chemistry, I assembled a synthetic 3-enzyme cascade on a microchip for conversion of methyl cycle intermediates into homocysteine, an electrochemically readable molecule. The modular construction approach and the scaffold materials that I developed can enable facile assembly of multi-subunit bio-components and diversify the range of metabolites that can be detected on a microchip for use in biosensing applications. Next, I focused on mediating communication from electronics to specific genes in the genome of biological systems. An electrogenetic promoter that is responsive to the electrical stimuli was reported in E. coli. In this work, I integrated the precise gene targeting capabilities of the CRISPR-Cas9 system with the electrogenetic promoter to target specific host transcriptional processes. I displayed temporal silencing of several host defense mechanisms against the electrical signals resulting in an overall positive feedback for electrogenicity in E. coli. A more sophisticated control of host transcriptional processes by the CRISPR-Cas9 system is a valuable addition to the existing electrogenetic toolbox, one that could enhance the interoperability of electrogenetic systems and mediate bio-electronic communication between strains and species.