Theses and Dissertations from UMD
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Item BIOMATERIALS REPROGRAM ANTIGEN PRESENTING CELLS TO PROMOTE ANTIGEN-SPECIFIC TOLERANCE IN AUTOIMMUNITY(2023) Eppler, Haleigh B; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune system is tightly regulated to balance the killing of disease-causing organisms while protecting host tissue from accidental damage. When this balance is disrupted, immune dysfunctions such as autoimmune diseases occur. Autoimmune diseases like type 1 diabetes and multiple sclerosis (MS) develop when self-tissue is mistakenly attacked and damaged by immune cells. For example, during MS, the immune system mistakenly attacks the myelin sheath that insulates neurons, causing loss of motor function and burdening patients and caregivers. Recent advances in immunotherapies offer exciting new treatments; however, even monoclonal antibody therapies cannot differentiate between healthy and disease-causing cells. Biomaterials provide powerful capabilities to help address these shortcomings. In particular, control over the concentration, duration, location, and combination of signals that are received by immune cells could be transformative in developing more selective immunotherapies that are safe and promote antigen-specific tolerance during autoimmune disease. This dissertation uses two biomaterial approaches to deliver regulatory cargo to antigen presenting cells (APCs). An important APC function is to detect disease-causing organisms by sensing pathogen associated molecular patterns (PAMP) through motif-specific receptors. CpG rich motifs are PAMPs that activate toll-like receptor 9 (TLR9) on DCs and B cells. TLR9 signaling activates B cells and DCs. In MS, TLR9 signaling is aberrantly elevated on certain DCs contributing to systemic inflammation. In MS, B cells signaling through the TLR9 pathwway induced the expression of more inflammatory cytokines as compared to B cells from healthy controls. Controlling this overactive TLR signaling restrains inflammation and is a possible tolerogenic therapeutic approach in MS. The first part of this dissertation uses biomaterials-based polyelectrolyte multilayers (PEMs) to deliver tunable amounts of GpG – an oligonucleotide that inhibits TLR9 signaling – to dendritic cells (DCs). These studies demonstrate that PEMs inhibit DC activation and reduce pathway-specific inflammatory signaling. Furthermore, this work demonstrates that these changes to DCs promote tolerance in downstream T cell development as shown by increasing regulatory T cells. These studies demonstrate this biomaterial delivery system selectively inhibits TLR signaling and DC activation. These changes to DCs promote myelin-specific T cells to adopt a regulatory phenotype, demonstrating a potential approach to developing tolerance inducing antigen-specific immunotherapies for MS. The second part of this dissertation uses a degradable polymer microparticle (MP) system to control the local microenvironment of lymph nodes (LNs). LNs are key sites in the development of immune responses. LNs are composed of different microdomains that coordinate immune cell interactions such as germinal centers (GCs), where B cells develop. These MPs are loaded with myelin self-antigen (MOG35-55) and an mTOR inhibitor, rapamycin (rapa). The MPs are designed to be too large to passively diffuse from the LNs; instead, they slowly degrade releasing encapsulated immune cues to immune cells within the lymph node (LN). Our previous work demonstrates this treatment approach induces antigen-specific tolerance in a preclinical model of MS, but the role of APCs – including DCs and B cells - has not been elucidated. This dissertation reveals that MP treatment alters key LN structural components responsible for interactions between cells in GCs. In addition, MPs alter interactions between B cells/DCs and T cells, as measured by presentation of encapsulated antigen and inhibition of T cell costimulatory molecules by encapsulated rapa. These changes inhibit myelin-specific T cell proliferation and promote regulatory T cells. Finally, B cells from MOG/rapa and MOG MP treated lymph nodes transfer myelin-specific efficacy to mice induced with EAE. These findings illustrate how LN and cellular processes can be regulated by MPs to promote myelin-specific tolerance informing the development of myelin-specific immunotherapies for MS. Together, this body of work provides insight into how biomaterials can be designed to exploit native LN and immune cell functions in the design of next-generation approaches to safely induce myelin-specific tolerance during MS or other autoimmune diseases.Item LEVERAGING SELF-ASSEMBLY AND BIOPHYSICAL DESIGN TO BUILD NEXT-GENERATION IMMUNOTHERAPIES(2022) Froimchuk, Yevgeniy; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune system has evolved mechanisms to respond not only to specific molecular signals, but also to biophysical cues. Interestingly, research at the interface of biomaterials and immunology has also revealed that the biophysical properties and form of vaccines and immunotherapies impact immunological outcomes. For example, the intermolecular distance between antigen molecules on the surface of nanoparticles can impact formation of T cell receptor clusters that are critical during T cell activation. Despite the importance of biophysical cues in tuning the immune response, the connections between these parameters and immunological outcomes are poorly understood in the context of immunotherapy. Immunotherapies harness an individual’s immune system to battle diseases such as autoimmunity. During autoimmune disease, the immune system malfunctions and mistakenly attacks self-tissue. Immunotherapies can help tailor and guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. To overcome these challenges, experimental strategies are attempting to co-deliver self-antigens and modulatory cues to reprogram dysfunctional responses against self-antigens without hindering normal immune function. These strategies have shown exciting potential in pre-clinical models of autoimmune disease but are unproven in clinical research. Understanding how biophysical features are linked to immunological mechanisms in these settings would add a critical dimension to designing translatable, antigen-specific immunotherapies. Self-assembling materials are a class of biomaterials that spontaneously assemble in aqueous solution. Self-assembling modalities are useful technologies to study the links between biophysical parameters and immune outcomes because they offer precise control and uniformity of the biophysical properties of assembled moieties. Our lab leveraged the benefits of self-assembly to pioneer development of “carrier-free” immunotherapies composed entirely of immune signals. The therapies are composed of self-antigens modified with cationic amino acid residues and anionic, nucleic acid based modulatory cues. These signals are self-assembled into nanostructured complexes via electrostatic interactions. The research in this dissertation utilizes this platform as a tool to understand how tuning the biophysical properties of self-antigens impacts molecular interactions during self-assembly and in turn, how changes in biophysical features are linked to immunological outcomes. Surface plasmon resonance studies revealed that the binding affinity between signals can be tuned by altering overall cationic charge and charge density of self-antigen, and by anchoring the self-antigen with arginine or lysine residues. For example, the binding affinity between signals can be increased by increasing the total cationic charge on the self-antigen, and by anchoring the self-antigen with arginine residues rather than lysine residues. Computational modeling approaches generated insights into how molecular interactions between signals, such as hydrogen bonding, salt-bridges, and hydrophobic interactions, change with different design parameters. In vitro assays revealed that a lower binding affinity between self-assembled signals was associated with greater reduction of inflammatory gene expression in dendritic cells and more differentiation of self-reactive T cells towards regulatory phenotypes that are protective during autoimmunity. Taken all together, these insights help intuit how to use biophysical design to improve modularity of the self-assembly platform to incorporate a range of antigens for distinct disease targets. This granular understanding of nanomaterial-immune interactions contributes to more rational immunotherapy design.Item Functionalized 3D DNA Crystals through Core-Shell and Layer-by-Layer Assembly(2019) McNeil, Ronald; Paukstelis, Paul; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A fundamental goal of DNA nanotechnology has been assembly of DNA crystals for use as molecular scaffolds to organize arrays of guest molecules. We use previously described 3D DNA crystals to demonstrate core-shell and layer-by-layer assembly of DNA crystals capable of accommodating tethered guest molecules within the crystals’ pervasive solvent channel network. We describe the first example of epitaxial biomacromolecular core-shell crystallization through assembly of the crystals in two or more discrete layers. The solvent channels also allow post-crystallization guest conjugation with layer-specific addressability. We present microfluidics techniques for core-shell crystal growth which unlock greater potential for finely tunable layer properties and assembling complex multifunctional crystals. We demonstrate assembly of these DNA crystals as nanoscale objects much smaller than previously observed. These techniques present new avenues for using DNA to create multifunctional micro- and nanoscale periodic biomaterials with tunable chemical and physical properties.Item Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence(2016) Rhoads, Melissa Katherine; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.Item Engineering Biodegradable Vascular Scaffolds for Congenital Heart Disease(2015) Melchiorri, Anthony John; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The most common birth defects worldwide are congenital heart defects. To treat these malformations in a child’s cardiovascular system, synthetic grafts have been used as a primary intervention. However, current grafts suffer from deficiencies such as minimal biological compatibility, inability to grow and adapt, and high failure rates. Additionally, the grafts are not customized to the patient, which may lead to graft failure given that defects may vary significantly from patient to patient. The work presented here aims to adapt tissue engineering paradigms to develop customizable vascular grafts for congenital heart defects using to reduce the long-term risk and the number of surgeries experienced by patients. The first component of this research focuses on solvent-cast vascular grafts. This system of fabrication was used to explore how various strategies and graft modifications affect the graft’s performance in vivo. Grafts were fabricated with the mechanical properties necessary to withstand the stresses of a physiological environment and support neotissue formation. To improve tissue formation, the grafts were modified with bioactive molecules to improve vascular tissue growth. In addition, the grafts were combined with a tissue perfusion bioreactor. The bioreactor applied fluid flow to support cell seeding, differentiation, and growth of endothelial progenitor cells on the grafts, demonstrating a robust strategy for tissue formation prior to implantation. The second component of this research centers on the development of a biomaterial for 3D printing. 3D printing offers unparalleled customizability, as a graft can be designed based on medical images of a patient, tested via computer modeling, and then printed for implantation. A resin was developed consisting to produce grafts that were mechanically compatible with native blood vessels and maintained patency and tissue formation six months after implantation. The library of 3D printed vascular graft materials was also expanded by creating a novel copolymer resins, which varied in mechanical properties and degradation profiles. In addition, the concepts and strategies of biofunctionalization developed in the solvent-cast vascular grafts can be combined with the 3D printed graft strategies. Grafts designed, printed, and modified using these combinatorial approaches can greatly improve the long-term outcomes of treating congenital heart disease.Item Bioengineered conduits for directing digitized molecular-based information(2015) Terrell, Jessica Lynn; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular recognition is a prevalent quality in natural biological environments: molecules- small as well as macro- enable dynamic response by instilling functionality and communicating information about the system. The accession and interpretation of this rich molecular information leads to context about the system. Moreover, molecular complexity, both in terms of chemical structure and diversity, permits information to be represented with high capacity. Thus, an opportunity exists to assign molecules as chemical portrayals of natural, non-natural, and even non-biological data. Further, their associated upstream, downstream, and regulatory pathways could be commandeered for the purpose of data processing and transmission. This thesis emphasizes molecules that serve as units of information, the processing of which elucidates context. The project first strategizes a biocompatible assembly process that integrates biological componentry in an organized configuration for molecular transfer (e.g. from a cell to a receptor). Next, we have explored the use of DNA for its potential to store data in richer, digital forms. Binary data is embedded within a gene for storage inside a cell carrier and is selectively conveyed. Successively, a catalytic relay is developed to transduce similar data from sequence-based DNA storage to a delineated chemical cue that programs cellular phenotype. Finally, these cell populations are used as mobile information processing units that independently seek and collectively categorize the information, which is fed back as fluorescently ‘binned’ output. Every development demonstrates a transduction process of molecular data that involves input acquisition, refinement, and output interpretation. Overall, by equipping biomimetic networks with molecular-driven performance, their interactions serve as conduits of information flow.Item Defining Critical Parameters for Producing and Modulating Inflammation Caused by Cell Encapsulating Alginate Microspheres(2007-09-11) Breger, Joyce Catherine; Wang, Nam Sun; Lyle, Dan B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Minimizing induced inflammation, particularly nitric oxide (NO) production, is critical to optimal function or failure of implanted encapsulated cells. The purpose of this study is to define critical factors that affect toxic NO production from the macrophage cell line RAW264.7 in response to alginate microcapsules. The effects of the following were determined: 1) concentration of endotoxin (LPS) contamination; 2) presence of interferon-gamma (IFN-γ); 3) bead diameter and alginate volume; and 4) anti-inflammatory drugs in the alginate. A higher concentration (5 X) of LPS was required in alginate to produce the effect seen by LPS free in medium, sensitivity was enhanced by IFN-γ, bead diameter was inversely proportional to NO2 under low inflammatory conditions, and parthenolide in alginate significantly reduced inflammation. These results suggest that survival of implanted encapsulated cells may be improved by using highly purified alginate, avoiding ancillary inflammation, controlling surface area presentation, and incorporating anti-inflammatory drugs into the capsule matrix.Item A Novel Cyclic Acetal Biomaterial and Its Use in Cleft Palate Repair(2006-05-04) Moreau, Jennifer Lynn; Fisher, John P; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cleft lip and/or palate are the most prevalent congenital craniofacial birth defect in humans. While myriad surgical techniques have been described to repair orofacial clefts, several complications have been associated with the repair techniques. To overcome these complications, a tissue engineering strategy may be employed. In particular, we are investigating strategies for regenerating the alveolar bone that is often missing as a result of cleft palates. Numerous materials have been explored as biomaterials for bone tissue engineering, however there are disadvantages to these, including compromised mechanical properties and harmful degradation products. To overcome this issue, a novel class of biomaterials has been created. These materials are crosslinked networks of monomers of 5-ethyl-5-(hydroxymethyl)-beta,beta-dimethyl-1,3-dioxane-2-ethanol diacrylate. The study presented here was designed to determine the effects of the material's formulation scheme on several of its physical properties, so as to develop a novel bone tissue engineering material suitable for cleft palate repair.