Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item Investigation and development of induced pluripotent stem cell derived extracellular vesicle-based therapeutics(2024) Levy, Daniel H; Jay, Steven; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Due to their complex, multicomponent nature, extracellular vesicle (EV)-based therapeutics have arisen as an intriguing option for treatment of complex diseases that require the simultaneous modulation of distinct pathways. Due to their inherent regenerative properties, mesenchymal stem cell (MSC)-derived EVs have been the most heavily investigated and utilized in clinical trials for diseases including acute respiratory distress syndrome, wound healing and many more. While pre-clinical studies have demonstrated promise for such EV-based therapeutics, source cell limitations act as a hurdle to the widespread clinical translation of MSC EV therapies. MSCs and other cells reported to produce therapeutic EVs (cardiac progenitor cells, neural stem cells, etc.) have limited expansion capabilities ex vivo before cellular senescence, therefore limiting the amount of therapeutic EVs that can be produced by a single cell line. Due to these limited expansion capabilities, alternative, self-renewing therapeutic EV source cells are needed. One such source cell is induced pluripotent stem cells (iPSCs), which possess self-renewing capabilities. However, the baseline bioactivity of iPSC EVs have yet to be rigorously evaluated; in our work, we report for the first time that iPSC EVs possess robust anti-inflammatory properties in addition to confirming previous reports of their ability to promote vascularization in a murine diabetic wound healing model. Building off these baseline results, we sought to augment iPSC EV potency by utilizing genetic approaches to load of bioactive RNAs including microRNA (miRNA) and long non-coding RNA (lncRNA) into iPSC EVs. In our miRNA loading studies, we effectively demonstrate that the natural biogenesis pathways of miRNA can be probed to facilitate export of bioactive miRNAs to secreted EVs, thereby enhancing their anti-inflammatory bioactivity. Lastly, we utilize a genetic engineering approach to enhance active sorting of lncRNAs into secreted EVs and test their therapeutic potential in a murine colitis model. The work described in this dissertation provides a foundation towards the clinical translation of iPSC EV-based therapeutics by benchmarking them against more established therapeutic EV sources (iPSC-derived MSC EVs) and developing strategies to enhance their bioactivity via RNA cargo loading.Item GENETICALLY ENGINEERED PROBIOTICS FOR DIAGNOSTICS AND DRUG DELIVERY: APPLICATIONS FOR CROHN’S DISEASE(2018) McKay, Ryan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the history of medicine, therapies have evolved while their mode of delivery has remained largely static. Generally, the active ingredient is formulated with an excipient to confer stability, and is ultimately delivered orally or intravenously in most applications. Crohn’s disease (CD), an illness with increasing global prevalence characterized by chronic inflammation of the intestines, is commonly treated with intravenously administered biologics. When these medicines spread throughout the body, only a small percentage acts at the desired site and side effects often arise. Thus, a targeted system is desired to localize treatment at sites of colonic inflammation. There is an entire field dedicated to localized delivery that typically employs drug-laden particles or capsules that can respond to local chemical or physical cues. We believe that bacteria can be “programmed” to respond analogously, and ultimately synthesize and deliver therapeutics. Nitric oxide (NO) levels are elevated at sites of intestinal inflammation, and thus serves as a targeting molecule that can attract programmed bacteria via a process called pseudotaxis. This is achieved by rewiring the native motility circuits of bacteria to respond to high NO levels. Additionally, localized treatment is attained by an NO- specific response whereby the designed bacteria produce and secrete a human protein reported to reduce inflammation in CD patients. This system may improve CD treatment via: 1) site-specific targeting to minimize side effects and increase efficacy, 2) in situ synthesis of the therapeutic avoids payload loss in the digestive tract and manufacturing obstacles associated with biologics, 3) probiotics are reported to provide innate benefits to CD patients, and 4) oral delivery is preferred by patients versus intravenous. We have also developed probiotics that fluoresce in response to NO which may serve as an ingestible biosensor for CD. We believe these reporter probiotics can assist in the diagnosis of CD by utilizing visualization of bacteria in a stool sample to reduce the need for invasive colonoscopies and biopsies. Overall, we have developed a platform of probiotic cells that respond to NO with applications for Crohn’s disease in mind, translating to noninvasive methods for both the diagnosis and treatment of CD.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 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 Bridging the biology-electronics communication gap with redox signaling(2015) Gordonov, Tanya; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Electronic and biological systems both have the ability to sense, respond to, and communicate relevant data. This dissertation aims to facilitate communication between the two and create bio-hybrid devices that can process the breadths of both electronic and biological information. We describe the development of novel methods that bridge this bi-directional communication gap through the use of electronically and biologically relevant redox molecules for controlled and quantitative information transfer. Additionally, we demonstrate that the incorporation of biological components onto microelectronic systems can open doors for improved capabilities in a variety of fields. First, we describe the original use of redox molecules to electronically control the activity of an enzyme on a chip. Using biofabrication techniques, we assembled HLPT, a fusion protein which generates the quorum sensing molecule autoinducer-2, on an electrodeposited chitosan film on top of an electrode. This allows the electrode to controllably oxidize the enzyme in situ through a redox mediator, acetosyringone. We successfully showed that activity decrease and bacterial quorum sensing response are proportional to the input charge. To engineer bio-electronic communication with cells, we first aimed for better characterizing an electronic method for measuring cell response. We engineered Escherichia coli (E.coli) cells to respond to autoinducer-2 by producing the β-galactosidase enzyme. We then investigated an existing electrochemical method for detecting β-galactosidase activity by measuring a redox-active product of the cleavage of the added substrate molecule PAPG. In our novel findings, the product, PAP, was found to be produced at a rate that correlated with the standard spectrophotometric method for measuring β-galactosidase, the Miller assay, in both whole live and lysed cells. Conversely, to translate electronic signals to something cells can understand, we used pyocyanin, a redox drug which oxidizes the E.coli SoxR protein and allows transcription from the soxS promoter. We utilized electronic control of ferricyanide, an electron acceptor, to amplify the production of a reporter from soxS. With this novel method, we show that production of reporter depends on the frequency and amplitude of electronic signals, and investigate the method’s metabolic effects. Overall, the work in this dissertation makes strides towards the greater goal of creating multi-functional bio-hybrid devices.