Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item DEVELOPMENT OF GLYCOSAMINOGLYCAN MIMICKING NANOGEL TECHNOLOGIES FOR CONTROLLED RELEASE OF THERAPEUTICS TO TREAT RETINAL DISEASES IN DIFFERENT AGE GROUPS(2024) Kim, Sangyoon; Lowe, Tao L.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Retinal diseases, such as diabetic retinopathy, glaucoma, macular degeneration, and retinoblastoma, affect around 13 million people worldwide, with projections indicating a rise to 20 million by 2030. These conditions lead to irreversible vision loss and significant impairment in both adults and children, with an annual economic burden of $139 billion in the United States alone. Aging significantly increases the risk of certain retinal conditions, and with improvements in healthcare leading to increased life expectancy, these conditions are becoming more prevalent due to the natural aging process and associated physiological changes in the eye. Current treatments are either destructive or have low efficacy and are not optimized for the younger population. While therapeutics including small molecular drugs, proteins and antibodies show promise in treating these diseases by reducing inflammation and neuronal apoptosis, their effectiveness is hindered by short half-lives and inability to cross the blood-retinal barrier (BRB). Nanoparticles offer a potential solution by improving drug delivery across biological barriers, yet no nanoparticles have been developed to effectively transport intact proteins or small molecules across the BRB to the retina without toxicity, slow clearance and stability. Therefore, there is an unmet need to evaluate the physical and physiological property changes of the eye along development and develop nanoparticle systems that can control and sustain the release of therapeutics across the blood retinal barrier (BRB) to treat the retinal diseases. In this project, the thickness, rheological property, permeability and morphological property changes of ocular barriers including sclera, cornea and vitreous humor in the developing eye from preterm to adult were evaluated using porcine ex vivo model. Two glycosaminoglycan mimicking nanogel systems, poly(NIPAAm-co-DEXcaprolactoneHEMA) nanogels with and without positive or negative charges and β-cyclodextrin based poly(β-amino ester) (CD-p-AE) nanogels were developed for sustained release of intact proteins including insulin and anti-TNFα, and small hydrophobic drugs, respectively across the ex vivo porcine sclera and in vitro BRB models: human fetal retinal pigment epithelial (hfRPE), adult retinal pigment epithelial (ARPE-19) and human cerebral microvascular endothelial (hCMEC/D3) cell monolayers. Completion of this project will have a significant impact on developing novel personalized nanotherapeutics to treat retinal diseases in different age groups.Item Optical and Thermal Systems for Automation of Point-of-Care Assays(2018) Goertz, John; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Modern medicine has detailed 70,000 different diagnoses; the 21st century challenge is bringing those diagnoses to over 7 billion people. This phenomenal feat requires precision biosensing strategies that minimize necessary training and manual effort while maximizing portability and affordability. Microfluidic strategies, both fabricated chips and paper-based devices, held the promise to facilitate point-of-care diagnostics but have been inadequate for many applications due to the trade-off between bulky pumps or limited control and complexity. This dissertation details novel strategies that control the progression of biochemical reactions with high functionality, portability, and ease-of-use. First, I will describe an amplified signaling reaction that leverages both positive and negative feedback loops to achieve optically-regulated control. This assay, termed “Peroxidyme-Amplified Radical Chain Reaction” enables naked-eye detection of catalytic reporter DNA structures at concentrations across five orders of magnitude down to 100 pM while eliminating the need for manual addition of hydrogen peroxide common to other such detection reactions. Next, I will describe the development of a platform for thermal regulation of generic reactions. To address the need for a broadly capable automation platform that provides equal utility in the lab and field alike, we recently developed “phase-change partitions”. In our system, purified waxes segregate reagents until incremental heating melts the partitions one by one, causing the now-liquid alkane to float and allowing the desired reagents to interact with the sample on demand. This tight control over reaction progression enabled us to construct hands-free detection systems for isothermal DNA amplification, heavy metal contamination, and antibiotic resistance profiling. My work has demonstrated a broadly capable suite of assay control systems with the potential to enable simple, inexpensive automation of a broad array of chemical and biological analysis across human medicine, environmental surveillance, and industrial chemical synthesis.Item PROTEIN AND PEPTIDE ENGINEERING FOR IMPROVING THERAPIES FOR APPLICATIONS IN HUMAN HEALTH(2018) Moghaddam-Taaheri, Parisa; Karlsson, Amy J; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The work in this dissertation focuses on protein and peptides engineering for improving therapies for applications in human health. First, we describe a directed evolution approach to engineer antibody fragments to bind to intracellular targets. An antibody fragment library was displayed using the twin arginine translocation inner-membrane display pathway, in order to allow only antibodies that are well-folded in the reducing cytoplasmic environment to be screened for binding. Displayed libraries were screened for binding to the apoptosis inhibitor survivin, and scFv cytoplasmic solubility and specificity was characterized. Though the antibodies isolated through this method displayed strong intracellular folding and high binding to survivin, they exhibited non-specific binding as well. We improved the screening approach by using whole-plasmid PCR to recover sequences of isolated antibodies. Additional improvements to the screening process to increase stringency will allow better isolation of antibodies with high affinity and specificity for their target. In a rational design approach, we designed an antimicrobial peptide-based approach for the treatment of candidiasis. Candida albicans is a commensal organism that resides asymptomatically in the body. This opportunistic pathogen can overgrow and cause potentially fatal bloodstream infections. C. albicans biofilms that colonize implanted devices exhibit increased resistance to antimicrobial treatments and current antifungal treatments contribute to the rise of resistant strains of C. albicans or may cause toxicity. Thus, there is a clinical need for new or improved antifungal therapeutics to treat C. albicans infections. Histatin-5 (Hst-5) is an antimicrobial peptide secreted by the salivary glands that exhibits antifungal activity against C. albicans. Hst-5 can, however, be degraded by secreted aspartic proteases (Saps) produced by C. albicans cells, reducing its antifungal activity. Amino acid substitutions made to Hst-5 reduced the likelihood of proteolytic degradation to better maintain antifungal activity. Of these modifications, the K11R-K17R and E16R peptides showed enhanced antifungal activity in preventing C. albicans biofilm formation and eradicating preformed biofilms as compared to parent Hst-5. The improvements to methods and experimental findings in this research contribute to the improvement of therapies to treat human disease.Item Development, characterization and optimization of a novel mammalian protein expression system(2016) Sampey, Darryl; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biopharmaceutical manufacturing plays a critical role in global healthcare systems. Methods and techniques for protein expression in upstream bioprocesses can have the highest impact on product quality and safety and, ultimately, on delivering effective means of treating and curing life threatening diseases. In addition to novel products, development and regulatory approval of biosimilars require precise matching of the quality attributes between lots of reference innovator drug and those of the biosimilar candidate. With continuing pressure to reduce the cost of these lifesaving medicines, increased bioprocess yields are also key to reducing cost of manufacturing and allowing for lower prices at the pharmacy. With these goals in mind, a novel biomanufacturing platform was developed that harnesses the commercially established murine NS0 myeloma host cell in new ways to create stable, highly productive manufacturing cell lines. Cholesterol metabolic markers were screened to identify the optimal enzyme, Hsd17b7, to enable stable cell line selection and eliminate the need for cholesterol addition to the bioprocess. Enhancement of the expression vector promoters, isolation and cloning of highly efficient hybridoma-derived heavy and light chain signal peptides and optimization of coding sequences increased monoclonal antibody expression by 10-fold. The development of novel multiplex selection strategies that combines antibiotic selection with cholesterol and glutamine metabolic selection both sequentially and in parallel allowed for the rapid and consistent generation of commercial grade cell lines with greater than 1 g/L yield potential. The platform was demonstrated to be highly valuable in developing the most complex of next generation biologics and commercially significant for the production of biosimilar monoclonal antibodies whose reference drugs are currently manufactured in murine host cells.