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 Development of food polymer-based colloidal delivery systems for nutraceuticals(2012) Luo, Yangchao; Wang, Qin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Colloidal delivery systems have drawn increasing attention in food science area. Biopolymers, i.e. proteins and polysaccharides originated from foods, with low toxicity, high biocompatibility and biodegradability, are the ideal biomaterials to develop delivery systems for nutraceuticals. The present work is dedicated to develop delivery systems for nutraceuticals, using food derived biopolymers, e.g. chitosan and zein. In the first part of this study, different core-shell structured nanoparticles were developed for encapsulating both hydrophilic and hydrophobic nutracetuicals. For chitosan nanoparticles with zein coating, the hydrophilic nutraceutical, selenite, was encapsulated and the physicochemical properties was improved after zein coating. Then, zein nanoparticles with chitosan (CS) or carboxymethyl chitosan (CMCS) coating were developed to encapsulate hydrophobic nutraceuticals, including vitamin E, vitamin D3, indole-3-carbinol and diindolylmethane. The fabrication parameters were systematically studied and the effects of encapsulation on stabilities of nutraceuticals were investigated under different conditions. Subsequently, a novel approach to prepare CMCS hydrogel beads was developed. CMCS, a water-soluble derivative of CS, was known as unable to form hydrogel beads by itself in aqueous solution due to chain rigidity and inefficient entanglement. In this part, the formation of CMCS hydrogel beads was studied in aqueous-alcohol binary solutions. Chemical crosslinking was required to maintain its integrity upon drying. Different drying methods (i.e. freeze and air drying) were also investigated to understand their effects on swelling and release profile in simulated gastrointestinal conditions. Some possible mechanisms were discussed. Lastly, cellular evaluation of zein nanoparticles stabilized by caseinate was carried out. The zein-caseinate nanoparticles had a good redispersibility after freeze-drying and were able to maintain original particle size in different cell culture medium and buffer at 37°C over time. The zein-caseinate nanoparticles had no cytotoxicity at concentrations up to 1 mg/ml over 3 days. Then, coumarin 6, a fluorescent marker, was encapsulated into zein-caseinate nanoparticles to investigate their cell uptake and epithelial transport. The cell uptake was clearly visualized by fluorescent microscopy and the uptake mechanisms were investigated. The epithelial transport was investigated on Caco-2 cell monolayers. The results suggested caseinate not only stabilized zein nanoparticles in different buffers, but also improved cell uptake and epithelial transport.Item Blueprinting Self-Assembled Soft Matter: An `Easy' Approach to Advanced Material Synthesis in Drug Delivery and Wound Healing(2010) Dowling, Matthew Burke; Raghavan, Srinivasa R; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)From Jello to mayonnaise to silly putty to biological cells, our world is replete with "soft matter" - materials that behave as soft, deformable solids or highly viscoelastic liquids. Living systems, in particular, can be thought of as extremely sophisticated `soft' machines, with each cellular unit representing a touchstone for the functional potential of soft materials built via self-assembly. Drawing inspiration from biology, we blueprint soft biomaterial designs which rely upon self-assembly to achieve enhanced functionality. As opposed to complex synthesis schemes often used to develop improved biomaterials, we take an `easy' approach by allowing relatively simple molecules orchestrate themselves into advanced machines. In this dissertation, we describe four separate "soft" systems, all constructed by self-assembly of amphiphilic molecules under designed and/or triggered conditions in aqueous media. These systems revolve around a common theme: the structural tandem of (1) vesicles and (2) biopolymers, and the resulting interactions between the two. Our blueprints show promise in several important biomedical applications including controlled drug release, tissue engineering, and wound care. In the first part of this study, we blueprint a biopolymer gel that entraps pH-sensitive vesicles. The vesicles are formed by the self-assembly of a single-tailed fatty acid surfactant. We show that the gel has pH-responsive properties imparted upon it via the embedded vesicle nanostructures. Specifically, when the gel is brought in contact with a high pH buffer, the diffusion of buffer into the gel disrupts the vesicles and transforms them into micelles. Accordingly, a vesicle-micelle front moves through the gel, and this can be visually seen by a difference in color. The disruption of vesicles means that their encapsulated solutes are released into the bulk gel, and in turn these solutes can rapidly diffuse out of the gel. Thus, we can use pH to tune the release rate of model drug molecules from these vesicle-loaded gels into the external solution. In the second part, we have blueprinted hybrid biopolymer capsules containing drug-loaded vesicles by means of a one-step self-assembly process. These capsules are called "motherships" as each unit features a larger container, the polymer capsule, carrying a payload of smaller vesicular containers, or "babyships," within its lumen. These motherships are self-assembled via electrostatic interactions between oppositely charged polymers/surfactants at the interface of the droplet. Capsule size is simply dictated by drop size, and capsules of sizes 200-5000 µm are produced here. Lipid vesicles, i.e. the babyships, are retained inside motherships due to the diffusional barrier created by the capsule shell. The added transport barrier provided by the vesicle bilayer in addition to the capsule shell provides sustained drug release from the motherships. Furthermore, this one-step drop method allows for the rapid synthesis of soft materials exhibiting structural features over a hierarchy of length scales, from nano-, to micro- to macro-. Thirdly, we have therapeutically functionalized biopolymer films by simply passing a solution of vesicles over the film surface. We deposit films of an associating biopolymer onto patterned solid substrates. Subsequently, these polymer films are able to spontaneously capture therapeutically-loaded vesicles from solution; this is demonstrated both for surfactant as well as lipid vesicles (liposomes). Importantly, it is verified that the vesicles are intact - this is shown both by direct visualization of captured vesicles (via optical and cryo-transmission electron microscopy) as well as through the capture and subsequent disruption of drug filled vesicles. Such therapeutically-functionalized films may be of use in the treatment of chronic wounds and burns. Lastly, we have demonstrated that the addition of a certain biopolymer transforms a suspension of whole blood into a gel. This blueprint is inspired from previous research in our group on the biopolymer-induced gelation of vesicles, which are structurally similar to cells. Upon mixture with heparinized human whole blood, this amphilic biopolymer rapidly forms into an "artificial clot." These mixtures have highly elastic character, with the mixtures able to hold their own weight upon vial inversion. Moreover, the biopolymer shows significant hemorrhage-controlling efficacy in animal injury models. Such biopolymer-cell gelation processes are shown to be reversed via introduction of an amphiphilic supramolecule, thus introducing the novel concept of the "revesible hemostat." Such a hemostatic functionality may be of large and unprecedented use in clinical the treatment of problematic hemorrhage both in trauma and routine surgeries.Item ENZYME INHIBITION IN MICROFLUIDICS FOR RE-ENGINEERING BACTERIAL SYNTHESIS PATHWAYS(2009) LARIOS BERLIN, DEAN EDWARD; RUBLOFF, GARY W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Enzyme-functionalized biological microfluidic (EF-BioMEMS) systems are an emerging class of lab-on-chip devices that manipulate enzymatic pathways by localizing reaction sites in a microfluidic network. An EF-BioMEM system was fabricated to demonstrate biochemical enzyme inhibition. Further, design optimizations to the EF-BioMEM system have been proposed which improve system sensitivity and performance. The pfs enzyme is part of the quorum-sensing pathway that ultimately produces the bacterial signaling molecule AI-2. An EF-BioMEM system was fabricated to investigate the pfs conversion activity in the presence of a transition state analogue inhibitor. A reduction in enzyme conversion was measured in microfluidics for increasing inhibitor concentration that was comparable to the response expected on a larger scale. This EF-BioMEMS testbed is capable of investigating other compounds that inhibit quorum sensing. Design improvements were demonstrates that improve overall system responsiveness by minimizing unintended reactions from non-specifically bound enzyme. EF-BioMEMS signal-to-background performance increased from 0.72 to 2.43.Item Programmable Biomolecule Assembly and Activity in Prepackaged BioMEMS(2008-10-21) Luo, Xiaolong; Rubloff, Gary W.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Antibiotic resistance is an increasing public health concern and few new drugs for bacterial pathogenesis have been obtained without addressing this resistance. Quorum sensing (QS) is a newly-discovered system mediated by extracellular chemical signals known as "autoinducers", which can coordinate population-scale changes in gene regulation when the number of cells reaches a "quorum" level. The capability to intercept and rewire the biosynthesis pathway of autoinduer-2 (AI-2), a universal chemical signaling molecule, opens the door to discover novel antimicrobial drugs that are able to bypass the antibiotic resistance. In this research, chitosan-mediated in situ biomolecule assembly has been demonstrated as a facile approach to direct the assembly of biological components into a prefabricated, systematically controlled bio-microelectromechanical system (bioMEMS). Our bioMEMS device enables post-fabricated, signal-guided assembly of labile biomolecules such as proteins and DNA onto localized inorganic surfaces inside microfluidic channels with spatial and temporal programmability. Particularly, the programmable assembly and enzymatic activity of the metabolic pathway enzyme Pfs, one of the two AI-2 synthases, have been demonstrated as an important step to reconstruct and interrogate the AI-2 synthesis pathway in the bioMEMS environment. Additionally, the bioMEMS has been optimized for studies of metabolic pathway enzymes by implementing a novel packaging technique and an experimental strategy to improve the signal-to-background ratio of the site-specific enzymatic reactions in the bioMEMS device. I envision that the demonstrated technologies represent a key step in progress toward a bioMEMS technology suitable to support metabolic engineering research and development.Item Assembly of Quorum Sensing Pathway Enzymes onto Patterned Microfabricated Devices(2007-07-31) Lewandowski, Angela; Bentley, William; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)I report patterned protein assembly onto microfabricated devices using our unique assembly approach. This approach is based on electrodeposition of the aminopolysaccharide chitosan onto a selected electrode pattern of the device, and covalent conjugation of a target protein to chitosan upon biochemical activation of a genetically fused C-terminal pentatyrosine "pro-tag." With this approach, assembly is "spatially selective", occurring only at selected electrode patterns, and the entire process occurs under mild experimental conditions. Additionally, assembly is reversible and the devices reusable, as the deposited chitosan can be removed by simple incubation in dilute acid. Finally, the protein is covalently and robustly linked to chitosan through the pro-tag versus the native tyrosines, and thus our approach confers "orientational control". I have examined patterned assembly of metabolic pathway enzymes onto both flat microfabricated chips and into 3-dimensional microfluidic devices. The assembled enzymes retain reproducible catalytic activities and protein recognition capabilities for antibody binding. Additionally, catalytic activity is retained over multiple days, demonstrating enzyme stability over extended time. Finally, substrate catalytic conversion can be controlled and manipulated through the assembly patterned area, or in the case of microfluidic devices, through the substrate flow rate over the assembled enzyme. I specifically examined the patterned assembly of Pfs and LuxS enzymes, members of the bacterial autoinducer-2 (AI-2) biosynthesis pathway. AI-2 is a small signaling molecule that mediates interspecies bacterial communication termed type II "quorum sensing", which is involved in regulating the pathogenesis of a bacterial population. Significantly, this is the first time that Pfs and LuxS have been assembled onto devices. More significantly, Pfs and LuxS have both been assembled onto the same chip; that is, the quorum sensing pathway has been assembled onto a single device. This device could be used to screen inhibitors of AI-2 biosynthesis and discover novel "anti-pathogenic" drugs. In summary, I have demonstrated patterned enzyme assembly onto microfabricated devices. The assembled enzymes retain reproducible catalytic activities and are capable of recognizing and binding antibodies. Importantly, patterned device-assembly of multiple enzymes representing a metabolic pathway is possible. I envision many potential biosensing, bioMEMS, drug screening, and metabolic engineering applications.Item Microcantilever Biosensors with Chitosan for the Detection of Nucleic Acids and Dopamine(2007-05-07) Koev, Stephan; Ghodssi, Reza; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microcantilever biosensors allow label-free detection of analytes within small sample volumes. They are, however, often limited in sensitivity or specificity due to the lack of proper bio-interface layers. This thesis presents the use of the biopolymer chitosan as a bio-interface material for microcantilevers with unique advantages. Sensors coated with chitosan were designed, fabricated, and functionalized to demonstrate two distinct applications: detection of DNA hybridization and detection of the neurotransmitter dopamine. The first demonstration resulted in signals from DNA hybridization that exceed by two orders of magnitude values previously published for sensors coated with SAM (self assembled monolayer) interface. The second application is the first reported demonstration of using microcantilevers for detection of the neurotransmitter dopamine, and it is enabled by chitosan's response to dopamine electrochemical oxidation. It was shown that this method can selectively detect dopamine from ascorbic acid, a chemical that interferes with dopamine detection in biological samples.Item Characterization of Electrodeposited Chitosan Films by Atomic Force Microscopy and Raman Spectroscopy(2006-05-08) Dreyer, Erin C; Rubloff, Gary W; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chitosan has served as a robust and reproducible scaffold for biological reactions by electrodeposition at specific sites in microfluidic channels. However, its growth and properties are not well understood as a function of deposition parameters. To better understand the materials and process science, in-vitro characterization techniques and post-deposition measurements of air-dried films were performed. AFM images of dried films depicted variable, rough morphology not directly correlated to deposition conditions while hydration increased surface homogeneity. Dry roughness increased logarithmically with thickness supporting growth by nucleation. In-vitro fluorescence images showed fairly smooth distribution of chitosan, whereas dried films were much rougher, indicating non-uniform collapse of structure during drying. Raman spectroscopy revealed the presence of primary amine groups active in biofunctionalization and served as a technique for evaluating the spatial selectivity of chitosan by electrodeposition. Further study of hydrated films is needed to fully understand chitosan as a platform for biotechnology applications.