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 Overcoming the Extracellular Matrix Barrier to Nanoparticle Transport(2024) Cahn, Devorah; Duncan, Gregg A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The extracellular matrix (ECM) is a major component of the tumor microenvironment which poses a significant barrier to nanoparticle (NP) transport, preventing delivery of therapeutic cargo. Studies have shown that PEGylation offers an effective strategy for improving NP transport in ECM. However, these studies have generally used ECM models that are not wholly representative of the native matrix. Furthermore, while ECM characteristics and composition varies across organs, it is unclear to what extent these tissue-specific characteristics affect NP transport through the ECM and how NP surface chemistry impacts ECM penetration in distinct tissues. The overall objective of this dissertation is to identify key factors of NP transport through the tumor microenvironment, facilitating the development of strategies to improve NP distribution throughout the tumor microenvironment. We hypothesized that PEG branching will enhance stability and mobility of NPs in ECM and that ECM source impacts NP transport. We further hypothesized that PEG architecture significantly affects NP mobility in ECM as well as biodistribution and tumor accumulation in vivo. Our first aim was to determine the effects of PEG branching on NP stability and transport through in vitro basement membrane model. We found that branched PEG significantly increased both the stability and mobility of NPs in Matrigel, a basement membrane model. We then assessed the impact of tissue source on NP transport through an in vitro ECM model. We decellularized porcine lung, liver, and small intestine submucosa to form tissue specific hydrogels and found NP mobility was significantly impacted by tissue source where low molecular weight linear PEG generally provided the greatest benefit to NP mobility within the different matrices. Finally, we evaluated how PEG branching affects biodistribution, immune cell infiltration, and NP uptake in tumors in vivo. We found that NPs coated with branched PEG increased NP accumulation within tumors and PEGylation significantly impacted immune cell infiltration within these tumors. This work provides additional insight into the transport mechanisms of NPs throughout the tumor microenvironment as well as additional considerations for the design of efficient NP delivery systems.Item ENGINEERING NANOPARTICLES FOR IMPROVED LYMPHATIC DELIVERY AND ELUCIDATING MECHANISMS REGULATING NANOPARTICLE TRANSPORT INTO LYMPHATICS(2023) McCright, Jacob Connor; Maisel, Katharina; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Immune modulatory therapies usually need to be effectively delivered to lymph nodes to enhance therapeutic effectiveness. Lymphatic vessels exist throughout the body and can transport 10 – 250 nm therapeutic nanoparticles to lymph nodes, however, nanoparticle formulations required to maximize this transport, and the mechanisms governing this transport are poorly understood. Here, we probed the effect of surface charge, surface poly(ethylene glycol) (PEG) density, shape, and size on nanoparticle transport across LECs (LECs) and lymph node delivery. Using an established in-vitro lymphatic transport model, we found PEGylation improved the transport of 100 and 40 nm nanoparticles across LECs 50-fold compared to non-PEGylated nanoparticles and that transport is maximized when the PEG is in a dense brush conformation corresponding to a high grafting density (Rf/D = 4.9). PEGylating 40 nm nanoparticles improved transport efficiency across LECs 68-fold compared to unmodified nanoparticles, demonstrating that the addition of PEG improves transport in a size-independent manner. We injected these nanoparticle formulations intradermally into C57Bl/6J mice and found that PEGylated 100 nm and 40 nm nanoparticles accumulate in lymph nodes within 4 hours, while unmodified nanoparticles accumulated minimally. Densely PEGylated nanoparticles also traveled furthest from the injection site. In this thesis, we also determined that nanoparticles are transported via both paracellular and transcellular mechanisms, and that both PEG conformation and nanoparticle size and shape modulates the cellular transport mechanisms. We also expanded our in-vitro lymphatic transport model to model important physiological conditions including transmural flow and found that the presence of this flow increased transport across lymphatic barriers in a shape and mechanism-dependent manner. To further investigate the mechanisms regulating nanoparticle transport, we generated a computational kinetic transport model that was able to quantify the contributions of both paracellular and transcellular transport mechanisms, as well as predict transport efficiency as a function of nanoparticle characteristics including size and surface chemistry. Using transport inhibitors, we can expand our system of equations to describe precise uptake and transport mechanisms, and the relation between nanoparticle formulation and mechanism. This computational model is one of the first to describe transport across lymphatic vessels, and offers some of the first definitions for coefficients used to quantitatively describe nanoparticles transport across LECs (i.e., permeability). Our computational, in-vitro, and in-vivo results indicate that nanoparticle surface charge, PEG conformation, and size are key criteria for nanoparticle design for effective lymphatic delivery with a dense, neutrally charged coating of PEG maximizing transport across LEC barriers and transport to lymph nodes. Optimizing nanoparticle formulation and surface characteristics, including PEG density, has the potential to enhance immunotherapeutic and vaccine outcomes.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 ACTUATION OF MULTIFUNCTIONAL HARD NANOPARTICLES FOR ACTIVELY CONTROLLED DRUG RELEASE(2019) Sangtani, Ajmeeta; Delehanty, James B; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Systemic drug delivery relies on repeated dosing of large concentrations of poorly targeted drug leading to off-target toxicity. Recently, nanoparticle (NP)-mediated drug delivery (NMDD) has been developed as an approach to overcome the limitations of traditional drug delivery. The unique size-dependent properties of NPs and their ability to augment the activity of attached/loaded cargos makes them attractive drug delivery vectors. NPs are classified into two categories (soft or hard depending on their material composition) and our understanding of how to load and control soft NP materials currently surpasses that of hard NPs. In this dissertation we seek to further our fundamental knowledge of hard NP-based drug delivery systems. In Aim 1 we utilize a quantum dot (QD)-cell uptake peptide complex as a central scaffold to append various responsive peptide-drug constructs in order to modulate the toxicity of one of the most widely used chemotherapeutics, doxorubicin. By doing a comparative study of four chemical linkages, we determine the role played by attachment chemistry in controlling drug release. In Aim 2, we utilize the knowledge gained from Aim 1 to develop a system capable of overcoming multidrug resistance in cancer cells, which is known to severely limit the efficacy of chemotherapeutics. Our hard NP conjugate system is unique as it is one of the few systems reported in the literature to bypass multidrug resistance pumps without the need for exogenous drugs. Finally, in Aim 3 we append a peptide for membrane targeting and a photosensitizing drug capable of generating reactive oxygen species to the QD. This multifunctional system displays augmented therapeutic efficacy of the appended photosensitizer by delivering it to the membrane of cells and controlling its actuation using energy transfer. The work described here details basic concepts for the design of “smart” hard NP materials for internally and externally-triggered, active release of surface-appended drug cargos. Additionally, we hope to elucidate the important design considerations that must be taken into account when designing hard NP systems for controlled drug delivery.Item CATANIONIC SURFACTANT VESICLES: TECHNOLOGY FOR VACCINE DEVELOPMENT AND TARGETED DRUG DELIVERY APPLICATIONS(2013) Stocker, Lenea Hope; DeShong, Philip; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Catanionic surfactant vesicles have gained attention due to their structural similarities to liposomes and their robust properties in biological media. Catanionic vesicles are formed from oppositely charged surfactants and can be exploited for applications in vaccine production and drug delivery. The focus of my research has been on the preparation, characterization, and application of functionalized catanionic surfactant vesicles. Chapter 2 describes the preparation and characterization of catanionic vesicles containing sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT). Vesicle solutions were determined to be stable for greater than 6 months, formed vesicles with two populations of 80 and 160 nm, and had a membrane surface charge similar to human cells, -56 mV. Furthermore, vesicles were stable between a pH of 2 and 12, in saline solutions up to 0.6 M NaCl, and after autoclaving. Next, I report the loading of various molecules into the vesicle leaflet and the characterization of the resulting functionalized systems. Hydrophobic molecules were readily incorporated into the hydrophobic region of the leaflet. Lipid conjugates of hydrophilic molecules were anchored in the vesicle bilayer. Chapters 3 and 4 report the loading of biological materials (i.e. liposaccharides and proteins) into catanionic vesicles for the development of bacterial vaccines. Initial studies, discussed in Chapter 3, pertain to the loading of the pure components lipooligosaccharide (LOS) and C12 -Pan DR helper T cell epitope (PADRE) conjugate into catanionic vesicles. A single dose of these vesicles generated a large IgG antibody titer in mice. Next, in Chapter 4, we focus on the extraction of cellular membrane components from cells for their direct incorporation into catanionic vesicles. Vesicles were prepared by adding surfactants in the presence of Neisseria gonorrhoeae cells. Vesicle extracts contained pathogen-derived LOS F62ΔlgtD and a subset of proteins from the outer membrane of the bacterium, including porin and OPA. Lastly, Chapter 5 describes catanionic vesicles in drug delivery. Vesicles were loaded with 88 μg/mL of doxorubicin and shown to retain the drug over 15 days. Doxorubicin loaded into catanionic vesicles were shown to be less toxic as compared to the free drug, IC 50 = 51 μg/mL and 0.16 μg/mL, respectively.Item CHARACTERIZATION OF BRANCHED HISTIDINE-LYSINE POLYPEPTIDES USED FOR NUCLEIC ACID DELIVERY AND THEIR COMPLEXES WITH DNA AND SMALL INTERFERING RNA.(2013) Tricoli, Lucas James; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA and siRNA must be packaged for protection prior to transfection in vivo when they are administered to humans or other animals. Dr. A. J. Mixson's laboratory at the University of Maryland, Baltimore Medical School has developed a family of branched histidine-lysine (HK) peptides that confer improved transfection in mice compared to naked nucleic acid. The branched polymer denoted H3K4b has a superior ability to transfect siRNA in vitro compared to H3K(+G)4b. H3K(+G)4b, made by the addition of two glycines to each of the original H3K4b branches, is presumably a more flexible polymer, and it allows for better transfection of plasmid DNA than H3K4b. Biophysical characterization of the HK-DNA and HK-siRNA complexes is aimed at understanding how the structures of both peptides affect their biological activity. This characterization was performed using ethidium bromide exclusion from nucleic acid, DNase I plasmid degradation, dynamic light scattering, circular dichroism, isothermal titration calorimetry and atomic force microscopy. Results from the characterization suggest that H3K4b forms condensed regions of packaged plasmid with some repeating accessibility of the DNA, compared to a more even coating of plasmid by H3K(+G)4b. Based on these results a "coating versus clumping" model was developed to relate the transfection efficiency of each peptide to its binding of plasmid DNA. A specific model for packaging of siRNA with these peptides was not developed, but we believe that characteristics that lead to effective transfection of plasmid are not key to siRNA delivery. A better understanding of characteristics important to peptide-nucleic acid complex formation may lead to the development of improved transfection agents.Item Study of Electrostatic Interaction between Charged Surfactant Vesicles and Ionic Molecules by Bulk and Fluorescence Correlation Spectroscopy Measurements(2007-09-28) wang, xiang; English, Douglas; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Phospholipid vesicles (Liposomes) for controlled release applications such as drug and gene delivery have attracted great interest. However, lack of long term stability and low solute encapsulation efficiency limit the usage of liposomes in many areas. In this thesis, charged surfactant vesicles that are formed spontaneously in mixtures of single-tailed surfactants are investigated as an alternative for liposomes in applications where improved long-term capture of charged organic molecules is desirable. The system of interest is dilute solutions of cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS). It is shown that charged surfactant vesicles have a high efficiency for encapsulating oppositely charged probe molecules with extremely slow release rates. Several probe molecules, both anionic and cationic, were studied including the cancer chemotherapeutic drug doxorubicin (Dox). All probe molecules were captured at high efficiency (ca. 20-70%) when the vesicle bilayer was of opposite charge from the probe molecule; when the charge of vesicle and probe molecule was the same, encapsulation was diminished (ca. 0-8%). Strong electrostatic interaction between surfactant vesicles and charged molecules are responsible for the extremely high encapsulation efficiency. The vesicle/probe formulations are stable for weeks to months due to the inherent stability of these vesicles which form spontaneously and are believed to be equilibrium structures. These properties allow surfactant vesicles to be used to selectively separate oppositely charged dye molecules, and this is demonstrated. Fluorescence correlation spectroscopy (FCS) was used to gain deeper understanding into the role of electrostatics in the capture of charged probe molecules by charged surfactant vesicles. FCS measures the diffusion of fluorescent probe molecules in aqueous solutions at very low concentrations (10-9-10-8 M) and distinguishes between rapidly diffusing single molecules and slowly diffusing molecules that are adsorbed on a vesicle bilayer. This method is sensitive enough to rapidly determine the fraction of probe molecules bound to the bilayer interface in a given sample. Binding isotherms were constructed from FCS measurements in which a series of solutions were measured by holding the dye concentration constant while increasing the vesicle concentrations. The resulting isotherm yields a measure of binding energy. Comparisons of binding energies show that probe/bilayer interactions are mainly governed by charge-charge interactions but may also depend on the size and structure of the surfactant counter ions. Our findings provide useful guidelines for implementing surfactant vesicles in biotechnological applications and also serve as an intriguing example of charge-mediated bilayer interactions.