UMD Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/3

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 given thesis/dissertation in DRUM.

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    IN VIVO BIODISTRIBUTION, LUNG TARGETING, AND PARAMETRIC MODULATION OF A DNA-BASED DRUG DELIVERY SYSTEM ADDRESSED TO ICAM-1
    (2020) Roki, Niksa; Muro, Silvia; Bentley, William; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The design goal of ligand-targeted nanoparticles (NPs) is to achieve site-specific targeting to specific biological targets, which can maximize therapeutic efficacy and safety. However, site-specific delivery remains suboptimal due to biological barriers, particularly non-specific interactions and sequestration of NPs by the immune system and anatomic structures of clearance organs in vivo. This formidable challenge prompted the exploration of novel ligand-based NP designs. Due to their exceptional precision, versatility, and biocompatibility, NPs composed of DNA (DNA-NPs) and targeted via ligands, have emerged as a promising strategy to deliver therapeutic effects with unique precision. One such formulation is anti-ICAM/3DNA, a multibranched DNA-made nanocarrier (3DNA®) functionalized with antibodies (Abs) against intercellular adhesion molecule-1 (ICAM-1), a cell surface glycoprotein accessible for targeting from the bloodstream and overexpressed in the lungs in many diseases. In particular, a prototype formulation of anti-ICAM/3DNA had demonstrated high cell-specific targeting and therapeutic potential in vitro. In this dissertation, we explored the kinetics, biodistribution, and lung-specific targeting in vivo of a new anti-ICAM/3DNA design that enabled precise surface functionalization with Abs to provide and modulate targeting. In Aim 1, we modified a radiotracing-based method to correct 125I-NP biodistribution results by separating the signal arising from the free 125I label, providing more accurate measurements of the NP biodistribution. In Aim 2, intravenous injection of anti-ICAM/3DNA in mice resulted in profuse and specific lung targeting, which had an unprecedently high specificity index over non-specific control. In Aim 3, we demonstrated that below the lung delivery saturation conditions and within the parametric range tested, anti-ICAM density on 3DNA played a key role in modulating lung specificity compared to the dose concentration and size of anti-ICAM/3DNA. Additionally, we estimated how this would impact targeting of drugs that can be intercalated into the DNA carrier core or linked to carrier outer arms. Overall, this study demonstrates that anti-ICAM/3DNA bio-physicochemical properties allow for efficient, specific, and tunable lung targeting. This new knowledge will help guide future DNA-NP designs for targeted therapeutic delivery and set the basis for investigational applications aimed at the treatment of pulmonary diseases.
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    Investigation of Vesicular-Mediated Transport of Intercellular Adhesion Molecule-1-Targeted Carriers for Treatment of Lysosomal Storage Disorders
    (2017) Manthe, Rachel Lee; Muro, Silvia; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Numerous cellular processes and therapeutic interventions rely on vesicular-mediated endocytosis to gain entry into cells and sub-cellular compartments, as well as for transcellular transport across biological barriers such as found at the blood-brain interface. Yet, endocytic behavior can be altered in disease, representing an additional hurdle in the design of effective therapeutic strategies. Lysosomal storage disorders (LSDs), characterized by lysosomal accumulation of undigested substrates as a result of deficient enzymatic activity, illustrate this paradigm. Currently, intravenous infusion of recombinant lysosomal enzymes to replace those deficient is the standard clinical approach for these disorders. However, clathrin-mediated endocytosis utilized by replacement enzymes for cellular uptake and lysosomal trafficking is altered, thereby impacting treatment efficacy as recently demonstrated in acid sphingomyelinase-deficient type A Niemann-Pick disease (NPD). Therefore, alternative means to bypass defunct routes is warranted. Therapeutic delivery via polymer nanocarriers targeting intercellular adhesion molecule-1 (anti-ICAM NCs), a cell-surface molecule overexpressed in endothelial and subjacent tissue cells during inflammation, such as in LSDs, represents a viable option since it permits uptake, intra- and transcellular transport via a unique endocytic route called the cell adhesion molecule (CAM) pathway. In this dissertation, cell culture and animal models were used to examine the (1) endocytic activity of the CAM pathway and other clathrin-independent routes in type A NPD, (2) role of targeting valency (i.e., density of ICAM-1-targeting molecules on the NC surface) in regulating the CAM pathway, and (3) effects induced via engagement of ICAM-1 on cells by anti-ICAM NCs. The results herein demonstrate the CAM pathway is more active in diseased cells compared to other classical endocytic pathways, making it the most amenable route for therapeutic enzyme replacement. Further, modulating targeting valency of NCs optimized this strategy for enhanced enzyme delivery to the brain, a target organ for type A NPD. Lastly, anti-ICAM NCs attenuated endothelial release of soluble ICAM-1, an inflammatory regulator, representing a secondary benefit of this system. Overall, this work validates utility of anti-ICAM NCs for enzyme replacement to treat NPD and likely other LSDs, and provides insight into biological processes and design parameters that influence the therapeutic efficacy of targeted drug carriers.
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    Investigation of Intercellular Adhesion Molecule-1 Targeted Drug Transport Across the Gastrointestinal Epithelium
    (2015) Ghaffarian, Rasa; Muro, Silvia; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Contrary to systemic injection of therapeutics, oral formulations represent clear advantages to patients, healthcare systems, and pharmaceutical companies including safety, low cost and patient compliance. However, oral delivery remains a major obstacle due to (1) drug instability in the harsh environment of the gastrointestinal (GI) tract owing to low gastric pH and enzymatic hydrolysis; (2) low permeability through the mucus layer and subsequent adhesion to the GI epithelium; and (3) suboptimal transport into or across the GI epithelium- the cell barrier responsible for selective absorption of substances into the circulation, for local or systemic delivery. While encapsulation methods have been developed to overcome barriers to stability and adhesion to the GI epithelium, safe and effective transport into and across this lining has not yet been achieved for several drugs, especially biotherapeutics. Hence, our goal is to overcome these challenges for delivery of therapeutics (including biotherapeutics) via the oral route. For this purpose, we targeted drugs to intercellular adhesion molecule-1 (ICAM-1), a protein expressed on the GI epithelium and other cell types. We previously demonstrated, that polymer nanocarriers (NCs) coated with antibodies to bind multiple copies of ICAM-1 (multimeric targeting) triggered uptake and transport across cultured GI epithelial cells, enabling intracellular and transcellular drug delivery. To implement this strategy in vivo, we successfully encapsulated antibody-coated NCs in chitosan-alginate microspheres for gastric protection of labile targeting antibodies, site-specific release in the intestinal environment (the site of drug absorption) and retention of targeting ability following release in vitro, in cell culture, and in vivo. Furthermore, to expand the utility of the ICAM-1 targeting approach, we explored a novel drug delivery system that binds only one to two molecules of ICAM-1 (monomeric targeting), which provides distinct advantages for oral drug delivery compared with multimeric strategies. In order to elucidate the advantages offered by this monomeric targeting approach, we compared the uptake and intracellular trafficking of ICAM-1 targeted monomeric antibodies vs. multimeric antibody-coated NCs in cultured endothelial cells, a commonly used cellular model to study ICAM-1 transport. We then revealed that the distinct itinerary of transport offered by monomeric ICAM-1 targeted antibodies led to enhanced uptake and transport across cultured GI epithelial cells, showing promise for oral delivery. Finally, in order to exploit this transport pathway for oral drug delivery, we conjugated a model drug cargo to monomeric ICAM-1 targeted antibodies, which was shown to endow drug targeting and delivery into and across cultured GI epithelial cells, while preserving the functional activity of the drug cargo. These findings demonstrate that monomeric vehicles serve as a viable alternative to multimeric strategies, expanding the range of oral delivery applications afforded by ICAM-1 targeting. Taken together, the work performed in this dissertation advocates the potential of ICAM-1 targeting strategies for improving oral absorption of therapeutics, and provides a foundation for studying these strategies in vivo.
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    ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY
    (2014) Serrano, Daniel; Muro, Silvia; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β2 integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.
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    ROLE OF ACID SPHINGOMYELINASE IN ICAM-1/NHE1-DEPENDENT ENDOCYTOSIS: IMPLICATIONS IN LEUKOCYTE TRANSMIGRATION
    (2010) Serrano, Daniel; Muro, Silvia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Engagement of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells (ECs) by anti-ICAM coated beads generates vesiculization via cell adhesion molecule (CAM)-mediated endocytosis, a clathrin-/caveolae-independent pathway involving Na+/H+ exchanger 1 (NHE1). ICAM-1 itself plays a key role in transendothelial migration (TEM) of leukocytes, particularly via the transcellular route. This involves endothelial endocytic vesicles that coalesce into transmigration pores, through which leukocytes transmigrate without disrupting EC junctions. The contribution of CAM-mediated endocytosis to the formation of docking sites and vesicular structures supporting TEM was explored in this study. Results show that the ICAM-1/NHE1-dependent CAM-mediated pathway associates with acid sphingomyelinase and ceramide. This supports plasmalemma deformability and cytoskeleton rearrangement, bridging these events to the formation of endothelial docking structures and vesicles involved in leukocyte transmigration.