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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Item 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.Item Functionalized 3D DNA Crystals through Core-Shell and Layer-by-Layer Assembly(2019) McNeil, Ronald; Paukstelis, Paul; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A fundamental goal of DNA nanotechnology has been assembly of DNA crystals for use as molecular scaffolds to organize arrays of guest molecules. We use previously described 3D DNA crystals to demonstrate core-shell and layer-by-layer assembly of DNA crystals capable of accommodating tethered guest molecules within the crystals’ pervasive solvent channel network. We describe the first example of epitaxial biomacromolecular core-shell crystallization through assembly of the crystals in two or more discrete layers. The solvent channels also allow post-crystallization guest conjugation with layer-specific addressability. We present microfluidics techniques for core-shell crystal growth which unlock greater potential for finely tunable layer properties and assembling complex multifunctional crystals. We demonstrate assembly of these DNA crystals as nanoscale objects much smaller than previously observed. These techniques present new avenues for using DNA to create multifunctional micro- and nanoscale periodic biomaterials with tunable chemical and physical properties.Item A New Role for the CYT-18 N-Terminus and Three-Dimensional DNA Crystals as Vehicles for Biocatalysis(2014) Geng, Chun; Paukstelis, Paul J; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (N. crassa mt TyrRS; CYT-18 protein) promotes the splicing of multiple group I introns by stabilizing the catalytically active intron structures. CYT-18, and mt TyrRS's from related fungal species, have evolved to promote group I intron splicing partly by accumulation of three N-terminal domain insertions that create a structure-stabilizing scaffold for critical tertiary interactions between the two major group I intron domains. The primarily alpha-helical N-terminal insertion, H0, contributes to protein stability and is necessary for splicing the N. crassa ND1 intron, but is dispensable for splicing the N. crassa mt LSU intron. Herein, I show CYT-18 with a complete H0 deletion retains residual ND1 intron splicing activity and addition of the missing N-terminus in trans restores a significant portion of its splicing activity. This peptide complementation assay revealed important characteristics of the CYT-18/group I intron interaction including the stoichiometry of H0 in intron splicing and the importance of specific H0 residues. Evaluation of truncated H0 peptides in this assay also suggests a previously unknown structural role of the first five N-terminal residues of CYT-18. These residues interact directly with another splicing insertion, making H0 a central structural element responsible for connecting all three N-terminal splicing insertions. Transitioning to a separate study, I have demonstrated that enzymes retain catalytic activity when captured in the solvent channels of three-dimensional (3D) DNA crystals. Using RNase A as a model enzyme system this work shows that crystals infused with enzyme can cleave a fluorescent dinucleotide substrate with similar kinetic restrictions as other immobilized enzyme systems, mainly limited by diffusion of substrate. This new vehicle for immobilized enzymes, created entirely from biomolecules, provides a platform for developing modular solid-state catalysts that could be both biocompatible and biodegradable.