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

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    PHYSICAL CHARACTERIZATION OF DNA CONDENSED WITH CATIONIC AGENTS
    (2016) Salgado, Eddy; Briber, Robert M; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Gene therapy using non viral vectors remains a challenging problem of maximizing efficiency while minimizing risks due to the multiple biological hurdles for a carrier agent to deliver its genetic cargo. The precise connection between the physical properties of the vectors and their transfection behaviors remains to be fully realized. We have used atomic force microscopy as well as dynamic light scattering and zeta potential measurements in order to image and characterize DNA complexes with polyethylenimine (PEI), histidine-lysine (HK) peptide, and triethylenetetramine (TETA)-functionalized gold nanoparticles. The resulting complex structures are analyzed as a function of amine to phosphate (N/P) ratios and as a function of sample preparation protocols. This work aims to not only characterize these specific complexes, but to aid in the general understanding of complex formation and how it relates to transfection observations to promote a more rational design of future gene delivery agents.
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    Development and Biophysical Characterization of HK Polymer for siRNA Delivery to Tumor in a Mouse Model
    (2014) Chou, Szu-Ting; Mixson, Archibald J; Seog, Joonil; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Delivery has been the major obstacle for nucleic acid therapeutics, including the RNA interference (RNAi) approach. Mixson's lab has been focused on the development of a non-viral peptide-based delivery system, HK (histidine-lysine) polymers, which have shown promise as carriers of plasmids and small interference RNA (siRNA) in several cell lines and in tumor-bearing models. In a previous study, a four-branched peptide, denoted H3K(+H)4b, with the predominant repeating -HHHK- sequence in the branch, has been shown to be the most effective and least toxic carrier in vitro and in vivo. Building on these results, I utilized different approaches including several structure and stability molecular characterization methods to study polyplex and to develop more effective carriers for improved therapy with siRNAs targeting malignancies. To understand the role of histidine in the stability of the H3K(+H)4b/siRNA polyplex, the physicochemical properties were investigated. With the use of isothermal titration calorimetry and heteronuclear single quantum coherence NMR, histidines were shown to form hydrogen bonds with siRNA, which enhanced the stability and biological activity of the polyplexes. In addition, to enhance resistance to nucleases and to target the tumors selectively, H3K(+H)4b was chemically modified with different patterns of polyethylene glycol (PEG) and cyclic RGD (Arg-Gly-Asp, cRGD) peptide conjugates. The luciferase marker gene expressed stably by tumor xenografts in mouse models was targeted in order to evaluate the efficacy of HK carriers of siRNA that differed in location and number of cRGD-PEG attachments. The most effective carrier was (RGD-PEG)4H3K(+H) (RP-HK), which has a cRGD-PEG on each of its four terminal branches. Consistent with its prolonged stability, as observed by pharmacokinetic studies, the RP-HK polyplex down-regulated luciferase activity in tumor xenografts by nearly 70% compared with the untreated group. Subsequently, the RP-HK polyplex was used to target the Raf-1 oncogene, an important mediator of tumor cell growth and angiogenesis. As in the luciferase studies, the polyplex reduced Raf-1 mRNA by more than 75%, and more importantly, the treatment inhibited the tumor growth by 60% in a mouse model. We anticipate that further design and engineering of HK carriers will improve the predictability and therapeutic activity of siRNA polyplexes in cancer treatment.