Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item CONFINED PHOTOTHERMAL HEATING OF NANOPARTICLE DISPLAYED BIOMATERIALS(2021) Hastman, David A; Medintz, Igor L; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Controlling the temperature of biological systems has long been utilized as a tool for regulating their subsequent biological activity. Recently, photothermal heating of gold nanoparticles (AuNPs) has emerged as an efficient and remote method to heat proximal biological materials. Moreover, this technique has tremendous potential for controlling biological systems at the subcellular level, as specific components within the system can be heated while the larger system remains unaffected. The small size, biocompatiblilty, and optical properties of AuNPs make them attractive nanoscale heat sources for controlling biological systems. While the utility of photothermal heating has significantly advanced through the optimization of AuNP size, shape, and composition, the choice of incident light source utilized has largely been unexplored. One of the more interesting excitation sources is a femtosecond (fs) pulsed laser, as the subsequent temperature increase lasts for only a few nanoseconds and is confined to the nanoscale. However, it is not yet clear how biological materials respond to these short-lived and ultra-confined nanoscale spaciotemporal temperature increases. In this dissertation, we utilize fs laser pulse excitation to locally heat biological materials displayed on the surface of AuNPs in order to understand the corresponding heating profiles and, in turn, interpret how this can be used to modulate biological activity. Due to its unique temperature sensitive hybridization properties, we exploit double-stranded deoxyribonucleic acid (dsDNA) as our prototypical biological material and demonstrate precise control over the rate of dsDNA denaturation by controlling the laser pulse radiant exposure, dsDNA melting temperature, bulk solution temperature, and the distance between the dsDNA and AuNP surface. The rate of dsDNA denaturation was well fit by a modified DNA dissociation equation from which a “sensed” temperature value could be obtained. Evaluating this sensed temperature in the context of the theoretical temperature profile revealed that the ultra-high temperatures near the AuNP surface play a significant role in denaturation. Additionally, we evaluate this technique as a potential means to enhance enzyme activity and report that enhancement is governed by the laser repetition rate, pulse width, and the enzyme’s inherent turnover number. Overall, we demonstrate that the confined and nanosecond duration temperature increase achievable around AuNPs with fs laser pulse excitation can be used to precisely control biological function and establish important design considerations for coupling this technique to more complex biological systems.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 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.Item 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.