Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item PREPARATION OF A NANOSUSPENSION OF THE PHOTOSENSITIZER VERTEPORFIN FOR PHOTODYNAMIC AND LIGHT-INDEPENDENT THERAPY IN GLIOBLASTOMA(2024) Quinlan, John Andrew; Huang, Huang-Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photodynamic therapy (PDT) using verteporfin (VP) has treated ocular disease for over 20 years, but recent interest in VP’s light-independent properties has reignited interest in the drug, particularly in glioblastoma (GBM) (NCT04590664). Separate efforts to apply PDT to GBM using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) have also garnered attention (NCT03048240), but, unfortunately, clinical trials using 5-ALA-induced PpIX-PDT have yet to yield a survival benefit. Previous studies have shown VP to be a superior PDT agent than 5-ALA-induced PpIX. Our lab has shown that 690 nm light activates VP up to 2 cm into the brain, while 635 nm light only activates PpIX at depths <1 cm into the brain. Additionally, VP is a more effective photosensitizer than PpIX because it has a higher singlet oxygen yield and is active in the vasculature as well as target tumor cells. However, the hydrophobicity of VP limits effective delivery of the drug to the brain for treatment of GBM.In this context, this thesis aims to re-evaluate the delivery method for VP. VP traditionally requires lipids for delivery as Visudyne. Recent shortages of Visudyne and potential drawbacks of liposomal carriers motivated our development of a carrier-free nanosuspension of VP, termed NanoVP. Previous work has shown that cellular uptake of VP is greater when delivered as NanoVP rather than liposomal VP, resulting in improved cell killing after light activation. This thesis builds on this previous work by (1) evaluating synthesis and storage parameters for NanoVP, (2) determining the pharmacokinetics, biodistribution, and brain bioavailability of NanoVP, and (3) evaluating the potential efficacy of NanoVP as a PDT and a chemotherapy agent, and by supporting development of a zebrafish model of the blood-brain barrier (BBB) for mechanistic studies of improved drug delivery to the brain.Item 3D ENGINEERING OF VIRUS-BASED PROTEIN NANOTUBES AND RODS: A TOOLKIT FOR GENERATING NOVEL NANOSTRUCTURED MATERIALS(2018) Brown, Adam Degen; Culver, James N; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Technological innovation at the nanometer scale has the potential to improve a wide range of applications, including energy storage, sensing of environmental and medical signals, and targeted drug delivery. A key challenge in this area is the ability to create complex structures at the nanometer scale. Difficulties in meeting this challenge using traditional fabrication methods have prompted interest in biological processes, which provide inspiration for complex structural organization at nanometer to micrometer length scales from self-assembling components produced inexpensively from common materials. From that perspective, a system of targeted modifications to the primary amino acid structure of Tobacco mosaic virus (TMV) capsid protein (CP) has been developed that induces new self-assembling behaviors to produce nanometer-scale particles with novel architectures. TMV CPs contain several negatively charged carboxylate residues which interact repulsively with those of adjacent CP subunits to destabilize the assembled TMV particle. Here, the replacement of these negatively charged carboxylate residues with neutrally charged or positively charged residues results in the spontaneous assembly of bacterially expressed CP into TMV virus-like particles (VLPs) with a range of environmental stabilities and morphologies and which can be engineered to attach perpendicularly to surfaces and to display functional molecular patterns such as target-binding peptide chains or chemical groups for attachment of functional targets. In addition, the distinct electrostatic surface charges of these CP variants enable the higher-level coassembly of TMV and VLP into continuous rod-shaped nanoparticles with longitudinally segregated distribution of functionalities and surface properties. Furthermore, the unique, novel, environmentally responsive assembly and disassembly behaviors of the modified CPs are shown to act as simple mechanisms to control the fabrication of these hierarchically structured functional nanoparticles.Item Development of Nanoparticle-Based Intracellular Dual Sensing and Actuation Modalities(2017) Field, Lauren D.; White, Ian M; Medintz, Igor L; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The integration of therapeutics with diagnostic agents, or theranostics, is vital for the development of novel and effective disease treatments. To effectively design new and efficient theranostic materials, a thorough understanding of the carrier ensemble, the interactions within the construct components, and the surrounding environment is required. This dissertation focuses on the development of new strategies to produce an effective ‘toolbox’ of nanoscale theranostics, namely through the use of a central NP scaffold and the visualization technique of Förster Resonance Energy Transfer (FRET). The NP scaffold used throughout this work, the semiconductor quantum dot (QD), is ideal for visualizing sensing modalities due to their high quantum yield (QY), tunable, narrow and symmetric emission profiles with broad, far-UV excitation, and resistance to photobleaching - making them optimal FRET donors. We first examined the intracellular assembly of QDs to proteins by injecting 545 nm emitting QDs, coated with various capping ligands, into cells transfected to express mCherry at two distinct intracellular locations: the cytosol and the plasma membrane. We found that the small, zwitterionic capping ligand CL4 and the cytosolically located mCherry protein assembled into the most efficient FRET complexes. We used this knowledge to design and implement a novel intracellular actuation modality for drug delivery that used a 520 nm emitting QD with the carrier maltose binding protein appended to the surface and carrying drug or dye conjugated to a maltose analog, -cyclodextrin in the binding pocket. Rather than relying on intracellular environmental changes or external stimuli to actuate release, the addition of the innocuous sugar maltose to the medium induced cargo actuation and could be visualized via FRET. Finally, the same methods were implemented to develop a novel pH sensor to report on the extracellular changes that occur in tumor development where the physiological pH is lowered dramatically. Using a 464 nm QD scaffold conjugated to pH-responsive FITC, we successfully monitored changes in extracellular pH and accurately determined unknown pH values. With the work in this thesis, we believe we have contributed greatly to the advancement and development NPs for the design and implementation of sensing and actuation complexes.