Fischell Department of Bioengineering Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/6628

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ENGINEERING TARGETED LIGHT ACTIVATABLE NANOPLATFORMS TO MANAGE RECURRENT CANCERS
(2024) Pang, Sumiao; Huang, Huang Chiao HH; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cancer recurrence poses a significant challenge in various malignancies that adverselyaffect long-term survival and quality of life. Glioblastoma (GBM) and ovarian cancer exhibit particularly high recurrence rates. For GBM, tumor recurrence is nearly universal (90%) within 10 months post initial treatment due to its invasive characteristics, limited delivery of therapeutic agents, and persistent drug resistance, resulting in a 5-year survival rate of <10%. While standard chemotherapy and surgery can temporarily alleviate symptoms for both diseases, there has been no significant improvement in long-term disease management or survival extension over several decades. Therefore, it is critical to develop targeted therapies that integrates well with current standards of care strategies. Photomedicine is a promising treatment modality, and the two main phototherapies are photodynamic therapy (PDT) which involves photosensitizer administration followed by light activation resulting in non-thermal chemical damage and photothermal therapy (PTT) which involves exogenous or endogenous sensitizing agents followed by light activation resulting in thermal damage. Clinical applications of both modalities have shown its feasibility and safety; however, they face challenges due to (i) limited cancer selectivity, (ii) heterogenous treatment response, and (iii) low monotherapy treatment efficacy. Leveraging strategic therapeutic targets to advance the current sensitizing agents for targeted delivery is a potential solution to overcome these limitations. The overall objective of this dissertation is to advance and evaluate targeted light-activatable nanoplatforms for phototherapy delivery with considerations for the current clinical workflow of GBM and advanced ovarian cancer. This is achieved through the following goals, (1) engineering a novel Fn14 receptor-directed gold nanorods (DART-GNRs) to assess selectivity and PTT efficacy for GBM, and (2) evaluate safety and long-term efficacy of targeted light-activatable multi-agent nanoplatform (tLAMP) to deliver targeted PDT for peritoneal carcinomatosis. First, this work establishes a reproducible synthesis protocol for DART-GNRs, characterizes its photothermal properties, and demonstrate high selectivity towards the Fn14 receptor of cancer cells. Second half of this dissertation established and investigated a two-fiber tissue optical property (TOP) monitoring method for liquid phantoms and for peritoneal carcinomatosis mouse model to enable safer light dosimetry during PDT, established an irinotecan active loading method to reproducibly synthesize tLAMP, and determined tLAMP tumor nodule penetration depth for enhanced targeted PDT combination therapy with adjuvant chemotherapy to enhance long-term survival for ovarian cancer.
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.
DEVELOPMENT OF GLYCOSAMINOGLYCAN MIMICKING NANOGEL TECHNOLOGIES FOR CONTROLLED RELEASE OF THERAPEUTICS TO TREAT RETINAL DISEASES IN DIFFERENT AGE GROUPS
(2024) Kim, Sangyoon; Lowe, Tao L.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Retinal diseases, such as diabetic retinopathy, glaucoma, macular degeneration, and retinoblastoma, affect around 13 million people worldwide, with projections indicating a rise to 20 million by 2030. These conditions lead to irreversible vision loss and significant impairment in both adults and children, with an annual economic burden of $139 billion in the United States alone. Aging significantly increases the risk of certain retinal conditions, and with improvements in healthcare leading to increased life expectancy, these conditions are becoming more prevalent due to the natural aging process and associated physiological changes in the eye. Current treatments are either destructive or have low efficacy and are not optimized for the younger population. While therapeutics including small molecular drugs, proteins and antibodies show promise in treating these diseases by reducing inflammation and neuronal apoptosis, their effectiveness is hindered by short half-lives and inability to cross the blood-retinal barrier (BRB). Nanoparticles offer a potential solution by improving drug delivery across biological barriers, yet no nanoparticles have been developed to effectively transport intact proteins or small molecules across the BRB to the retina without toxicity, slow clearance and stability. Therefore, there is an unmet need to evaluate the physical and physiological property changes of the eye along development and develop nanoparticle systems that can control and sustain the release of therapeutics across the blood retinal barrier (BRB) to treat the retinal diseases. In this project, the thickness, rheological property, permeability and morphological property changes of ocular barriers including sclera, cornea and vitreous humor in the developing eye from preterm to adult were evaluated using porcine ex vivo model. Two glycosaminoglycan mimicking nanogel systems, poly(NIPAAm-co-DEXcaprolactoneHEMA) nanogels with and without positive or negative charges and β-cyclodextrin based poly(β-amino ester) (CD-p-AE) nanogels were developed for sustained release of intact proteins including insulin and anti-TNFα, and small hydrophobic drugs, respectively across the ex vivo porcine sclera and in vitro BRB models: human fetal retinal pigment epithelial (hfRPE), adult retinal pigment epithelial (ARPE-19) and human cerebral microvascular endothelial (hCMEC/D3) cell monolayers. Completion of this project will have a significant impact on developing novel personalized nanotherapeutics to treat retinal diseases in different age groups.
Functionalized Nanoparticles for the Controlled Modulation of Cellular Behavior
(2023) Pendragon, Katherine Evelyn; Fisher, John; Delehanty, James; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The ability to control cellular behavior at the single-cell level is of great importance for gaining a nuanced understanding of cellular machinery. This dissertation focuses on the development of novel hard nanoparticle (NP) bioconjugate materials, specifically gold nanoparticles (AuNPs) and quantum dots (QDs), for the controlled modulation of cellular behavior. These hard NPs offer advantages such as small size on the order of 1 – 100 nm, high stability, unique optical properties, and the ability to load cargo on a large surface area to volume ratio, making them ideal tools for understanding and controlling cell behavior. In Aim 1, we demonstrate the use of AuNPs to manipulate cellular biological functions, specifically the modulation of membrane potential. We present the conception of anisotropic-shaped AuNPs, known as gold nanoflowers (AuNFs), which exhibit broad absorption extending into the near-infrared region of the spectrum. We demonstrate the effectiveness of utilizing the plasmonic properties AuNFs for inducing plasma membrane depolarization in rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells. Importantly, this is achieved with temporal control and without negatively impacting cellular viability. Aim 2 explores the use of QDs as an optical, trackable scaffold for the multivalent display of growth factors, specifically erythropoietin (EPO), for the enhanced induction of protein expression of aquaporin-4 (AQPN-4) within human astrocytes. This results in enhanced cellular water transport within human astrocytes, a critical function in the brain's glymphatic system. We show that EPO-QD-induced augmented AQPN-4 expression does not negatively impact astrocyte viability and augments the rate of water efflux from astrocytes by approximately two-fold compared to cells treated with monomeric EPO, demonstrating the potential of EPO-NP conjugates as research tools and prospective therapeutics for modulating glymphatic system function. Overall, the body of work presented in this dissertation develops new NP tools, namely solid anisotropic AuNFs and growth factor-delivering QDs, for the understanding and control of cell function. These new functional nanomaterials pave the way for the continued development of novel NP-based tools for the precise modulation of cellular physiology.
LEVERAGING SELF-ASSEMBLY AND BIOPHYSICAL DESIGN TO BUILD NEXT-GENERATION IMMUNOTHERAPIES
(2022) Froimchuk, Yevgeniy; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The immune system has evolved mechanisms to respond not only to specific molecular signals, but also to biophysical cues. Interestingly, research at the interface of biomaterials and immunology has also revealed that the biophysical properties and form of vaccines and immunotherapies impact immunological outcomes. For example, the intermolecular distance between antigen molecules on the surface of nanoparticles can impact formation of T cell receptor clusters that are critical during T cell activation. Despite the importance of biophysical cues in tuning the immune response, the connections between these parameters and immunological outcomes are poorly understood in the context of immunotherapy. Immunotherapies harness an individual’s immune system to battle diseases such as autoimmunity. During autoimmune disease, the immune system malfunctions and mistakenly attacks self-tissue. Immunotherapies can help tailor and guide more effective responses in these settings, as evidenced by recent advances with monoclonal antibodies and adoptive cell therapies. However, despite the transformative gains of immunotherapies for patients, many therapies are not curative, work only for a small subset of patients, and lack specificity in distinguishing between healthy and diseased cells, which can cause severe side effects. To overcome these challenges, experimental strategies are attempting to co-deliver self-antigens and modulatory cues to reprogram dysfunctional responses against self-antigens without hindering normal immune function. These strategies have shown exciting potential in pre-clinical models of autoimmune disease but are unproven in clinical research. Understanding how biophysical features are linked to immunological mechanisms in these settings would add a critical dimension to designing translatable, antigen-specific immunotherapies. Self-assembling materials are a class of biomaterials that spontaneously assemble in aqueous solution. Self-assembling modalities are useful technologies to study the links between biophysical parameters and immune outcomes because they offer precise control and uniformity of the biophysical properties of assembled moieties. Our lab leveraged the benefits of self-assembly to pioneer development of “carrier-free” immunotherapies composed entirely of immune signals. The therapies are composed of self-antigens modified with cationic amino acid residues and anionic, nucleic acid based modulatory cues. These signals are self-assembled into nanostructured complexes via electrostatic interactions. The research in this dissertation utilizes this platform as a tool to understand how tuning the biophysical properties of self-antigens impacts molecular interactions during self-assembly and in turn, how changes in biophysical features are linked to immunological outcomes. Surface plasmon resonance studies revealed that the binding affinity between signals can be tuned by altering overall cationic charge and charge density of self-antigen, and by anchoring the self-antigen with arginine or lysine residues. For example, the binding affinity between signals can be increased by increasing the total cationic charge on the self-antigen, and by anchoring the self-antigen with arginine residues rather than lysine residues. Computational modeling approaches generated insights into how molecular interactions between signals, such as hydrogen bonding, salt-bridges, and hydrophobic interactions, change with different design parameters. In vitro assays revealed that a lower binding affinity between self-assembled signals was associated with greater reduction of inflammatory gene expression in dendritic cells and more differentiation of self-reactive T cells towards regulatory phenotypes that are protective during autoimmunity. Taken all together, these insights help intuit how to use biophysical design to improve modularity of the self-assembly platform to incorporate a range of antigens for distinct disease targets. This granular understanding of nanomaterial-immune interactions contributes to more rational immunotherapy design.
ENGINEERING LIGHT-ACTIVATABLE NANOCOMPLEX TO OVERCOME MULTIDRUG RESISTANCE AND IMPROVE DRUG DELIVERY
(2022) Liang, Barry; Huang, Huang-Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Chemotherapy remains the main strategy for combating cancer, despite significant advances in alternative treatment modalities. It has been estimated that up to 90% of cancer-related deaths are caused by chemotherapy failure due to cancer multidrug resistance (MDR). MDR is a cellular phenomenon where cells are able to evade drug-induced cell death by developing resistance to multiple structurally and mechanistically distinct therapeutic compounds. Insufficient drug delivery, activation of compensatory survival pathways, and enhanced drug efflux by ATP-binding cassette (ABC) drug transporters are the primary challenges underlying MDR. As a result, an ideal cancer treatment strategy should involve selective delivery, retention, and activation of multiple therapeutic agents at the diseased site.Photodynamic therapy (PDT) is a photochemistry-based treatment modality that has shown promise in overcoming cancer drug resistance due to its unparalleled spatiotemporal control over treatment induction using light. The overall objective of this dissertation is to combine engineering strategies and PDT to overcome the existing challenges of MDR. The findings from this dissertation reveal PDT photochemically inactivates ABC drug transporters via functional (i.e., ATPase activity) inhibition and protein structural damage in a dose dependent manner. Our data suggest conjugation of a photosensitizer to conformation-sensitive antibody enables selective photosensitizer delivery to drug-resistant cancer cells and fluorescence visualization of functionally active ABC drug transporters. Our findings further show that targeted nanotechnology can improve photosensitizer delivery and allow for multidrug packaging for PDT-based combination treatment. Lastly, we leverage a dual fluorescence-guided approach to monitor the biodistribution of a targeted nanoformulation and customize intraoperative PDT dosimetry in vivo. Together, these findings from this dissertation advance the current understanding on using a light-activatable strategy to combat cancer drug resistance in three major ways: 1) elucidating the mechanism underlying photochemical inactivation of ABC drug transporters, 2) providing novel engineering strategies to improve multidrug delivery to cancer cells, and 3) demonstrating fluorescence-guided drug delivery and PDT light dosimetry.
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.
Formulation and Delivery of Enhanced Extracellular Vesicles for Wound Repair
(2021) Born, Louis Joseph; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. Extracellular vesicles (EVs) have recently emerged as an alternative approach to address this issue. In particular, EVs derived from mesenchymal stem/stromal cells (MSCs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. However, despite their clearly established potential, EVs have limitations that limit clinical relevancy, including low potency and rapid clearance from the body. Additionally, the ability to sustainably deliver EVs may enhance their efficacy in wound healing. Here, we leveraged the capability of EVs to be engineered via producer cell modification to investigate the therapeutic potential of EVs from MSCs transfected to overexpress a well-established pro-angiogenic long non-coding RNA HOX transcript antisense RNA (HOTAIR). We established that HOTAIR was able to be successfully loaded into MSC EVs (HOTAIR-MSC EVs) and delivered to endothelial cells in vitro with increased functional angiogenic activity. HOTAIR-MSC EVs injected intradermally around excisional wounds also showed increased angiogenic activity in vivo in two different species of rodents and improved wound healing in diabetic mice. We further report biomaterial-enabled sustained release of EVs using injectable hydrogel nanoparticles containing a composite of thiolated hyaluronic acid, maleimide functionalized poly(ε-caprolactone), and polyethylene glycol tetraacryalte as well as 3D-printed hydrogel discs composed of gelatin methacrylate for topical application. EVs released from the formulation of both of these biomaterials retained angiogenic bioactivity. Nanoparticles containing HOTAIR-MSC EVs were injected intradermally around an excisional wound in diabetic mice and were able to increase angiogenesis and improve wound healing. EVs released from 3D-printed EV-loaded GelMa hydrogels retained bioactivity in an in vitro endothelial scratch assay. Overall, these data suggest increasing the content of lncRNA HOTAIR in MSC EVs as a promising wound healing therapeutic. Additionally, establishing a biomaterial-enabled sustained release therapeutic represents a promising translational product for clinical implementation.
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.
Colloid Assembly Strategies For Structurally Colored Materials And Protease Detection
(2019) Torres, Leopoldo; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The goal of this dissertation is to better understand a mechanism that produces large color changes in a protease responsive nanoparticle hydrogel (PRNH) with structural color. The outcomes of this research can lead in the development of a peptide-based hydrogel optical sensor for the detection of toxic proteases in solution to prevent public exposure by means of water or food source contamination, and a potential terrorist event. Towards this application, a structural color changing SiO2 nanoparticle hydrogel film was made with a 4-arm poly(ethylene glycol) terminated with carboxylic acid norbornene (4PEGN), and a degradable dicysteine peptide. To fabricate the PRNHs, a rapid and tunable centrifugation-based assembly was developed. The color of centrifuged colloids of a single particle diameter was precisely controlled within 50 nm by modulating the particle concentration. The peak wavelength reflected by the material was further tuned by altering the centrifugal rate and assembly time. When placed in a protease solution, the peptide crosslinks degrade causing electrostatic binding and adsorption of the polymer to the particle surface which leads to the assembly of particles into compact amorphous arrays with structural color. Only PRNHs with highly negative particle surface charge exhibit color changes after degradation. Ultra-small angle x-ray scattering revealed that the particles become coated in polymer after degradation, producing a material with less order compared to the initial state. Altering the particle diameter modulates the composites' color, and all sizes investigated (178–297 nm) undergo the degradation-directed assembly. Varying the amount of 4PEGN adjusts the swollen PRNH color and has no effect on the degradation-directed assembly. Next, a botulinum neurotoxin (Botox) responsive nanoparticle hydrogel was developed. Its stability, optical properties, and response time were characterized and optimized for detecting 10 µg/mL of BoTox in solution. Last, a new method to produce bright full-spectrum structurally colored fluids that are non-iridescent is presented. The color was modulated by altering the particle volume fraction and a model predicting the peak wavelength reflected by the colloid was developed. Collectively, this body of work advances the development of responsive structurally colored detection platforms and particle assembly strategies for the production of structural color.

Fischell Department of Bioengineering Theses and Dissertations

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