Fischell Department of Bioengineering Theses and Dissertations

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    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.
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    Additive Manufacturing for Recapitulating Biology in vitro and Establishing Cellular & Molecular Communication
    (2023) Chen, Chen-Yu; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Recapitulating biological systems within laboratory devices, particularly those with analytical instrumentation, has enhanced our ability to understand biology. Especially useful are systems that provide data at the length and time scales characteristic of the assembled biological systems. In this dissertation, we have employed two advanced technologies — additive manufacturing and electrobiofabrication to create systems that both recapitulate biology and provide ready access to molecular data. First, we utilized two-photon direct laser writing (DLW) and digital light processing (DLP) 3D printing to reconstruct morphologies of human gut villi. Our constructs enable small molecule diffusion through pores and enable epithelial cell growth and differentiation, as in the gastrointestinal (GI) tract. We also developed a cell/particle alignment methodology that applies a vacuum on the underside of a device to rapidly facilitate attachment to 3D printed scaffolds. These simple demonstrations of additive manufacturing show how one can better tailor geometric features of organ-on-a-chip and other in vitro models. We then added electrobiofabrication as a means create functionalized surfaces that rapidly assemble biological components, noted for their labile nature, onto devices with just an applied voltage. In one example, we show how a thiolated polyethylene glycol (PEG) can be electroassembled as a sensor interface that includes antibody binding proteins for both titer and glycan analysis. Rapid assessment of titer and glycan structure is important for biopharmaceuticals development and manufacture. While the interface and sensing methodology was performed using standard laboratory instrumentation, we show that the methodology can be streamlined and operated in parallel by incorporating into a microfluidic sensor platform. Additionally, we show how the combination of optical and electrochemical (redox) based measurements can be combined in a simplified insert that “fits” nearly any microplate reader or other fairly standardized laboratory spectrophotometric unit. We believe that by adapting transformative electrochemical analytical methods so they can augment more traditional optical techniques, we might ultimately generate devices that provide a far more comprehensive picture of the target, promoting better investigation. Specifically, we show how three important biological and chemical systems can be interrogated using both optical measurements and electrochemistry: the oxidation state of proteins including monoclonal antibodies, redox status of hydrogel materials, and electrobiofabrication and electrogenetic induction. Lastly, we demonstrate how electrobiofabrication can be used to create designer communities of bacteria — artificial biofilms — the study of which is important for understanding phenomena from infectious disease to food contamination. That is, we discovered that by varying the applied voltage, surface area, and composition of the to-be-assembled hydrogel solution, we can precisely control the intercellular environment among bacterial populations. In sum, this dissertation integrates advances in assembly, through additive manufacturing, electrobiofabrication, with advances in electrochemical analysis to bring to the fore an electronic understanding of complex biological phenomena. We believe that the capability of translating biological information into a processible digital language opens tremendous opportunities for advancing our understanding of nature’s amazing systems, potentially enabling electronic means to control her subsystems.
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    Photoimmunotherapy-Based Combination Regimens and Drug Delivery Systems for Ovarian Cancer Treatment
    (2023) Sorrin, Aaron; Huang, Huang Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Ovarian cancer is among the deadliest gynecologic malignancies, accounting for over 13,000 deaths and nearly 20,000 new cases each year in the United States alone. The lethality of this disease results from several fundamental challenges, including diagnosis at advanced stages, development of resistance to standard-of-care chemotherapies, and extensive metastasis throughout the peritoneal cavity. Photodynamic therapy (PDT) is a promising treatment modality which enables spatiotemporally controlled cancer ablation upon light-activation of specialized drugs (photosensitizers). Clinical studies have demonstrated the feasibility and safety of PDT for women with peritoneally disseminated ovarian cancer, though treatment outcomes were limited by off-target toxicities and the heterogenous cellular uptake of photosensitizer. The use of antibody-conjugated photosensitizers (photoimmunoconjugates) has the potential to overcome these prior limitations, making the targeted version of PDT (photoimmunotherapy, PIT) a valuable tool for ovarian cancer treatment.The overarching objective of this dissertation is to develop PIT-based strategies for ovarian cancer management through three complimentary goals: 1) overcome metastatic behaviors in ovarian cancer using PIT-based combination therapies; 2) bolster photosensitizer drug delivery using a clinically-relevant, fluid flow-based drug delivery approach; and 3) enhance cytotoxic effects of PIT through developing a new nanocomplex for photochemotherapy. This work establishes novel PIT-based combination treatments that incorporate clinically relevant therapies, including prostaglandin E2 receptor 4 (EP4) antagonism, poly(ADP-Ribose) polymerase (PARP) inhibition, and epidermal growth factor receptor (EGFR)-targeted antibodies. Results from this dissertation reveal pronounced combination effects of PIT and EP4 antagonism, leading to cooperative reductions in metastasis- related behaviors and cell signaling in vitro. The findings of this work further demonstrate that fluid flow enhances photoimmunoconjugate delivery, modulates subcellular photosensitizer localization, and enhances the phototoxicity to ovarian cancer cells in a pump system. Lastly, we developed 1) a targeted nanocomplex for combination of PIT and PARP inhibitors; and 2) a 3- dimentional (3D) ovarian tumor spheroid coculture model for the longitudinal quantification of treatment effects and the development of multidrug resistance.
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    (2023) Eppler, Haleigh B; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The immune system is tightly regulated to balance the killing of disease-causing organisms while protecting host tissue from accidental damage. When this balance is disrupted, immune dysfunctions such as autoimmune diseases occur. Autoimmune diseases like type 1 diabetes and multiple sclerosis (MS) develop when self-tissue is mistakenly attacked and damaged by immune cells. For example, during MS, the immune system mistakenly attacks the myelin sheath that insulates neurons, causing loss of motor function and burdening patients and caregivers. Recent advances in immunotherapies offer exciting new treatments; however, even monoclonal antibody therapies cannot differentiate between healthy and disease-causing cells. Biomaterials provide powerful capabilities to help address these shortcomings. In particular, control over the concentration, duration, location, and combination of signals that are received by immune cells could be transformative in developing more selective immunotherapies that are safe and promote antigen-specific tolerance during autoimmune disease. This dissertation uses two biomaterial approaches to deliver regulatory cargo to antigen presenting cells (APCs). An important APC function is to detect disease-causing organisms by sensing pathogen associated molecular patterns (PAMP) through motif-specific receptors. CpG rich motifs are PAMPs that activate toll-like receptor 9 (TLR9) on DCs and B cells. TLR9 signaling activates B cells and DCs. In MS, TLR9 signaling is aberrantly elevated on certain DCs contributing to systemic inflammation. In MS, B cells signaling through the TLR9 pathwway induced the expression of more inflammatory cytokines as compared to B cells from healthy controls. Controlling this overactive TLR signaling restrains inflammation and is a possible tolerogenic therapeutic approach in MS. The first part of this dissertation uses biomaterials-based polyelectrolyte multilayers (PEMs) to deliver tunable amounts of GpG – an oligonucleotide that inhibits TLR9 signaling – to dendritic cells (DCs). These studies demonstrate that PEMs inhibit DC activation and reduce pathway-specific inflammatory signaling. Furthermore, this work demonstrates that these changes to DCs promote tolerance in downstream T cell development as shown by increasing regulatory T cells. These studies demonstrate this biomaterial delivery system selectively inhibits TLR signaling and DC activation. These changes to DCs promote myelin-specific T cells to adopt a regulatory phenotype, demonstrating a potential approach to developing tolerance inducing antigen-specific immunotherapies for MS. The second part of this dissertation uses a degradable polymer microparticle (MP) system to control the local microenvironment of lymph nodes (LNs). LNs are key sites in the development of immune responses. LNs are composed of different microdomains that coordinate immune cell interactions such as germinal centers (GCs), where B cells develop. These MPs are loaded with myelin self-antigen (MOG35-55) and an mTOR inhibitor, rapamycin (rapa). The MPs are designed to be too large to passively diffuse from the LNs; instead, they slowly degrade releasing encapsulated immune cues to immune cells within the lymph node (LN). Our previous work demonstrates this treatment approach induces antigen-specific tolerance in a preclinical model of MS, but the role of APCs – including DCs and B cells - has not been elucidated. This dissertation reveals that MP treatment alters key LN structural components responsible for interactions between cells in GCs. In addition, MPs alter interactions between B cells/DCs and T cells, as measured by presentation of encapsulated antigen and inhibition of T cell costimulatory molecules by encapsulated rapa. These changes inhibit myelin-specific T cell proliferation and promote regulatory T cells. Finally, B cells from MOG/rapa and MOG MP treated lymph nodes transfer myelin-specific efficacy to mice induced with EAE. These findings illustrate how LN and cellular processes can be regulated by MPs to promote myelin-specific tolerance informing the development of myelin-specific immunotherapies for MS. Together, this body of work provides insight into how biomaterials can be designed to exploit native LN and immune cell functions in the design of next-generation approaches to safely induce myelin-specific tolerance during MS or other autoimmune diseases.
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    (2023) Frank, Eric; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The use of Brillouin spectroscopy for noninvasive probing of the mechanical properties of biologically relevant materials shows great promise. Stimulated Brillouin scattering (SBS) spectroscopy has the potential to significantly improve measurement speed and resolution by amplifying the scattered signal resonantly. However, current SBS spectrometers have been limited by fundamental and practical constraints in detection parameters. Here, we develop and demonstrate a novel LabVIEW-automated SBS instrumentation scheme in which a number of instruments that otherwise operate independently are automatized and synchronized from a singular LabVIEW program with emphasis on the user interface. Additionally, localization theory, originating from fluorescence-based super resolution microscopy techniques, is applied to the acquisition of SBS spectra, and experimentally demonstrated using this instrumentation scheme, resulting in spectra being acquired an order of magnitude faster while maintaining performances in terms of signal to noise ratio (SNR) and measurement precision.