Fischell Department of Bioengineering Theses and Dissertations

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Metabolic Profiling of Brain Microvascular Endothelial Cells: Investigating the Role of Sex, Stress, APOE Genotype, and Exercise in Alzheimer's Disease Risk
(2024) Weber, Callie; Clyne, Alisa M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Alzheimer’s disease (AD) is the 7th leading cause of death in the United States, yet there are still no effective treatments to prevent or slow the progression of the disease. AD develops from a combination of genetic and lifestyle risk factors including female sex, elevated stress hormone exposure, the apolipoprotein (APOE) ε4 genotype, and a sedentary lifestyle. In order to better identify the manifestations of AD, it is vital to understand how each of these risk factors impact brain health and lead to neurological dysfunction associated with AD. Brain microvascular endothelial cells (BMEC) line the blood vessels of the brain and have specialized tight junctions designed to strictly regulate nutrient and waste transfer between the blood and the brain. Two of the early indicators of AD development are breakdown of the tight junctions and whole brain glucose hypometabolism. Since BMEC form the first line of defense for the brain against neurotoxic compounds in the blood and are responsible for glucose transport to the rest of the brain, the overarching goal of this thesis is to understand how female sex, elevates stress hormone exposure, the APOE ε4 genotype, and a sedentary lifestyle induce breakdown of tight junction proteins and glucose hypometabolism in BMEC. I first demonstrate that female sex exacerbates endothelial dysfunction in response to high levels of a stress hormone, Angiotensin II (AngII). Specifically, I show that in response to AngII, female endothelial cells increase oxidative stress and inflammatory responses while male endothelial cells do not. Next, I used CRISPR/Cas9 to generate a set of induced pluripotent stem cells (iPSC) homozygous for the APOE ε3 and ε4 genotype and differentiated them into BMEC (hiBMEC). Using the hiBMEC I showed the APOE ε4 genotype induces barrier deficiencies that are partially mediated through reduced levels of protein deacetylase Sirtuin 1 (SIRT1), and that the APOE ε4 genotype causes glucose hypometabolism through decreased insulin signaling. Finally, by adding serum from sedentary and exercise trained individuals to genotype-matched hiBMEC, I show that APOE ε3 and ε4 hiBMEC have divergent responses to treatment with serum from sedentary and exercise trained individuals. Treatment with exercise trained serum increases SIRT1 and glycolytic enzymes compared to sedentary serum, while exercise trained serum decreases SIRT1 and glycolytic enzymes in APOE ε4 hiBMEC compared to sedentary serum. The work described in this thesis gives a fundamental, mechanistic understanding to the roles of female sex, stress hormone exposure, the APOE ε4 genotype, and a sedentary lifestyle in BMEC dysfunction and hypometabolism, giving insight into how these factors contribute to AD development and progression.
TUNABLE ATOMIC LINE MONOCHROMATORS FOR BRILLOUIN SPECTROSCOPY
(2024) Hutchins, Romanus Joshua; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Brillouin microscopy, a non-contact, spatially-resolved imaging method, provides insights into the mechanical information of samples. The first generation of Brillouin microscopes combined confocal microscopes and etalon-based spectrometers. In this setup, a confocal microscope scans a laser across the sample pixel-by-pixel, while the etalon spectrometer measures the Brillouin shift frequency at each pixel. Despite the extended image acquisition times in biological samples (>20 ms/pixel), advancements have been made in the field to enhance the overall speed of Brillouin imaging. For example, line-scan Brillouin spectrometers use orthogonal detection to measure the Brillouin scattering at a row of pixels in a single shot. The pixel multiplexing in one-dimension (1D) improved the Brillouin imaging speeds 20-fold. Further multiplexing to two dimensions, or full-field spectroscopy, where the frequency domain is sequentially acquired but all the pixels in the field of view are simultaneously measured at each frequency, can further improve the average image acquisition time. However, there are currently no solutions for sub-picometer (sub-GHz) spectral resolution, two-dimensional (2D) multiplexing of Brillouin images. Here, I use the laser induced circular dichroism (LICD) effect in atomic vapors to create monochromators for 2D multiplexing at high spectral resolutions. These atomic line monochromators possess spectral resolutions dependent on the linewidth of the atomic resonance (~MHz), and they are ideal for pixel multiplexing because they have spectral analysis capabilities that do not depend on the spatial separation of spectral components. First, I present a full characterization of a tunable atomic line monochromator. I measure the transmission, spectral resolution, and spectral tunability of the device, as well as demonstrate whole-image transmission through the atomic line monochromator. Next, for practical implementations of the device to Brillouin spectroscopy, I created an atomic line monochromator based on a ladder-type atomic transition. This iteration of the device suffers from less noise than the previous version, leading to the first Brillouin measurements with this device. Finally, I present the first full-field Brillouin microscope by demonstrating whole Brillouin imaging with orthogonal detection with an atomic line monochromator.
SPECTROSCOPY-BASED METHODS FOR BIOLOGICAL APPLICATIONS
(2024) Zhou, Xuewen; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Spectroscopy plays a crucial role in biological measurements by offering functional insights across various biological scales, from cellular to organ levels. It has become an important complement to imaging technologies. The thesis focuses on enhancing spectroscopy-based methods, addressing both technical advancements and application-oriented improvements. The first part of the research presents the development of standardized test methods for multispectral photoacoustic imaging (MPAI), a phantom-based test method specific for evaluating MPAI performance in breast cancer detection. This test method contributes to standardizing the evaluation of multispectral photoacoustic imaging across different research and clinical settings with better reliability and reproducibility. The next segment of the thesis introduces an innovative 2D-dispersion spectrometer, utilizing an etalon and grating setup. This instrument significantly improves the sensitivity in detecting Whispering Gallery Mode (WGM) microlasers by providing spectral resolution less than 0.3 pm. The enhanced sensitivity allows for the measurement of hyperfine refractive index and absorption changes in liquid environments, which expands the application limit of microlasers as biosensors. In the final part, the thesis extends the application of the 2D-dispersion spectrometer to detect Brillouin and low-frequency Raman signals. For the best of our knowledge, this is the first setup demonstrating simultaneous Brillouin and low-frequency Raman measurement, which can provide mechanical and chemical information of a sample such as the viscoelasticity and intermolecular dynamic.
MULTISCALE TECHNOLOGIES FOR ENGINEERING AND CRYOPRESERVING OVARIAN TISSUES AND HUMAN IPSCs
(2023) Stewart, Samantha; He, Xiaoming; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
An estimated 9.8 million reproductive-age people with ovaries in the United States are impactedby fertility issues, oftentimes caused by the dysregulation of the tightly controlled process of ovarian follicle development. Impaired fertility can arise from disorders like polycystic ovarian syndrome (PCOS) or premature ovarian insufficiency (POI), which affect the function of the ovary, an integral reproductive organ that houses the ovarian follicles. POI can also negatively impact endocrine function, decreasing estrogen and leading to increased risk of osteoporosis, cardiovascular disease, and neurological disorders. Novel fertility preservation and restoration strategies, like ovarian tissue engineering, have emerged to address these effects of ovarian dysregulation and offer alternatives for those who wish to delay childbearing. Human induced pluripotent stem cells (hiPSCs) hold tremendous potential for tissue engineering and cell-based medicine, as they have the capacity of differentiating into ectodermal, mesodermal, endodermal, and germ cell lineages. In recent years, research into differentiating hiPSCs into cells like those that make up the ovary has garnered much interest, highlighting these cells as a promising source for ovarian tissue engineering and other types of cell-based medicine and research. This work addresses critical challenges associated with engineering ovarian tissue for reproductive and cellbased medicine: (1) engineering the microenvironment for the cell/microtissue and (2) cryopreservation of the cells/microtissues. To understand the microenvironment of the ovary for informed tissue engineering system design, we spatially characterize the micromechanical properties of ovarian tissue from domestic cats to reveal both elastic and viscoelastic property heterogeneities, correlating these findings with the distribution of key extracellular matrix (ECM) molecules. We then developed a novel cryopreservation technology to enhance cryopreservation of ovarian follicles and hiPSCs, using sand to seed ice in the extracellular solution at high subzero temperatures during cooling. Together, this work investigates multiscale strategies for advancing ovarian tissue engineering, contributing to the advancement of reproductive medicine approaches for treating infertility and related endocrine dysfunction.
Deep Learning-Reinforced Engineering of Islets with Micro and Nano Biomaterials for Type 1 Diabetes Treatment
(2023) White, Alisa; He, Xiaoming; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
An estimated 1.6 million Americans live with type 1 diabetes (T1D). The most common treatment method involves daily blood glucose monitoring and insulin injections, which can negatively impact quality of life and lead to severe health complications. Whole pancreatic transplantation, a more permanent treatment option, entails invasive surgery with associated risks and a high morbidity rate. Furthermore, lifelong immunosuppressant use, which can be detrimental to health, is necessary for transplant recipients. Islet transplantation has emerged as a promising alternative for T1D treatment, offering a less invasive approach, though it still requires the use of immunosuppressants to prevent graft rejection. Encapsulation of islets in biomaterials has shown potential for mitigating immune responses post-transplantation while facilitating islet survival and insulin production. However, despite its promise, this method faces several challenges. First, a significant issue is the generation of numerous empty microcapsules during islet encapsulation, which requires an efficient method for their removal due to the limited space for the transplanted islets in patients. Second, microcapsules are typically suspended in an oil phase after generation with microfluidic devices, whereas they must be transferred into an aqueous solution for further culture or transplantation, posing technical difficulties. Third, conventional microcapsules do not provide a tissue-like environment for islets which is detrimental to islet health, and microcapsule design flaws can result in a lack of insulin production and islet cell death due to post-transplantation immune response. Furthermore, islets experience hypoxia and increased amounts of reactive oxygen species post-isolation and transplantation, resulting in islet death. This work focuses on addressing the challenges mentioned above by enhancing islet encapsulation methods through a deep learning-based on-chip detection and sorting system, enabling the creation of highly pure samples of islet-laden core-shell hydrogel microcapsules that mimic the structure and microenvironment of the pancreas. We also investigate the use of nanoparticles to encapsulate hydrophobic antioxidants for improving their delivery into islet cells to enhance islet viability after isolation and hypoxic stress. We address critical challenges in islet transplantation by investigating deep learning-enabled selective extraction, core-shell hydrogel microencapsulation, and nanoparticle-mediated antioxidant delivery. This novel multiscale biomaterials-engineering strategy has great potential for future clinical translation, contributing to the advancement of type 1 diabetes treatment.
The Effect of Glycemic Condition on Substrate Stiffness-Mediated Mechanosensitivity in Macrophages
(2023) Johnson, Courtney Dashawn; Aranda-Espinoza, Helim; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Diabetes is a disease that plagues over 463 million people globally. About 40 million of these patients have Type 1 diabetes, and the global incidence is increasing up to 5% per year. Type 1 diabetes is characterized by the body's immune system targeting the pancreas with antibodies, leading to a disruption in insulin production. While existing treatments, such as exogenous insulin injections, are both successful and popular, the exorbitant insulin costs and need for meticulous administration are driving factors for alternative long-term solutions to glucose dysregulation caused by diabetes. Encapsulated islet transplantation (EIT) is a tissue-engineered solution to address the challenges raised by diabetes. Donor islets are encapsulated in a semi-permeable hydrogel, allowing the diffusion of oxygen, glucose, and insulin but preventing leukocyte infiltration. Despite its successes in small animal models, EIT is still far from commercialization due to the requirements of long-term systemic immunosuppressants and consistent immune rejection from the foreign body response. While most published research has focused on tailoring the characteristics of the capsule material to promote clinical viability, many studies have had a limited scope centered on biochemical changes. Current mechanobiology studies on the effect of substrate stiffness on the function of leukocytes, especially macrophages – primary foreign body response orchestrators, show promise in tailoring a favorable response to tissue-engineered therapies such as EIT. However, before successful integration into the EIT design, it is imperative to determine the impact of external glucose concentrations on substrate stiffness-mediated mechanosensitivity. Glucose plays a critical role in macrophage functionality, impairing or enhancing their function in certain situations. Immunometabolism literature demonstrates that decreasing or inhibiting the uptake of glucose via impairing glycolysis can result in a significant decline in functionality in macrophages. Patients suffering from diabetes experience dysregulation in glycemic maintenance, ranging from hypo-, normo-, and hyperglycemic conditions. As a result, it is imperative to assess whether these changes in external glucose conditions will affect macrophage mechanosensitivity in response to EIT biomaterials to use substrate stiffness as a design parameter for EIT effectively This project investigates the role of glycemic conditions on macrophage mechanosensitivity, considering substrate stiffness factors including morphology, phenotype, phagocytic and inflammatory functionality. These parameters aim to mimic the early stages of foreign body response, particularly the initiation and acute inflammation phases. This work demonstrates that glycemic condition significantly influences the severity of substrate stiffness-mediated mechanosensitivity in reference to macrophage phagocytosis and pro-inflammatory functionality. This study serves to advance the understanding of macrophage functionality, bridging the fields of mechanobiology and immunometabolism. Understanding the role of glycemic conditions on substrate stiffness-mediated mechanosensitivity will assist in EIT design to enhance the clinical viability of the therapy and prevent immune rejection by pericapsular fibrotic overgrowth.
ENHANCING BIOPRINTING STRATEGIES TOWARDS THE DEVELOPMENT OF BIOMIMETIC OSTEOCHONDRAL TISSUE ENGINEERING SCAFFOLDS
(2023) Choe, Robert; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that affects approximately 900,000 Americans annually and is characterized by the progressive breakdown of the articular cartilage and remodeling of the subchondral bone in the synovial joint. During early-stage osteoarthritis, the articular cartilage begins to degrade, the synovial joint space narrows, and the subchondral bone undergoes rapid bone turnover, leading to insufficient bone mineralization and compromised matrix integrity. While decades of research have revealed that an intricate balance between the bone and cartilage layers influences biochemical and biomechanical changes experienced within the osteochondral unit, most osteochondral tissue engineering scaffolds have not achieved clinical viability. Tissue engineering (TE) strategies, such as 3D bioprinting (3DP), offer a new avenue to help develop novel osteochondral tissue engineering scaffolds to regenerate healthy and diseased osteochondral joints. In this project, our immediate goal is to expand the repertoire of osteochondral bioprinting strategies toward developing a biomimetic, 3D-printed osteochondral scaffold that can be implanted into acute focal cartilage defects during early-stage OA. We will explore the designs and fabrication strategies of various 3D-printed biomimetic osteochondral interface scaffolds with enhanced mechanics guided by computational simulations. Additionally, we will examine the potential of utilizing osteoblast- and osteoclast-lineage cell co-cultures to improve regenerative outcomes at the bone scaffold layer of osteochondral tissue engineering scaffolds. The long-term goal of this work is to aid in developing a biomimetic 3D printed osteochondral scaffold that has enhanced load-bearing properties and elevated regeneration potential to recreate the unique osteochondral architecture at each distinct tissue layer.
ENGINEERING NANOPARTICLES FOR IMPROVED LYMPHATIC DELIVERY AND ELUCIDATING MECHANISMS REGULATING NANOPARTICLE TRANSPORT INTO LYMPHATICS
(2023) McCright, Jacob Connor; Maisel, Katharina; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Immune modulatory therapies usually need to be effectively delivered to lymph nodes to enhance therapeutic effectiveness. Lymphatic vessels exist throughout the body and can transport 10 – 250 nm therapeutic nanoparticles to lymph nodes, however, nanoparticle formulations required to maximize this transport, and the mechanisms governing this transport are poorly understood. Here, we probed the effect of surface charge, surface poly(ethylene glycol) (PEG) density, shape, and size on nanoparticle transport across LECs (LECs) and lymph node delivery. Using an established in-vitro lymphatic transport model, we found PEGylation improved the transport of 100 and 40 nm nanoparticles across LECs 50-fold compared to non-PEGylated nanoparticles and that transport is maximized when the PEG is in a dense brush conformation corresponding to a high grafting density (Rf/D = 4.9). PEGylating 40 nm nanoparticles improved transport efficiency across LECs 68-fold compared to unmodified nanoparticles, demonstrating that the addition of PEG improves transport in a size-independent manner. We injected these nanoparticle formulations intradermally into C57Bl/6J mice and found that PEGylated 100 nm and 40 nm nanoparticles accumulate in lymph nodes within 4 hours, while unmodified nanoparticles accumulated minimally. Densely PEGylated nanoparticles also traveled furthest from the injection site. In this thesis, we also determined that nanoparticles are transported via both paracellular and transcellular mechanisms, and that both PEG conformation and nanoparticle size and shape modulates the cellular transport mechanisms. We also expanded our in-vitro lymphatic transport model to model important physiological conditions including transmural flow and found that the presence of this flow increased transport across lymphatic barriers in a shape and mechanism-dependent manner. To further investigate the mechanisms regulating nanoparticle transport, we generated a computational kinetic transport model that was able to quantify the contributions of both paracellular and transcellular transport mechanisms, as well as predict transport efficiency as a function of nanoparticle characteristics including size and surface chemistry. Using transport inhibitors, we can expand our system of equations to describe precise uptake and transport mechanisms, and the relation between nanoparticle formulation and mechanism. This computational model is one of the first to describe transport across lymphatic vessels, and offers some of the first definitions for coefficients used to quantitatively describe nanoparticles transport across LECs (i.e., permeability). Our computational, in-vitro, and in-vivo results indicate that nanoparticle surface charge, PEG conformation, and size are key criteria for nanoparticle design for effective lymphatic delivery with a dense, neutrally charged coating of PEG maximizing transport across LEC barriers and transport to lymph nodes. Optimizing nanoparticle formulation and surface characteristics, including PEG density, has the potential to enhance immunotherapeutic and vaccine outcomes.
Hinge-Bill Orientation Techniques for Automated Oyster Processing
(1977) Gird, John; Wheaton, F.W.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)
The width and thickness dimensions of oysters and an inclined V-shaped trough were studied as means for achieving end orientation. Two series of experiments were conducted on 2,430 oysters sampled from three different locations in the Chesapeake Bay. Both width and thickness were measured every 0.2 inch along the oyster length from the hinge to the bill end. A width to thickness ratio was found to be the best dimensional combination for distinguishing between the hinge and bill ends. Less than 0.50 percent of all oysters failed the ratio test conditions. Statistical analysis on five width to thickness ratio tests with failure rates between 0.25 and 0.49 percent showed there to be no differences in the percent oyster failure over all bars and across all tests. Results indicate that comparable oyster orienting efficiencies can be attained by width to thickness ratios with orienting points located 0.4 to 1.0 inches in from the oyster ends. Negative results occurred when an inclined V-shaped trough was used for orienting oysters. There were significant differences in the proportion of hinge and bill leading oysters exiting the trough for each trough loading position over all bars and oyster axes. The tendency for the oyster axes to behave differently explained some of the differences in the trough's orienting efficiency. However, there were no significant relationships between orienting efficiency and oyster axes.
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.
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.
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.
BIOMATERIALS REPROGRAM ANTIGEN PRESENTING CELLS TO PROMOTE ANTIGEN-SPECIFIC TOLERANCE IN AUTOIMMUNITY
(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.
INSTRUMENTATION AND AUTOMATION FOR STIMULATED BRILLOUIN SPECTROSCOPY
(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.
Enhanced Throughput Single-Cell Capillary Electrophoresis Mass Spectrometry
(2023) Mendis, John Udara; Nemes, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mass spectrometry (MS) has allowed for the analysis of small molecules and metabolites with high specificity and sensitivity. Capillary Electrophoresis mass spectrometry (CE-MS) is an ultrasensitive analytical technique to process amount-limited samples. Robust high-throughput ultrasensitive CE-MS methods and technologies are needed to be developed to comprehensively study the metabolome or proteome of a sample with a limited amount of material. In this study, we developed an enhanced-throughput multi field amplified sample stacking (M-FASS) method. The resulting approach has a sample processing throughput of 5–10 times that of conventional CE methods. FASS voltage duration and strength were optimized for peak area and peak resolution. The M-FASS CE-MS method was then applied for single cell analysis (SCA) of metabolic differences and gradients in the developing embryo of Xenopus Laevis. The statistical analysis: PCA and Fuzzy-c means clustering analysis revealed cell-to-cell differences among D11, V11, D12, and V12 cells and uncovered 6 distinct metabolite gradients between the four cells in X. Laevis 16-cell embryos. The findings showcase inherent metabolic gradients in the developing embryo.
Simplifying assay chemistry via complex sample preparation integration for point-of-care diagnostics
(2023) Everitt, Micaela Luisa; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Although therapeutics are undeniably important in the fight against any disease, a crucial step in making timely and effective therapeutic choices is diagnostics. Early treatments often lead to better patient outcomes, however early treatments are only possible with accessible diagnostics. Additionally, in public health emergencies, tracking the spread of disease via diagnostic tools is crucial to slowly and stopping it. Currently, standardized diagnostic testing is typically limited to central laboratories, requiring expensive, bulky instrumentation, trained technicians to perform these diagnostic tests, and the means to collect samples from patients, deliver the samples to the central laboratory, then return the results to the patient. In resource-limited settings (e.g., rural clinics), near-patient settings (e.g., at home), and in time-sensitive settings (e.g., emergency care), where accessibility and speed to a diagnosis are critical, the current central laboratory approach to diagnostic testing slows or even prevents an informed diagnosis and thus proper treatment. Subsequently, a large sector of the diagnostic field has focused away from a central laboratory approach and instead toward a near-patient paradigm, where samples can be testing immediately at the site of the patient such that results are rapidly available, allowing for faster treatment response. The diagnostic community refers to this idea of near-patient testing as point-of-care. Although point-of-care diagnostics have seen some commercial progress, the field is still struggling to develop tests for complex sample mediums. For complex samples, it is crucial that any sample preparation steps are integrated into the overall device to be truly considered point-of-care. Specifically, a diagnostic that can produce an easy-to-interpret result with little effort exerted by the user once the sample has been input is considered to be sample-to-answer. This dissertation highlights scientific advancements in the field of sample-to-answer diagnostics through optimized approaches that integrate higher order alkanes acting as pseudo-valves with point-of-care assay techniques. These higher order alkanes are solid at ambient temperatures and liquify when warmed and these alkanes can act as either as (1) a breachable barrier to allow for hands-free, controlled reagent mixing as the alkane melts or (2) as a permeable barrier that remains in place to separate assay regions, while magnetic beads can be pulled through. The behavior can be controlled by modifying the specific geometry in which the assay is confined. Developments involving thermally responsive alkane partitions enable complex sample processing and assay integration with intervention-free operation in low-cost, easily manufacturable cartridges. The work presented in this dissertation aims to address the need for sample-to-answer diagnostics, especially for complex samples, using these alkane partitions in three sequential avenues. This dissertation first details a point-of-care method to detect proteins, specifically histones, in whole blood, which can be a biomarker for severe internal trauma. This work demonstrates a hands-free technique for temperature-controlled reagent mixing as well as automated blood separation to allow for quantitative fluorescent measurements from whole blood samples. Although this work contributes to the field of sample-to-answer diagnostics, by integrating blood sample preparation, there was a need to generalize the diagnostic device to detect not just histones, but a broader category of protein biomarkers, like antibodies. Next, this dissertation assesses a sample-to-answer immunoassay, designed to quantify antibodies from whole blood which is useful to not only help track community spread of a disease, but also aid in validating vaccines and determining immunity. Although this diagnostic device was optimized to be as sensitive as its gold standard, central laboratory equivalent, in order to branch into nucleic acid biomarker detection, there was a need to include an exponential amplification step. Thus, finally, this dissertation looks at a sample-to-answer approach to monitor viral RNA genome in an additional complex sample, wastewater, in order to monitor the spread of disease community-wide. These sample-to-answer advancements to the field of point-of-care diagnostics enable low-cost, user-friendly solutions that increase overall accessibility to healthcare.
DEVELOPMENT OF AN ELECTROCHEMICAL-BASED TOOLKIT FOR IMPROVED BIOPROCESSING APPLICATIONS
(2023) Motabar, Dana; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Recombinant antibody therapeutics have become an important class of biopharmaceuticals that have shown effectiveness in treating a variety of diseases such as cancer, infection, and inflammation due to their high binding affinity and specificity. Importantly, process conditions established during the development and manufacture of antibodies dramatically impacts their quality, clinical efficacy, and safety. For process monitoring and control purposes, analytical technologies that enable rapid and cost-effective assessment of therapeutics are needed as they trim development time and costs. To address this need, we developed electrochemical-based analytical technologies that will enable low volume, near real-time monitoring of product quality attributes and process parameters. First, we demonstrate the development of thiolated PEG-based sensor interfaces for the detection of antibody titer and N-linked glycosylation. The interfaces couple electrochemical techniques with molecular recognition-based elements and a novel spectroelectrochemical reporter to provide rapid assessment of titer and galactosylation. Next, we demonstrate successful integration of the sensor interfaces with a microfluidic device in order to enable rapid, low volume sampling that is amenable to on-line monitoring. Lastly, we apply a mediated electrochemical probing (MEP) approach that uses redox mediators to quantitatively characterize redox-based quality information of antibodies that have undergone reduction or oxidation events. We believe that these technologies can provide fast, quantifiable results for bioprocessing applications and offer advantages in their simplicity, rapid response, and connectivity to electronics.
FEEDBACK-CONTROLLED BIOELECTRONIC HYBRID SYSTEM ENABLED BY ELECTROGENETIC CRISPR
(2023) Wang, Sally Patricia; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
With the rise of concepts like the “internet of things” and the advances in electronic technologies, our lives have now been occupied with smart devices that easily communicate with one another. These devices, however, lack the ability to freely exchange information with the world of biology, since electronics and biology possess very different communication modalities. Recently, the field of “electrogenetics” was introduced by enlisting redox mediators like hydrogen peroxide as a novel signaling medium to facilitate the connection between electronics with biology. In this dissertation, we expanded the electrogenetic framework and established a complete network of Bio-Nano Things, which collectively allowed automated, algorithm-based feedback control of electrogenetic CRISPR activity. First, we engineered the abiotic/biotic interface in order to improve information transfer between electronics and biological systems. Inspired by nature, we created an “artificial biofilm” that immobilized living cells on the surface of the electrode by electrochemically assembling bacteria and thiolated polyethylene glycol (PEG-SH) to form a thin film. We then endowed the PEG-SH hydrogel with redox capabilities via conjugation to generate an interactive material that can autonomously synthesize hydrogen peroxide to initiate communication with a bacterial population. Additionally, a polycysteine-tagged Streptococcal protein G was introduced for PEG-SH hydrogel surface decoration to enable the recognition of cells and other biological molecules. Next, we developed oxyRS-based electrogenetic CRISPR to broaden the bandwidth of electrochemical signaling, allowing multiplexed transcriptional regulation on various genetic targets. These include two crucial quorum sensing genes that controlled the relay of electrochemical signals to a broader yet selective audience of microbial populations through quorum sensing communication. We then integrated the engineered interface with eCRISPR-mediated transcriptional regulation to present “Biospark”, a full electrogenetic system including custom-made hardware and software, for algorithm-governed automated control of gene expression. Finally, we demonstrated a network of Bio-Nano Things by connecting the Biospark system with another custom bio-electrochemical device and even users to achieve remote feedback control of eCRISPR activity and more importantly, multidirectional communication between living systems regardless of physical distance. Together, we believe this work represents a huge leap toward making “smarter” devices and networks that can seamlessly guide biological processes with electronic input and can spawn various applications in the fields of biotechnology.
UNDERSTANDING FUNCTIONAL BEHAVIORS OF ORGANOTROPIC TRIPLE NEGATIVE BREAST CANCER CELLS
(2023) DeCastro, Ariana Joy; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
11.7% of all cancer cases consist of breast cancer worldwide according to global cancer statistics. Triple negative breast cancer (TNBC) is subtype of breast cancer that has no expression of common hormonal receptors - estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Due to this, TNBC is insensitive to endocrine or molecular targeted therapy and chemotherapy is the most effective treatment. Additionally, TNBC patients have reoccurrence within 3 years of diagnosis. Going further, due to the non-specific targeting of chemotherapy, cancer cells can develop drug resistance. The gold standard does not work in conjunction with microenvironmental factors to reduce disease progression and drug resistance. Not only is this disease lacking in effective treatments but is associated with a health disparity being most prevalent in pre-menopausal and African American women. There is clearlya need to understand the mechanisms of TNBC metastasis because of the impact not only on women in general but on women in historically marginalized communities. A significant innovation in determining cancer treatment is the use of genomic sequencing to identify mutations associated with metastasis. However, tumor heterogeneity puts limitations on fully understanding genomic landscape of TNBC, a highly mutational disease, using sequencing. Further, even when mutations are identified they may not be targetable, or patients may not respond to treatments. While genomic sequencing can be beneficial in improving treatment outcomes, they require further downstream validation of genetic expression to completely understand tumor biology and metastatic progression. This is where understanding the functional behavior of tumor cells with respect to their preferred secondary microenvironment can be advantageous in supplementing genomics data to get a comprehensive understanding of TNBC metastasis. The overall goal of this dissertation is to address this gap by quantifying tumor cell functional behavior and their response to microenvironmental cues. We evaluate three different physical and biochemical behaviors of TNBC tumor cells. In Chapter 3, the effect of TNBC secretome on endothelial barrier properties and function is explored. Chapter 4 quantifies the morphological and migratory phenotypes of brain and bone-seeking TNBC cells in response to ECM protein substrates found in their relevant microenvironments. Lastly, Chapter 5 will quantify the TNBC incorporation in response to brain relevant microenvironmental cues. Quantifying these functional behaviors could provide indicators of brain and bone tropic metastatic behavior and have broader impacts in creating a complete physical profile of organotropic TNBC metastasis.
Modeling, Targeting, and Optimization of BMPS for Environmental Health in a Coupled Human-Natural System
(2023) Zhang, Zeshu; Montas, Hubert; Shirmohammadi, Adel; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Pollution is a severe problem throughout the world. For control purposes, it may be classified based on how it enters the environment as either point source pollution or nonpoint source (NPS) pollution. Point source pollution refers to situations where pollutants enter the environment from a single place, like a pipe or a smokestack, and is best controlled at or before that discharge point. NPS pollution, on the contrary, refers to situations where pollutants come from spatially distributed and unconfined locations. NPS pollution is a severe problem worldwide. In the Chesapeake Bay region, surface runoff, sediment, nitrogen, and phosphorus are among the most critical pollution parameters. To increase water quality and control NPS pollution, Best Management Practices (BMPs), such as conservation tillage, rain gardens, or porous pavement, are being implemented in various areas of the Chesapeake Bay basin, but improvements are not as significant as expected, and some areas show degrading trends. Hydrologic models have been developed to simulate the processes that govern the generation and movement of NPS pollutants, and to provide specific guidance on the location and type of BMPs best suited for efficiently improving water quality and conserving water. The approach has shown promise in agricultural areas, but several research questions remain to enhance its broader applicability. One major question is how to translate the approach to suburban and urban areas common in the Chesapeake Bay basin, and whether the spatial distribution of polluting regions may differ from that in agricultural zones. Another question is the degree to which selected BMPs are the most cost-effective for a given targeted pollution reduction and whether an alternative allocation can reduce costs. A third research question is about BMP adoption by stakeholders: can adoption likelihood of BMPs be predicted based on socioeconomic factors, and can it be increased to meet pollution reduction goals?This dissertation investigates the above questions and generates guidance on cost-effective BMPs and social intervention strategies for BMP adoption across diverse land use types. First, the study characterizes how the spatial distribution of NPS hotpots changes among natural, agricultural, suburban, and ultra-urban watersheds typical of the region, using a hydrologic and water quality model, SWAT (Soil Water Assessment Tool). The results indicate that the spatial distribution of NPS constituents becomes increasingly uniform as urbanization increases. It is found that the spatial distribution of NPS constituents is a function of the major landcover categories in a study site, and that control measures should be adopted accordingly. Second, this study evaluates the cost-effectiveness of eight pre-selected BMPs capable of controlling NPS constituents in different land covers using random, targeted, and optimized allocation methods. Results show that the optimized selection of BMPs at optimized locations achieves the highest-performing BMP allocation plan across different landscape types. In the areas where NPS constituents are highly concentrated (natural or agricultural areas), a lower computational cost method: targeting NPS hotspots by criterion-based methods, is also broadly applicable. Lastly, this research explores the physical and socioeconomic factors that affect stakeholders' BMP adoption and quantifies the impact of these factors based on the RiverSmart Home BMP adoption data for Washington, D.C. The best regression model (random forest regression: R2=0.67, PBIAS=7.2%) shows that distance to the nearest BMP has the most significant impact on BMP adoption. Other features like median household income, education level, and existing green space area contribute less to BMP adoption. Spatio-temporal simulation of BMP adoption provides social intervention guidelines for increasing adoption locally, thus helping to achieve NPS pollutant reduction targets.

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