Fischell Department of Bioengineering Theses and Dissertations

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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.
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.
Acquired Platelet And Neutrophil Dysfunction Due To High Mechanical Shear Stress
(2022) Arias, Katherin; Wu, Zhongjun; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heart failure (HF) is a public health burden. In the next ten years, 8 million Americans are expected to have HF. A subset of these patients will develop advanced HF. They are refractory to medical therapies and have limited treatment options, including heart transplantation or a left ventricular assist device (LVAD). Heart transplants for all advanced HF patients are impractical due to the scarcity of donors. LVAD therapy is the sole viable option for advanced HF patients as a bridge to transplant, a temporary treatment while the heart recovers, and a long-term destination therapy. Over the last two decades, significant progress in LVADs have been made through various iterations. Advances in LVADs have been due to redesign focused on lowering adverse events. However, bleeding and infections are still the most prevalent adverse complications. LVADs and other mechanical circulatory support devices induce damage to blood cells and plasma components due to the high mechanical shear stress (HMSS) generated. Therefore, there must be a link between LVAD-induced cellular damage and the adverse events experienced in LVAD patients. This dissertation aimed to investigate the relationship between cellular blood damage and LVAD-associated complications and qualify the extent of cellular damage/defects and functional alterations.The overall objective of this dissertation was to investigate the acquired cellular defects of platelets and neutrophils in blood after shear stress exposure. This objective was accomplished through in-vivo, in-vitro, and in-silico studies. The in-vivo studies examined the shear stress-induced injury of platelets in LVAD recipients and linked the adverse bleeding events (Chapter 3). The in-vitro studies explored the shear stress-induced injury of leukocytes (Chapter 4). The extent of the structural damage and functional alterations related to shear stress level and the exposure time was quantified (Chapter 5 and Chapter 6). Finally, the in-silico studies developed a simulation of leukocyte function with experimental data that was used to predict the extent of the shear stress-induced leukocyte function change (Chapter 6). The damaging effects of the high shear stress produced by mechanical circulatory support devices such as LVADs were conveyed through an integrated biological and engineering approach.
Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles
(2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.
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.
Quantitative Motion Analysis of the Upper Limb: Establishment of Normative Kinematic Datasets and Systematic Comparison of Motion Analysis Systems
(2022) Wang, Sophie Linyi; Kontson, Kimberly L; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Upper limb prosthetic devices with advanced capabilities are currently in development. With these advancements brings to light the importance of objectively and quantitatively measuring effectiveness and benefit of these devices. Recently, the application of motion capture (i.e., digital tracking of upper body movements in space) to performance-based outcome measures has gained traction as a possible tool for human movement assessment that could facilitate optimal device selection, track rehabilitative progress, and inform device regulation and review. While motion capture shows promise, the clinical, regulatory, and industry communities would benefit from access to large clinical and normative datasets from different motion capture systems and a better understanding of advantages and limitations of different motion capture approaches. The first objective of this dissertation is to establish kinematic datasets of normative and upper-limb prosthesis user motion. The normative kinematic distributions of many performance-based outcome measures are not established, and it is difficult to determine departures from normative patterns without relevant clinical expertise. In Specific Aim 1, normative and clinically relevant datasets were created using a gold standard motion capture system to record participants performing standardized tasks from outcome measures. Without kinematic data, it is also difficult to identify informative kinematic features and tasks that exhibit characteristic differences from normative motion. The second objective is to identify salient kinematic characteristics associated with departures from normative motion. In Specific Aim 2, an unsupervised K-means machine learning algorithm was applied to the previously collected data to determine motions and tasks that distinguish between normative and prosthesis user movement. The third objective is to compare three commonly used motion capture systems that vary in motion tracking mechanisms. The most informative tasks and kinematic characteristics previously identified will be used to evaluate the detection of these differences for several motion capture systems with varying tracking methods in Specific Aim 3.

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