Fischell Department of Bioengineering Theses and Dissertations

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

Browse

Now showing 1 - 20 of 224

Overcoming the Extracellular Matrix Barrier to Nanoparticle Transport
(2024) Cahn, Devorah; Duncan, Gregg A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The extracellular matrix (ECM) is a major component of the tumor microenvironment which poses a significant barrier to nanoparticle (NP) transport, preventing delivery of therapeutic cargo. Studies have shown that PEGylation offers an effective strategy for improving NP transport in ECM. However, these studies have generally used ECM models that are not wholly representative of the native matrix. Furthermore, while ECM characteristics and composition varies across organs, it is unclear to what extent these tissue-specific characteristics affect NP transport through the ECM and how NP surface chemistry impacts ECM penetration in distinct tissues. The overall objective of this dissertation is to identify key factors of NP transport through the tumor microenvironment, facilitating the development of strategies to improve NP distribution throughout the tumor microenvironment. We hypothesized that PEG branching will enhance stability and mobility of NPs in ECM and that ECM source impacts NP transport. We further hypothesized that PEG architecture significantly affects NP mobility in ECM as well as biodistribution and tumor accumulation in vivo. Our first aim was to determine the effects of PEG branching on NP stability and transport through in vitro basement membrane model. We found that branched PEG significantly increased both the stability and mobility of NPs in Matrigel, a basement membrane model. We then assessed the impact of tissue source on NP transport through an in vitro ECM model. We decellularized porcine lung, liver, and small intestine submucosa to form tissue specific hydrogels and found NP mobility was significantly impacted by tissue source where low molecular weight linear PEG generally provided the greatest benefit to NP mobility within the different matrices. Finally, we evaluated how PEG branching affects biodistribution, immune cell infiltration, and NP uptake in tumors in vivo. We found that NPs coated with branched PEG increased NP accumulation within tumors and PEGylation significantly impacted immune cell infiltration within these tumors. This work provides additional insight into the transport mechanisms of NPs throughout the tumor microenvironment as well as additional considerations for the design of efficient NP delivery systems.
HIGH THROUGHPUT STIMULATED BRILLOUIN SCATTERING SPECTROSCOPY
(2024) Rosvold, Jake Robert; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Brillouin light scattering arises from the coupled interaction between light and material acoustic phonons. The measurand of Brillouin scattering is the characteristic frequency difference between incident and scattered light which depends on the local longitudinal modulus of the material. Spontaneous Brillouin scattering has been used in combination with confocal microscopy to provide non-contact, label-free mapping at micron-scale resolution in biological media. To date, spontaneous Brillouin microscopy has reached the speed limit (~20-50ms per spectrum) as determined by the theoretical scattering efficiency. While a great deal of research has been directed to speeding up Brillouin microscopy acquisition times, spontaneous Brillouin scattering is fundamentally an inefficient process thus limiting the ability to study faster biological phenomena and rapid processes. To combat this limitation, its nonlinear counterpart, stimulated Brillouin scattering (SBS) has been proposed for microscopy applications. For decades, stimulated Brillouin scattering has been used in fiber sensing and all-optical pulse control and leverages a nonlinear interaction where two counterpropagating light beams stimulate a more efficient scattering relationship. However, the small interaction volumes and photodamage constraints presented in microscopy have hindered the translation of stimulated Brillouin scattering into the biological realm. Recently, continuous wave stimulated Brillouin microscopy has led to competitive acquisition times (~5ms per spectrum) when compared to the spontaneous alternative but has yet to be widely adopted. Due to a plethora of factors, such as an inefficient power balance between pump and probe beams, lack of proper commercial laser sources, and nonoptimal detection schemes, the complete picture of what SBS spectroscopy has to offer has yet to be revealed. As such, there is a need to customize light sources and detection schemes in order to fully take advantage of the enhanced Brillouin efficiency possible in SBS. Herein we introduce novel methodology to improve the acquisition speed of Brillouin microscopy by designing and developing proper laser sources and detection schemes for efficient SBS spectroscopy. First, we showcase the potential utility of our state-of-the-art continuous wave SBS technology in a flow cytometry application, highly suitable for the counterpropagating geometry of SBS where the laser position is fixed while the sample is being moved at high speeds. Additionally, we will present an optimized receiver design based on polarization detection which enables 100x faster spectral measurements in the low-gain regime relevant to biological materials. Finally, we demonstrate an optimal pulsed laser source specifically designed for SBS Brillouin microscopy.
BIOMATERIAL BASED STRATEGIES FOR VIRAL AEROSOL CAPTURE AND PREVENTION OF RESPIRATORY INFECTIONS
(2024) Doski, Shadin; Duncan, Gregg; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In the 2022-2023 flu season, the Center for Disease Control (CDC) estimated 21,000 deaths and 31 million symptomatic illnesses in the United States. Current FDA approved antivirals for influenza are grouped into three categories, matrix protein 2 (M2) inhibitors, neuraminidase inhibitors (NAI) and polymerase acidic protein cap-dependent endonuclease (CEN) inhibitors. However, limitations of these treatments have been evident. For example, NAI inhibitors require early treatment to be efficacious and some influenza strains can develop resistance to both NAI and CEN inhibitors. Thus, there is a need for new classes of antivirals as well as better understanding of influenza transmission and monitoring of influenza to inform development of efficacious interventions. In chapter 2 we describe how we design biomaterials inspired by the physiological characteristics of mucus to capture and trap pathogens. We performed studies to establish this material as a suitable substrate for viral capture and release after collection using advanced aerosol capture technology. In chapter 3, we formulate an antiviral based around polyinosinic polycytidylylic acid (polyIC). PolyIC is commonly used in research as an adjuvant in vaccine delivery through its targeting of Toll like receptor 3 (TLR3). This pathway also results in type 1 and 3 interferon production, which in turn stimulate a range of antiviral mechanisms. Because of this, it has also been investigated as a prophylactic or treatment to various viruses, including hepatitis B virus, human immunodeficiency virus and rhinovirus. However, due to stability and toxicity concerns, it has not been implemented as an inhaled treatment to induce local immunity in the lungs at the site of infection. Towards this end, we used polyethylene imine-polyethylene glycol (PEI-PEG) copolymer to condense PolyIC into nanoparticles to enhance their bioavailability in target cells. By combining the two, we can utilize the antiviral capabilities of Poly(IC) while minimizing the dosage concentration to therapeutic levels.
MECHANICAL SIGNATURES OF BRILLOUIN SPECTROSCOPY
(2024) Rodriguez Lopez, Raymundo; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Brillouin light spectroscopy (BLS) has recently emerged as a tool for noncontact, nonperturbative and label-free characterization of biomechanical properties. BLS probes the longitudinal modulus of material while traditional techniques for biomechanical characterization aim to quantify Young’s or shear modulus. However, empirical correlations between the different moduli have been observed in several biological materials, correlations that are not yet universally established. The objective of this thesis is to advance the understanding of these correlations and their limitations with controlled systematic comparisons of longitudinal modulus and gold-standard modulus of hydrogels and corneal tissue. First, using polymer hydrogels as model of study, experimental data and theoretical models were used to demonstrate that the correlation between longitudinal and shear moduli is due to their common dependence on underlying physico-chemical parameters of the polymer system. This dependence allowed to predict one modulus from the other when enough information of the system is available. Furthermore, the limitation of thiscorrelation was studied when hydrogels absorb water, finding that hydration affects both moduli but in different manner and thus, their correlation. Having established hydration as an important variable for biomechanical properties, the correlation between modulus in the corneal tissue and crosslinking procedure (CXL) was studied. CXL is the gold-standard treatment for corneal ectatic disorders, and its success is due to the strengthening of the mechanical properties of the cornea as a result of photochemical induced collagen crosslinking and dehydration of the tissue. However, most mechanical characterization ex vivo, does not factor in the tissue dehydration effect, overestimating the effect in the clinical situation. With experimental data obtained by gold-standard methods and established theoretical models, the modulus after hydration changes after the CXL was systematically characterized. Finally, another scenario where studying the correlation between moduli is important is the nonlinear mechanical behavior of the cornea. Effect that has been observed with different techniques, but BLS has failed to capture so far. This works proves that the reason of this discrepancy has to do with the mechanical anisotropy of the cornea and the nature of BLS, which is a purely uniaxial measurement of mechanical properties. Considering these factor, it is proven that BLS has the ability to measure the nonlinear mechanical properties of the corneal tissue.
SAMPLE-TO-ANSWER POINT-OF-CARE VIRUS DIAGNOSTIC SYSTEM USING THERMALLY RESPONSIVE ALKANE PARTITIONS
(2024) Boegner, David John; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Many viral infections can be accurately diagnosed using today’s most sophisticated detection systems. Unfortunately, many of these detection systems fail to benefit society as a whole, but rather favor select areas of the world that are able to install and maintain the infrastructure such diagnostics require. Thus, in an effort to eliminate the barrier of access to diagnosis and treatment in low-and-middle-income areas, portable point-of-care devices are fabricated such that rapid results can be obtained without the need for bulky lab equipment or skilled technicians. An ideal point-of-care diagnostic device can easily collect an untampered sample and limits a patient’s encounter with a clinician to a single visit for both the diagnosis and the treatment. Many so-called point-of-care diagnostics for blood-borne viruses first require blood sample preparation (e.g. centrifugation) prior to testing in the device. Other point-of-care devices sacrifice diagnostic accuracy in favor of speed and portability. Both cases demonstrate our inability to properly distribute the benefits of sophisticated diagnostics worldwide.I present a solution in the form of an affordable handheld diagnostic device with the sensitivity and specificity of benchtop lab equipment and built-in automatic sample preparation. Automatic sample preparation will be achieved using thermally responsive alkane partitions, which are solid at ambient temperatures and liquid at moderately elevated temperatures. When liquid, the alkane partitions allow passage of magnetically activated microbeads coated with material that captures viruses. Despite magnetic beads with virus particles passing through, the alkane partition continues to prevent unwanted sample components (e.g. blood cells, DNases, etc.) from interfering with the virus-detecting mechanism on the other side. To address the lack of sensitivity in many point-of-care diagnostics, the virus-detecting mechanism will feature isothermal amplification which enables detection of attomolar concentrations of virus within 30 minutes without expensive thermo-cycling equipment that standard detection systems require. The novel technology described here is demonstrated in a platform which detects SARS-CoV-2 from blood, a capability currently unachievable in point-of-care settings.
Synthetic Mucus Hydrogels for Antimicrobial Peptide Delivery and Treatment of Bacterial Infections
(2024) Yang, Sydney; Duncan, Gregg A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Antibiotic resistant infections have the propensity to form biofilms that contribute to chronic infections and result in hyper-inflammatory response in tissues. Recent studies pose antimicrobial peptides (AMPs) as alternatives to antibiotics and to modulate inflammatory response. However, AMPs have a short half-life due to rapid clearance and degradation reducing AMP bioavailability and efficacy. In the human body, AMPs interact and may associate with mucins which result in the sequestering of AMPs within mucus. Previously, we have developed a synthetic mucus (SM) hydrogel inspired by the innate properties of mucins. The objective of this work was to evaluate the SM hydrogel as a tool for local antimicrobial peptide delivery of LL37 to enhance the treatment for infection and inflammation. To study this, we (1) assessed the release of LL37 and antimicrobial activity of LL37 loaded SM (LL37-SM) hydrogels on Pseudomonas aeruginosa, (2) evaluated the antibiofilm activity of LL37-SM hydrogel treatment on Pseudomonas aeruginosa biofilms, and (3) determined the impact of LL37-SM hydrogel treatment on RAW 264.7 macrophage activation and phagocytic activity. The association of LL37 to SM hydrogels enabled the sustained release of LL37 over 8 hours and retained antimicrobial activity. Treatment with LL37-SM hydrogels for 24 hours disrupted biofilm growth and resulted in a mixed inflammatory response in macrophages. Our results highlight the antimicrobial, antibiofilm, and potentially inflammatory modulating capabilities of SM hydrogels which can further inform the use of mucins in bioactive biomaterials for biomedical applications.
Investigation and development of induced pluripotent stem cell derived extracellular vesicle-based therapeutics
(2024) Levy, Daniel H; Jay, Steven; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Due to their complex, multicomponent nature, extracellular vesicle (EV)-based therapeutics have arisen as an intriguing option for treatment of complex diseases that require the simultaneous modulation of distinct pathways. Due to their inherent regenerative properties, mesenchymal stem cell (MSC)-derived EVs have been the most heavily investigated and utilized in clinical trials for diseases including acute respiratory distress syndrome, wound healing and many more. While pre-clinical studies have demonstrated promise for such EV-based therapeutics, source cell limitations act as a hurdle to the widespread clinical translation of MSC EV therapies. MSCs and other cells reported to produce therapeutic EVs (cardiac progenitor cells, neural stem cells, etc.) have limited expansion capabilities ex vivo before cellular senescence, therefore limiting the amount of therapeutic EVs that can be produced by a single cell line. Due to these limited expansion capabilities, alternative, self-renewing therapeutic EV source cells are needed. One such source cell is induced pluripotent stem cells (iPSCs), which possess self-renewing capabilities. However, the baseline bioactivity of iPSC EVs have yet to be rigorously evaluated; in our work, we report for the first time that iPSC EVs possess robust anti-inflammatory properties in addition to confirming previous reports of their ability to promote vascularization in a murine diabetic wound healing model. Building off these baseline results, we sought to augment iPSC EV potency by utilizing genetic approaches to load of bioactive RNAs including microRNA (miRNA) and long non-coding RNA (lncRNA) into iPSC EVs. In our miRNA loading studies, we effectively demonstrate that the natural biogenesis pathways of miRNA can be probed to facilitate export of bioactive miRNAs to secreted EVs, thereby enhancing their anti-inflammatory bioactivity. Lastly, we utilize a genetic engineering approach to enhance active sorting of lncRNAs into secreted EVs and test their therapeutic potential in a murine colitis model. The work described in this dissertation provides a foundation towards the clinical translation of iPSC EV-based therapeutics by benchmarking them against more established therapeutic EV sources (iPSC-derived MSC EVs) and developing strategies to enhance their bioactivity via RNA cargo loading.
MULTISCALE BIOMATERIALS-ENABLED GLIOBLASTOMA STEM CELL-TARGETED THERAPY WITH microRNA TO OVERCOME CANCER RECURRENCE
(2024) Shamul, James George; He, Xiaoming; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Glioblastoma (GBM) is one of the most lethal diseases in the world with a dismal ~7.2% 5-year survival rate and median survival time of ~15 months. More than 90% of GBM patients experience recurrence, which is primarily attributed to the presence of a rare subpopulation of cancer cells inside tumors called glioblastoma stem cells (GSCs). These GSCs are highly drug-resistant and tumorigenic. To address recurrence in GBM, it is crucial to isolate true GSCs, which possess two main abilities of all classical stem cells: self-renewal and multilineage differentiation. Current isolation technologies for GSCs, and all cancer stem cells (CSCs), predominantly include 1) sorting based on the expression of putative markers for CSCs, and 2) 3D suspension culture in defined, serum-free medium. However, these methods are unreliable because there is no definitive marker for CSCs and there is merging of cells in 3D suspension culture. Due to the deficiencies of these methods, no GSCs have been previously isolated that include the capability of both self-renewal and multi-lineage differentiation. Using our previously developed microfluidics-enabled 1-cell culture method, we demonstrated that true breast cancer stem cells with the ability for self-renewal and multi-lineage differentiation can be isolated. With the same method, we isolated patient GSCs that are capable of self-renewal and multi-lineage differentiation, which has never been demonstrated before. With microRNA (miR) sequencing, we identified miRs-10a/b as candidates that are differentially expressed in the GSCs from 1-cell culture compared to “GSCs” from conventional 3D suspension culture. These miRs were encapsulated inside nitric oxide (NO) precursor-laden, fucoidan-decorated nanoparticles. These nanoparticles generate NO gas and disassemble under low pH conditions, and are thus named “nanobombs” due to this explosive effect. The fucoidan is decorated on the nanoparticle surface for targeting of both activated blood vessels adjacent to GBM cells, and GBM cells directly. The NO-releasing, fucoidan-decorated nanobombs demonstrate efficient blood-brain barrier (BBB) crossing in vitro and endosomal escape into the cytosol of GBM cells. After treatment with miR-10a/b-laden, fucoidan-decorated nanobombs, GBM cells show diminished stemness marker expression and self-renewal capability. Moreover, miR-10a/b-laden, fucoidan-decorated nanobombs and temozolomide (TMZ) co-treatment eliminates recurrence in vitro. However, recurrence occurs in GBM neurospheres that are administered with all other control treatments, including miR-scrambled-laden, fucoidan-decorated nanobombs plus TMZ, miR-10a-, miR-10b-, and miR-scrambled-laden, fucoidan-decorated nanobombs, free TMZ, and DMSO. Using a multi-scale approach that includes 1) microfluidics-enabled GSC isolation, 2) fucoidan-decorated nanobombs which are NO gas-generating, BBB-penetrating and endosomal-escaping, and 3) miR target identification in GSCs, there is promise to target true GSCs and inhibit recurrence in GBM patients.
BRAIN ENDOTHELIAL BARRIER, METABOLIC, AND TRANSPORT DYSFUNCTION IN NIEMANN-PICK DISEASE TYPE C: MECHANISMS AND THERAPEUTIC STRATEGIES
(2024) Moiz, Bilal; clyne, Alisa; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Brain microvascular endothelial cells (BMECs) form the blood-brain barrier, which protects the brain from neurotoxic elements and simultaneously transports glucose and other vital nutrients into the brain. Neurovascular dysfunction is implicated in pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease; however, little is known about how neurovascular changes contribute to rare inherited neurogenetic disorders such as Niemann-Pick Disease (NP-C). NP-C is caused by mutations in the intracellular cholesterol trafficking proteins NPC1 and NPC2, which leads to endolysosomal cholesterol accumulation and membrane cholesterol depletion. Clinical manifestations vary by age and genetic factors but include neurological symptoms such as developmental delay, cognitive impairment, ataxia, and seizures. Current clinical management strategies are challenged by diagnostic difficulties and poor therapeutic efficacy. Hydroxypropyl-beta-cyclodextrin (HPβCD), an agent believed to release accumulated cholesterol, has shown promising clinical results; however, its efficacy is limited due to poor brain penetration. The major objective of this thesis was to determine how NPC1-deficiency impacts BMEC barrier function, metabolism, and nanoparticle uptake. I found that NPC1 deficiency diminishes barrier integrity in BMECs by disrupting claudin-5 and occludin morphology. Using isotope labeling, mass spectrometry, and computational flux analysis, I also observed that NPC1 inhibition leads to systemic metabolic changes, including increased glycolytic flux, elevated activity in peripheral glycolytic pathways, and reduced mitochondrial respiration. HPβCD treatment attenuated barrier function changes and partially restored BMEC metabolic phenotype. Finally, I found that isoproprylacrylamide (NIPAA-m) nanogels loaded with HPβCD were transported across NPC1-deficient BMECs, suggesting their potential for HPβCD delivery to the brain. This thesis demonstrates a unique, integrated computational-translational approach that unveils the role of BMEC in NP-C pathology, possibly leading to improved therapeutic strategies. In addition, this thesis improves our understanding of how variants in cholesterol metabolism and trafficking, as well as in proteins such as NPC1, which has been implicated in Alzheimer’s, diabetes, obesity, and atherosclerosis, contribute to brain endothelial dysfunction.
DEVELOPMENT OF GLYCOSAMINOGLYCAN MIMICKING NANOGEL TECHNOLOGIES FOR CONTROLLED RELEASE OF THERAPEUTICS TO TREAT RETINAL DISEASES IN DIFFERENT AGE GROUPS
(2024) Kim, Sangyoon; Lowe, Tao L.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Retinal diseases, such as diabetic retinopathy, glaucoma, macular degeneration, and retinoblastoma, affect around 13 million people worldwide, with projections indicating a rise to 20 million by 2030. These conditions lead to irreversible vision loss and significant impairment in both adults and children, with an annual economic burden of $139 billion in the United States alone. Aging significantly increases the risk of certain retinal conditions, and with improvements in healthcare leading to increased life expectancy, these conditions are becoming more prevalent due to the natural aging process and associated physiological changes in the eye. Current treatments are either destructive or have low efficacy and are not optimized for the younger population. While therapeutics including small molecular drugs, proteins and antibodies show promise in treating these diseases by reducing inflammation and neuronal apoptosis, their effectiveness is hindered by short half-lives and inability to cross the blood-retinal barrier (BRB). Nanoparticles offer a potential solution by improving drug delivery across biological barriers, yet no nanoparticles have been developed to effectively transport intact proteins or small molecules across the BRB to the retina without toxicity, slow clearance and stability. Therefore, there is an unmet need to evaluate the physical and physiological property changes of the eye along development and develop nanoparticle systems that can control and sustain the release of therapeutics across the blood retinal barrier (BRB) to treat the retinal diseases. In this project, the thickness, rheological property, permeability and morphological property changes of ocular barriers including sclera, cornea and vitreous humor in the developing eye from preterm to adult were evaluated using porcine ex vivo model. Two glycosaminoglycan mimicking nanogel systems, poly(NIPAAm-co-DEXcaprolactoneHEMA) nanogels with and without positive or negative charges and β-cyclodextrin based poly(β-amino ester) (CD-p-AE) nanogels were developed for sustained release of intact proteins including insulin and anti-TNFα, and small hydrophobic drugs, respectively across the ex vivo porcine sclera and in vitro BRB models: human fetal retinal pigment epithelial (hfRPE), adult retinal pigment epithelial (ARPE-19) and human cerebral microvascular endothelial (hCMEC/D3) cell monolayers. Completion of this project will have a significant impact on developing novel personalized nanotherapeutics to treat retinal diseases in different age groups.
INVESTIGATION OF THE IMPACT OF ACOUSTIC FORCING ON NEURAL NETWORK ACTIVITY
(2024) Dutta, Arijit; Losert, Wolfgang; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The increase in the prevalence of neurological disorders, now approximated to affect 15% of the worldwide population, has highlighted the need to fundamentally understand how neuronal networks function in normal and diseased conditions. Acoustic forcing has been identified as a method to advance the fabrication and understanding of how neural networks function via cell patterning and neuromodulation. This phenomenon occurs as acoustic waves at ultrasonic frequencies impart a force on a target through a medium. Cell patterning via acoustic forcing has led to the development of biomimetic neural organoids. Neuromodulation, via acoustic forcing, has been shown to stimulate neuronal activity and is popularly researched in transcranial focused ultrasound. A concern in transcranial focused ultrasound is standing wave formation, and thus pressure gradients, in the brain. The mechanism of how standing wave formation impacts neuronal network activity remains unknown. This thesis elucidates the impact of acoustic standing waves on large-scale neuronal activity through the fabrication and application of a standing-wave generating device on assembled ReNcell human neural progenitor-derived neural networks. The preliminary results showed younger networks to have large decreases in activity while older networks saw minimal change following acoustic forcing application. Additionally, varying intensity from did not affect network activity of older networks during acoustic forcing application.
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.

Browse

Recent Submissions