Theses and Dissertations from UMD
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Item 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.Item Design and Assembly of Block Copolymer-Modified Nanoparticles into Supracolloidal, Molecular Mimics(2023) Webb, Kyle; Fourkas, John T; Nie, Zhihong; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Large strides have been achieved in nanoparticle self-assembly, using various strategies to achieve ordered, supracolloidal structures, ranging from dimers to chains and vesicles to 3-D lattices. However, these methods, while expanding the scope and accessibility of design, face inherent limitations in targeting complex structures with high yields, particularly when using isotropic building blocks (e.g. gold nanoparticles and polystyrene nanoparticles). Additionally, research studying the reversibility of nanoparticle assemblies is mostly limited to small-ligand-modified particles rather than polymer-modified nanoparticles. Polymers are particularly advantageous as they provide a higher degree of functionality to the nanoparticle surface and allow for increased control in directing particle interactions. This control is necessary to continue furthering the advancement of gold nanoparticles in plasmonics, sensors, and catalysts. Here, we introduce two strategies to assemble gold nanoparticles into supracolloidal nanostructures. Gold nanoparticles are modified with complementary, functionalized-block-copolymers that drive the assembly of the nanoparticles. The first strategy uses a diblock copolymer composed of a hydrophilic outer block and an acid or base-functionalized inner block. Upon mixing, particles are assembled due to the acid–base neutralization between the complementary block copolymers. The resultant supracolloids consist of nanoparticles precisely arranged in space, which mimic the geometries of small molecules. The particle interactions are fine tuned by varying the size and feeding ratio of the nanoparticles, along with the length and composition of the block copolymers. Careful tuning of these parameters yields nanostructures with different valences that were produced in high yield. Additionally, the implementation of a long outer, hydrophilic polymer block provided the assembled nanostructures stability when transferred from THF to water. Colloidal stability in an aqueous medium could allow for expanded use of these nanostructures in cellular uptake studies and biomedical applications. The second strategy uses a diblock copolymer composed of a hydrophilic outer block and an inner block containing either complementary host or guest moieties. Particularly, we take advantage of the well-established interactions between β-cyclodextrin and adamantane as the host and guest molecules. Upon the slow addition of water, particles assemble due to the host–guest interactions between the complementary block copolymers, as the hydrophobic adamantane moieties are driven within the β-cyclodextrin macrocycles. Fine tuning of the nanoparticle sizes and feeding ratios and the block copolymer lengths and compositions results in high yields of targeted supracolloids that also mimic the geometries of molecules. Interestingly, the size difference between the host and guest-modified particles led to different types of nanostructures. In addition, due to the reversibility of the host–guest interactions, we demonstrate the ability of our system to reorder in response to competitive host moieties. Upon addition of free β-cyclodextrin, the host–guest interactions are disrupted, resulting in disassembly of the nanostructures, which we could reassemble upon removal of the free cyclodextrin. Finally, due to the strength of the nanoparticle interactions, we also tested the selectivity of the nanoparticle interactions by assembling the host building block with different guest building blocks. We showed that when assembled with competing guest building blocks, the β-cyclodextrin building blocks preferentially interact the adamantane building blocks due to the stronger particle interactions. This reversibility and selectivity make our system a potential candidate for use in biosensors.Item 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.Item DEVELOPMENT OF HORDEIN-PECTIN NANOPARTICLE COMPLEX FOR THE ENCAPSULATION OF BIOACTIVE COMPOUNDS FOR ENHANCED FUNCTIONALITIES(2023) Tarwa, Kevin; Wang, Qin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Nanoparticle delivery systems composed of food polymers are a sustainable and eco-friendly approach to protect functional ingredients and promote healthier food options. In this research, a hordein-pectin nanoparticle complex (HP-NPC) was fabricated using an anti-solvent precipitation and electrostatic deposition (pH 4) method for the encapsulation of hydrophobic bioactive compounds to enhance their functionalities. First, hordein was extracted from whole barley grains to obtain a dried powder to synthesize hordein nanoparticles (HNP). Then pectin with a degree of esterification (DE) around 71% was applied as a coating material. The average particle size of the freshly prepared nanoparticle complex was relatively small (~246 ± 11 nm), and Fourier transform infrared spectroscopy (FTIR) indicated that cationic hordein interacted with anionic pectin mainly though newly formed hydrogen bonds and electrostatic interaction as indicated by their opposite surface charges. Scanning electron microscopy (SEM) revealed that the morphology of the nanoparticle complex was spherical with a smooth surface. The pectin coating was shown to have a protective effect against pH (3.0-9.0), heat (80 °C for 0-120 mins), and salt (0-100 µM) which are all factors known to degrade proteins. Second, lutein, a hydrophobic bioactive xanthophyll was encapsulated into HP-NPC to develop a lutein-hordein/pectin nanoparticle complex (L-HP-NPC). Since lutein has low water solubility and low bioavailability in the gastrointestinal tract (GIT), the effect of the encapsulation system on the functional properties of lutein was investigated. The loading capacity (LC%) and encapsulation efficiency (EE%) was around 15.5 and 82%, respectively. In vitro digestion resulted in a higher bioaccessibility of lutein for encapsulated HP-NPC (~22.3%), which is defined as the percentage of lutein accessible for absorption in the simulated intestinal fluid (SIF) compared to lutein encapsulated into HNP (~9%). The ability of pectin to produce gels in acidic media was shown to have a significant effect against gastric enzymes that can degrade both hordein and lutein. Also, lyophilization (an important step in food processing) had no significant effect on the stability of L-HP-NPC. This encapsulation system could potentially be used as a functional ingredient in the food industry to develop healthy and nutritious foods for consumers. Third, carvacrol, a phenolic monoterpene known for its antimicrobial properties was encapsulated into HP-NPC to develop a carvacrol-hordein/pectin nanoparticle complex (CA-HP-NPC). Special focus was on the solubility of encapsulate carvacrol due to its known low solubility in aqueous solutions. The antimicrobial effectiveness of the encapsulated nanoparticle complex was tested against non-pathogenic gram-positive L. innocua and gram-negative E. coli K12. CA-HP-NPC was still able to maintain a relatively small particle size (~207 ± 8 nm) after being dispersed into water post-lyophilization. Carvacrol was shown to be effective against the two bacteria, however, CA-HP-NPC did not show antimicrobial effectiveness. Although carvacrol was successfully encapsulated into the nanoparticle complex, further studies on their release properties need to be investigated to further understand their functional properties for food applications.Item New Nanoparticle Characterization Techniques by Differential Mobility Analysis(2022) Duelge, Kaleb John; Zachariah, Michael R; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Nanoparticles are being used increasingly in new fields for new applications. Thoroughcharacterization of particle properties such as size, aggregation, and mass are key to understanding particle behavior. In this dissertation I discuss a variety of new measurement approaches using the aerosol-based technique: differential mobility analysis (DMAS). This technique consists of the combination (hyphenation) of several components, the primary being an ion mobility chamber for the spatial separation of nanoparticles. The specific aerosol source and detector used is flexible and allows for wide applicability of the technique. The applications discussed here relate to a variety of everyday scenarios. Medicinal protein particles are studied to improve the health outcomes associated with this growing class of medicine. Nanoparticle catalysts are studied to improve the activity and repeatability, analogous to how a catalytic converter is used in cars to reduce combustion emissions. Detailed size measurements are made for gold nanoparticles, a class of particles that have been used for cancer treatments and as carrier particles, for example to transport medicine to a particular location within the body. Finally, determination of nanoparticle size is studied by comparing results from different instruments, as determining size in the nanometer scale is more complicated than an analogous measurement of a macroscopic sphere (for example, measuring the length with a ruler and comparing the result to the length derived from the mass of a sphere with a known density). In the second chapter, I demonstrate protein aggregation kinetics measurements by DMAS and asymmetrical flow field flow fractionation. Thermal aggregation was conducted in traditional formulation buffers and good agreement was determined between the two techniques. These are potential alternative instruments to the gold standard, size exclusion chromatography, used by the biopharmaceutical industry. In the third chapter, I demonstrate a calibration technique for mass distribution measurements by DMAS using inductively coupled plasma mass spectrometry for detection. Determination of the total mass of a sprayed ionic standard was sufficient to calibrate measurements of monodisperse gold nanoparticles of various shapes. A disagreement was determined between the ionic standard and a polydisperse distribution of titania coated with small gold nanoparticles. A multiple charge correction was applied that significantly improved the agreement, though the issue remained. In the fourth chapter, comparison measurements are presented for monodisperse gold nanoparticles made in two operational modes of DMAS: step voltage mode and scan voltage mode. The step voltage mode remains at each voltage for a certain dwell time (on the order of 30 s), while the scan voltage mode continuously changes voltages. Good agreement was determined for the two approaches when calibrated using a nanoparticle size standard. Additionally, the scan voltage mode data were analyzed with an alternative calibration method: a direct measurement of the sheath volumetric flow rate. The data from the two scan voltage mode calibrations bracket the measurement made in step voltage mode. This agreement suggests that scan voltage mode measurements for certification of nanoparticle size standards could be used in the future if a few additional uncertainty terms are explored. In the fifth chapter, traceable measurements with quantitative uncertainty analyses are compared for a size standard. The measurement by DMAS is compared to atomic force microscopy, scanning electron microscopy, and electro-gravitational aerosol balance. The measurement by DMAS was bracketed by the other techniques, with microscopy measuring a slightly smaller size and electro-gravitational aerosol balance measuring a slightly larger size. The measurements all agreed within 3%, but some of the differences exceeded the 95% confidence intervals of the measurements. These differences may be significant if these techniques are used to develop future size standards.Item CONFINED PHOTOTHERMAL HEATING OF NANOPARTICLE DISPLAYED BIOMATERIALS(2021) Hastman, David A; Medintz, Igor L; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Controlling the temperature of biological systems has long been utilized as a tool for regulating their subsequent biological activity. Recently, photothermal heating of gold nanoparticles (AuNPs) has emerged as an efficient and remote method to heat proximal biological materials. Moreover, this technique has tremendous potential for controlling biological systems at the subcellular level, as specific components within the system can be heated while the larger system remains unaffected. The small size, biocompatiblilty, and optical properties of AuNPs make them attractive nanoscale heat sources for controlling biological systems. While the utility of photothermal heating has significantly advanced through the optimization of AuNP size, shape, and composition, the choice of incident light source utilized has largely been unexplored. One of the more interesting excitation sources is a femtosecond (fs) pulsed laser, as the subsequent temperature increase lasts for only a few nanoseconds and is confined to the nanoscale. However, it is not yet clear how biological materials respond to these short-lived and ultra-confined nanoscale spaciotemporal temperature increases. In this dissertation, we utilize fs laser pulse excitation to locally heat biological materials displayed on the surface of AuNPs in order to understand the corresponding heating profiles and, in turn, interpret how this can be used to modulate biological activity. Due to its unique temperature sensitive hybridization properties, we exploit double-stranded deoxyribonucleic acid (dsDNA) as our prototypical biological material and demonstrate precise control over the rate of dsDNA denaturation by controlling the laser pulse radiant exposure, dsDNA melting temperature, bulk solution temperature, and the distance between the dsDNA and AuNP surface. The rate of dsDNA denaturation was well fit by a modified DNA dissociation equation from which a “sensed” temperature value could be obtained. Evaluating this sensed temperature in the context of the theoretical temperature profile revealed that the ultra-high temperatures near the AuNP surface play a significant role in denaturation. Additionally, we evaluate this technique as a potential means to enhance enzyme activity and report that enhancement is governed by the laser repetition rate, pulse width, and the enzyme’s inherent turnover number. Overall, we demonstrate that the confined and nanosecond duration temperature increase achievable around AuNPs with fs laser pulse excitation can be used to precisely control biological function and establish important design considerations for coupling this technique to more complex biological systems.Item ACTUATION OF MULTIFUNCTIONAL HARD NANOPARTICLES FOR ACTIVELY CONTROLLED DRUG RELEASE(2019) Sangtani, Ajmeeta; Delehanty, James B; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Systemic drug delivery relies on repeated dosing of large concentrations of poorly targeted drug leading to off-target toxicity. Recently, nanoparticle (NP)-mediated drug delivery (NMDD) has been developed as an approach to overcome the limitations of traditional drug delivery. The unique size-dependent properties of NPs and their ability to augment the activity of attached/loaded cargos makes them attractive drug delivery vectors. NPs are classified into two categories (soft or hard depending on their material composition) and our understanding of how to load and control soft NP materials currently surpasses that of hard NPs. In this dissertation we seek to further our fundamental knowledge of hard NP-based drug delivery systems. In Aim 1 we utilize a quantum dot (QD)-cell uptake peptide complex as a central scaffold to append various responsive peptide-drug constructs in order to modulate the toxicity of one of the most widely used chemotherapeutics, doxorubicin. By doing a comparative study of four chemical linkages, we determine the role played by attachment chemistry in controlling drug release. In Aim 2, we utilize the knowledge gained from Aim 1 to develop a system capable of overcoming multidrug resistance in cancer cells, which is known to severely limit the efficacy of chemotherapeutics. Our hard NP conjugate system is unique as it is one of the few systems reported in the literature to bypass multidrug resistance pumps without the need for exogenous drugs. Finally, in Aim 3 we append a peptide for membrane targeting and a photosensitizing drug capable of generating reactive oxygen species to the QD. This multifunctional system displays augmented therapeutic efficacy of the appended photosensitizer by delivering it to the membrane of cells and controlling its actuation using energy transfer. The work described here details basic concepts for the design of “smart” hard NP materials for internally and externally-triggered, active release of surface-appended drug cargos. Additionally, we hope to elucidate the important design considerations that must be taken into account when designing hard NP systems for controlled drug delivery.Item Design of Self-Assembling Nanostructures to Promote Immune Tolerance(2018) Hess, Krystina; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In autoimmune diseases, which affect more than 23 million Americans, the immune system mistakenly attacks healthy tissue. This occurs when the process that normally controls self-reactive inflammatory cells (i.e. tolerance) fails. In multiple sclerosis (MS), the myelin sheath, which insulates nerves, is recognized as a foreign antigen. Demyelination by immune cells results in serious symptoms of neurodegeneration. Current treatments for MS are not curative, but rather manage symptoms by broadly suppressing the immune system, leaving patients unable to fight infection. New therapies that are more specific and effective could greatly improve the quality of life for patients. Biomaterials offer specific advantages for generating antigen-specific tolerance, such as cargo protection, targeted delivery, and controlled release of signals. Additionally, recent reports demonstrate that materials themselves can be intrinsically immunogenic. Two promising biomaterials-based strategies for combating autoimmunity involve: 1) delivery of self-antigen with a regulatory molecule or 2) delivery of self-antigen alone. Aim 1 of this dissertation focuses on the first strategy, creating a novel delivery system for myelin peptide and GpG, an immunomodulatory oligonucleotide. This approach involves electrostatic self-assembly of the two immune signals, eliminating the need for a carrier that could exacerbate inflammation, while still offering attractive features of biomaterials, such as co-delivery. The goal is for immune cells to encounter both signals simultaneously, biasing the response towards tolerance. This work represents the first studies using self-assembled materials to target toll-like receptor signaling, recently shown to be implicated in many autoimmune diseases. Aim 2 of this dissertation is based on the second strategy above, which relies on evidence that changing the trafficking and processing of a self-antigen can impact the development of inflammation or tolerance. Quantum dots, NPs that are intrinsically fluorescent and rapidly drain to lymph nodes, can be decorated with a large and controllable number of myelin peptides. These key features of QDs were exploited to reveal that parameters of self-antigen display (i.e. dose, density) impact biodistribution and immune cell uptake, and are directly correlated to the level of tolerance induced. Together, the described nanotechnologies offer opportunities to probe important questions towards the design of antigen-specific therapies.Item Optical and magnetic measurements of a levitated, gyroscopically stabilized graphene nanoplatelet(2017) Coppock, Joyce Elizabeth; Wellstood, Frederick; Kane, Bruce; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)I discuss the design and operation of a system for levitating a charged, $\mu$m-scale, multilayer graphene nanoplatelet in a quadrupole electric field trap in high vacuum. Levitation decouples the platelet from its environment and enables sensitive mechanical and magnetic measurements. First, I describe a method of generating and trapping the nanoplatelets. The platelets are generated via liquid exfoliation of graphite pellets and charged via electrospray ionization. Individual platelets are trapped at a pressure of several hundred mTorr and transferred to a trap in a second chamber, which is pumped to UHV pressures for further study. All measurements of the trapped platelet's motion are performed via optical scattering. Second, I present a method of gyroscopically stabilizing the levitated platelet. The rotation frequency of the platelet is locked to an applied radio frequency (rf) electric field $\bm{E}_{\mathrm{rf}}$. Over time, frequency-locking stabilizes the platelet so that its axis of rotation is normal to the platelet and perpendicular to $\bm{E}_{\mathrm{rf}}$. Finally, I present optical data on the interaction of a multilayer graphene platelet with an applied magnetic field. The stabilized nanoplatelet is extremely sensitive to external torques, and its low-frequency dynamics are determined by an applied magnetic field. Two mechanisms of interaction are observed: a diamagnetic polarizability and a magnetic moment proportional to the frequency of rotation. A model is constructed to describe this data, and experimental values are compared to theory.Item Controlled Delivery of a Glutamate Receptor Modulator to Promote Regulatory T cells and Restrain Autoimmunity(2015) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Autoimmunity occurs when the immune system incorrectly recognizes and attacks self-molecules. Current therapies involve broad immunosuppressants that are not curative and leave patients immunocompromised. Dendritic cells (DCs) are a target for new therapies because DCs influence the differentiation of immune effector cells. N-Phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), a glutamate receptor enhancer, modulates DC cytokine profiles to polarize T cells toward regulatory phenotypes (TREG ) that are protective in multiple sclerosis (MS). However, PHCCC treatment is limited by poor solubility, a short half-life, and toxicity. We hypothesized that controlled delivery of PHCCC from nanoparticles would alter DC function with reduced treatment frequency. PHCCC nanoparticles attenuated DC activation and promoted TREGs while reducing toxicity 30-fold. In mouse models of MS, these particles delayed disease and reduced severity compared to an equivalent dosing schedule of soluble drug. This outcome demonstrates controlled delivery of metabolic modulators can promote tolerance, suggesting a new route to improve autoimmune therapy.