Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item ENGINEERING TARGETED LIGHT ACTIVATABLE NANOPLATFORMS TO MANAGE RECURRENT CANCERS(2024) Pang, Sumiao; Huang, Huang Chiao HH; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cancer recurrence poses a significant challenge in various malignancies that adverselyaffect long-term survival and quality of life. Glioblastoma (GBM) and ovarian cancer exhibit particularly high recurrence rates. For GBM, tumor recurrence is nearly universal (90%) within 10 months post initial treatment due to its invasive characteristics, limited delivery of therapeutic agents, and persistent drug resistance, resulting in a 5-year survival rate of <10%. While standard chemotherapy and surgery can temporarily alleviate symptoms for both diseases, there has been no significant improvement in long-term disease management or survival extension over several decades. Therefore, it is critical to develop targeted therapies that integrates well with current standards of care strategies. Photomedicine is a promising treatment modality, and the two main phototherapies are photodynamic therapy (PDT) which involves photosensitizer administration followed by light activation resulting in non-thermal chemical damage and photothermal therapy (PTT) which involves exogenous or endogenous sensitizing agents followed by light activation resulting in thermal damage. Clinical applications of both modalities have shown its feasibility and safety; however, they face challenges due to (i) limited cancer selectivity, (ii) heterogenous treatment response, and (iii) low monotherapy treatment efficacy. Leveraging strategic therapeutic targets to advance the current sensitizing agents for targeted delivery is a potential solution to overcome these limitations. The overall objective of this dissertation is to advance and evaluate targeted light-activatable nanoplatforms for phototherapy delivery with considerations for the current clinical workflow of GBM and advanced ovarian cancer. This is achieved through the following goals, (1) engineering a novel Fn14 receptor-directed gold nanorods (DART-GNRs) to assess selectivity and PTT efficacy for GBM, and (2) evaluate safety and long-term efficacy of targeted light-activatable multi-agent nanoplatform (tLAMP) to deliver targeted PDT for peritoneal carcinomatosis. First, this work establishes a reproducible synthesis protocol for DART-GNRs, characterizes its photothermal properties, and demonstrate high selectivity towards the Fn14 receptor of cancer cells. Second half of this dissertation established and investigated a two-fiber tissue optical property (TOP) monitoring method for liquid phantoms and for peritoneal carcinomatosis mouse model to enable safer light dosimetry during PDT, established an irinotecan active loading method to reproducibly synthesize tLAMP, and determined tLAMP tumor nodule penetration depth for enhanced targeted PDT combination therapy with adjuvant chemotherapy to enhance long-term survival for ovarian cancer.Item EVALUATING THE PHOTODYNAMIC AND SONODYNAMIC POTENTIAL OF CLINICALLY RELEVANT PHOTOSENSITIZERS AND DYES(2024) Vig, Shruti; Chiao Huang, Huang; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Light-activable agents such as fluorophores and photosensitizers are used for fluorescenceimaging and photodynamic therapy (PDT) applications in the clinic. These agents can absorb light at specific wavelengths and generate fluorescence and/or cytotoxic reactive molecular species (RMS). Photosensitizers can also interact with ATP-binding cassette (ABC) transporters on target cells. This interaction can affect the intracellular accumulation of photosensitizers and thereby influence imaging and treatment efficacy and outcomes. Currently, there are no well-established methods for screening photoactive agents for potential phototoxicity, creating a need for reliable iii testing methods. Comprehensive screening methods are essential for ensuring safe and effective imaging and therapeutic outcomes with light activable agents. Moreover, photosensitizers are currently being explored for sonodynamic therapy (SDT) with ultrasound in patients. Just like PDT, photosensitizers are thought to be activated by ultrasound-mediated light generation (sonoluminescence) to generate RMS. However, no evidence supporting this mechanism has been published with safe, monitorable, and reproducible SDT effects. Thus, rigorous test methods must be developed to evaluate photochemical activation of photosensitizers using clinically relevant SDT parameters. The results obtained through the studies in this dissertation resulted in (1) A modified invitro test method for assessing the photo-cytotoxic potential of light-activable agents at clinically relevant concentrations and illumination parameters, (2) Updated the ABC transporter substrate status of clinically relevant using in-vitro extraction and flowcytometry methods. (3) Confirmed lack of photochemical activation of clinically relevant photosensitizers during SDT as a potential mechanism of action using a phantom model. A comprehensive understanding of the mechanisms and factors affecting the safety and efficacy of fluorophores and photosensitizers is essential for advancing the field of fluorescence imaging, PDT, and SDT for cancer and other diseases.Item PREPARATION OF A NANOSUSPENSION OF THE PHOTOSENSITIZER VERTEPORFIN FOR PHOTODYNAMIC AND LIGHT-INDEPENDENT THERAPY IN GLIOBLASTOMA(2024) Quinlan, John Andrew; Huang, Huang-Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photodynamic therapy (PDT) using verteporfin (VP) has treated ocular disease for over 20 years, but recent interest in VP’s light-independent properties has reignited interest in the drug, particularly in glioblastoma (GBM) (NCT04590664). Separate efforts to apply PDT to GBM using 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) have also garnered attention (NCT03048240), but, unfortunately, clinical trials using 5-ALA-induced PpIX-PDT have yet to yield a survival benefit. Previous studies have shown VP to be a superior PDT agent than 5-ALA-induced PpIX. Our lab has shown that 690 nm light activates VP up to 2 cm into the brain, while 635 nm light only activates PpIX at depths <1 cm into the brain. Additionally, VP is a more effective photosensitizer than PpIX because it has a higher singlet oxygen yield and is active in the vasculature as well as target tumor cells. However, the hydrophobicity of VP limits effective delivery of the drug to the brain for treatment of GBM.In this context, this thesis aims to re-evaluate the delivery method for VP. VP traditionally requires lipids for delivery as Visudyne. Recent shortages of Visudyne and potential drawbacks of liposomal carriers motivated our development of a carrier-free nanosuspension of VP, termed NanoVP. Previous work has shown that cellular uptake of VP is greater when delivered as NanoVP rather than liposomal VP, resulting in improved cell killing after light activation. This thesis builds on this previous work by (1) evaluating synthesis and storage parameters for NanoVP, (2) determining the pharmacokinetics, biodistribution, and brain bioavailability of NanoVP, and (3) evaluating the potential efficacy of NanoVP as a PDT and a chemotherapy agent, and by supporting development of a zebrafish model of the blood-brain barrier (BBB) for mechanistic studies of improved drug delivery to the brain.Item DEVELOPMENT OF AN X-RAY AND ULTRASOUND IMAGEABLE POLOXAMER-BASED GEL FOR IMAGE-GUIDED LOCAL PERCUTANEOUS DRUG DELIVERY(2024) Delgado Jimenez, Jose Francisco; Wood, Bradford J; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Intratumoral dosing of anti-cancer agents using long needles under image guidance faces challenges due to the invisibility and unpredictable leakage of low viscous agents that leads to adverse effects. To address this, an ultrasound and x-ray imageable drug-loaded gel was developed and tailored for liver delivery. The hypothesis is that by incorporating highly viscous materials and imageable components into drug formulations, we can improve the visibility and reproducibility of the intratumoral drug delivery process. This thesis tested the hypothesis with ex vivo and in vivo tissue. First, 4mL of a pre-heated (37°C) ex vivo bovine liver was injected with gel containing iodine, microbubbles, and doxorubicin as x-rays, ultrasound, and drug respectively. The gel was imageable under ultrasound and x-rays. The gels in tissue were highly localized with circularities of 0.7, and sphericities of 0.9 (1 makes a perfect circle and sphere). Both imaging modalities were able to be predictable for each milliliter injected with a circular pattern growing from the needle tip. The area of the injected 4mL was 3.3cm2. Direct injections with iodine (without gel) were amorphous with high degree of leakage and were not predictable. The acoustic intensity from ultrasound imaging was 150 times higher than the intensity of formulations without gel which contrast clearly from tissue. The in vivo testing involved the dual imageable gel testing in swine liver to assess gel with doxorubicin distribution as well as pharmacokinetics compared to free injected doxorubicin. The results showed high degree of localization for gel containing doxorubicin under perfusionconditions with 0.7 sphericities and circularities from ultrasound and x-ray imaging, respectively, with minimal leakage to vessels. The volume localized without leakage was 5 times higher in the gel with doxorubicin than free doxorubicin. Pharmacokinetic parameters acquired using high performance liquid chromatography equipment showed that doxorubicin concentration in tissue was one million times at the liver injection site compared to liver, spleen, heart, and lung for gel with doxorubicin and free doxorubicin. In addition, gel with doxorubicin formulation provided 3 times lower doxorubicin concentration in kidney than free doxorubicin. The area under the curve and maximum concentration in blood over 4 h was 4 times higher in blood for swine treated with free doxorubicin which may indicate lower systemic exposure of doxorubicin mitigating side effects. In conclusion, gel with doxorubicin were visible under ultrasound and x-rays with high degree of localization in vivo and ex vivo, opening the possibility towards a predictable, precise, and safer delivery of anticancer agents.Item PHOTODYNAMIC THERAPIES AND ETHANOL ABLATION FOR UNRESECTABLE TUMORS AND ASSAY DEVELOPMENT(2024) Ma, Chen-Hua; Huang, Huang-Chiao; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Many cancers pose significant challenges due to their low survival rates, especially when tumors are deemed unresectable. These tumors are either spread diffusely or located in areas that make surgical intervention risky or impossible. This dissertation addresses innovative approaches to treating unresectable cancers, including solid and peritoneal metastasis, by using photodynamic therapy (PDT)-based combination therapy. The initial section of the dissertation reviews the atypical administration routes of photosensitizers, particularly through intratumoral, intraperitoneal, and intra-arterial injections. Next, the study evaluates the Light-Activatable Sustained-Exposure Ethanol Injection Technology (LASEIT)’s performance in xenograft models of hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). The photochemical properties of LASEIT in tissue-mimicking agarose, ex vivo swine liver, and xenograft animal models were confirmed, demonstrating its ability to maintain fluorescence and extend light propagation. Tumor-killing efficacy was shown in both single- and multi-cycle treatments, positioning LASEIT as a promising alternative for solid tumor therapy. The dissertation also investigates the potential of NanoVP, a carrier-free photosensitizer, for the treatment of peritoneal cancers. We confirmed that NanoVP was detectable in swine peritoneal organs using the ML7710 medical laser system, indicating its potential for personalized PDT in the future. However, we also discovered challenges in detecting photobleaching in pigmented organs like the spleen and liver. Finally, the dissertation employs quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to quantify peritoneal cancer cell metastasis in a xenograft ovarian cancer model, confirming the technique’s reliability in detecting low numbers of human cells in mouse tissues.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 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.Item 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.Item 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.Item 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.