The research presented in this dissertation is the result of our laboratory's effort to develop a microfluidic platform to interrogate, manipulate, isolate, and enrich rare mammalian cells dispersed within heterogeneous populations. Relevant examples of these target cells are stem cells within a differentiated population, circulating tumor cells (CTCs) in the blood stream, and tumor initiating cells (TICs) in a population of benign cancer cells. The ability to isolate any of these rare cells types with high efficiency will directly lead to advances in tissue engineering, cancer detection, and individualized medicine. This work lead directly to the development of a new cell manipulation technique, termed contactless dielectrophoresis (cDEP). In this technique, cells are isolated from direct contact with metal electrodes by employing fluid electrode channels filled with a highly conductive media. Thin insulating barriers, approximately 20 μ­m, serve to isolate the fluid electrode channels from the low conductivity sample buffer. The insulating barriers in a fluid-electrical system create a number of unique and interesting challenges from an electrical engineering standpoint. Primarily, they block the flow of DC currents and create a non-constant frequency response which can confound experimental results attempting to characterize the electrical characteristics of cells. Due to these, and other, considerations, the use of high-voltage high-frequency signals are necessary to successfully manipulate cells. The effect of these high frequency fields on cells are studied and applied to microfluidic and tissue level applications. In later chapters, theoretical and experimental results show how high frequency and pulsed electric fields can ablate cells and tissue. Understanding the parameters necessary to electroporate cells aids in the development of cDEP devices where this phenomenon is undesirable and gives insight towards the development of new cancer ablation therapies where targeted cell death is sought after. This work shows that there exists a finite frequency spectrum over which cDEP devices can operate in which cells are minimally affected by the induced electric fields. Finally, lessons learned in the course of the development of cDEP were adapted and applied towards cancer ablation therapies where electroporation are favorable. It was found that bursts of high frequency pulsed electric fields can successfully kill cells and ablate tissue volumes through a process termed High Frequency Irreversible Electroporation (H-FIRE). This technique is advantageous as these waveforms mitigate or eliminate muscle contractions associated with traditional IRE technologies. Similarly, the use of fluid electrodes in cDEP inspired the use of an organs vascular system as the conductive pathway to deliver pulses. This novel approach allows for the ablation of large volumes of tissue without the use of puncturing electrodes. These techniques are currently undergoing evaluation in tissue engineering applications and pre-clinical evaluation in veterinary patients. / Ph. D.

Irreversible electroporation (IRE) is an energy directed focal ablation technique. This procedure typically involves the placement of two or more electrodes into, or around, a region of interest within the tissue and administering a sequence of short, intense, pulsed electric fields (PEFs). The application of these PEFs results in an increase in the transmembrane potential of all cells within the electric field above a critical value, destabilizing the lipid bilayer of the cellular membrane and increasing the cell-tissue permeability. For years, many have used this phenomenon to assist the transport of macromolecules typically unable to penetrate the cell membrane with the intent of avoiding cell necrosis or irreversible electroporation. More recently, however, irreversible electroporation has proven to be a successful alternative for the treatment of cancer. Proper tuning of the pulse parameters has allowed for a targeted treatment of localized tumors, and has shown immense value in the treatment of surgically inoperable tumors located near major blood vessels and nerves. While it is critical to ensure sufficient treatment of the target tissue, it can be equally vital to the treatment and patients overall outcome that the pulsing conditions are set to moderate the associated thermal effects with the electroporation of biological tissue. The development of thermal mitigation strategies for IRE treatment is the focus of this dissertation. Herein, the underlying theory and thermal considerations of tissue electroporation in various scenarios are described. Additionally, new thermal mitigation approaches with the intention of maintaining tissue temperature below a thermally damaging threshold, while also preserving or improving IRE lesion volume are detailed. Further, numerical models were developed and ex vivo tissue experiments performed using a perfused organ model to examine three thermal mitigation strategies in their ability to moderate temperature. Tests conducted using thermally mitigating treatment delivery on live tissue confirm the capacity to deliver more energy to the tissue at a thermally acceptable temperature, and provide the potential for a replete IRE lesion. / Doctor of Philosophy / Irreversible electroporation (IRE) is a minimally invasive therapy utilized to treat a variety of cancers. This procedure involves the delivery energy in the form of pulsed electric fields (PEFs) through two or more needle electrodes. These PEFs destabilize the cell membrane, increase the cell-tissue permeability, and ultimately induce cell death for any given cell within the targeted treatment region. Over the years, this treatment modality has shown a great deal of promise in the treatment of unresectable tumors in which the tumor is positioned near or around sensitive regions making the surgical removal of the tumor impossible and thermal ablation techniques limited in their ability to treat without irrevocably damaging the underlying tissue architecture and other critical surrounding structures. Thus, it can be vital to the treatment and patients overall outcome that the IRE therapy is set to moderate any associated thermal effects with the electroporation of biological tissue. However, the design of an electric field that simultaneously maps the entire region of interest for a single treatment and avoids undesirable thermal effects can be challenging when treating larger or irregularly shaped volumes of tissue. Thus, in this dissertation, we demonstrate various treatment delivery methods/ enhancements to reduce temperature rise during IRE therapy. The underlying theory of tissue electroporation and associated thermal considerations are described to provide a foundation and general context. Additionally, novel approaches to tissue electroporation therapy with the intention of maintaining tissue temperature below a thermally damaging threshold throughout treatment are detailed.

Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models. / Master of Science

Irreversible electroporation (IRE) is a focal ablation technique that has been shown in recent clinical trials to be effective in treating pancreatic cancer. The technique uses short, high voltage pulses to induce nanoscale pores in the target cell membranes, leading to cell death. Due to its non-thermal mechanism, IRE is particularly well suited for treating a tumor that is unresectable due to its close location to crucial structures such as blood vessels and nerves. Predicting the region of treatment is critical for optimal treatment of the tumor. The only predictive tools clinicians currently rely on for IRE treatment planning are computer tomography (CT), ultrasound (US) imaging, and real-time resistance measurement is used to monitor treatment progress. However, there is currently no method to plan optimal pulse parameters such as voltage, pulse duration, pulse number, and electrode spacing prior to treatment. Computational treatment planning models aim to perform this prediction in 3D, however, the electric field region relies on the electrical response of human tissue during IRE. This work quantifies this response for the first time and implements human tissue properties in a patient-specific, 3D treatment planning model. / Master of Science / Pancreatic cancer results in 40,000 deaths every year in the U.S, making it one of the most challenging diseases to treat. The current treatments for this disease fall short and have failed to significantly extend patient life expectancy. A technique called irreversible electroporation (IRE) has been shown in recent clinical trials to be effective in treating pancreatic cancer. IRE excels at treating tumors that are located near important blood vessels, nerves, and other important structures. However, clinicians do not have a way to visualize the region of treatment before surgery. In the research setting, 3D computational models aim to predict this area, but so far these models have been based on animal tissue, often of the incorrect organ type. This work applies IRE to human tissue samples, quantifies its electrical behavior, and implements that information in a personalized, predictive 3D model.

Osteosarcoma (OS) is the most common primary bone tumor with an annual incidence rate of 3-4 individuals per million particularly affecting children and young adults. The 5-year survival rate stands at 60-80% with the current standard of care for human OS patients who do not have metastatic disease at presentation, but this drops to 20% for patients with metastatic disease which frequently occurs in the lungs. OS is much more common in canines, with metastasis being the major contributor to mortality, the same as in humans. Metastatic OS warrants novel treatment strategies to improve prognosis and survival. High-frequency irreversible electroporation (H-FIRE) is a promising, non-thermal, minimally invasive technique that induces cell death by applying pulsed electric fields in targeted regions, potentially triggering an anti-tumor immune response that could also target and prevent metastases. Such a dual functionality of H-FIRE is uniquely suited to treat pulmonary metastatic OS. The goal of this thesis was to study the ablative and immunomodulatory effects of H-FIRE on OS in-vitro with the overall hypothesis that H-FIRE completely ablates OS cells, induces the release of damage-associated molecular patterns (DAMPs), and promotes pro-inflammatory immune activating signatures in macrophages and T cells. Using an in-vitro model, my master's thesis focused on 1) Determining the electric field strength that completely ablates OS cells 2) Evaluating the immunomodulatory effects of H-FIRE by co-culturing H-FIRE treated OS cells with macrophages and T cells separately. Our study has utilized murine, canine, and human OS and immune cells, thus demonstrating a unique cross-species approach, 3) Evaluating DAMPs (ATP, calreticulin, and HMGB1) post-H-FIRE ablation of human OS cells. Overall, our study showed that H-FIRE successfully ablated OS cells in-vitro, induced the release of DAMPs from treated cells, and promoted activation signatures in immune cells. This thesis provides foundational data for future investigations developing H-FIRE as an immunomodulatory strategy for treating metastatic OS. / Master of Science / Osteosarcoma (OS) is the most common primary bone tumor that majorly affects young adults and children with an incidence rate of 3-4 individuals per million per year. When metastasis occurs (i.e. OS spreads from its site of origin to other organs in the body), most frequently to the lungs, patients experience poor chances of recovery and survival. Currently, the treatment protocol followed for patients with metastatic OS largely includes complete surgical removal and chemotherapy both of which can be very grueling for patients. No significant improvement in the overall 5-year survival rate with current mainstay treatment has led to the urgent need of novel treatment modalities for treating patients with pulmonary metastatic OS. High-Frequency Irreversible Electroporation (H-FIRE) is a novel non-thermal tumor ablation strategy that utilizes electrical pulses to create pores on the cell membrane, thus leading to irreversible damage and cell death. These dying tumor cells release certain molecules and proteins that send danger signals to activate the body's own immune system against the tumor. H-FIRE with its dual function of destroying the targeted tumor region via electroporation and distant metastases via activating immune system is uniquely suited to treat pulmonary metastatic OS. This thesis is the first to investigate H-FIRE ablation and immunomodulation for OS. We hypothesized that H-FIRE can completely destructs OS cells, promotes the release of danger signals, and causes immune activation. Using an in-vitro model, this thesis focused on 1) Determining the electric field strength needed for complete OS cell destruction by H-FIRE 2) Evaluating the immune activation potential of H-FIRE by exposing these H-FIRE treated cells to immune cells like macrophages and T cells separately. We utilized human, mouse, and dog-derived OS cells to increase the biological and clinical relevance of our study. 3) Evaluating certain proteins that act as danger signals post-H-FIRE treatment of human OS cells. Overall, our results indicated that H-FIRE can successfully destruct OS cells in-vitro, promotes the release of danger signals, and induces immune activation. This thesis contributes to providing crucial preliminary data in the development of H-FIRE as a novel ablation and immunomodulation treatment strategy for pulmonary metastatic OS.

Breast cancer (BC) is the second most common cause of cancer-related deaths for women in the United States, estimated to affect 1 in 8 women. Difficulties arise in BC treatment due to the hormone sensitivity and heterogeneity of the malignancies, and the poor prognosis after metastases. Due to the immense physical and psychological effects of conventional surgical methods, minimally invasive, non-thermal, focal electroporation-based ablation therapies are being investigated for the treatment of BC. Irreversible Electroporation (IRE) delivers a series of long, monopolar electrical pulses via electrodes inserted directly into the targeted tissue which disrupt cellular membranes by creating nano-scale pores, killing the cells via loss of homeostasis while promoting an immune response. However, IRE requires cardiac synchronization and a full-body paralytic to mitigate unwanted muscle contractions, which motivated the creation of second generation High-Frequency IRE or H-FIRE. H-FIRE delivers short, bipolar pulses to destroy cancer cells without muscle contractions and nerve excitation, and allows for more tunable treatment parameters. Throughout my thesis, I discuss investigations of H-FIRE for the treatment of triple-negative and hormone-sensitive BC cell lines and compare efficacy to IRE outcomes. To further establish the translation and understanding of H-FIRE for BC applications, my master's thesis focuses on: (1) determining the lethal electric field threshold of both cell lines in a 3D hydrogel matrix after H-FIRE and IRE; and (2) employ those values in a single bipolar probe numerical model to simulate in vivo treatments. The culmination of this thesis advances the use of H-FIRE in breast tissues, as well as demonstrates how in vitro data can be used to develop clinically relevant numerical models to better predict in vivo treatment outcome. / Master of Science / Breast cancer (BC) is one of the most deadly forms of cancer for women in the United States, affecting every 1 in 8 women. Difficulties arising in the treatment of BC include the hormone sensitivity of malignancies, metastatic tendencies, and the diversity of the tissue that characterizes the breast. Surgical options like mastectomy or lumpectomy are most often used when treating BC; however, these are incredibly taxing on the patient. This reason has sparked investigations of focused ablation modalities for the treatment of BC, specifically non-thermal mechanisms like electroporation-based therapies. Electroporation explains the phenomenon that cells subjected to a high enough electric field will result in increased membrane permeability, allowing for the entrance of therapeutic agents in reversible mechanisms, or cell death beyond an irreversible point. Irreversible Electroporation (IRE) has shown success for the treatment of prostate, liver, kidney, and pancreas. However, due to some drawbacks, second generation High-Frequency IRE (H-FIRE) is increasingly being investigated for certain cancer types and is the main focus of this thesis project. Within this thesis, I discuss investigations of H-FIRE with applications to treat malignant breast cell lines. Specifically, my thesis focuses on: (1) determining the point at which cancer cells damage is irreversible; and (2) incorporate those values into a numerical model used to simulate electroporation treatment if a tumor were embedded in a layer of fatty connective breast tissue. The culmination of this thesis enhances our understanding of H-FIRE in the breast, with the hopes of future transition of application into animal studies and ultimately the clinic.

Conventional methods of cancer therapy have been severely limited by inefficient delivery of therapeutic doses without incidence of harsh and toxic side effects in normal tissues. Consequently, countless new methods for early detection and drug delivery have been investigated in the area of nanoparticles and hydrogels. Although many of these methods are promising, the complex nature of cancer increases the difficultly for the development of the perfect system. Cellulose nanocrystals (CNCs) have been studied widely for a variety of applications. Despite their advantages, investigations of their abilities in the biomedical field have not been explored. The goal of this project is to delve into the potential uses of CNCs in detection, targeted drug delivery, and potentiation of irreversible electroporation (IRE)-induced cell death in folate receptor (FR)-positive cancers. To accomplish this task we have prepared stable and reproducible CNCs from wood pulp via sulfuric acid hydrolysis. Furthermore, we have functionalized the surface of these nanoparticles and conjugated them with the targeting ligand folic acid (FA) and the fluorescent imaging agent fluorescein-5\'-isothiocyanate (FITC) to create FITC-CNC-FA; CNCs have also been conjugated with doxorubicin (DOX), a potent chemotherapeutic (DOX-ALAL-CNC-FA). We have determined FITC-CNC-FA's and DOX-ALAL-CNC-FA's ability to specifically target FR-positive cancer cells in vitro; meanwhile non-targeted CNCs (FITC-CNC) were shown unable to bind to these cell types. In addition, we have investigated FITC-CNC-FA's pharmacokinetic activity in vivo. To properly model the CNC conjugate's activity in vivo, a physiologically based pharmacokinetic (PBPK) model has been constructed. We have also examined CNCs' ability to potentiate a new technique for tumor ablation, IRE. Pre-incubation with FA-conjugated CNCs (CNC-FA) have shown an increase in cytotoxicity in FR-positive cancer cells induced by IRE. In addition, CNC-FA did not potentiate IRE-induced cytotoxicity in a FR-negative cancer cell type. For a more comprehensive understanding of CNC-FA's ability to potentiate IRE induced cytotoxicity, we optimized a 3D in vitro hydrogel system. Preliminary data suggest this method of experimentation will be more realistic to in vivo studies to be completed in the future. Together, these studies showcase CNCs as efficient and effective nano-carriers in tumor detection and treatment. / Ph. D.

Emerging technologies in the healthcare industry encompass revolutionary devices or drugs that have the potential to change how healthcare will be practiced in the future. While there are several emerging healthcare technologies in the pipeline, a few key innovations are slated to be implemented clinically sooner based on their mass appeal and potential for healthcare breakthroughs. This thesis will focus on specific topics in the emerging technological fields of nanotechnology for photothermal cancer therapy, 3D printing for irreversible electroporation applications, and imageable biomaterials. While these general areas are receiving significant attention, we highlight the potential opportunities and limitations presented by our select efforts in these fields. First, in the realm of nanomedicine, we discuss the optimization and characterization of sodium thiosulfate facilitated gold nanoparticle synthesis. While many nanoparticles have been examined as agents for photothermal cancer therapy, we closely examine the structure and composition of these specific nanomaterials and discuss key findings that not only impact their future clinical use, but elucidate the importance of characterization prior to preclinical testing. Next, we examine the potential use of 3D printing to generate unprecedented multimodal medical devices for local pancreatic cancer therapy. This additive manufacturing technique offers exquisite design detail control, facilitating tools that would otherwise be difficult to fabricate by any other means. Lastly, in the field of imageable biomaterials, we demonstrate the development of composite catheters that can be visualized with near infrared imaging. This new biomaterial allows visualization with near infrared imaging, offering potentially new medical device opportunities that alleviate the use of ionizing radiation. This collective work emphasizes the need to thoroughly optimize and characterize emerging technologies prior to preclinical testing in order to facilitate rapid translation. / Master of Science

High-frequency irreversible electroporation (H-FIRE) is a non-thermal ablation technique that uses intense, short, bipolar electrical pulses to induce cell death in cancerous tissues. It's being studied for treating hepatocellular carcinoma (HCC) in dogs. Previous in vitro research suggests H-FIRE may impact the release of extracellular vesicles (EVs). This study aims to explore how H-FIRE affects peripheral extracellular vesicle (EV) dynamics, potentially providing insights into its broader systemic effects and implications for biomarker development in canine liver cancer treatment. Dogs diagnosed with HCC were enrolled in a clinical trial. H-FIRE was applied to tumors, followed by surgical resection at three different time points. Peripheral blood samples were collected before and immediately after H-FIRE treatment. Plasma was isolated, aliquoted, and stored at -20°C. EVs were enriched from plasma via filtration and ultracentrifugation. Nanoparticle Tracking Analysis (NTA) quantified EV concentration and size distribution. Ten patients provided pre- and post-treatment plasma samples. The median EV concentration in peripheral blood increased from 2.56 x 10^11 particles/ml pre-treatment to 2.68 x 10^11 particles/ml post-treatment (p = 0.0048). The mean EV size decreased from 99.32 nm pre-treatment to 87.82 nm post-treatment (p = 0.007). The mode of EV size decreased from 83 nm pre-treatment to 70.5 nm post-treatment (p = 0.0076). The results of this study raise intriguing questions on the significance of changes in extracellular vesicle size and concentration post-treatment, as well as the potential clinical implications of these changes. / Master of Science / High-frequency irreversible electroporation (H-FIRE) is a new method to destroy cancer cells without using heat. It's being tested for treating liver cancer in dogs. Previous lab studies suggest H-FIRE might affect the release of small structures known as extracellular vesicles (EVs). This study aims to see how H-FIRE affects EVs in the blood of dogs with liver cancer. Understanding these changes could help develop new ways to diagnose and treat the disease in dogs and humans. Dogs with liver cancer were part of a study. They received H-FIRE treatment followed by surgery, and blood samples were taken before and right after treatment. The plasma was separated and stored. EVs were collected from plasma using special methods, including Nanoparticle Tracking Analysis (NTA) to help measure the number and size of EVs.

Since its conception in 2005, irreversible electroporation (IRE), a non-thermal tumor ablation modality, was investigated for safety and efficacy in clinical applications concerning different organs. IRE utilizes high voltage (~3kV), short duration (~100us) pulses to create transient nanoscale defects in the plasma membrane to cause cell death due to irreversible defects, osmotic imbalances and ATP loss. More recently, high-frequency irreversible electroporation (H-FIRE), which employs narrow bipolar pulses (~0.5-10us) delivered in bursts (on time ~100us), was invented to provide benefits such as the mitigation of intense muscle contractions associated with IRE-based treatments. Furthermore, H-FIRE exhibits the potential to improve lesion predictability in homogeneous and heterogeneous tissue masses. Therapeutic IRE and H-FIRE utilize source and sink electrodes inserted into or around the tumor to deliver the treatment. Prediction of the ablation size, for a set of parameters, can be achieved by the use of pre-treatment planning algorithms that calculate the induced electric field distribution in the target tissue. An electric field above a certain threshold induces cell death and parameters are tuned to ensure complete tumor coverage while sparing the nearby healthy tissue. IRE studies have shown that the underlying field is influenced by the increase in tissue conductivity due to enhanced membrane permeability, and treatment outcome can be improved when this nonlinearity is accounted for in numerical models. Since IRE pulses far exceed the time constant of the cell (~1us), the tissue response can be treated as essentially DC a static approximation can be used to predict the field distribution. Alternately, as H-FIRE pulses are on the order of the time constant of the membrane, the tissue response can no longer be treated as DC. The complexity of the H-FIRE-induced field distribution is further enhanced due to the dispersion and non-linearity in biological tissue impedance during treatment. In this dissertation, we have studied the electromagnetic fields induced in tissue during H-FIRE using several experimental and modeling techniques. In addition, we have characterized the nonlinearity and dispersion in tissue impedance during H-FIRE treatments and proposed simpler methods to predict the field distribution to enable easier translation to the clinic. / Ph. D. / Development within urbanized regions increase impervious surfaces, which further cause significant storm events in watersheds. The increased impervious surfaces result in hotter stormwater particularly during hot summers, which has diverse effects on aquatic health of downstream receiving streams. The main objective of the current study is to evaluate the thermal impact of urbanization on aquatic health habitats in Stroubles Creek Watershed, Blacksburg, Virginia. To aim this goal and achieve the thermal evaluation of the highly urbanized Stroubles Creek Watershed, a U.S. Environmental Protection Agency’s Storm Water Management Model (SWMM) and a Minnesota Urban Heat Export Tool (MINUHET) models from scratch of the Stroubles Creek watershed, using Town of Blacksburg and Virginia Tech Physical Facility information were developed. This necessitated combining information from a wide variety of sources, including geologic maps, geodatabases, hydraulic models, computer-aided design (CAD) files, and scanned as-built information. In addition to the models, a hybrid model was developed that combines SWMM and MINHET outputs. The temperatures and heat loads at the downstream of the watershed were predicted using SWMM, MINUHET, and Hybrid models for two summer periods of 2016 and 2015, and the predicted temperature were compared to the criteria for survival of aquatic health such as trout. Furthermore, a number of thermal mitigation strategies such as bioretentions systems, concrete pavements (which has lighter color compared to asphalt pavements), and increased vegetation canopies were simulated within the MINUHET and SWMM models configurations to reduce simulated temperatures and heat loads at the watershed scale. The simulated temperatures and heat loads represented that concrete pavements results in better performance of thermal mitigation within watersheds than bioretention systems, and increased vegetation canopies.