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.

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

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.

This masteral thesis describes phenomenon of electroporation and it’s use to deal with occluded self-expandable metalic stent. Thesis briefly summarizes theory of electroporation, currently used medical treatments of occluded stents. The next part of the thesis is dedicated to current state of mathematical simulations of electroporation. The core of the thesis are simulations of electroporation effects on tissue of occluded metal stents.

The heterogeneity of pancreatic cancer renders many available general therapies ineffective holding the five-year survival rate close to 10% for decades. Surgical resection eligibility, resistance to chemotherapy and limited efficacy of immunotherapy emphasize the dire need for diverse and innovative treatments to combat this challenging disease. This study evaluates co-therapy strategies that combine non-thermal, minimally invasive ablation technology and targeted drug delivery to enhance treatment efficacy. Our research begins by uncovering the multifaceted potential of Irreversible Electroporation (IRE), a cutting-edge non-thermal tumor ablation technique. This study demonstrates IRE-mediated ability to trigger programmed necrotic cell death, induce cell cycle arrest, and modulate immune cell populations within the tumor microenvironment. This transformation from a pro-tumor state to a proinflammatory milieu, enriched with cytotoxic T lymphocytes and neutrophils. IRE-induced proinflammation in the tumor site renders immunologically "cold" tumor into immunologically "hot" tumor and holds significant promise of improving treatment efficacy. Notably, IRE-treated mice exhibited an extended period of progression-free survival, implying clinical potential. The transient nature of these effects suggests potential mechanisms of tumor recurrence highlighting the need for further studies to maximize the efficacy of IRE. Our mechanistic studies evaluated the IFN-STAT1-PD-L1 feedback loop as a possible reason for pancreatic tumor recurrence. Our data also suggest a stronger IFN-PD-L1 feedback loop compared to mammary, osteosarcoma and glioblastoma tumors rendering pancreatic cancer immunologically "cold". This study also investigates the use of histotripsy (a non-thermal, noninvasive, nonionizing ultrasound-guided ablation modality) to treat pancreatic cancer utilizing a novel immunocompromised swine model. We successfully generated human orthotopic pancreatic tumors in the immune deficient pigs, which allowed for consequent investigation of clinical challenges presented by histotripsy. While rigorous clinical studies are indispensable for validation, the promise of histotripsy offers new hope for patients. In parallel, we used our immunocompromised swine model of orthotopic pancreatic cancer to investigate the SonoTran® system, which employs ultrasound-activated oscillating particles to enhance drug delivery within hard-to-reach tumors. Our study demonstrates that SonoTran® significantly enhances the intratumoral penetrance of therapeutic agents, including commonly used chemotherapy drugs like paclitaxel and gemcitabine. Additionally, SonoTran® improved delivery of the anti-epidermal growth factor (EGFR) monoclonal antibody, cetuximab- which is frequently used in cancer immunotherapy. Together, our findings address challenges in the delivery of a range of therapeutics while simultaneously exposing challenges like off-target damage. In conclusion, this study presents a multifaceted approach to confront the complex characteristics of pancreatic cancer. Given the variations in patient response and the complexity of the disease, it is clear that a singular solution is unlikely. Our research, which combines IRE, histotripsy, and SonoTran®, to interrogate a promising array of tools to tackle different challenges to provide tailored treatments. In the ever-evolving landscape of pancreatic cancer therapy, this research opens new avenues to investigate deeper into molecular mechanisms, co-therapy treatment options, future preclinical and clinical studies which eventually encourage the potential for improved patient outcomes. / Doctor of Philosophy / Pancreatic cancer is a formidable disease, known for its late-stage diagnosis and limited treatment options with a poor 5-year survival rate of ~10%. However, a promising frontier in the battle against this lethal disease has emerged through combining mechanical, cell based and immunotherapies to attack the cancer from multiple angles at once. In my PhD research, I explored novel approaches to transform the landscape of pancreatic cancer treatment. We began by investigating Irreversible Electroporation (IRE), a non-thermal method to ablate tumors. Beyond its known function of reducing tumor size, IRE initiated programmed necrotic cell death, halted tumor cell division, and triggered changes in the immune landscape within the tumor. In response to IRE treatment, the immune environment shifted from pro-tumor to proinflammatory state, showing potential for clinical use. Mice treated with IRE experienced extended cancer progression-free survival temporarily, followed by eventual relapse. During relapse, we found that immune cells reverted back to their original, pre- IRE treated state. This observation logically implies combining IRE and immune checkpoint inhibitors aimed towards maintaining the IRE-altered immunological environment. Next, we developed and used novel pig models that closely resemble human pancreatic cancer patients to test histotripsy, a first phase toward making histotripsy as a non-invasive treatment approach for pancreatic cancer. Use of orthotopic tumor in a large animal model and clinical device allowed us to expose some challenges of ultrasound guidance of histotripsy. Notably, the treatment results in partial ablation and a reduction in stroma materials, which play a role in the tumor's resistance to commonly used treatments. While rigorous clinical studies are needed for validation, this approach offers hope in the quest for innovative pancreatic cancer treatment. Another promising approach we investigated involves SonoTran® particles, ultrasound-activated oscillating particles that can increase drug absorption in a targeted fashion. Our study demonstrated increased concentrations of commonly used therapeutic agents within tumors through SonoTran®-facilitated delivery, providing an effective means to overcome drug delivery issues within pancreatic tumors. There is no one size fits all treatment to address the complexity of pancreatic cancer. The future of treatment lies in the integration of IRE, histotripsy and SonoTran® into clinical practice. In summary, this PhD research identified promising novel technologies and combinations of treatments for pancreatic cancer, reaffirming the importance of exploring innovative solutions to combat pancreatic cancer. The dynamic nature of the pancreatic tumor microenvironment underscores the importance of further research to extend the positive impacts of these treatments and improve tumor debulking.