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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.

Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency. / Master of Science

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.

No / This work investigated the effect of electroporation on human epidermal penetration of a model neutral lipophilic compound (estradiol) from saturated aqueous solution and when encapsulated in ultradeformable liposomes. Total amount penetrated and skin deposition were compared with values obtained from passive diffusion. The effect of electrical pulsing on liposome size was investigated. The action of phosphatidylcholine on skin that was structurally altered by such pulses was determined. Electroporation did not affect liposome size. Skin pulsing considerably increased estradiol penetration and skin deposition from solution, relative to passive delivery, with subsequent partial recovery of skin resistance to molecular penetration. Surprisingly, with liposomes, electroporation did not markedly affect estradiol skin penetration. Importantly, liposomal phosphatidylcholine applied during or after pulsing accelerated skin barrier repair, i.e. provided an anti-enhancer or retardant effect.

The objective of this research is the development of an electrically active microneedle array that can deliver biomolecules such as DNA and drugs to epidermal cells by means of electroporation. Properly metallized microneedles could serve as microelectrodes essential for electroporation. Furthermore, the close needle-to-needle spacing of microneedle electrodes provides the advantage of utilizing reduced voltage, which is essential for safety as well as portable applications, while maintaining the large electric fields required for electroporation. Therefore, microneedle arrays can potentially be used as part of a minimally invasive, highly-localized electroporation system for cells in the epidermis layer of the skin. This research consists of three parts: development of the 3-D microfabrication technology to create the microneedle array, fabrication and characterization of the microneedle array, and the electroporation studies performed with the microneedle array. A 3-D fabrication process was developed to produce a microneedle array using an inclined UV exposure technique combined with micromolding technology, potentially enabling low cost mass-manufacture. The developed technology is also capable of fabricating 3-D microstructures of various heights using a single mask. The fabricated microneedle array was then tested to demonstrate its feasibility for through-skin electrical and mechanical functionality using a skin insertion test. It was found that the microneedles were able to penetrate skin without breakage. To study the electrical properties of the array, a finite element simulation was performed to examine the electric field distribution. From these simulation results, a predictive model was constructed to estimate the effective volume for electroporation. Finally, studies to determine hemoglobin release from bovine red blood cells (RBC) and the delivery of molecules such as calcein and bovine serum albumin (BSA) into human prostate cancer cells were used to verify the electrical functionality of this device. This work established that this device can be used to lyse RBC and to deliver molecules, e.g. calcein, into cells, thus supporting our contention that this metallized microneedle array can be used to perform electroporation at reduced voltage. Further studies to show efficacy in skin should now be performed.