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

No / Electrically assisted skin delivery of liposomal estradiol; phospholipid as damage retardant.

Cell-level and tissue-level mechanical properties are key to healthy biological functions, and many diseases and disorder arise or progress due to altered cell and tissue mechanics. Pulse electric field (PEFs), which employ intense external electric fields to cause electroporation, a phenomenon characterized by increased cell membrane permeability, also can cause significant changes to cell and tissue mechanics. Here, we investigate the mechanics of brain and brain cancer cells, specifically focusing on how PEFs impact cell mechanics and PEF-induced blood-brain barrier disruption. In our first study, we investigate single-cell mechanical disruption of glioblastoma cells after reversible electroporation using Nanonet Force Microscopy (NFM). A precise network of extracellular-matrix mimicking nanofibers enabled cell attachment and contraction, resulting in measurable fiber deflections. Cell contractile forces were shown to be temporarily disrupted after reversible electroporation, in an orientation and field-dependent manner. Furthermore, we found that cell response is often a multi-stage process involving a cell-rounding stage, biphasic stage, and a cell re-spreading stage. Additionally, cell viability post-PEFs was orientation-dependent. In another study, we investigated the mechanical properties of brain cancer for various-grade glioma cells (healthy astrocytes, grade II, grade III, and grade IV (glioblastoma) cells). A microfluidic constriction channel caused cell deformation as cells, driven by hydrostatic pressure, entered a narrow constriction. Finite element models of cell deformation and a neural network were used to convert experimental results (cell entry time and cell elongation within the channel) into elastic modulus values (kPa). We found that the that low-grade glioma cells showed higher stiffnesses compared to healthy and grade IV glioma cells, which both showed similar values. These results warrant future studies to investigate these trends further. PEFs can induce Blood-brain barrier (BBB) disruption, an effect we studied using a multiplexed, PDMS microdevice. A monolayer of human cerebral endothelial cells on a semi-permeable membrane was used to model the BBB, and permeability was assessed by the diffusion of a fluorescent dye from an upper to lower channel. A custom tapered channel and branching channel design created a linear gradient in the electric field within the device that enabled six electric field strengths to be tested at once against two unexposed (control) channels. Normalization of permeability by the control channels significantly removed experimental noise. We found that after high-frequency bipolar irreversible electroporation (HFIRE) electric pulses, permeability transiently increased within the first hour after electroporation, in a voltage- and pulse-number dependent manner. However, we found significant electrofusion events after pulsing at high voltages, which reduced monolayer permeability below baseline values. This device enables efficient exploration of a wide range of electroporation parameters to identify the optimal conditions for blood-brain barrier disruption. In another blood-brain barrier study, we incorporate dense, polystyrene nanofiber networks to create ultra-thin, ultra-porous basement-membrane-mimics for In vitro blood-brain barrier models. Fiber networks are fabricated using the non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. Endothelial cells cultured on one side of the fiber network are in close contact with supporting cell types (pericytes) cultured on the backside of the fibers. Contact-orientation co-cultures have been shown to increase blood-brain barrier integrity, and our nanofiber networks increase the physiological realism of basement-membrane mimics for improve modeling. Finally, we investigate how cell viability post-electroporation is impacted by cell morphology. The impact of cell morphology (shape and cytoskeletal structure) on cell survival after electroporation is not well understood. Linking specific morphological characteristics with cell susceptibility to electroporation will enhance fundamental knowledge and will be widely useful for improving electroporation techniques where cell viability is desirable (gene transfection, electrofusion, electrochemotherapy) or where cell viability is undesirable (tumor ablation, cardiac ablation). Precise control of cell shape and orientation enabled by nanofiber scaffolds provides a convenient and expedient platform for investigating a wide variety of factors (morphological and experimental) on cell viability. Altogether, these investigations shed new light on cell mechanical changes due to disease and pulsed electric fields, and suggest opportunities for improving brain cancer therapies. / Doctor of Philosophy / In biology, structure and function are interrelated. Cells and tissue have structures that enable them to perform their proper function. In the case of disease, cell and tissue properties are altered, leading to dysfunction. Alternatively, healthy structures sometime hinder effective treatments, and therefore can be therapeutically disrupted to improve treatments. In this study, we investigate single-cell and multi-cellular mechanical change due to disease or after pulsed electric fields (PEFs), with a specific focus on the brain. Pulsed electric fields (PEFs) use electrodes to deliver short, intense pulses of electrical energy to disrupt cell membranes and change cell mechanics. We studied as single-cell contractility, cancer cell stiffness, and blood-brain barrier (BBB) disruption by PEFs. We found that PEFs cause significant change to cell shape and mechanics, and can disrupt the BBB. By studying several grades of brain cancers, we found that low-grade brain cancer (gliomas) showed increased stiffness compared to healthy and highly diseased (grade IV) cells. To mimic the BBB, we used microfluidic devices to grow specialized brain cells (endothelial cells) on permeable membranes and nanofibers networks and showed that these devices can mimic structures found in animals/humans. Finally, we studied how cell properties (such as shape) determine whether cells will survive PEFs. Taken together, our investigations improve the understanding of brain mechanics during disease and after PEFs, and suggest the usefulness of PEFs for improved brain cancer therapies.

Glioblastoma (GBM) is the most common adult malignant glioma (MG) variant, and the median survival of persons with GBM is about 2 years, even with aggressive treatments. Dogs and humans are the only species in which brain tumors commonly develop spontaneously, with an estimated post-mortem frequency of primary brain tumors approximating 2% in both species. Gliomas represent about 35% of all canine primary brain tumors, with high-grade oligodendroglioma and astrocytoma phenotypes accounting for about 70% of all canine gliomas. Canine gliomas are also treated using surgical, radiotherapeutic, and chemotherapeutic regimens similar to those used in humans. The efficacy of these therapies in dogs with MG is also poor, with median survival times ranging from 3-8 months, which closely mirrors the dismal prognosis associated with human GBM. Thus, treatment of MG represents a current and critically unmet need in both human and veterinary medicine. In this work, we investigate minimally invasive methods to access the brain for the purposes of ultimately improving the diagnosis and treatment of malignant brain tumors. Chapter 1 reviews the current clinical challenges associated with the treatment of GBM, highlights the value of using the spontaneous canine glioma model in translational brain tumor studies, and introduces High-Frequency Irreversible Electroporation (H-FIRE) and Convection Enhanced Delivery (CED), which are two novel treatment platforms for GBM being developed in our lab. In Chapter 2, we demonstrate that definitive diagnosis of brain tumors, a critical first step in patient management, can be safely and accurately performed in dogs with naturally occurring brain tumors using a stereotactic brain biopsy procedure. Chapter 3 evaluates the in vivo safety and biocompatibility of fiberoptic microneedle devices, a major technical component of our convection-enhanced thermotherapy catheter system (CETCS), chronically implanted in the rodent brain. The CETCS is a novel technology being developed and used in our laboratory to improve the delivery of drugs to brain tumors using CED. This study provides regulatory data fundamental to the commercialization of the CETCS device for brain tumor treatment by illustrating that the device did not cause clinically significant neurological complications and resulted in mild pathologic changes in brain tissue, similar to other types of devices designed and approved for use in the brain. In Chapters 4 and 5 we explore possible bystander effects of H-FIRE on glutamate metabolism in the brain. H-FIRE has been shown to be able to both ablate brain tumors as well as disrupt the blood-brain barrier (BBB). As these therapeutic effects of H-FIRE are dependent on applying electrical fields to the tissue that either reversibly permeabilize the cell membrane, allowing treated cells to survive, or permanently disrupt the structure of the cell membrane, causing cell death, we hypothesized that altering the membrane permeability with HFIRE would increase the extracellular glutamate concentrations and contribute to excitotoxic brain tissue damage. Chapters 4 used in vitro brain cell culture systems and in vivo experiments in normal and glioma-bearing rat brains to determine if glutamate release in the brain occurs as a bystander effect following H-FIRE treatment, identify concentrations of glutamate necessary to induce death of cells or BBB disruption, and characterize glutamatergic gene expression in response to H-FIRE treatment. Chapter 5 describes the use of magnetic resonance spectroscopic and spatial transcriptomic methods to further quantify the in vivo effects of H-FIRE treatment on glutamate release and metabolism in dogs with spontaneous brain tumors. The in vitro results indicated that the magnitude of glutamate release following H-FIRE is insufficient to induce cytotoxicity in normal or neoplastic brain cell lines, and also did not increase the permeability of the BBB. In our in vivo model systems, we documented significant, transient post-H-FIRE increases in glutamate to concentrations previously associated with excitotoxicty, with upregulation of the expression of genes involved with ionotropic and metabotropic glutamatergic receptor signaling. A contemporaneous upregulation of genes associated with glutamate uptake and recycling were also noted, indicating an adaptive, protective response to the glutamate release. Our work summarily demonstrates that the diagnosis and potential treatment of malignant brain tumors can be achieved through the use of minimally invasive techniques that provide local access to brain tissue. While complications will always be possible anytime the brain is manipulated surgically, and further investigations are required to characterize the spectrum and mechanisms of adverse events that can occur following CETCS CED and H-FIRE treatment, our results support the continued development of these novel therapeutic platforms for the treatment of GBM. / Doctor of Philosophy / Glioblastoma (GBM) is the most common adult malignant glioma (MG) variant, and the median survival of persons with GBM is about 2 years, even with aggressive treatments. Dogs and humans are the only species in which brain tumors commonly develop spontaneously, with an estimated post-mortem frequency of primary brain tumors approximating 2% in both species. Gliomas represent about 35% of all canine primary brain tumors, with high-grade oligodendroglioma and astrocytoma phenotypes accounting for about 70% of all canine gliomas. Canine gliomas are also treated using surgical, radiotherapeutic, and chemotherapeutic regimens similar to those used in humans. The efficacy of these therapies in dogs with MG is also poor, with median survival times ranging from 3-8 months, which closely mirrors the dismal prognosis associated with human GBM. Thus, treatment of MG represents a current and critically unmet need in both human and veterinary medicine. In this work, we investigate minimally invasive methods to access the brain for the purposes of ultimately improving the diagnosis and treatment of malignant brain tumors. Chapter 1 reviews the current clinical challenges associated with the treatment of GBM, highlights the value of using the spontaneous canine glioma model in translational brain tumor studies, and introduces High-Frequency Irreversible Electroporation (H-FIRE) and Convection Enhanced Delivery (CED), which are two novel treatment platforms for GBM being developed in our lab. In Chapter 2, we demonstrate that definitive diagnosis of brain tumors, a critical first step in patient management, can be safely and accurately performed in dogs with naturally occurring brain tumors using a stereotactic brain biopsy procedure. Chapter 3 evaluates the in vivo safety and biocompatibility of fiberoptic microneedle devices, a major technical component of our convection-enhanced thermotherapy catheter system (CETCS), chronically implanted in the rodent brain. The CETCS is a novel technology being developed and used in our laboratory to improve the delivery of drugs to brain tumors using CED. This study provides regulatory data fundamental to the commercialization of the CETCS device for brain tumor treatment by illustrating that the device did not cause clinically significant neurological complications and resulted in mild pathologic changes in brain tissue, similar to other types of devices designed and approved for use in the brain. In Chapters 4 and 5 we explore possible bystander effects of H-FIRE on glutamate metabolism in the brain. H-FIRE has been shown to be able to both ablate brain tumors as well as disrupt the blood-brain barrier (BBB). As these therapeutic effects of H-FIRE are dependent on applying electrical fields to the tissue that either reversibly permeabilize the cell membrane, allowing treated cells to survive, or permanently disrupt the structure of the cell membrane, causing cell death, we hypothesized that altering the membrane permeability with HFIRE would increase the extracellular glutamate concentrations and contribute to excitotoxic brain tissue damage. Chapters 4 used in vitro brain cell culture systems and in vivo experiments in normal and glioma-bearing rat brains to determine if glutamate release in the brain occurs as a bystander effect following H-FIRE treatment, identify concentrations of glutamate necessary to induce death of cells or BBB disruption, and characterize glutamatergic gene expression in response to H-FIRE treatment. Chapter 5 describes the use of magnetic resonance spectroscopic and spatial transcriptomic methods to further quantify the in vivo effects of H-FIRE treatment on glutamate release and metabolism in dogs with spontaneous brain tumors. The in vitro results indicated that the magnitude of glutamate release following H-FIRE is insufficient to induce cytotoxicity in normal or neoplastic brain cell lines, and also did not increase the permeability of the BBB. In our in vivo model systems, we documented significant, transient post-H-FIRE increases in glutamate to concentrations previously associated with excitotoxicty, with upregulation of the expression of genes involved with ionotropic and metabotropic glutamatergic receptor signaling. A contemporaneous upregulation of genes associated with glutamate uptake and recycling were also noted, indicating an adaptive, protective response to the glutamate release. Our work summarily demonstrates that the diagnosis and potential treatment of malignant brain tumors can be achieved through the use of minimally invasive techniques that provide local access to brain tissue. While complications will always be possible anytime the brain is manipulated surgically, and further investigations are required to characterize the spectrum and mechanisms of adverse events that can occur following CETCS CED and H-FIRE treatment, our results support the continued development of these novel therapeutic platforms for the treatment of GBM.

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.

Electroporation-based treatments are motivated by the response of biological membranes to high- intensity pulsed electric fields. These fields rearrange the membrane structure to enhance the membrane's diffusive permeability, or the degree to which a membrane allows molecules to diffuse through it, is impacted by the structure, composition, and environment in which the cell resides. Tracer molecules have been developed that are unable to pass through intact cell membranes yet enter permeabilized cells. This dissertation investigates the hypothesis that the flow of such molecules may be used to quantify the effects of the electrical stimulus and environmental conditions leading to membrane electroporation. Specifically, a series of electrical pulses that alternates between positive and negative pulses permeabilizes cells more symmetrically than a longer pulse with the same total on-time. However, the magnitude of this symmetric entry decreases for the shorter alternating pulses. Furthermore, a method for quantitatively measuring the permeability of the cell membrane was proposed and validated. From data near the electroporation threshold, the response of cells varies widely in the manner in which cells become permeabilized. This method is applied to study the transient cell membrane permeability induced by electroporation and is used to demonstrate that the cell membrane remains permeable beyond 30 min following treatment. To analyze these experimental findings in the context of physical mechanisms, computational models of molecular uptake were developed to simulate electroporation. The results of these simulations indicate that the cell's local environment during electroporation facilitates the degree of molecular uptake. We use these models to predict how manipulating both the environment of cells during electroporation affects the induced membrane permeability. These experimental and computational results provide evidence that supports the hypothesis of this dissertation and provide a foundation for future investigation and simulation of membrane electroporation. / PHD / Electroporation is a biophysical process in which intense electric fields permeabilize bilayer membranes. The degree to which a membrane allows molecules to diffuse through it is called its diffusive permeability, and is impacted by the structure, composition, and environment in which the cell resides. This dissertation investigates the hypothesis that the flow of molecules into cells through their membranes may be used to quantitatively study the effects of the electrical stimulus and environmental conditions leading to membrane disruption. Here, I demonstrate that the cellular response to pulsed electric fields is affected by the waveform of the applied electrical stimulus. Specifically, a series of electrical pulses that alternates between positive and negative pulses permeabilizes cells more symmetrically than a longer pulse with the same total energized time. However, the total molecular uptake decreases for the shorter alternating pulses over the longer pulse. A method for quantitatively measuring the permeability of the cell membrane using a fluorescent tracer molecule is also developed and validated. This method is applied to show how cell membrane permeability changes following electroporation. To analyze these findings, computational models of molecular flow through the cell membrane are developed. These simulations indicate that the cell’s surrounding environment during electroporation dramatically impacts the degree of molecular uptake. We use these models to predict how manipulating both the environment of cells during electroporation affects the induced membrane permeability. These experimental and computational results provide a foundation for future investigation and simulation of membrane electroporation.

This dissertation thesis describes a phenomenon called electroporation. It is about its theoretical aspects as well as about modeling of processes in the tissue during electroporation. Further, it describes the technical design of two developed unique experimental generators of DC and AC pulses for electroporation purposes. It also includes a description of experiments which were done using discussed generators.

L'électroporation est un procédé physique qui consiste à appliquer des impulsions de champ électrique pour perméabiliser de manière transitoire ou permanente la membrane plasmique. Ce phénomène est d'un grand intérêt dans le domaine clinique ainsi que dans l'industrie en raison de ses diverses applications, notamment l'électrochimiothérapie qui combine les impulsions électriques à l'administration d'une molécule cytotoxique, dans le cadre du traitement des tumeurs. L'analyse de ce phénomène est traditionnellement réalisée à l'aide des méthodes optique et biochimique (microscopie, cytométrie en flux, test biochimique). Elles sont très efficaces mais nécessitent l'utilisation d'une large gamme de fluorochromes et de marqueurs dont la mise en œuvre peut être laborieuse et coûteuse tout en ayant un caractère invasif aux cellules. Durant ces dernières années, le développement de nouveaux outils biophysiques pour l'étude de l'électroporation a pris place, tels que la diélectrophorèse et la spectroscopie d'impédance (basse fréquence). Outre une facilité de mise en œuvre, ces méthodes représentent un intérêt dans l'étude des modifications membranaires de la cellule. De là vient l'intérêt d'opérer au-delà du GHz, dans la gamme des micro-ondes, pour laquelle la membrane cytoplasmique devient transparente et le contenu intracellulaire est exposé. L'extraction de la permittivité relative suite à l'interaction champ électromagnétique/cellules biologiques reflète alors l'état cellulaire. Cette technique, la spectroscopie diélectrique hyperfréquence, se présente comme une méthode pertinente pour analyser les effets de l'électroporation sur la viabilité cellulaire. De plus, elle ne nécessite aucune utilisation des molécules exogènes (non-invasivité) et les mesures sont directement réalisées dans le milieu de culture des cellules. Deux objectifs ont été définis lors de cette thèse dont les travaux se situent à l'interface entre trois domaines scientifiques : la biologie cellulaire, l'électronique hyperfréquence et les micro-technologies. Le premier objectif concerne la transposition de l'électroporation conventionnelle à l'échelle micrométrique, qui a montré une efficacité aussi performante que la première. La deuxième partie du travail concerne l'étude par spectroscopie diélectrique HyperFréquence de cellules soumises à différents traitements électriques (combinés ou non à une molécule cytotoxique). Ces travaux présentent une puissance statistique et montrent une très bonne corrélation (R2 >0 .94) avec des techniques standards utilisées en biologie, ce qui valide 'biologiquement' la méthode d'analyse HF dans le contexte d'électroporation. Ces travaux montrent en outre que la spectroscopie diélectrique hyperfréquence s'avère être une technique puissante, capable de révéler la viabilité cellulaire suite à un traitement chimique et/ou électrique. Ils ouvrent la voie à l'analyse 'non-invasive' par spectroscopie diélectrique HyperFréquence de cellules électroporées in-situ. / Electroporation is a physical process that consists in applying electric field pulses to transiently or permanently permeabilize the plasma membrane. This phenomenon is of great interest in the clinical field as well as in the industry because of its various applications, in particular electrochemotherapy which combines electrical pulses with the administration of a cytotoxic molecule in the treatment of tumors. The evaluation of this phenomenon is raditionally carried out using optical and biochemical methods (microscopy, flow cytometry, biochemical test). They are very effective but require the use of a wide range of fluorochromes and markers, which can be laborious and costly to implement, while being invasive to the cells. In recent years, the development of new biophysical tools for the study of electroporation has taken place, such as dielectrophoresis and impedance spectroscopy (low frequency). In addition to the ease of implementation, these methods are of interest in the study of membrane modifications of the cell. Hence the advantage of operating beyond the GHz, in the range of microwaves, for which the cytoplasmic membrane becomes transparent and the intracellular content is exposed. The extraction of the relative permittivity as a result of the electromagnetic field / biological cell interaction then reflects the cell state. This technique, microwave dielectric spectroscopy, is a relevant method for analyzing the effects of electroporation on cell viability. Moreover, it does not require any use of the exogenous molecules (non-invasive) and the measurements are directly carried out in the culture medium of the cells. Two objectives were defined during this thesis whose work is located at the interface between three scientific fields: cellular biology, microwave electronics and micro-technologies. The first objective concerns the transposition of conventional electroporation to the micrometric scale, which has shown an efficiency as efficient as the first. The second part of the work concerns the study by HighFrequency dielectric spectroscopy of cells subjected to different electrical treatments (combined or not with a cytotoxic molecule). This work presents a statistical power and shows a very good correlation (R2> 0.94) with standard techniques used in biology, which biologically validates the HF analysis method in the context of electroporation. This work also shows that microwave dielectric spectroscopy proves to be a powerful technique capable of revealing cell viability following chemical and / or electrical treatment. They open the way to 'non-invasive' analysis by hyper-frequency dielectric spectroscopy of electroporated cells in situ.

This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials. / Master of Science / Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.