A novel in vitro model of clinical cryoablation to investigate the transition zone for focal tumor ablation.

Cryoablation (CA) of solid tumors is highly effective at reducing tumor burden and eliminating small, early stage tumors. However, complete ablation is difficult to achieve and cancer recurrence is a significant barrier to treatment of larger tumors compared to resection. In this study, we explored the relationship between temperature, ice growth, and cell death using a novel in vitro model of clinical CA with the Visual-ICE (Boston Scientific) system, a clinically approved and widely utilized device. We found that increasing the duration of freezing from 1 to 2 min increased ice radius from 3.44 ± 0.13 mm to 5.29 ± 0.16 mm, and decreased the minimum temperature achieved from -22.8 ± 1.3 °C to -45.5 ± 7.9 °C. Furthermore, an additional minute of freezing increased the amount of cell death within a 5 mm radius from 42.5 ± 8.9% to 84.8 ± 1.1%. Freezing at 100% intensity leads to faster temperature drops and a higher level of cell death in the TRAMP-C2 mouse prostate cancer cell line, while lower intensities are useful for slow freezing, but result in less cell death. The width of transition zone between live and dead cells decreased by 0.4 ± 0.2 mm, increasing from one to two cycles of freeze/thaw cycles at 100% intensity. HMGB-1 levels significantly increased with 3 cycles of freeze/thaw compared to the standard 2 cycles. Overall, a longer freezing duration, higher freezing intensity, and more freeze thaw cycles led to higher levels of cancer cell death and smaller transition zones. These results have the potential to inform future preclinical research and to improve therapeutic combinations with CA.

High-Frequency Irreversible Electroporation Using 5,000-V Waveforms to Create Reproducible 2- and 4-cm Ablation Zones-A Laboratory Investigation Using Mechanically Perfused Liver.

PURPOSE: To investigate if high-frequency irreversible electroporation (H-FIRE) treatments can be delivered at higher voltages and with greater energy delivery rates than currently implemented in clinical irreversible electroporation protocols. MATERIALS AND METHODS: Treatments using 3,000 V and 5,000 V were administered to mechanically perfused ex vivo porcine liver via a single applicator and grounding pad (A+GP) as well as a 4-applicator array (4AA). Integrated energized times (IET) 0.01-0.08 seconds and energy delivery rates 25-300 µs/s were investigated. Organs were preserved at 4°C for 10-15 hours before sectioning and gross analysis using a metabolic stain to identify the size and shape of ablation zones. RESULTS: A+GP ablations measured between 1.6 cm and 2.2 cm, which did not increase when IET was increased from 0.02 seconds to 0.08 seconds (P > .055; range, 1.9-2.1 cm). Changes in tissue color and texture consistent with thermal damage were observed for treatments with energy delivery rates 50-300 µs/s, but not for treatments delivered at 25 µs/s. Use of the 4AA with a 3-cm applicator spacing resulted in ablations measuring 4.4-4.9 cm with energy delivery times of 7-80 minutes. CONCLUSIONS: H-FIRE treatments can rapidly and reproducibly create 2-cm ablations using an A+GP configuration. Treatments without thermal injury were produced at the expense of extended treatment times. More rapid treatments resulted in ablations with varying degrees of thermal injury within the H-FIRE ablation zone. Production of 4-cm ablations is possible using a 4AA.

Reduction of Muscle Contractions during Irreversible Electroporation Therapy Using High-Frequency Bursts of Alternating Polarity Pulses: A Laboratory Investigation in an Ex Vivo Swine Model.

PURPOSE: To compare the intensity of muscle contractions in irreversible electroporation (IRE) treatments when traditional IRE and high-frequency IRE (H-FIRE) waveforms are used in combination with a single applicator and distal grounding pad (A+GP) configuration. MATERIALS AND METHODS: An ex vivo in situ porcine model was used to compare muscle contractions induced by traditional monopolar IRE waveforms vs high-frequency bipolar IRE waveforms. Pulses with voltages between 200 and 5,000 V were investigated, and muscle contractions were recorded by using accelerometers placed on or near the applicators. RESULTS: H-FIRE waveforms reduced the intensity of muscle contractions in comparison with traditional monopolar IRE pulses. A high-energy burst of 2-µs alternating-polarity pulses energized for 200 µs at 4,500 V produced less intense muscle contractions than traditional IRE pulses, which were 25-100 µs in duration at 3,000 V. CONCLUSIONS: H-FIRE appears to be an effective technique to mitigate the muscle contractions associated with traditional IRE pulses. This may enable the use of voltages greater than 3,000 V necessary for the creation of large ablations in vivo.

Irreversible electroporation for the ablation of pancreatic malignancies: A patient-specific methodology.

BACKGROUND AND OBJECTIVES: Irreversible Electroporation (IRE) is a focal ablation technique highly attractive to surgical oncologists due to its non-thermal nature that allows for eradication of unresectable tumors in a minimally invasive procedure. In this study, our group sought to address the challenge of predicting the ablation volume with IRE for pancreatic procedures. METHODS: In compliance with HIPAA and hospital IRB approval, we established a pre-treatment planning methodology for IRE procedures in pancreas, which optimized treatment protocols for individual cases of locally advanced pancreatic cancer (LAPC). A new method for confirming treatment plans through intraoperative monitoring of tissue resistance was also proved feasible in three patients. RESULTS: Results from computational models showed good correlation with experimental data available in the literature. By implementing the proposed resistance measurement system 210 ± 26.1 (mean ± standard deviation) fewer pulses were delivered per electrode-pair. CONCLUSION: The proposed physics-based pre-treatment plan through finite element analysis and system for actively monitoring resistance changes can be paired to significantly reduce ablation times and risk of thermal effects during IRE procedures for LAPC.

Production of Spherical Ablations Using Nonthermal Irreversible Electroporation: A Laboratory Investigation Using a Single Electrode and Grounding Pad.

PURPOSE: To mathematically model and test ex vivo a modified technique of irreversible electroporation (IRE) to produce large spherical ablations by using a single probe. MATERIALS AND METHODS: Computed simulations were performed by using varying voltages, electrode exposure lengths, and tissue types. A vegetable (potato) tissue model was then used to compare ablations created by conventional and high-frequency IRE protocols by using 2 probe configurations: a single probe with two collinear electrodes (2EP) or a single electrode configured with a grounding pad (P+GP). The new P+GP electrode configuration was evaluated in ex vivo liver tissue. RESULTS: The P+GP configuration produced more spherical ablation volumes than the 2EP configuration in computed simulations and tissue models. In prostate tissue, computed simulations predicted ablation volumes at 3,000 V of 1.6 cm(3) for the P+GP configurations, compared with 0.94 cm(3) for the 2EP configuration; in liver tissue, the predicted ablation volumes were 4.7 times larger than those in the prostate. Vegetable model studies verify that the P+GP configuration produces larger and more spherical ablations than those produced by the 2EP. High-frequency IRE treatment of ex vivo liver with the P+GP configuration created a 2.84 × 2.21-cm ablation zone. CONCLUSIONS: Computer modeling showed that P+GP configuration for IRE procedures yields ablations that are larger than the 2EP configuration, creating substantial ablation zones with a single electrode placement. When tested in tissue models and an ex vivo liver model, the P+GP configuration created ablation zones that appear to be of clinically relevant size and shape.

Joule heating effects on particle immobilization in insulator-based dielectrophoretic devices.

In this work, the temperature effects due to Joule heating obtained by application of a direct current electric potential were investigated for a microchannel with cylindrical insulating posts employed for insulator-based dielectrophoresis. The conductivity of the suspending medium, the local electric field, and the gradient of the squared electric field, which directly affect the magnitude of the dielectrophoretic force exerted on particles, were computationally simulated employing COMSOL Multiphysics. It was observed that a temperature gradient is formed along the microchannel, which redistributes the conductivity of the suspending medium leading to an increase of the dielectrophoretic force toward the inlet of the channel while decreasing toward the outlet. Experimental results are in good agreement with simulations on the particle-trapping zones anticipated. This study demonstrates the importance of considering Joule heating effects when designing insulator-based dielectrophoresis systems.

Integrated Time Nanosecond Pulse Irreversible Electroporation (INSPIRE): Assessment of Dose, Temperature, and Voltage on Experimental and Clinical Treatment Outcomes.

OBJECTIVE: This study sought to investigate a novel strategy using temperature-controlled delivery of nanosecond pulsed electric fields as an alternative to the 50-100 microsecond pulses used for irreversible electroporation. METHODS: INSPIRE treatments were carried out at two temperatures in 3D tumor models using doses between 0.001 s and 0.1 s. The resulting treatment zones were quantified using viability staining and lethal electric field intensities were determined numerically. Computational modeling was then used to determine parameters necessary for INSPIRE treatments to achieve equivalent treatment zones to clinical electroporation treatments and evaluate the potential for these treatments to induce deleterious thermal damage. RESULTS: Lethal thresholds between 1109 and 709 V/cm were found for nominal 0.01 s treatments with pulses between 350 ns and 2000 ns at physiological temperatures. Further increases in dose resulted in significant decreases in lethal thresholds. Given these experimental results, treatment zones comparable to clinical electroporation are possible by increasing the dose and voltage used with nanosecond duration pulses. Temperature-controlled simulations indicate minimal thermal cell death while achieving equivalent treatment volumes to clinical electroporation. CONCLUSION: Nanosecond electrical pulses can achieve comparable outcomes to traditional electroporation provided sufficient electrical doses or voltages are applied. The use of temperature-controlled delivery may minimize thermal damage during treatment. SIGNIFICANCE: Intense muscle stimulation and the need for cardiac gating have limited irreversible electroporation. Nanosecond pulses can alleviate these challenges, but traditionally have produced significantly smaller treatment zones. This study suggests that larger ablation volumes may be possible with the INSPIRE approach and that future in vivo studies are warranted.

Treatment of Spontaneous Tumors With Algorithmically Controlled Electroporation.

OBJECTIVE: To study the safety and efficacy of algorithmically controlled electroporation (ACE) against spontaneous equine melanoma. METHODS: A custom temperature sensing coaxial electrode was paired with a high voltage pulse generation system with integrated temperature feedback controls. Computational modeling and ex vivo studies were conducted to evaluate the system's ability to achieve and maintain target temperatures. Twenty-five equine melanoma tumors were treated with a 2000V protocol consisting of a 2-5-2 waveform, 45ºC temperature set point, and integrated energized times of 0.005 s, 0.01 s, or 0.02 s (2500x, 5000x, and 10000x 2 µs pulses, respectively). Patients returned 20-50 days post treatment to determine the efficacy of the treatment. RESULTS: ACE temperature control algorithms successfully achieved and maintained target temperatures in a diverse population of spontaneous tumors with significant variation in tissue impedance. All treatments were completed successfully without and without adverse events. Complete response rates greater than 93% were achieved in all treatment groups. CONCLUSION: ACE is a safe and effective treatment for spontaneous equine melanoma. The temperature control algorithm enabled rapid delivery of electroporation treatments without prior knowledge of tissue electrical or thermal properties and could adjust to real time changes in tissue properties. SIGNIFICANCE: Real time temperature control in electroporation procedures enables treatments near critical structures where thermal damage is contraindicated. Unlike standard approaches, ACE protocols do not require extensive pretreatment planning or knowledge of tissue properties to determine an optimal energy delivery rate and they can account for changes in tissue state (e.g. perfusion) in real time to simultaneously minimize treatment time and potential for thermal damage.

Investigation of integrated time nanosecond pulse irreversible electroporation against spontaneous equine melanoma.

Introduction: Integrated time nanosecond pulse irreversible electroporation (INSPIRE) is a novel tumor ablation modality that employs high voltage, alternating polarity waveforms to induce cell death in a well-defined volume while sparing the underlying tissue. This study aimed to demonstrate the in vivo efficacy of INSPIRE against spontaneous melanoma in standing, awake horses. Methods: A custom applicator and a pulse generation system were utilized in a pilot study to treat horses presenting with spontaneous melanoma. INSPIRE treatments were administered to 32 tumors across 6 horses and an additional 13 tumors were followed to act as untreated controls. Tumors were tracked over a 43-85 day period following a single INSPIRE treatment. Pulse widths of 500ns and 2000ns with voltages between 1000 V and 2000 V were investigated to determine the effect of these variables on treatment outcomes. Results: Treatments administered at the lowest voltage (1000 V) reduced tumor volumes by 11 to 15%. Higher voltage (2000 V) treatments reduced tumor volumes by 84 to 88% and eliminated 33% and 80% of tumors when 500 ns and 2000 ns pulses were administered, respectively. Discussion: Promising results were achieved without the use of chemotherapeutics, the use of general anesthesia, or the need for surgical resection in regions which are challenging to keep sterile. This novel therapeutic approach has the potential to expand the role of pulsed electric fields in veterinary patients, especially when general anesthesia is contraindicated, and warrants future studies to demonstrate the efficacy of INSPIRE as a solid tumor treatment.

Multilayer contactless dielectrophoresis: theoretical considerations.

Dielectrophoresis (DEP), the movement of dielectric particles in a nonuniform electric field, is of particular interest due to its ability to manipulate particles based on their unique electrical properties. Contactless DEP (cDEP) is an extension of traditional and insulator-based DEP topologies. The devices consist of a sample channel and fluid electrode channels filled with a highly conductive media. A thin insulating membrane between the sample channel and the fluid electrode channels serves to isolate the sample from direct contact with metal electrodes. Here we investigate, for the first time, the properties of multilayer devices in which the sample and electrode channels occupy distinct layers. Simulations are conducted using commercially available finite element software and a less computationally demanding numerical approximation is presented and validated. We show that devices can be created that achieve a similar level of electrical performance to other cDEP devices presented in the literature while increasing fluid throughput. We conclude, based on these models, that the ultimate limiting factors in device performance resides in breakdown voltage of the barrier material and the ability to generate high-voltage, high-frequency signals. Finally, we demonstrate trapping of MDA-MB-231 breast cancer cells in a prototype device at a flow rate of 1.0 mL/h when 250 V(RMS) at 600 kHz is applied.

Polymer Nanoparticles Enhance Irreversible Electroporation In Vitro.

Expanding the volume of an irreversible electroporation treatment typically necessitates an increase in pulse voltage, number, duration, or repetition. This study investigates the addition of polyethylenimine nanoparticles (PEI-NP) to pulsed electric field treatments, determining their combined effect on ablation size and voltages. U118 cells in an in vitro 3D cell culture model were treated with one of three pulse parameters (with and without PEI-NPs) which are representative of irreversible electroporation (IRE), high frequency irreversible electroporation (H-FIRE), or nanosecond pulsed electric fields (nsPEF). The size of the ablations were compared and mapped onto an electric field model to describe the electric field required to induce cell death. Analysis was conducted to determine the role of PEI-NPs in altering media conductivity, the potential for PEI-NP degradation following pulsed electric field treatment, and PEI-NP uptake. Results show there was a statistically significant increase in ablation diameter for IRE and H-FIRE pulses with PEI-NPs. There was no increase in ablation size for nsPEF with PEI-NPs. This all occurs with no change in cell media conductivity, no observable degradation of PEI-NPs, and moderate particle uptake. These results demonstrate the synergy of a combined cationic polymer nanoparticle and pulsed electric field treatment for the ablation of cancer cells. These results set the foundation for polymer nanoparticles engineered specifically for irreversible electroporation.

Contactless dielectrophoretic spectroscopy: examination of the dielectric properties of cells found in blood.

The use of non-invasive methods to detect and enrich circulating tumor cells (CTCs) independent of their genotype is critical for early diagnostic and treatment purposes. The key to using CTCs as predictive clinical biomarkers is their separation and enrichment. This work presents the use of a contactless dielectrophoresis (cDEP) device to investigate the frequency response of cells and calculate their area-specific membrane capacitance. This is the first demonstration of a cDEP device which is capable of operating between 10 and 100 kHz. Positive and negative dielectrophoretic responses were observed in red blood cells, macrophages, breast cancer, and leukemia cells. The area-specific membrane capacitances of MDA-MB231, THP-1 and PC1 cells were determined to be 0.01518 ± 0.0013, 0.01719 ± 0.0020, 0.01275 ± 0.0018 (F/m(2)), respectively. By first establishing the dielectrophoretic responses of cancerous cells within this cDEP device, conditions to detect and enrich tumor cells from mixtures with non-transformed cells can be determined providing further information to develop methods to isolate these rare cells.

Selective concentration of human cancer cells using contactless dielectrophoresis.

This work is the first to demonstrate the ability of contactless dielectrophoresis (cDEP) to isolate target cell species from a heterogeneous sample of live cells. Since all cell types have a unique molecular composition, it is expected that their dielectrophoretic (DEP) properties are also unique. cDEP is a technique developed to improve upon traditional and insulator-based DEP devices by replacing embedded metal electrodes with fluid electrode channels positioned alongside desired trapping locations. Through the placement of the fluid electrode channels and the removal of contact between the electrodes and the sample fluid, cDEP mitigates issues associated with sample/electrode contact. MCF10A, MCF7, and MDA-MB-231 human breast cells were used to represent early, intermediate, and late-staged breast cancer, respectively. Trapping frequency responses of each cell type were distinct, with the largest difference between the cells found at 20 and 30 V. MDA-MB-231 cells were successfully isolated from a population containing MCF10A and MCF7 cells at 30 V and 164 kHz. The ability to selectively concentrate cells is the key to development of biological applications using DEP. The isolation of these cells could provide a workbench for clinicians to detect transformed cells at their earliest stage, screen drug therapies prior to patient treatment, increasing the probability of success, and eliminate unsuccessful treatment options.

High-frequency irreversible electroporation (H-FIRE) for non-thermal ablation without muscle contraction.

BACKGROUND: Therapeutic irreversible electroporation (IRE) is an emerging technology for the non-thermal ablation of tumors. The technique involves delivering a series of unipolar electric pulses to permanently destabilize the plasma membrane of cancer cells through an increase in transmembrane potential, which leads to the development of a tissue lesion. Clinically, IRE requires the administration of paralytic agents to prevent muscle contractions during treatment that are associated with the delivery of electric pulses. This study shows that by applying high-frequency, bipolar bursts, muscle contractions can be eliminated during IRE without compromising the non-thermal mechanism of cell death. METHODS: A combination of analytical, numerical, and experimental techniques were performed to investigate high-frequency irreversible electroporation (H-FIRE). A theoretical model for determining transmembrane potential in response to arbitrary electric fields was used to identify optimal burst frequencies and amplitudes for in vivo treatments. A finite element model for predicting thermal damage based on the electric field distribution was used to design non-thermal protocols for in vivo experiments. H-FIRE was applied to the brain of rats, and muscle contractions were quantified via accelerometers placed at the cervicothoracic junction. MRI and histological evaluation was performed post-operatively to assess ablation. RESULTS: No visual or tactile evidence of muscle contraction was seen during H-FIRE at 250 kHz or 500 kHz, while all IRE protocols resulted in detectable muscle contractions at the cervicothoracic junction. H-FIRE produced ablative lesions in brain tissue that were characteristic in cellular morphology of non-thermal IRE treatments. Specifically, there was complete uniformity of tissue death within targeted areas, and a sharp transition zone was present between lesioned and normal brain. CONCLUSIONS: H-FIRE is a feasible technique for non-thermal tissue ablation that eliminates muscle contractions seen in IRE treatments performed with unipolar electric pulses. Therefore, it has the potential to be performed clinically without the administration of paralytic agents.

Electro-Thermal Therapy Algorithms and Active Internal Electrode Cooling Reduce Thermal Injury in High Frequency Pulsed Electric Field Cancer Therapies.

Thermal tissue injury is an unintended consequence in current irreversible electroporation treatments due to the induction of Joule heating during the delivery of high voltage pulsed electric fields. In this study active temperature control measures including internal electrode cooling and dynamic energy delivery were investigated as a process for mitigating thermal injury during treatment. Ex vivo liver was used to examine the extent of thermal injury induced by 5000 V treatments with delivery rates up to five times faster than current clinical practice. Active internal cooling of the electrode resulted in a 36% decrease in peak temperature vs. non-cooled control treatments. A temperature based feedback algorithm (electro-thermal therapy) was demonstrated as capable of maintaining steady state tissue temperatures between 30 and 80 °C with and without internal electrode cooling. Thermal injury volumes of 2.6 cm3 were observed for protocols with 60 °C temperature set points and electrode cooling. This volume reduced to 1.5 and 0.1 cm3 for equivalent treatments with 50 °C and 40 °C set points. Finally, it was demonstrated that the addition of internal electrode cooling and active temperature control algorithms reduced ETT treatment times by 84% (from 343 to 54 s) vs. non-cooled temperature control strategies with equivalent thermal injury volumes.

Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP).

Contactless dielectrophoresis (cDEP) is a recently developed method of cell manipulation in which the electrodes are physically isolated from the sample. Here we present two microfluidic devices capable of selectively isolating live human leukemia cells from dead cells utilizing their electrical signatures. The effect of different voltages and frequencies on the gradient of the electric field and device performance was investigated numerically and validated experimentally. With these prototype devices we were able to achieve greater than 95% removal efficiency at 0.2-0.5 mm s(-1) with 100% selectivity between live and dead cells. In conjunction with enrichment, cDEP could be integrated with other technologies to yield fully automated lab-on-a-chip systems capable of sensing, sorting, and identifying rare cells.

Towards the creation of decellularized organ constructs using irreversible electroporation and active mechanical perfusion.

BACKGROUND: Despite advances in transplant surgery and general medicine, the number of patients awaiting transplant organs continues to grow, while the supply of organs does not. This work outlines a method of organ decellularization using non-thermal irreversible electroporation (N-TIRE) which, in combination with reseeding, may help supplement the supply of organs for transplant. METHODS: In our study, brief but intense electric pulses were applied to porcine livers while under active low temperature cardio-emulation perfusion. Histological analysis and lesion measurements were used to determine the effects of the pulses in decellularizing the livers as a first step towards the development of extracellular scaffolds that may be used with stem cell reseeding. A dynamic conductivity numerical model was developed to simulate the treatment parameters used and determine an irreversible electroporation threshold. RESULTS: Ninety-nine individual 1000 V/cm 100-µs square pulses with repetition rates between 0.25 and 4 Hz were found to produce a lesion within 24 hours post-treatment. The livers maintained intact bile ducts and vascular structures while demonstrating hepatocytic cord disruption and cell delamination from cord basal laminae after 24 hours of perfusion. A numerical model found an electric field threshold of 423 V/cm under specific experimental conditions, which may be used in the future to plan treatments for the decellularization of entire organs. Analysis of the pulse repetition rate shows that the largest treated area and the lowest interstitial density score was achieved for a pulse frequency of 1 Hz. After 24 hours of perfusion, a maximum density score reduction of 58.5 percent had been achieved. CONCLUSIONS: This method is the first effort towards creating decellularized tissue scaffolds that could be used for organ transplantation using N-TIRE. In addition, it provides a versatile platform to study the effects of pulse parameters such as pulse length, repetition rate, and field strength on whole organ structures.

Thermochromic Tissue Phantoms for Evaluating Temperature Distribution in Simulated Clinical Applications of Pulsed Electric Field Therapies.

Background: Irreversible electroporation (IRE) induces cell death through nonthermal mechanisms, however, in extreme cases, the treatments can induce deleterious thermal transients. This study utilizes a thermochromic tissue phantom to enable visualization of regions exposed to temperatures above 60°C. Materials and Methods: Poly(vinyl alcohol) hydrogels supplemented with thermochromic ink were characterized and processed to match the electrical properties of liver tissue. Three thousand volt high-frequency IRE protocols were administered with delivery rates of 100 and 200 µs/s. The effect of supplemental internal applicator cooling was then characterized. Results: Baseline treatments resulted thermal areas of 0.73 cm2, which decreased to 0.05 cm2 with electrode cooling. Increased delivery rates (200 µs/s) resulted in thermal areas of 1.5 and 0.6 cm2 without and with cooling, respectively. Conclusions: Thermochromic tissue phantoms enable rapid characterization of thermal effects associated with pulsed electric field treatments. Active cooling of applicators can significantly reduce the quantity of tissue exposed to deleterious temperatures.

Irreversible electroporation is a thermally mediated ablation modality for pulses on the order of one microsecond.

Irreversible electroporation (IRE) is generally considered to be a non-thermal ablation modality. This study was designed to examine the relative effect of temperature on IRE ablation sizes for equivalent dose treatments with constitutive pulses between 1 and 100 µs. 3D in-vitro brain tumor models maintained at 10 °C, 20 °C, 30 °C, or 37 °C were exposed to 500 V treatments using a temperature control algorithm to limit temperature increases to 5 °C. Treatments consisted of integrated energized times (doses) of 0.01 or 0.1 s. Pulse width, electrical dose, and initial temperature were all found to significantly affect the size of ablations and the resulting lethal electric field strength. The smallest ablations were created at 10 °C and ELethal were calculated to be 1729, 1359, 929, 777, 483 V/cm for 0.01 s treatments with 1, 2, 4, 8, and 100 µs pulses, respectively. At 37 °C these values decreased to 773, 614, 507, 462, and 394 V/cm, respectively. Increasing the dose from 0.01 to 0.1 s at 37 °C resulted in statistically significant decreases (p < 0.001) in ELethal for all treatments except for the 100 µs group. This study found that IRE is a thermally mediated, dose-dependent ablation modality for pulses on the order of one microsecond. Tissue temperatures are not accounted for when determining ablative boundaries in treatment planning algorithms. This work demonstrates that data generated at room temperature may not be predictive of ablation volumes in-vivo and that local temperatures should be accounted for in treatment planning.

Algorithmically Controlled Electroporation: A Technique for Closed Loop Temperature Regulated Pulsed Electric Field Cancer Ablation.

OBJECTIVE: To evaluate the effect of a closed-loop temperature based feedback algorithm on ablative outcomes for pulsed electric field treatments. METHODS: A 3D tumor model of glioblastoma was used to assess the impact of 2 µs duration bipolar waveforms on viability following exposure to open and closed-loop protocols. Closed-loop treatments evaluated transient temperature increases of 5, 10, 15, or 22 °C above baseline. RESULTS: The temperature controlled ablation diameters were conditionally different than the open-loop treatments and closed-loop treatments generally produced smaller ablations. Closed-loop control enabled the investigation of treatments with steady state 42 °C hyperthermic conditions which were not feasible without active feedback. Baseline closed-loop treatments at 20 °C resulted in ablations measuring 9.9 ± 0.3 mm in diameter while 37 °C treatments were 20% larger (p < 0.0001) measuring 11.8 ± 0.3 mm indicating that this protocol induces a thermally mediated biological response. CONCLUSION: A closed-loop control algorithm which modulated the delay between successive pulse waveforms to achieve stable target temperatures was demonstrated. Algorithmic control enabled the evaluation of specific treatment parameters at physiological temperatures not possible with open-loop systems due to excessive Joule heating. SIGNIFICANCE: Irreversible electroporation is generally considered to be a non-thermal ablation modality and temperature monitoring is not part of the standard clinical practice. The results of this study indicate ablative outcomes due to exposure to pulses on the order of one microsecond may be thermally mediated and dependent on local tissue temperatures. The results of this study set the foundation for experiments in vivo utilizing temperature control algorithms.