Robert H. Williamson

Objective: The objective of this study was to investigate the effect of electrical dose on in vivo INSPIRE treatments which administer high voltage ultrashort alternating polarity electrical pulses with active temperature control. Methods: INSPIRE was administered to healthy swine liver in vivo via a percutaneous single applicator and grounding pad approach. Using 45 °C temperature control, 6000 V waveforms consisting of 750 ns, 1000 ns, or 2000 ns bipolar pulses were administered to examine the effect of pulses approximately shorter than, equal to, and longer than the cell membrane charging time. Treatment volumes were assessed one week post treatment via computed tomography and cardiac safety was assessed via serum troponin analysis. Results: Pulse duration did not significantly affect treatment volumes, however, dose was found to be a critical factor affecting treatment outcomes. For 0.0025 s doses, treatment volumes of 1.3±0.6 cm³ (2.4 × 0.9 cm) were created in 0.3 minutes. This increased to 12.8±4.8 cm³ (9.7 minutes, 3.9 × 2.5 cm) for 0.04 s doses. No significant changes in troponin levels were found. Conclusion: This study demonstrated the in vivo safety of high voltage INSPIRE treatments without cardiac synchronization. There is a strong dose dependent effect on treatment volumes. Optimal treatment efficiency was found for treatment doses between 0.01 and 0.02 s with treatment times between 2-4 minutes. Significance: Single applicator INSPIRE treatments significantly simplify treatment planning and clinical implementation versus traditional two to six applicator approaches. This study demonstrates that INSPIRE protocols can rapidly produce large spherical treatment zones while reducing treatment times by an order of magnitude compared to existing electroporation approaches.

Objective: To study the safety and reproducibility of high-voltage integrated nanosecond pulse irreversible electroporation (INSPIRE) administered through a single applicator and grounding pad approach in a healthy liver model. Methods: A percutaneous approach to the liver was made under ultrasound guidance in female pigs weighing between 25 and 35 kg. INSPIRE treatments at 3000 V, 4500 V, and 6000 V using 1000 ns or 2000 ns waveforms, with a 0.02 s dose and a 45°C temperature set point were delivered using an actively cooled single applicator and distal grounding pad. Ablation size, muscle stimulation, and cardiac safety were evaluated. Results: All INSPIRE treatments were completed successfully without cardiac synchronization or break-through muscle stimulation. Ablations were visible on ultrasound shortly after treatments were complete. Treatments were completed within approximately 2–8 minutes. The largest ablations, achieved with the 2000 ns waveform at 6000 V, measured 4.4 ± 0.7 cm by 2.9 ± 0.1 cm. Conclusion: INSPIRE can be safely used to achieve significantly larger ablations significantly faster than current irreversible electroporation (IRE) technologies using a simplified single-applicator and grounding pad approach. Significance: INSPIRE overcomes technical and procedural challenges facing IRE including ablation size limitations, muscle stimulation, the need for cardiac synchronization, long procedure times, and the lack of visualization during procedures.

Objectives: There is a critical need for effective focal therapies for patients with inoperable or anatomically complex tumors where conventional ablation techniques pose high risk or are ineffective. Integrated Nanosecond Pulsed Irreversible Electroporation (INSPIRE) is a novel non-thermal ablation modality which uses real time temperature feedback during pulse delivery to safely treat tumors near critical structures. This study evaluated the impact of exposed electrode length on ablation zone size, reproducibility, and cardiac safety in a large animal model. Methods: INSPIRE treatments were performed in an in vivo healthy porcine liver model. All treatments administered 6000 V 1000 ns pulses with a 45 °C temperature set point. Treatments were administered percutaneously via an electrode and grounding pad approach using an internally cooled electrode applicator. The exposed electrode region at the distal end of the applicator was set to either 0.5, 1.0, 1.5, or 2.0 cm. Ablation zones were assessed via ultrasound, contrast-enhanced CT, and gross pathology one week post-treatment. Cardiac safety was evaluated by measuring pre- and post-treatment serum Troponin levels. Results: All treatments were completed without adverse events. Troponin levels remained stable (pre: 0.249 ng/mL; post: 0.224 ng/mL), indicating no measurable cardiac injury. The 1.5 cm exposure length produced the largest and most consistent ablation volumes, with a mean volume of 12.8 ± 2.6 cm³ and average dimensions of 3.7 × 2.7 cm in under 6 min. Increasing exposure length beyond 1.5 cm introduced greater variability and reduced treatment volumes. Conclusions: INSPIRE enables safe, large-volume, single-applicator ablation without a need for electrical pulse synchronization with R wave in cardiac rhythm. The 1.5 cm exposure length offers optimal balance between energy delivery and treatment consistency. These findings support further clinical investigation of INSPIRE for non-thermal ablation of inoperable tumors.

Electroporation is a promising technology utilizing electrical pulses for macromolecule delivery and soft-tissue ablation, with applications that include next-generation prophylactics and the treatment of genetic diseases such as cancer. This study demonstrates a high-throughput capable 3D tissue culture model for quantification of the reversible and irreversible electroporation thresholds for a given electroporation protocol. By using a non-uniform electric field and analyzing the spatial distribution of transfected cells, both reversible and irreversible thresholds can be identified within a single sample, increasing the efficiency at which electroporation protocols can be characterized, especially for in vivo translation. To show this capability, 3D tissue mimics containing HEK293 cells were transfected using a ring and pin electrode to deliver a GFP-encoding plasmid. Electroporation thresholds were then derived based on fluorescent microscopy images of the transfected samples. This model demonstrates potential for use as a means for high-throughput evaluation of electroporation protocols, a key advantage over current methods to evaluate these thresholds, which tend to be time-intensive and are less representative of in vivo conditions.

Electroporation is a promising technology utilizing electrical pulses for macromolecule delivery and soft-tissue ablation, with applications that include next-generation prophylactics and the treatment of genetic diseases such as cancer. This study demonstrates a high-throughput capable 3D tissue culture model for quantification of the reversible and irreversible electroporation thresholds for a given electroporation protocol. By using a non-uniform electric field and analyzing the spatial distribution of transfected cells, both reversible and irreversible thresholds can be identified within a single sample, increasing the efficiency at which electroporation protocols can be characterized, especially for in vivo translation. To show this capability, 3D tissue mimics containing HEK293 cells were transfected using a ring and pin electrode to deliver a GFP-encoding plasmid. Electroporation thresholds were then derived based on fluorescent microscopy images of the transfected samples. This model demonstrates potential for use as a means for high-throughput evaluation of electroporation protocols, a key advantage over current methods to evaluate these thresholds, which tend to be time-intensive and are less representative of in vivo conditions.

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 2000 V protocol consisting of a 2-5-2 waveform, 45 °C 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.

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.