An Investigation for Large Volume, Focal Blood-Brain Barrier Disruption with High-Frequency Pulsed Electric Fields

The treatment of CNS disorders suffers from the inability to deliver large therapeutic agents to the brain parenchyma due to protection from the blood-brain barrier (BBB). Herein, we investigated high-frequency pulsed electric field (HF-PEF) therapy of various pulse widths and interphase delays for BBB disruption while selectively minimizing cell ablation. Eighteen male Fisher rats underwent craniectomy procedures and two blunt-tipped electrodes were advanced into the brain for pulsing. BBB disruption was verified with contrast T1W MRI and pathologically with Evans blue dye. High-frequency irreversible electroporation cell death of healthy rodent astrocytes was investigated in vitro using a collagen hydrogel tissue mimic. Numerical analysis was conducted to determine the electric fields in which BBB disruption and cell ablation occur. Differences between the BBB disruption and ablation thresholds for each waveform are as follows: 2-2-2 μs (1028 V/cm), 5-2-5 μs (721 V/cm), 10-1-10 μs (547 V/cm), 2-5-2 μs (1043 V/cm), and 5-5-5 μs (751 V/cm). These data suggest that HF-PEFs can be fine-tuned to modulate the extent of cell death while maximizing peri-ablative BBB disruption. Furthermore, numerical modeling elucidated the diffuse field gradients of a single-needle grounding pad configuration to favor large-volume BBB disruption, while the monopolar probe configuration is more amenable to ablation and reversible electroporation effects.

A Microdosimetry Analysis of Reversible Electroporation In Scattered, Overlapping, And Cancerous Cervical Cells

In this paper,a numerical method for studying reversible electroporation on normal and cancerous cervical cells is introduced. This microdosimetry analysis has been done by a unique approach for extracting contours of free and overlapping cervical cells in the cluster from the External Depth field images19. The algorithm used for extracting the contours is a joint optimization of multiple level set function along with the Gaussian mixture model and Maximally Stable Extremal Regions. This contour is then imported a multiphysics domain solver, where variable frequency pulsed electric field is applied. The Trans-Membrane Voltage (TMV) developed across the cell membrane is then calculated using the Maxwell equation coupled with a statistical approach employing the asymptotic Smoluchowski equation, which calculates the generated temporal pore density. The numerical model was validated by successful replication of existing experimental approach that employed low-frequency uni-polar pulses on the overlapping cells to obtain reversible electroporation. Using several overlapping clumps of cervical cells, simulations are performed to match the experimental data. For high-frequency calculation, a combination of normal and cancerous cells is introduced to the computational domain. The cells are assumed to be dispersive and the Debye dispersion equation is a second-order partial derivative equation used for further calculations. The difference in time duration for reaching the threshold value of electroporation is seen between the normal and cancerous cervical cells due to their size and conductivity change. The drug and dye uptake modulation during the high-frequency electric field electroporation is advocated by a mathematical model.

Evaluation of electroporated area using 2,3,5-triphenyltetrazolium chloride in a potato model

Irreversible electroporation (IRE) is a tissue ablation method, uses short high electric pulses and results in cell death in target tissue by irreversibly permeabilizing the cell membrane. Potato is commonly used as a tissue model for electroporation experiments. The blackened area that forms 12 h after electric pulsing is regarded as an IRE-ablated area caused by melanin accumulation. Here, the 2,3,5-triphenyltetrazolium chloride (TTC) was used as a dye to assess the IRE-ablated area 3 h after potato model ablation. Comparison between the blackened area and TTC-unstained white area in various voltage conditions showed that TTC staining well delineated the IRE-ablated area. Moreover, whether the ablated area was consistent over time and at different staining times was investigated. In addition, the presumed reversible electroporation (RE) area was formed surrounding the IRE-ablated area. Overall, TTC staining can provide a more rapid and accurate electroporated area evaluation.

Study and modulation of cardiac electroporation with a novel model utilizing human induced pluripotent stem cells

Background Cardiac electroporation is a promising novel non-thermal ablation method, gaining significant interest with recent first-in-man data suggesting effective cardiac lesion generation with no collateral damage. Nevertheless, significant knowledge gaps exist regarding its electrophysiological consequences in cardiomyocytes, including; cell specificity, protocol optimization, irreversibility threshold, recovery time-constants, and the mechanistic nature of its cytolytic and anti-arrhythmic properties. Purpose Establishing an innovative in-vitro model for the study of cardiac electroporation-ablation, utilizing human induced pluripotent stem cells (hiPSCs). Methods and results Healthy-control hiPSC-derived cardiomyocytes were enzymatically dissociated and seeded as circular cell sheets (hiPSC-CCSs). Electroporation-ablation experiments were performed using a custom designed high-frequency electroporation (HF-EP) generator. Two needle-shaped electrodes were used for HF-EP delivery (Figure 1). Subsequently, detailed voltage- and Ca2+-mapping studies of the hiPSC-CCSs were conducted (Figure 2). HF-EP application resulted in the generation of electrically isolated lesions within the hiPSC-CCSs (Figure 3). Further characterization of the temporal changes and electrophysiological properties following electroporation revealed that; (1) lesions persisted over prolonged periods of time (days), indicating irreversible electroporation, (2) a temporal decrease in lesion dimensions was noted, consistent with a significant reversible electroporation component (Figures 3–5), (3) most tissue recovery had occurred within the first 15 minutes following electroporation, with little recovery beyond that time-frame, (4) increasing pulse-number augmented lesion area as well as the proportion of irreversible damage, and (5) electroporation sensitization was achieved by increasing extracellular Ca2+, indicating its crucial role in electroporation cytolysis, potentially via direct cellular toxicity and apoptosis facilitation (Figures 5–6). Finally, evaluating for HF-EP anti-arrhythmic properties, we targeted multiple rotors or focal triggered-activity generated in the hiPSC-CCSs. HF-EP application generated sustained line-blocks, isolating arrhythmogenic substrates within the hiPSC-CCSs while blocking the propagation of arrhythmic wavefronts (Figure 7). Conclusion Our results demonstrate the ability to study cardiac electroporation utilizing hiPSC-derived cardiomyocytes, provide novel insights into its temporal and electrophysiological characteristics, facilitate electroporation protocol optimization, screen for potential electroporation sensitizers, and to study its mechanistic nature and anti-arrhythmic properties. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Division of Cardiology, and Tamman Cardiovascular Research Institute, Leviev Heart Center, Sheba Medical Center - Tel Hashomer, Ramat-Gan, Israel Figures 1–4 Figures 5–7

Micromotor-based localized electroporation and gene transfection of mammalian cells

Herein, we studied localized electroporation and gene transfection of mammalian cells using a metallodielectric hybrid micromotor that is magnetically and electrically powered. Much like nanochannel-based, local electroporation of single cells, the presented micromotor was expected to increase reversible electroporation yield, relative to standard electroporation, as only a small portion of the cell’s membrane (in contact with the micromotor) is affected. In contrast to methods in which the entire membrane of all cells within the sample are electroporated, the presented micromotor can perform, via magnetic steering, localized, spatially precise electroporation of the target cells that it traps and transports. In order to minimize nonselective electrical lysis of all cells within the chamber, resulting from extended exposure to an electrical field, magnetic propulsion was used to approach the immediate vicinity of the targeted cell, after which short-duration, electric-driven propulsion was activated to enable contact with the cell, followed by electroporation. In addition to local injection of fluorescent dye molecules, we demonstrated that the micromotor can enhance the introduction of plasmids into the suspension cells because of the dielectrophoretic accumulation of the plasmids in between the Janus particle and the attached cell prior to the electroporation step. Here, we chose a different strategy involving the simultaneous operation of many micromotors that are self-propelling, without external steering, and pair with cells in an autonomic manner. The locally electroporated suspension cells that are considered to be very difficult to transfect were shown to express the transfected gene, which is of significant importance for molecular biology research.

Electrochemotherapy In The Treatment of Kaposi’s Sarcoma: A Single Centre Cohort Study And A Comparison With Literature Data

: Kaposi's sarcoma (KS) is a tumor of endothelial derivation, which primarily affects the skin and is mainly related to the type 8 human herpesvirus (HHV8). Its onset is favored by immunosuppression, although the most common form is the classic or sporadic KS mainly developing in elderly men of Mediterranean and Eastern European origin. Different therapeutic options are available, depending on the clinical variant, progression pattern, and comorbidities. The treatment of localized forms includes surgical excision, laser treatment, cryosurgery, radiotherapy, imiquimod 5%, and intra-lesion injection of cytotoxic drugs; on the other hand, the treatment of widespread disease encompasses radiotherapy and chemotherapy. In this scenario, electrochemotherapy (ECT), has shown to be an effective alternative to traditional treatment for disseminated KS skin lesions. The rationale of ECT relies on the local application of short, high-voltage electric pulses, able to open transient pores in the cell membrane (reversible electroporation, that increases the delivery of some poorly permeant cytotoxic agents into the cytosol. Herein we performed a retrospective analysis on 9 KS patients treated with ECT at our center between June 2016 and January 2020. The rate of Complete Response (CR) was 77.8% after the first cycle of treatment and 88.9% after the second course, with an overall response (OR) of 100%. Sustained local control of treated lesions was present in 77.8% of patients 6 months after the treatment and all of them reported only mild local toxicity, together with an excellent functional and cosmetic outcome, in agreement with data obtained from the comparison with the recent literature.

Electrochemotherapy and Other Clinical Applications of Electroporation for the Targeted Therapy of Metastatic Melanoma

Electrochemotherapy (ECT) is an effective bioelectrochemical procedure that uses controlled electrical pulses to facilitate the increase of intracellular concentration of certain substances (electropermeabilization/ reversible electroporation). ECT using antitumor drugs such as bleomycin and cisplatin is a minimally invasive targeted therapy that can be used as an alternative for oncologic patients not eligible for surgery or other standard therapies. Even though ECT is mainly applied as palliative care for metastases, it may also be used for primary tumors that are unresectable due to size and location. Skin neoplasms are the main clinical indication of ECT, the procedure reporting good curative results and high efficiency across all tumor types, including melanoma. In daily practice, there are many cases in which the patient’s quality of life can be significantly improved by a safe procedure such as ECT. Its popularity must be increased because it has a safe profile and minor local adverse reactions. The method can be used by dermatologists, oncologists, and surgeons. The aim of this paper is to review recent literature concerning electrochemotherapy and other clinical applications of electroporation for the targeted therapy of metastatic melanoma.

Ablation Modalities for Therapeutic Intervention in Arrhythmia-Related Cardiovascular Disease: Focus on Electroporation

Targeted cellular ablation is being increasingly used in the treatment of arrhythmias and structural heart disease. Catheter-based ablation for atrial fibrillation (AF) is considered a safe and effective approach for patients who are medication refractory. Electroporation (EPo) employs electrical energy to disrupt cell membranes which has a minimally thermal effect. The nanopores that arise from EPo can be temporary or permanent. Reversible electroporation is transitory in nature and cell viability is maintained, whereas irreversible electroporation causes permanent pore formation, leading to loss of cellular homeostasis and cell death. Several studies report that EPo displays a degree of specificity in terms of the lethal threshold required to induce cell death in different tissues. However, significantly more research is required to scope the profile of EPo thresholds for specific cell types within complex tissues. Irreversible electroporation (IRE) as an ablative approach appears to overcome the significant negative effects associated with thermal based techniques, particularly collateral damage to surrounding structures. With further fine-tuning of parameters and longer and larger clinical trials, EPo may lead the way of adapting a safer and efficient ablation modality for the treatment of persistent AF.