Irreversible Electroporation Research Articles

Long-term follow-upexperience with adjuvant therapy after irreversible electroporation of locally advanced pancreatic cancer.

Irreversible electroporation (IRE) expands the surgical options for patients with unresectable pancreatic cancer. This study evaluated for differences in survival stratified by type of IRE and receipt of adjuvant chemotherapy. Patients with locally advanced pancreatic cancer treated by IRE (2012-2020) were retrospectively included. Overall survival (OS) and recurrence-free survival (RFS) were compared by type of IRE (in situ for local tumor control or IRE of potentially positive margins with resection) and by receipt of adjuvant chemotherapy. Thirty-nine patients had IRE in situ, 61 had IRE for margin extension, and 19 received adjuvant chemotherapy. Most (97.00%) underwent induction chemotherapy. OS was 28.71 months (interquartile range [IQR] 19.17, 51.19) from diagnosis, with no difference by IRE type (hazard ratio [HR] 1.05 for margin extension [p = 0.85]) or adjuvant chemotherapy (HR 1.14 [p = 0.639]). RFS was 8.51 months (IQR 4.95, 20.17) with no difference by IRE type (HR 0.90 for margin extension [p = 0.694]) or adjuvant chemotherapy (HR 0.90 [p = 0.711]). These findings suggest that adjuvant therapy may have limited benefit for patients treated with induction chemotherapy followed by local control with IRE for unresectable pancreatic cancer. Further study of the duration and timing of systemic therapy is warranted to maximize benefit and limit toxicity.

Early Single-Center Experience With Irreversible Electroporation for Stage 2, 3, and 4 Pancreatic Adenocarcinomas.

Irreversible electroporation (IRE) is an ablation technology that uses electrical energy delivered between electrodes. If the electrodes are placed atraumatically, there is little to no risk of collateral injury, making IRE appealing for the treatment of pancreatic tumors. We report on 20 patients with pancreatic adenocarcinoma (PAC) who underwent 21 IRE in our center. There were 6 IRE for stage 2 PAC, 11 for stage 3 PAC, 1 for stage 4 PAC, and 2 patients treated with IRE for recurrence after pancreaticoduodenectomy. One patient had local progression 18 months after IRE and received a second IRE treatment. Using propensity score matching (age, sex, stage, tumor size, and chemotherapy), cases were matched 2 to 1 with patients from the Surveillance, Epidemiology, and End Results database. A total of 7 cases experienced 8 complications; 4 complications were mild, and 4 were severe. Significant survival benefit was seen for patients with stage 3 PAC (27.5 vs 14.6 months for the matched group, P = 0.003); for stage 2, median survival was 15 months, and the single stage 4 patient survived 9 months after IRE treatment. Pancreatic cancers were safely and effectively treated with image-guided IRE in our medium-sized center.

Irreversible Electroporation Balloon Therapy for Palliative Treatment of Obstructive Urethral Transitional Cell Carcinoma in Dogs.

Progression of transitional cell carcinoma (TCC) in dogs often leads to urinary obstruction. This observational pilot study aimed to evaluate the safety and efficacy of irreversible electroporation (IRE) balloon therapy for the palliative treatment of TCC with partial urethral obstruction. Three client-owned dogs diagnosed with TCC causing partial urethral obstruction were enrolled. After ultrasonographic and cystoscopic examination, IRE pulse protocols were delivered through a balloon catheter device inflated within the urethral lumen. After the procedure, the patients were kept overnight for monitoring and a recheck was planned 28 days later. No complication was observed during the procedure and postprocedural monitoring. After 28 days, one dog had a complete normalization of the urine stream, one dog had stable stranguria, and one dog was presented with a urethral obstruction secondary to progression of the TCC. On recheck ultrasound, one dog had a 38% diminution of the urethral mass diameter whereas the other two dogs had a mass stable in size. IRE balloon therapy seems to be a feasible and apparently safe minimally invasive novel therapy for the palliative treatment of TCC causing urethral obstruction. Further studies are needed to better characterize the safety, efficacy, and outcome of this therapy.

An overview of the irreversible electroporation for the treatment of liver metastases: When to use it.

Tumour ablation is an established therapy for local treatment of liver metastases and hepatocellular carcinoma. Most commonly two different kind of thermic ablation, radiofrequency ablation and microwave ablation, are used in clinical practice. The aim of both is to induce thermic damage to the malignant cells in order to obtain coagulative necrosis of the neoplastic lesions. Our main concerns about these procedures are the collateral thermic damage to adjacent structures and heat-sink effect. Irreversible electroporation (IRE) is a recently developed, non-thermal ablation procedure which works applying short pulses of direct current that generate an electric field in the lesion area. The electric field increase the transmembrane potential, changing its permeability to ions.Irreversible electroporation does not generate heat, giving the chance to avoid the heat-sink effect and opening the path to a better treatment of all the lesions located in close proximity to big vessels and bile ducts. Electric fields produced by the IRE may affect endothelial cells and cholangiocytes but they spare the collagen matrix, preserving re-epithelization process as well as the function of the damaged structures. Purpose of the authors is to identify the different scenarios where CT-guided percutaneous IRE of the liver should be preferred to other ablative techniques and why.

A Solid-State Pulse Adder for High-Voltage Short Pulses and Low-Voltage Long Pulses

The synergistic effect of high-voltage short pulses and low-voltage long pulse on cell electroporation can expand the tissue ablation area and enhance the irreversible electroporation effect. To meet the requirements of electroporation for pulse waveform, this article proposes a pulse generator which can output high-voltage short pulses and low-voltage long pulses cooperatively. The generator only uses one resonant power supply and uses magnetic rings with separate windings to control different charging voltages. The structure is relatively simple, and the output waveform can meet the experimental requirements. Finally, a prototype is built, in which the main circuit includes 14 switch tubes and 14 spring mud capacitors. The whole system can generate high-voltage short pulses with amplitude of 0–3 kV, a pulsewidth of 1– <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$4 ~\mu \text{s}$ </tex-math></inline-formula> , and low-voltage long pulses with amplitude of 0–1 kV, a pulsewidth of 30– <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$150 ~\mu \text{s}$ </tex-math></inline-formula> . The delay between high voltage pulses and low voltage pulses is adjustable. At the same time through the control of driving timing, a seven-level step wave with amplitude of 2 kV can be generated.

Irreversible electroporation for stage III locally advanced pancreatic cancer: Single-center experience

Introduction: Irreversible Electroporation (IRE) is a non-thermal ablation technique with promising results for treating locally advanced pancreatic cancer (LAPC). This study was conducted to evaluate safety and efficacy of IRE in the management of LAPC. Methods: This was e retrospective single center study of ten patients with radiographic and biopsy proven stage III pancreatic head or body cancer that received open IRE with intraoperative ultrasound imaging. Perioperative complications at 90 days, tumor volume measurements, local recurrence and survival were recorded. Results: 20 patients, with a median age of 62, underwent IRE for locally advanced pancreatic head cancer (n=11) and body cancer (n=9). All patients were treated successfully with an open IRE approach. Nine patients experiences grade II (Clavien-Dindo) procedure related complications. There were no grade 3 to 5 complications. Median follow up was 30 monts. Tumor volume decrease at 6 months imaging follow up was found in 70% of patients (n=14). Local disease progression was observed in two patients, and there was evidence of metastatic disease in 7 patients. 8 patients died with a mean of 15, 2 months and 12 patients are still alive with a mean of 17,3 months. Median overall survival was 16,5 months. Conclusion: Our initial experience with IRE showed encouraging results regarding safety, feasibility and efficacy in patients with locally advanced pancreatic head or body cancer. Further investigation is needed.

Image-guided in situ cancer vaccination with combination of multi-functional nano-adjuvant and an irreversible electroporation technique

Cancer immunotherapy is a next-generation treatment strategy; however, its side effects limit its clinical translation. Here, a novel combination of a multi-functional nano-adjuvant (M-NA) prepared with an iron oxide/gold core and a cationic polymer shell via multilayer synthesis with CpG oligodeoxynucleotide (CpG-ODN) electrostatically complexed on its surface, and irreversible electroporation (IRE) technique was developed for effective image-guided in situ cancer vaccination. The M-NA can be retained long-term in the dense tumoral extracellular matrix after intratumoral injection and internalized by antigen-presenting cells (APCs). The IRE can induce immunogenic cell death. Indeed, in a mouse tumor model, the M-NA showed longer tumor retention time than free CpG-ODN. Compared with other treatments, the combined treatment significantly inhibited tumor growth with 100% survival rate for ∼60 days. The therapy induced the activation of cytotoxic lymphocytes and the maturation of APCs in vivo. This treatment could be effective in image-guided local cancer immunotherapy.

Effect of pulsed field ablation on solid tumor cells and microenvironment.

Pulsed field ablation can increase membrane permeability and is an emerging non-thermal ablation. While ablating tumor tissues, electrical pulses not only act on the membrane structure of cells to cause irreversible electroporation, but also convert tumors into an immune active state, increase the permeability of microvessels, inhibit the proliferation of pathological blood vessels, and soften the extracellular matrix thereby inhibiting infiltrative tumor growth. Electrical pulses can alter the tumor microenvironment, making the inhibitory effect on the tumor not limited to short-term killing, but mobilizing the collective immune system to inhibit tumor growth and invasion together.

Automated irreversible electroporated region prediction using deep neural network, a preliminary study for treatment planning

ABSTRACT The primary purpose of cancer treatment with irreversible electroporation (IRE) is to maximize tumor damage and minimize surrounding healthy tissue damage. Finite element analysis is one of the popular ways to calculate electric field and cell kill probability in IRE. However, this method also has limitations. This paper will focus on using a deep neural network (DNN) in IRE to predict irreversible electroporated regions for treatment planning purposes. COMSOL Multiphysics was used to simulate the IRE. The electric conductivity change during IRE was considered to create accurate data sets of electric field distribution and cell kill probability distributions. We used eight pulses with a pulse width of 100 μs, frequency of 1 Hz, and different voltages. To create masks for DNN training, a 90% cell kill probability contour was used. After data set creation, U-Net architecture was trained to predict irreversible electroporated regions. In this study, the average U-Net DICE coefficient on test data was 0.96. Also, the average accuracy of U-Net for predicting irreversible electroporated regions was 0.97. As far as we are aware, this is the first time that U-Net was used to predict an irreversible electroporated region in IRE. The present study provides significant evidence for U-Net’s use for predicting an irreversible electroporated region in treatment planning.

Cytoreduction with Hyperthermic Intraperitoneal Chemoperfusion for Pancreatic Cancer with Low-Volume Peritoneal Metastasis: Results from a Prospective Pilot Study.

Resection of oligometastatic pancreatic ductal adenocarcinoma (PDAC) has historically been ineffective, however modern systemic chemotherapy has improved survival. Thus, re-evaluating safety and outcomes of surgical resection in selected patients with limited peritoneal metastasis (PM) warrants consideration. From 2018 to 2021, patients with PDAC and positive cytology or limited PM without extraperitoneal metastasis and who had an objective response to ≥ 6 months of systemic chemotherapy were enrolled. Patients underwent laparoscopic hyperthermic intraperitoneal chemotherapy (HIPEC) with cisplatin/mitomycin C. If amenable to a complete cytoreduction, patients went on to cytoreduction and HIPEC. Overall, 18 patients were enrolled and received a median of 14 (interquartile range [IQR] 12-17) cycles of chemotherapy; 16 (89%) patients received chemoradiation. Laparoscopic HIPEC was completed in 17 patients, with a median length of stay of 1day, and no grade III complications or hematological toxicities were observed. All 18 patients subsequently underwent a complete cytoreduction (CC-0) along with definitive treatment of the primary tumor, with formal resection (7/18), irreversible electroporation (IRE; 10/18), or intraoperative radiation therapy (IORT; 1/18). Median PCI was 2 (IQR 0-4), median LOS was 7 days (IQR 6-8), and 7 (39%) patients were readmitted. Eight (44%) patients experienced grade 3 or higher complications, including one 30-day mortality. At a median follow-up of 16 months, the median progression-free survival was 20 months and the median overall survival was 26 months. Cytoreduction and HIPEC for selected patients with low-volume PMfrom PDAC is safe and feasible with favorable short-term outcomes. A phase II trial (NCT04858009) is now enrolling to further assess this multimodality approach in select patients.

Establishing electroporation thresholds for targeted cell specific cardiac ablation in a 2D culture model.

BackgroundIrreversible electroporation has emerged as a new modality to overcome issues associated with other energy sources for cardiac ablation. Strong evidence on the optimal, effective, and selective voltage threshold is lacking for both in vitro and preclinical in vivo studies. The aim of this study is to examine the optimal threshold for selective cell ablation on cardiac associated cell types.MethodsConventional monophasic and biphasic pulses of different field strength were delivered in a monolayer culture system of cardiomyocytes, neurons, and adipocytes. The dynamics of cell death mechanisms were examined at different time points.ResultsNeurons exhibit higher susceptibility to electroporation and cell death at higher field strength of 1250 V/cm in comparison to cardiomyocytes. Cardiac adipocytes showed lower susceptibility to electroporation in comparison to other cell types. A significant proportion of cardiomyocytes recovered after 24 h postelectroporation, while neuronal cell death remained consistent but with a significant delayed cell death at a higher voltage threshold. Caspase 3/7 activity was observed in both cardiomyocytes and neurons, with a higher level of activity in cardiomyocytes in response to electroporation. Biphasic and monophasic pulses showed no significant difference in both cell types, and significantly lower cell death in neurons when inter pulse interval was reduced.ConclusionsThis study presents important findings on the differences in the susceptibility of neurons and cardiomyocytes to irreversible electroporation. Cell type alone yielded selective and different dynamics in terms of the evolution and signaling mechanism of cell death in response to electroporation.

Safety and feasibility of irreversible electroporation for the pancreatic head in a porcine model

BACKGROUNDIrreversible electroporation (IRE) is a local non-thermal ablative technique which has been suggested as a potential cancer therapy. However, the specific anatomic characteristics of the pancreatic head make it challenging to perform any local ablation in this region. Therefore, the safety and feasibility of IRE in the pancreatic head region should be further explored.AIMTo evaluate the safety of IRE in pancreatic head region including its effects on pancreatic ducts, vessels, and adjacent gastrointestinal organs.METHODSEight landrace miniature pigs underwent IRE of pancreatic head tissue successfully, with a total of 16 lesions created. Laboratory testing including white blood cell (WBC) count and serum amylase before IRE with follow-up laboratory analysis and pathological examination at 1, 7, 14, and 28 d postablation were performed. RESULTSAll pigs tolerated the ablation procedure without serious perioperative complications. Transiently elevated WBC count and amylase were observed at 24 h post-IRE, suggesting an acute pancreatic tissue damage which was confirmed by pathological observations. Vascular endothelial cells and pancreatic duct epithelial cells in ablation zone were also positive in terminal deoxynucleotidyl transferase dUTP nick end labeling staining. There was extensive duodenum mucosa damage with local hemorrhage 24 h after ablation, while regeneration of new villous structures were observed at 7 and 28 d post-IRE. Masson’s trichromatic staining showed that the extracellular matrix was still intact in vessels and pancreatic ducts, and even in the duodenum.CONCLUSIONIRE ablation to the pancreatic head may be safe and feasible without long-term damage to the surrounding vital structures. However, risks of stress injuries in acute phase should be taken into consideration to prevent severe perioperative complications.

Modern Management of Localized Renal Cell Carcinoma- Is Ablation Part of the Equation?

While the gold-standard for management of localized renal cell carcinoma (RCC) is partial nephrectomy, recent ablative strategies are emerging as alternatives with comparable rates of complications and oncologic outcomes. Thermal ablation, in the form of radiofrequency ablation and cryoablation, is being increasingly accepted by professional societies, and is particularly recommended in patients with a significant comorbidity burden, renal impairment, old age, or in those unwilling to undergo surgery. Maturation of long-term oncologic outcomes has further allowed increased confidence in these management strategies. New and exciting ablation technologies such as microwave ablation, stereotactic body radiotherapy, and irreversible electroporation are emerging. In this article, we review the existing management options for localized RCC, with specific focus on the oncologic outcomes associated with the various ablation modalities.

Scanning electrochemical microscopy based irreversible destruction of living cells

In this research, scanning electrochemical microscopy combined with electrochemical impedance spectroscopy has been applied to irreversible electroporation of active yeast cells by causing cell death. This finding is important for the development of irreversible electroporation technique, which could be suitable for the curing of cancerous tissues, because during this research cell death has been achieved using relatively low ultramicro-electrode (UME) voltage, precisely of 2.0 V vs Ag/AgCl,Cl-sat. It was determined that the irreversibly electroporated area of immobilized yeast cells was located directly below the UME and was of approximately 20 times larger width than the diameter of the UME, leaving undamaged cells out of this area. The ability of SECM to move the UME with high accuracy in x, y, and z directions and the ability to use electrodes of various diameters as well as the fact that the diameter of the electroporated area depends on the diameter of the UME and on the distance between the UME and the surface, what offers the possibility to establish targeted electroporation systems for selective treatment of tissues.

New progress in tumor treatment based on nanoparticles combined with irreversible electroporation

AbstractNanoknife is a new tumor ablation technology. It forms nano permanent perforation on tumor cells by releasing high‐voltage pulses, destroying the intracellular balance and making cells apoptosis rapidly. So nano knife is also known as irreversible electroporation (IRE). IRE ablation technology and nanoparticles (NPs) have unique advantages in tumor treatment. Relevant studies have confirmed that NPs can enhance the effect of IRE ablation, expand the ablation boundary and reduce the threshold intensity of tumor cell perforation. Moreover, IRE combined with the drug‐loaded NPs can synergize tumor immunotherapy. Thus, the combination of NPs and IRE has broad research prospects. This paper reviews the mechanism of tumor treatment with NPs combined with IRE and discusses the future challenges and opportunities of combining these two technologies.

Induction of Bystander and Abscopal Effects after Electroporation-Based Treatments.

Simple SummaryThe delivery of electric field to tumor cells or nodules induces cell electroporation that allows intracellular delivery of cytotoxic agents and eventually inhibits tumor growth. In this study, we showed that intracellular delivery of calcium ions and anticancer drug bleomycin not only kills the cells but also has a negative bystander effect on indirectly treated cells. We also showed that, when directly applied to one tumor, these treatments can inhibit the growth of a second, non-electroporated tumor.Electroporation-based antitumor therapies, including bleomycin electrotransfer, calcium electroporation, and irreversible electroporation, are very effective on directly treated tumors, but have no or low effect on distal nodules. In this study, we aimed to investigate the abscopal effect following calcium electroporation and bleomycin electrotransfer and to find out the effect of the increase of IL-2 serum concentration by muscle transfection. The bystander effect was analyzed in in vitro studies on 4T1tumor cells, while abscopal effect was investigated in an in vivo setting using Balb/c mice bearing 4T1 tumors. ELISA was used to monitor IL-2 serum concentration. We showed that, similarly to cell treatment with bleomycin electrotransfer, the bystander effect occurs also following calcium electroporation and that these effects can be combined. Combination of these treatments also resulted in the enhancement of the abscopal effect in vivo. Since these treatments resulted in an increase of IL-2 serum concentration only in mice bearing one but not two tumors, we increased IL-2 serum concentration by muscle transfection. Although this did not enhance the abscopal effect of combined tumor treatment using calcium electroporation and bleomycin electrotransfer, boosting of IL-2 serum concentration had a significant inhibitory effect on directly treated tumors.

Multielectrode Contact Measurement Can Improve Long-Term Outcome of Pulmonary Vein Isolation Using Circular Single-Pulse Electroporation Ablation.

Background:Irreversible electroporation (IRE) ablation is generally performed with multielectrode catheters. Electrode-tissue contact is an important predictor for the success of pulmonary vein (PV) isolation; however, contact force is difficult to measure with multielectrode ablation catheters. In a preclinical study, we assessed the feasibility of a multielectrode impedance system (MEIS) as a predictor of long-term success of PV isolation. In addition, we present the first-in-human clinical experience with MEIS.Methods:In 10 pigs, one PV was ablated based on impedance (MEIS group), and the other PV was solely based on local electrogram information (EP group). IRE ablations were performed at 200 J. After 3 months, recurrence of conduction was assessed. Subsequently, in 30 patients undergoing PV isolation with IRE, MEIS was evaluated and MEIS contact values were compared to local electrograms.Results:In the porcine study, 43 IRE applications were delivered in 19 PVs. Acutely, no reconnections were observed in either group. After 3 months, 0 versus 3 (P=0.21) PVs showed conduction recurrence in the MEIS and EP groups, respectively. Results from the clinical study showed a significant linear relation was found between mean MEIS value and bipolar dV/dt (r2=0.49, P<0.001), with a slope of 20.6 mV/s per Ohm.Conclusions:Data from the animal study suggest that MEIS values predict effective IRE applications. For the long-term success of electrical PV isolation with circular IRE applications, no significant difference in efficacy was found between ablation based on the measurement of electrode interface impedance and ablation using the classical EP approach for determining electrode-tissue contact. Experiences of the first clinical use of MEIS were promising and serve as an important basis for future research.

Feasibility of Concentric Electrodes in Contact Irreversible Electroporation for Superficial Lesion Treatment.

Contact irreversible electroporation (IRE) is a method for ablating cells by applying electric pulses via surface electrodes in contact with a target tissue. To facilitate the application of the contact IRE to superficial lesion treatment, this study further extended the ablation depth, which had been limited to a 400-μm depth in our previous study, by using concentric electrodes. A prototype device of concentric electrodes was manufactured using a Teflon-coated copper wire inserted in a copper tube. The ablation area was experimentally determined using a tissue phantom comprising 3D cultured fibroblasts and compared with the electric field distribution obtained using numerical analyses. Experiments showed that cells 540 μm from the surface of the tissue phantom were necrotized by the application of 150 pulses at 100 V. The outline of the ablation area agreed well with the contour line of 0.4 kV/cm acquired by the analyses. The ablation depth predicted for the concentric electrode using this critical electric field was 1.4 times deeper than that for the parallel electrode. For the actual application of treatment, a multiple-electrode device that bundles several pairs of concentric electrodes was developed, and confirmed that to be effective for treating wide areas with a single treatment. The electric field estimated by the analyses with the experimentally determined threshold confirmed that concentric electrodes could attain a deeper ablation than parallel electrodes. Using the concentric electrodes, we were able to localize ablation to specific target cells with much less damage to neighboring cells.

A Drive Circuit With Overcurrent Protection for Solid-State Marx Generators

In recent years, all-solid-state high-voltage pulse generators have been widely used in irreversible electroporation for tumor ablation technology. However, high voltage may cause the breakdown of loads that leads to short circuits. The short circuit of loads will generate high short-circuit currents that will affect the performance of semiconductor switches. To improve the reliability and lifetime of solid-state pulse generators, a passive synchronous isolated gate drive circuit with short-circuit protection based on decreasing the gate–source voltage is proposed in this article. To verify the overcurrent protection during the breakdown of loads, a solid-state Marx generator prototype using the drive circuit was built and tests were carried out. The experimental results show that the 205-A short-circuit current was clamped with 90 A in less than 500 ns, while the transient voltage can be neglected. Besides, the response time, the maximum transient voltage, and the maximum current amplitude can all be adjusted.

Electroporation Parameters for Human Cardiomyocyte Ablation In Vitro.

Cardiac ablation with irreversible electroporation (IRE) is quickly being established as a modality of choice for atrial fibrillation treatment. While it has not yet been optimised, IRE has the potential to significantly limit collateral damage and improve cell-specific targeting associated with other energy sources. However, more tissue and cell-specific evidence is required to demonstrate the selective threshold parameters for human cells. The aim here is to determine the optimal ablation threshold parameters related to lesion size for human cardiomyocytes in 2D culture. Conventional biphasic pulses of different field strengths and on-times were delivered in a monolayer culture system of human AC16 cardiomyocytes. The dynamics of cell death and lesion dimensions were examined at different time points. Human cardiomyocytes are susceptible to significant electroporation and cell death at a field strength of 750 V/cm or higher with 100 μs pulses. Increasing the IRE on-time from 3 ms to 60 ms reduces the effective field threshold to 250 V/cm. Using very short pulses of 2 μs and 5 μs also causes significant cell death, but only at fields higher than 1000 V/cm. A longer on-time results in more cell death and induced greater lesion area in 2D models. In addition, different forms of cell death are predicted based on the evolution of cell death over time. This study presents important findings on the ability of different IRE parameters to induce human cardiomyocyte cell death. Lesion size can be tuned by appropriate choice of IRE parameters and cardiomyocytes display an upregulation of delayed cell death 24 h after electroporation, which is an important consideration for clinical practice.