Medicine and Health Sciences Commons^™

Control Of The Electroporation Efficiency Of Nanosecond Pulses By Swinging The Electric Field Vector Direction, Vitalii Kim, Iurii Semenov, Allen S. Kiester, Mark A. Keppler, Bennett L. Ibey, Joel N. Bixler, Ruben M. L. Colunga Biancatelli, Andrei G. Pakhomov

Bioelectrics Publications

Reversing the pulse polarity, i.e., changing the electric field direction by 180°, inhibits electroporation and electrostimulation by nanosecond electric pulses (nsEPs). This feature, known as “bipolar cancellation,” enables selective remote targeting with nsEPs and reduces the neuromuscular side effects of ablation therapies. We analyzed the biophysical mechanisms and measured how cancellation weakens and is replaced by facilitation when nsEPs are applied from different directions at angles from 0 to 180°. Monolayers of endothelial cells were electroporated by a train of five pulses (600 ns) or five paired pulses (600 + 600 ns) applied at 1 Hz or 833 kHz. Reversing …

Identification Of Proteins Involved In Cell Membrane Permeabilization By Nanosecond Electric Pulses (Nsep), Giedre Silkuniene, Uma Mangalanathan, Alessandra Rossi, Peter A. Mollica, Andrei G. Pakhomov, Olga N. Pakhomova

Bioelectrics Publications

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in …

Electroporation And Cell Killing By Milli- To Nanosecond Pulses And Avoiding Neuromuscular Stimulation In Cancer Ablation, Emily Gudvangen, Vitalii Kim, Vitalij Novickij, Federico Battista, Andrei G. Pakhomov

Bioelectrics Publications

Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns–1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2–4 h, dead cells were labeled with propidium. Electroporation and …

Impedance Analysis Of Tissues In Nspef Treatment For Cancer Therapy, Edwin Ayobami Oshin

Biomedical Engineering Theses & Dissertations

Nanosecond pulsed electric field (nsPEF) for cancer therapy is characterized by applications of high voltage pulses with low pulsed energy to induce non-thermal effects on tissues such as tumor ablation. It nonthermally treats tissues via electroporation. Electroporation is the increase in permeabilization of a cell membrane due to the application of high pulsed electric field. The objective of this study was to investigate the effect of nsPEF on tissue by monitoring the tissue’s impedance in real-time. Potato slices (both untreated and electroporated), and tumors extracted from female BALBc mice were studied. 100ns, 1-10kV pulses were applied to the tissues using …

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied By Immunogenic Cell Death In Murine Models Of Lymphoma And Colorectal Cancer, Alessandra Rossi, Olga N. Pakhomova, Peter A. Mollica, Maura Casciola, Uma Mangalanathan, Andrei G. Pakhomov, Claudia Muratori

Bioelectrics Publications

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of …

Intratumoral Delivery Of Plasmid Il12 Via Electroporation Leads To Regression Of Injected And Noninjected Tumors In Merkel Cell Carcinoma, Shailender Bhatia, Natalie V. Longino, Natalie J. Miller, Rima Kulikauskas, Jayasri G. Iyer, Dafina Ibrani, Astrid Blom, David R. Byrd, Upendra Parvathaneni, Christopher Twitty, Jean S. Campbell, Mai H. Le, Sharron Gargosky, Robert H. Pierce, Richard Heller, Adil Daud, Paul Nghiem

Bioelectrics Publications

Purpose: Interleukin-12 (IL12) promotes adaptive type I immunity and has demonstrated antitumor efficacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer.

Experimental Design: Fifteen patients with MCC with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N = 3) received one cycle before definitive surgery in week 4. …

Moderate Heat Application Enhances The Efficacy Of Nanosecond Pulse Stimulation For The Treatment Of Squamous Cell Carcinoma, Chelsea M. Edelblute, Sigi Guo, Embo Yang, Chunqi Jiang, Karl Schoenbach, Richard Heller

Bioelectrics Publications

Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times …

Tumor Cell Death After Electrotransfer Of Plasmid Dna Is Associated With Cytosolic Dna Sensor Upregulation, Katarina Znidar, Masa Bosnjak, Nina Semenova, Olga N. Pakhomova, Loree Heller, Maja Cemazar

Bioelectrics Publications

Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol. Their activation leads to the upregulation of pro-inflammatory cytokines and chemokines and can also induce cell death. The presence of cytosolic DNA sensors and inflammatory cytokines in TS/A murine mammary adenocarcinoma and WEHI 164 fibrosarcoma cells was demonstrated using real time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbent assay (ELISA). After electrotransfer of plasmid DNA (pDNA) using two pulse protocols, the upregulation of DNA-depended activator of interferon regulatory factor or Z-DNA binding …

Gadolinium Modifies The Cell Membrane To Inhibit Permeabilization By Nanosecond Electric Pulses, Elena C. Gianulis, Andrei G. Pakhomov

Bioelectrics Publications

Lanthanide ions are the only known blockers of permeabilization by electric pulses of nanosecond duration (nsEP), but the underlying mechanisms are unknown. We employed timed applications of Gd³⁺ before or after nsEP (600-ns, 20 kV/cm) to investigate the mechanism of inhibition, and measured the uptake of the membrane-impermeable YO-PRO-1 (YP) and propidium (Pr) dyes. Gd³⁺ inhibited dye uptake in a concentration-dependent manner. The inhibition of Pr uptake was always about 2-fold stronger. Gd³⁺ was effective when added after nsEP, as well as when it was present during nsEP exposure and removed afterward. Pores formed by nsEP in …

Electrotransfer Of Single-Stranded Or Double-Stranded Dna Induces Complete Regression Of Palpable B16.F10 Mouse Melanomas, Loree Heller, Vesba Todorovic, Maja Cemazar

Bioelectrics Publications

Enhanced tumor delivery of plasmid DNA with electric pulses in vivo has been confirmed in many preclinical models. Intratumor electrotransfer of plasmids encoding therapeutic molecules has reached Phase II clinical trials. In multiple preclinical studies, a reduction in tumor growth, increased survival or complete tumor regression have been observed in control groups in which vector or backbone plasmid DNA electrotransfer was performed. This study explores factors that could produce this antitumor effect. The specific electrotransfer pulse protocol employed significantly potentiated the regression. Tumor regression was observed after delivery of single-stranded or double-stranded DNA with or without CpG motifs in both …

Cell Responses Without Receptors And Ligands, Using Nanosecond Pulsed Electric Fields (Nspefs), Stephen J. Beebe

Bioelectrics Publications

No abstract provided.

Facilitation Of Electroporative Drug Uptake And Cell Killing By Electrosensitization, Olga N. Pakhomova, Betsy W. Gregory, Andrei G. Pakhomov

Bioelectrics Publications

Cell permeabilization by electric pulses (EP), or electroporation, is widely used for intracellular delivery of drugs and plasmids, as well as for tumour and tissue ablation. We found that cells pre-treated with 100-mus EP develop delayed hypersensitivity to subsequent EP applications. Sensitizing B16 and CHO cells by splitting a single train of eight 100-mus EP into two trains of four EP each (with 5-min. interval) decreased the LD(50) 1.5-2 times. Sensitization profoundly enhanced the electroporation-assisted uptake of bleomycin, a cell-impermeable cytotoxic agent accepted for killing tumours by electrochemotherapy. EP exposures that were not lethal per se caused cell death in …

Electroporation-Mediated Gene Transfer Directly To The Swine Heart, Barbara Hargrave, Harre Downey, Cathryn Lundberg, Annelise Israel, Yeong-Jer Chen, Richard Heller

Bioelectrics Publications

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on …

Nanosecond Pulsed Electric Field Induced Cytoskeleton, Nuclear Membrane And Telomere Damage Adversely Impact Cell Survival, Michael W. Stacey, P. Fox, S. Buescher, Juergen F. Kolb

Bioelectrics Publications

We investigated the effects of nanosecond pulsed electric fields (nsPEF) on three human cell lines and demonstrated cell shrinkage, breakdown of the cytoskeleton, nuclear membrane and chromosomal telomere damage. There was a differential response between cell types coinciding with cell survival. Jurkat cells showed cytoskeleton, nuclear membrane and telomere damage that severely impacted cell survival compared to two adherent cell lines. Interestingly, disruption of the actin cytoskeleton in adherent cells prior to nsPEF exposure significantly reduced cell survival. We conclude that nsPEF applications are able to induce damage to the cytoskeleton and nuclear membrane. Telomere sequences, regions that tether and …

Evaluation Of Toxicity Following Electrically Mediated Interleukin-12 Gene Delivery In A B16 Mouse Melanoma Model, Loree Heller, Kathleen Merkler, Jeffrey Westover, Yolmari Cruz, Domenico Coppola, Kaaron Benson, Adil Daud, Richard Heller

Bioelectrics Publications

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model.

EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically.

RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated …

Nanosecond Pulsed Electric Fields: A New Stimulus To Activate Intracellular Signaling, Stephen J. Beebe, Karl H. Schoenbach

Bioelectrics Publications

When new technologies are introduced into the scientific community, controversy is expected and both excitement and disappointment enrich the lives of those who initiate the new ideas. It becomes the mission of the “inventors” to embrace the burden of proof to establish their ideas and convince the skeptics and disbelievers who will undoubtedly temper their enthusiasm and test their patience. While open mindedness is generally a scientific motto, those who review patents, manuscripts, and grants do not always readily practice it, even when the evidence is convincingly presented; old ideas and concepts often die hard. So it has been and …

Simulations Of Nanopore Formation And Phosphatidylserine Externalization In Lipid Membranes Subjected To A High-Intensity, Ultrashort Electric Pulse, Q. Hu, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

A combined MD simulator and time dependent Laplace solver are used to analyze the electrically driven phosphatidylserine externalization process in cells. Time dependent details of nanopore formation at cell membranes in response to a high-intensity (100kV∕cm), ultrashort (10ns) electric pulse are also probed. Our results show that nanosized pores could typically be formed within about 5ns. These predictions are in very good agreement with recent experimental data. It is also demonstrated that defect formation and PS externalization in membranes should begin on the anode side. Finally, the simulations confirm that PS externalization is a nanopore facilitated event, rather than the …

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

Electric Field Enhanced Plasmid Delivery To Liver Hepatocellular Carcinomas, Richard Gilbert, Mark J. Jaroszeski, Loree Heller, Richard Heller

Bioelectrics Publications

Electric field enhanced molecular delivery for cancer research and treatment is a new technology that has demonstrated its effectiveness in clinical trials using bleomycin or cisplatin (Heller, R., Gilbert, R., Jaroszeski, M. J. Clinical applications of electrochemotherapy, Advanced Drug Delivery Reviews 35,119-129, 1999), as chemotherapeutic agents. The technology is being investigated in research applications for applicability as a method to enhance gene expression in a target tumor. Success is predicated on an appropriate effective electric field mediated delivery protocol that triggers significant appropriate gene expression duration and levels. An electric field mediated delivery protocol includes a set of conditions …

Il-12 Plasmid Delivery By In Vivo Electroporation For The Successful Treatment Of Established Subcutaneous B16.F10 Melanoma, M. Lee Lucus, Loree Heller, Domenico Coppola, Richard Heller

Bioelectrics Publications

Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six …

Improved Energy Model For Membrane Electroporation In Biological Cells Subjected To Electrical Pulses, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

A self-consistent model analysis of electroporation in biological cells has been carried out based on an improved energy model. The simple energy model used in the literature is somewhat incorrect and unphysical for a variety of reasons. Our model for the pore formation energy E(r) includes a dependence on pore population and density. It also allows for variable surface tension, incorporates the effects of finite conductivity on the electrostatic correction term, and is dynamic in nature. Self-consistent calculations, based on a coupled scheme involving the Smoluchowski equation and the improved energy model, are presented. It is shown that E(r) becomes …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.