Biotechnology Commons^™

Apoptosis Initiation And Angiogenesis Inhibition: Melanoma Targets For Nanosecond Pulsed Electric Fields, Xinhua Chen, Juergen F. Kolb, R. James Swanson, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Many effective anti-cancer strategies target apoptosis and angiogenesis mechanisms. Applications of non-ionizing, nanosecond pulsed electric fields (nsPEFs) induce apoptosis in vitro and eliminate cancer in vivo; however in vivo mechanisms require closer analysis. These studies investigate nsPEF-induced apoptosis and anti-angiogenesis examined by fluorescent microscopy, immunoblots, and morphology. Six hours after treatment with one hundred 300 ns pulses at 40 kV/cm, cells transiently expressed active caspases indicating that caspase-mediated mechanisms. Three hours after treatment transient peaks in Histone 2AX phosphorylation coincided with terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells and pyknotic nuclei, suggesting caspase-independent mechanisms on nuclei/DNA. Large …

Increased Perfusion And Angiogenesis In A Hindlimb Ischemia Model With Plasmid Fgf-2 Delivered By Noninvasive Electroporation, B. Ferraro, Y. L. Cruz, M. Baldwin, D. Coppola, R. Heller

Bioelectrics Publications

Gene therapy approaches delivering fibroblast growth factor-2 (FGF-2) have shown promise as a potential treatment for increasing blood flow to ischemic limbs. Currently, effective noninvasive techniques to deliver plasmids encoding genes of therapeutic interest, such as FGF-2, are limited. We sought to determine if intradermal injection of plasmid DNA encoding FGF-2 (pFGF) followed by noninvasive cutaneous electroporation (pFGFE+) could increase blood flow and angiogenesis in a rat model of hindlimb ischemia. pFGFE+ or control treatments were administered on postoperative day 0. Compared to injection of pFGF alone (pFGFE-), delivery of pFGFE+ significantly increased FGF-2 expression for 10 days. Further, the …

A New Pulsed Electric Field Therapy For Melanoma Disrupts The Tumor's Blood Supply And Causes Complete Remission Without Recurrence, Richard Nuccitelli, Xinhua Chen, Andrei G. Pakhomov, Wallace H. Baldwin, Saleh Sheikh, Jennifer L. Pomicter, Wei Ren, Chris Osgood, R. James Swanson, Juergen F. Kolb, Stephen J. Beebe, Karl H. Schoenbach

Bioelectrics Publications

We have discovered a new, ultrafast therapy for treating skin cancer that is extremely effective with a total electric field exposure time of only 180 mu sec. The application of 300 high-voltage (40 kV/cm), ultrashort (300 nsec) electrical pulses to murine melanomas in vivo triggers both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. None of these melanomas recurred during a 4-month period after the initial melanoma had disappeared. These pulses generate small, long-lasting, rectifying nanopores in the plasma membrane of exposed cells, resulting in increased membrane permeability to …

Electroporation For The Delivery Of Dna-Based Vaccines And Immunotherapeutics: Current Clinical Developments, Angela M. Bodles-Brakhop, Richard Heller, Ruxandra Draghia-Akli

Bioelectrics Publications

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. the recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. current research has revealed that EP can be successfully used in many species, including humans. clinical trials are …

Intradermal Delivery Of Plasmid Vegf(165) By Electroporation Promotes Wound Healing, Bernadette Ferraro, Yolmari Cruz, Domenico Coppola, Richard Heller

Bioelectrics Publications

Skin flaps are extensively used in reconstructive surgeries to repair large defects and deep wounds, but severe ischemia and necrosis often results in loss of the transplanted tissue. Thus, skin flap models are often used to study the biology of healing and necrosis of acute ischemic wounds. Delivery of exogenous vascular endothelial growth factor (VEGF) to areas of ischemia has shown promise for promoting therapeutic angiogenesis, but its expression must be tightly regulated to avoid adverse effects. In this study, plasmid DNA encoding VEGF₁₆₅ (pVEGF) was delivered to the ischemic skin of a rat skin flap model by intradermal …

Comparison Of Electrically Mediated And Liposome-Complexed Plasmid Dna Delivery To The Skin, Loree C. Heller, Mark J. Jaroszeski, Domenico Coppola, Richard Heller

Bioelectrics Publications

BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

RESULTS: Experiments …

Optimization Of Cutaneous Electrically Mediated Plasmid Dna Delivery Using Novel Electrode, L. C. Heller, M. J. Jaroszeski, D. Coppola, A. N. Mccray, J. Hickey, R. Heller

Bioelectrics Publications

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of …

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 …

Plasma Membrane Voltage Changes During Nanosecond Pulsed Electric Field Exposure, W. Frey, R. O. Price, P. F. Blackmore, R. P. Joshi, R. Nuccitelli, S. J. Beebe, K. H. Schoenbach, J. F. Kolb

Bioelectrics Publications

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of similar to 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat …

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 …

Selective Field Effects On Intracellular Vacuoles And Vesicle Membranes With Nanosecond Electric Pulses, Ephrem Tekle, Hammou Oubrahim, Sergey M. Dzekunov, Juergen F. Kolb, Karl H. Schoenbach

Bioelectrics Publications

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of …

The Effects Of Intense Submicrosecond Electrical Pulses On Cells, Jingdong Deng, Karl H. Schoenbach, E. Stephen Buescher, Pamela S. Hair, Paula M. Fox, Stephen J. Beebe

Bioelectrics Publications

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the submicrosecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs– 60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of …

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

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 …

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 …

Effect Of Electrically Mediated Intratumor And Intramuscular Delivery Of A Plasmid Encoding Ifn Α On Visible B16 Mouse Melanomas, Loree C. Heller, Stephanie F. Ingram, M. Lee Lucas, Richard A. Gilbert, Richard Heller

Bioelectrics Publications

Interferon α may be used as a single agent therapy for metastatic malignant melanoma or as an adjuvant to chemotherapy. Delivery of interferon α by gene therapy offers an alternative to recombinant protein therapy. Electrically mediated delivery enhances plasmid expression in a number of tissues, for instance skin, liver, muscle and tumors including melanomas. Here we compare the effect of delivery of a plasmid encoding mouse interferon α on growth of visible B16 mouse melanomas following electrically mediated delivery to muscle or directly to the tumor. Intratumoral delivery of interferon α plasmid not only slows melanoma growth, but induces complete, …

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 …

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.