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Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

ELECTROPORATION IN LABORATORY AND CLINICAL INVESTIGATIONS

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Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved. Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

ELECTROPORATION IN LABORATORY AND CLINICAL INVESTIGATIONS

ENRICO P. SPUGNINI AND

ALFONSO BALDI Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

EDITORS

Nova Science Publishers, Inc. New York

Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Electroporation in laboratory and clinical investigations / editors, Enrico P. Spugnini, Alfonso Baldi. p. ; cm. Includes bibliographical references and index. ISBN:  (eBook)

1. Electroporation. 2. Electroporation--Therapeutic use. I. Spugnini, Enrico P. II. Baldi, Alfonso. [DNLM: 1. Electroporation. 2. Cell Membrane--physiology. 3. Neoplasms--therapy. QY 95 E386 2010] QH585.5.E48E45 2010 571.6'4--dc22 2010001743

Published by Nova Science Publishers, Inc. † New York Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

CONTENTS

Preface

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Chapter 1

vii Mathematical-Physical Modeling and Biomedical Optimization of Cell Electropermeabilization: An Overview Alessandro Porrello and Andrea Giansanti

1

Chapter 2

Technical Aspects of the Electrochemotherapy Ivan Dotsinsky, Nicolay Mudrov and Tsvetan Mudrov

45

Chapter 3

Non-Thermal Irreversible Electroporation for Tissue Ablation Paulo A. Garcia, Robert E. Neal II and Rafael V. Davalos

63

Chapter 4

Mechanisms of Microorganism Inactivation by Pulsed Electric Fields Gintautas Saulis

85

Electrochemical Processes Occuring During Cell Electromanipulation Procedures Gintautas Saulis

99

Chapter 5

Chapter 6

Ultrastructural Modifications Induced by ―Electroporation‖ Agnese Molinari, Giuseppe Arancia and Enrico P. Spugnini

115

Chapter 7

Electroporation in Bacteria Maria Papagianni

133

Chapter 8

Electroporation of Plant Cells Alejandro Araya

155

Chapter 9

Anti-Tumoral Effects of Pulsed Low Electric Field Enhanced Chemotherapy: Lessons from Experimental Malignant Tumors Yona Keisari and Rafi Korenstein

Chapter 10

Electrogenetherapy: Electrogene Transfer Using Low Field Strength Feng Liu, Amber Frick, and Jue Wang

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vi

Contents

Chapter 11

Electroporation – Treating Mice or Men? Angela M. Bodles-Brakhop and Ruxandra Draghia-Akli

217

Chapter 12

Electrochemotherapy in Veterinary Medicine, Part I: Solid Tumors Enrico P. Spugnini, Gennaro Citro and Alfonso Baldi

245

Chapter 13

Electrochemotherapy in Veterinary Medicine, Part II: Round Cell Tumors Enrico P. Spugnini, Alfonso Baldi and Gennaro Citro

Chapter 14

Chapter 15

Histopathological Analysis of Canine and Feline Cancer Treated With Electrochemotherapy Alfonso Baldi, Feliciano Baldi, Pasquale Mellone, Alfredo D’Avino, Gennaro Citro, and Enrico P. Spugnini Clinical Application of Electrochemotherapy – An Adjunct to Surgery Declan M. Soden, Mira Sadadcharam, Patrick Forde, and Gerald C. O'Sullivan

Chapter 16

Electroporation in Chronic Lymphocytic Leukemia Femke Van Bockstaele†, Valerie Pede, Bruno Verhasselt and Jan Philippé

Chapter 17

Future Developments in Electroporation: Recombinant Clostridia as Viable and Targeted Tumour Therapeutics Tam H Nguyen, Siyu Cao, Shu-Feng Zhou, and Ming Q Wei

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Index

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265

273

299

315 329

PREFACE

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Electroporation is a widespread technique adopted to increase the uptake of molecules by biological targets. This approach is gaining momentum due to its low cost and feasibility both in basic and in applied science. Notwithstanding the raise in interest in this method at scientific and clinical level, there are very few books completely dedicated to this argument. Principal purpose of this book is a comprehensive and up to date overview on electroporation in mathematic modeling, bioengineering, molecular biology, plant biology, pathology, veterinary and human oncology. Several authors from different countries have been selected for the contributions, based on their recognized expertise in the different themes discussed. It is our wish that the book will serve the needs of investigators in the field of electroporation for teaching, research and professional reference. Nevertheless, we hope that it could also be an excellent reading for clinicians and PhD students and anyone that needs an in-depth study on this topic.

Rome, September 2009 Enrico P. Spugnini DVM, Ph D Diplomate ACVIM, (Oncology) Diplomate ECVIM-CA (Oncology) Regina Elena Cancer Institute Rome Italy

Alfonso Baldi, MD Associate Professor of Pathology Second University of Naples Naples Italy

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 1

MATHEMATICAL-PHYSICAL MODELING AND BIOMEDICAL OPTIMIZATION OF CELL ELECTROPERMEABILIZATION: AN OVERVIEW Alessandro Porrello 1, and Andrea Giansanti 2,3 1

Institute for Genome Sciences and Policy, Duke University, Durham, NC, U.S. 2 Department of Physics, Sapienza University of Rome, Rome, Italy 3 National Institute of Nuclear Physics (INFN), Division of Rome, Rome, Italy

ABSTRACT Copyright © 2010. Nova Science Publishers, Incorporated. All rights reserved.

Cell electropermeabilization is the process that occurs after prokaryotic and eukaryotic cells, whether in suspension or belonging to tissues and/or living organisms, are exposed to a proper electric field, and that generates an increased permeability of their plasma membranes. Both in vitro and in vivo, it happens when and where the transmembrane voltage, which is dependent on several physical variables, exceeds a critical threshold. This procedure is generally used to introduce different kinds of ions and molecules having biological or medical relevance into living cells. We refer to this biophysical event as electropermeabilization, even though most of this chapter is indeed devoted to the theory of electroporation, which explains the cell permeabilization in terms of opening of membrane pores. As a matter of fact, electroporation is the theory of electropermeabilization with the greatest scientific consensus, and also has the largest dedicated literature. The theoretical approaches analyzed rely on different types of mathematical models whose shared purpose is to assist researchers in this field in predicting, managing, optimizing, and interpreting the experimental results. In order to make this text more suitable for applications, several equations are accompanied by detailed qualitative descriptions of the experimental and/or clinical effects produced by the variables involved. Conversely, many biomedical results are discussed emphasizing their value for modelers. In the first section we briefly introduce the main models of electropermeabilization proposed. In the second and third sections we focus on the general and kinetic physical investigations concerning electroporation and on the 

Correspondence: [email protected]

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Alessandro Porrello and Andrea Giansanti electropore mathematical modeling. The fourth section is devoted to explain which role molecular simulations play in describing the electropermeabilization process and as a potential approach to study the core events of pore formation. In the fifth and last section, theoretical analyses, experimental studies and clinical research disseminated throughout a large number of publications are originally and systematically organized, in the attempt to look at this heterogeneous information in a comprehensive perspective, for the purposes of bioengineering and biomedical optimization. In particular, the final section discusses technical and instrumental issues, cell and tissue properties, electric field and heat generation, physiological mechanisms, molecular uptake, and clinical adverse events.

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INTRODUCTION The first article describing the use of electric pulses to enhance the gene transfer into murine cells in vitro was published by Neumann et al. (1, 2). At that time, the authors used the definition of ―electroporation model‖ to describe the induction and stabilization of permeation sites and the subsequent transport of DNA across the membrane as a consequence of the interaction between the external electric field and the lipid dipoles of a pore configuration. However, alternative models of electro-induced membrane permeabilization have been proposed by the scientific community (see the first section), and for this reason we chose to use in the title the term electropermeabilization, instead of electroporation, which makes less stringent assumptions about the underlying mechanism. Since more than 20 years ago, the use of electropermeabilization has been reported in bacterial, plant, and mammal cells (3-7), as well as in fungi and protozoans (8-10). Importantly, in the early ‗90s, studies on the in vivo electropermeabilization in rodents also came forth (11-15). The first clinical study on the use of electropermeabilization to increase the uptake of a chemotherapeutic agent in human tumors was reported by Belehradek et al. (16), and since then several investigations have been published on this issue in humans (1722) and in pet models (23-25). Nowadays, electropermeabilization has been proven effective for the targeted delivery to living cells of ions, dyes, radiotracers, drugs, antibodies, oligonucleotides, DNA, and RNA and is under consideration for virtually any molecule (26). Unless differently specified, for simplicity, from here on we will use the word ―molecule‖ in a broad sense, also for referring to ions and charged molecules. The cells of living organisms of the five kingdoms of life (i.e., Prokaryotae, Protoctista, Animalia, Fungi, and Plantae), which are all potential targets of the electropermeabilization modeling, are extraordinarily heterogeneous, as for size and biological properties (27). Not surprisingly, the clinical and experimental protocols that cope with this diversity, as well as the available instrumental options and biomedical key factors, are selected on a case by case basis and it is impractical for us to summarize them and their rationale in this introduction. Nonetheless, mostly to provide some benchmarks by a physics standpoint, which may allow a better understanding of the chapter sections, we report below some basic data referred to typical mammalian cells exposed to direct current (DC). It is known that their resting transmembrane potential differences are comprised between 50 and 70 mV and are due to the difference between the ionic strength inside and outside the cell. The transmembrane potential needed to induce their electropermeabilization is similar in different cell types and ranges, on average, from 250 to 350 mV. Cell electropermeabilization can be either transient or stable,

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depending on the magnitude and the time length of the imposed transmembrane potential, which in turn is related to the external applied field. Characteristic DC field strengths required for electropermeabilization are in the range of 1 kV·cm−1 for most of these cell types, whose size is typically comprised between 10 and 40 μm. The electropermeabilization kinetics is dependent on the voltage across the cell membrane, with time constants of the membrane charge (see the second section) in the 0.01-0.10 ms range at normal mammalian body temperatures (28). In the first section of this chapter, we discuss pore- and non-pore- based models of electropermeabilization; then, we focus on the main subject, which is an in-depth analysis of pore-based models of cell permeabilization. Specifically, we analyze the effects of electric fields onto cell membranes from a physical perspective (second section) and thereafter discuss the most interesting mathematical models that have been proposed to microscopically describe the electroporation events (third section). Later on, the contribution to this field of alternative methods of molecular simulation is assessed (fourth section). The last section, which is the longest, examines the issues associated with the mathematical-physical modeling and biomedical optimization of electropermeabilization in vivo. This final section is an original and systematic organization of theoretical analyses and results contained in many publications about electroporation from different scientific perspectives, and aims to be a stimulus to transfer experimental, clinical and instrumental complexities into consistent mathematical models by further investigation. Not surprisingly, due to the great heterogeneity of the investigations performed on cell electropermeabilization, we found divergent opinions and conflicting data and analyses about a number of issues, which we tried to report in a way that could help the reader to develop his/her personal point of view. Some results have a broad scope and others are restricted to specific applications or subfields: hopefully, the papers and books cited in this chapter should support a clear definition of these two categories of information. Since the application of electric fields in living bodies may involve phenomena and effects that go beyond the poration of plasma membranes (see, for example, the model of skin poration in the fifth section) some supplementary information is given, in order to make this publication more valuable for investigators interested in in vivo applications. The majority of models presented are properly applied when the cell system meets specific criteria: even though we discuss many limitations in the text, some are left out, for the sake of brevity, and can be found in the cited bibliography. Additionally, in the attempt to make each section appropriate for scientists with different backgrounds, the mathematical parts have been carefully selected and, where multiple ways existed to write the same equation, the most intuitive one has generally been our favored choice. For a similar purpose, mathematical formulations and analyses have often been matched with ad hoc qualitative descriptions of the role played by single variables. All the equations have been reported using standard mathematical notation and with minor style and symbol changes with respect to the originals, in order to help readers who wish to consult the listed references.

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I. ARE THERE ALTERNATIVE MODELS OF ELECTROPERMEABILIZATION? From its inception (1, 2), electroporation has been the most popular theory among scientists for explaining the membrane permeabilization after the cell is exposed to electric fields (29). This theory is supported by thermodynamic arguments: it is hypothesized that the electric field increases the surface tension of the plasmalemma and that, when pores appear as in a nucleation process, they lower the system‘s electrostatic energy (30), by reducing the available area and increasing the conductivity of the lipid bilayer (31). Interestingly, we do not yet have a clear proof that pores appear on the cell membranes (29), even though a number of investigations pointed towards a pore-based process. For instance, some experiments of electrical conductance and light scattering published in the late ‗90s detected the induction of membrane leaks under the application of electric fields (32 and references therein). Before them, a well-known article of Chang and Reese reported large post-pulse pores in red blood cells in hypoosmosis (33), but their results have been questioned by other investigations (32, 34, 35). More recently, ultrastructural modifications in the assembly of lipids and proteins in melanoma cells exposed to electric fields were described (36). Therefore, for the sake of completeness, and because multiple mechanisms and events may possibly be involved at the same time, at least four other electropermeabilization theories should be mentioned: the electrocompression, the electrocompression associated with phase transition, the electrohydrodynamic instability, and the wave instability model. Briefly, the first model postulates that the ―bilayer lipid membrane‖ behaves like an elastic capacitor with a constant elasticity and that a high transmembrane voltage can induce its breakdown by compression; nonetheless, this theory seems to have severe inconsistencies, such as the large over-estimation of the critical transmembrane potentials (37-40). The second model was designed for including temperature effects on phospholipid bilayers, as an extension of Jacobs‘ analysis (41). This theoretical description, while being similar to the first model, specifically assumes that a solid-liquid phase transition also happens and that, when the voltage across the membrane is above 280 mV, the membrane ruptures (42). The third model looks at the system as a planar layer of non-conducting liquid separated by two charged conducting liquids and describes how electric fields promote a faster coalescence of water droplets; this description has important caveats, in particular as for the role played by the increasing transmembrane potential in determining the bilayer lifetime (43). The fourth model, focused on the membrane stability in the presence of different wavelength perturbations, states that the increase in surface tension and viscosity enhances the membrane lifetime, but fails in describing the stochastic nature of the breakdown event as well as the direct relation between transmembrane voltage and bilayer susceptibility to the permeabilization events (44-46). More information about these models can be found in a review by Chen et al. (47), which inspired much of this section, and in Teissie et al. (32). Altogether, despite the fact that we have been clearly stating that electroporation is a theory (29), it is also, in our judgment, the best descriptive model that has been proposed at the present time and for this reason is the subject of the next four sections. For a graphical and detailed description of the steps that, according to Neumann‘s model, lead to the development of electroporative channels, see Somiari et al. (48).

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II. PHYSICS OF ELECTROPORATION FROM THE GENERAL AND KINETIC STANDPOINTS Based on a minireview dedicated to this subject by Teissie et al. (32 and references therein), the process of electroporation is conveniently split into five steps, namely: induction or trigger, expansion, stabilization, resealing, and memory effect. They happen on a timeframe of microseconds, milliseconds, milliseconds, seconds, and hours, respectively. In a simplified description, the cell is surrounded by a conducting medium and has an inner conducting cytoplasm, and the two compartments are separated by a lipid bilayer. Calling the external conductivity λext, the cytoplasmic conductivity λint, the membrane conductivity λmemb, and E the electric field intensity, the induced potential difference (ΔΨE) as a function of the time t can be expressed (32) as: ΔΨE(t) = g(λext, λmemb, λint)·E·k·r·cosθ·(1 − exp(− t/τ))

(2.1)

where k is a form factor accounting for the impact of the cell on the extracellular field distribution (32), r is the radius of the cell (assumed to be spherical) (32), θ is the angle between the normal to the cell membrane and the field direction (32), τ is a membrane charging time constant (28, 32), and g is a function of the three involved conductivities (32). Specifically, assuming that the membrane has thickness d, g can be calculated (32) as: g(λext, λmemb, λint) = [λext∙λint∙(2∙d/r)]/[(2∙λext + λint)·λmemb + + (2d/r)∙(λext − λmemb)·(λint − λmemb)].

(2.2)

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In the most general situation, τ can be physically defined as: τ = r·Cmemb∙(λint + 2∙λext)/[2∙λint∙λext + r∙λmemb∙(λint + 2∙λext)/d].

(2.3)

In (2.3), Cmemb is the membrane ―specific capacitance‖, which has the International System (SI) units of F∙m-2, where F is the abbreviation for farad and m for meter (see also equation (5.1)). Clearly, we can split formula (2.1) into three parts, the first including E and g (electrical variables), the second involving k, r and cosθ (geometrical variables), and the third (made by t and τ) accounting for the time evolution. The value of the factor k is 1.5 in the case of a spherical cell (26); however, this is a modeling simplification (49, 50) and, indeed, a generalization of these concepts is discussed in the subsection ―Cell and tissue properties‖, inside the fifth section. The use of equation (2.2) allows relaxing one of the basic assumptions of this model, i.e., that the cell membrane has to be a dielectric, and enables us to better deal with real cases, where leaks are present or appear in the membrane during the process (λmemb ≠ 0). After a reasonable time following the administration of the electric field (t→∞), the formula describing the steady state can be written (32) as: ΔΨE = g(λext, λmemb, λint)·E·k·r·cosθ.

(2.4)

The permeabilization occurs when and where the potential difference crosses a critical poration value (ΔΨp), which has been estimated as being not less than 200 mV (51). Clearly, Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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E controls the poration, and a critical threshold has been defined also for this variable (Ep), so that when E > Ep, the permeabilization becomes possible (51). According to equations (2.1) and (2.4), Ep is cell size-dependent, and indeed it roughly ranges from 0.1 kV·cm-1 for large cells (e.g. muscle cells) to 1-2 kV·cm-1 for small cells (e.g. bacteria) (51). Interestingly, taking also into account the role of the angle θ in these two equations, cases of cells incompletely porated, because some of their membrane regions are above and others are below the poration threshold ΔΨp, are not exceptional. The expansion and stabilization phases were described and experimentally analyzed in several papers (52-60). While the expansion is progressing, the membrane goes through a transition characterized by abnormally high conductance and permeability. During this phase, the electric field intensity controls the geometry of the part of the cell affected, while the pulse duration controls the density of the perturbation of the cell membrane. Interrupting the administration of the electric field induces the stabilization process, during which i) the electric field becomes subcritical (E < Ep), ii) there is a decrease of the conductance in the permeabilized part of the cell membrane, and iii) the plasmalemma shifts from highly permeable to variously leaky (32, 60). During the resealing, the membrane leaks are annihilated. As stated before, sealing kinetics takes seconds and is therefore generally much slower than the electric field relaxation, because pushing water out of the formed hydrophilic pore and the rearrangement of the lipid bilayer into the normal state require the overcoming of relatively high free energy barriers (28). Independent investigations showed that lowering the temperature slows down the cell resealing and raises ―the longevity of the porous membrane states‖ (61), with important consequences for the optimization of the molecular uptake (61, 62). Looking at the electroporation from a complementary perspective, Krassowska and coworkers (63) focused on the way the opening of a pore dynamically affects the transmembrane potential Vm. Vm is modeled, in an experimental setting having a uniformly polarized membrane, by using the following formula: C·dVm/dt + (1/Rs + 1/R)·Vm + Σj ip(rj, Vm) = V0/Rs.

(2.5)

Here, C and R are capacitance and resistance, and are respectively equal to Cm·A and Rm/A, being Cm, Rm, and A the surface capacitance, surface resistance and total area of the membrane; Rs is the series resistance of the experimental setup and V0 is the ―external stimulus‖ (i.e., the voltage applied to the membrane through Rs). In the above equation, the K total ip(rj, Vm) currents are the currents through the K pores, and the formula that depicts each of them is: ip(r, Vm) = Vm/(Rp + Ri).

(2.6)

According to Newman (64), Rp is the pore resistance and Ri is the input resistance; their values are defined as Rp = h/(π·g·r2) and Ri = 1/(2·g·r), where g is the conductivity of the solution and h is the membrane thickness. Equation (2.5) can be flexibly used: when the voltage is beyond the threshold ΔΨp, the number of pores (i.e., K) grows and their radii expand (i.e., there is a reduction of Rp and Ri) and this accounts for a steep increase of Σjip(rj, Vm). Instead, in the same framework, when the electric pulse is turned off, V m→0, r→rm and pores undergo the resealing process (63). Specifically, it is modeled that pores

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convert to a hydrophilic configuration and annihilate by fluctuations of the lipid bilayer when their size is rm. For a microscopic explanation of these events, see in the next section equations (3.2) and (3.3), keeping into account that during the resealing the pore density is greater than the equilibrium pore density (i.e., when Vm = 0) and the right-hand term of equation (3.2) is negative (63). A formula commonly used for describing the molecular diffusion, which is the most prominent mechanism involving small molecules that move across the lipid bilayer (65) and that is made possible from when membrane leaks appear until when the resealing is completed, expresses Fick‘s first law (32, 62): Φ(S) = − D·A·dS/dz

(2.7)

where Φ(S) is the flow of a solute S, D the diffusion coefficient of S across the membrane, A the membrane area subject to diffusion, and dS/dz the solute concentration gradient across the bilayer and along the direction z, which is perpendicular to the membrane surface. Using a finite difference instead of a differential notation, in the case of biological membranes the solute flow is also reported as:

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Φ(S) = P·A·ΔS

(2.8)

where P is ―the permeability coefficient of the solute S to the membrane‖, A is described as in (2.7), and ΔS is ―the concentration difference of S between cell interior and exterior‖ (62). However, investigators have shown that other mechanisms of molecular transport are also involved in the electroporation process, beside diffusion: they are discussed in the part entitled ―Molecular uptake‖, inside the last section. The memory effect refers to the fact that some cells, which escape lysis by resealing, still may go through cellular alterations (such as the asymmetrical lipid distribution) that, in the long term, are typically lethal (i.e., they usually induce cell death and apoptosis) (66-68). Because the variability of these cellular changes and/or damages among recovering cells seems to be important, and has the potential to affect the efficacy of electroporation, it would be interesting to see this last phase modeled as a stochastic process.

III. MATHEMATICAL MODELING OF ELECTROPORE NUCLEATION AND EVOLUTION The mathematical description of the creation of pores and of their time-dependent changes in size is particularly important for the optimization of biomedical protocols based on electroporation. Indeed, these microscopic models aim at giving clues about key experimental variables, such as the size of created pores, the time of pore existence, and the relationship between reversibility of the porated status and severity of the membrane injury (63). The pore dimension is critical because, for instance, when the delivered molecules are antibodies having sizes in the range of nanometers (69), pores need to accommodate for their bulk (70); similarly, in the case of transfections, a good DNA uptake is expected only if pores are larger than 9 nm, according to the research of Rybenkov et al. (71). The opening time is

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another paramount topic, because there is evidence that it is insufficient for the macromolecular uptake when it is shorter than one millisecond (72, 73). The poration reversibility instead deals with not inducing directly cell death (see also the memory effect of the previous section), since the whole process generally aims to allow a genetical reprogramming or a molecular interference (by drugs, proteins, etc.) with the cellular biological mechanisms and/or pathways, which requires the cell viability. Of course, this description is still valid for electrochemotherapy (ECT), where the killing effect is druginduced and, for the most part, not directly dependent on the electric field. Cell viability is a particularly interesting issue, since it differentiates the electropermeabilization protocols from the irreversible electroporation (IRE), which is used for drug-free tissue ablations and whose final goal is obtaining a direct and localized cell death (74, 75). Until some years ago, the most commonly used model for describing the pore concentration in the presence of an external electric field was based on the Smoluchowski's equation (76). This formula, using the notation of Barnett and Weaver (77) is:

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  2 n   n U   n  Dp  2    t r  kT r    r

(3.1)

where n = n(r, t) is the pore density function (these pores have radius between r and (r + dr) at time t), Dp is the diffusion constant for the pore radius, k is Boltzmann‘s constant, T is the temperature in the Kelvin scale, ∆U is the pore energy, and rmin ≤ r ≤ rmax. Notably, rmin is generally set at 1.0 nm, i.e., just above 0.7 nm, which is the approximate size of the hydrophilic head groups, while rmax is set at 2∙γ/Γ, with γ and Γ respectively defined as the edge energy of the pore and as the surface tension of the membrane-water interface. These two boundary conditions for r are chosen in order to allow the equation to more realistically deal with: i) the physical limitations of pore size in a bilayer membrane; ii) the existence of a population of stable pores; iii) the rate of pore diffusion. A thorough discussion of the mathematical studies related to this partial differential equation (PDE), including the parameters used, is beyond the scope of this chapter; we refer readers interested in a numerical solution to the above cited paper (77). Importantly, the Smoluchowski's equation was depicted as inadequate to comprehensively cope with the three issues mentioned at the beginning of this section (63). Indeed, although some discrepancies between the theoretical expectations derived from this formula and the experimental electroporation data have been partially explained by proposing a specific mechanism of interaction between carried molecule (e.g. DNA) and sets of small pores in the cell membrane (73, 78-81), other deficiencies depending on its mathematical behavior are not negligible (63). Therefore, an alternative elegant analysis that addresses this set of topics has been proposed by Smith et al., who used a nonlinear extension of the Smoluchowski's equation (63). Their multi-phase description is as follows: all pores are initially hydrophobic and convert into long-lived hydrophilic if their radius becomes greater than a critical level r* (approximately equal to 0.5 nm) (82). In order to model this generation by an ordinary differential equation (ODE) Krassowska and co-workers (63) assume that pores are created directly with a minimum-energy radius rm. The describing ODE is: dN/dt = α·eβ·{1 − N/[Neq(Vm)]}.

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(3.2)

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Specifically, the equilibrium pore density at Vm (i.e., Neq(Vm)) is given by the following formula: Neq(Vm) = N0eq·β.

(3.3)

In (3.2) and (3.3), N is the pore density, Vm is the transmembrane potential, Vep is the electroporation voltage, α is the pore creation rate coefficient, β = (Vm/Vep)2, N0 is the equilibrium pore density at Vm = 0, and q = (rm/r*)2. According to Neu and Krassowska (83) the hydrophilic pores originally having radius r  r* grow quickly, thus reducing the membrane resistance; consequently, the transmembrane voltage increase is slowed down and eventually the cell membrane potential is reduced. The evolution of electropores is based on the principle that they have to minimize the energy W of the lipid bilayer (63). Starting from rm, the time derivative of rj (radius of the j-th pore), for each of the K pores, is given by the following differential equation: drj/dt = − (D/k·T)·(∂W/∂rj)

(3.4)

where D is the diffusion coefficient of the pore radius, k is the Boltzmann constant, T is the temperature in Kelvin and, clearly, 1 ≤ j ≤ K; interestingly, the above mentioned work by Smith et al. contains also an analytical description of W (63). A reformulation of (3.4) by means of the advection velocity U is in an article of Krassowka and Filev (84). A complementary modeling, also based on U, focuses on n(r, t), pore density distribution, by means of the following advection-diffusion PDE (83):

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∂tn + ∂r{Un − D∂rn} = 0

(3.5)

with r ≥ r*, D diffusion coefficient, and ∂r(D∂rn) representing the random fluctuation of pore radii, which depends on the thermal energy; t stands for /t, the partial derivative with respect to time and r is /r, the partial derivative with respect to r. This equation is part of a boundary value problem that, in conditions like those of most experimental applications, is stiff and whose analysis can be found in the original article of Neu and Krassowka (83). In a perspective favorable to translational research, the parameters used by these two investigators (63, 83-87 and references therein), which are reported in several equations of this and the previous section, are generally based on pre-existing experimental data or on bona fide estimations and are referred to standard or average experimental conditions.

IV. A DIRECT VIEW ON ELECTROPORE FORMATION: THE PERSPECTIVE OF MOLECULAR SIMULATIONS A way of obtaining information on the molecular details of material processes is through atomistic/molecular simulation methods. Although the data coming from these simulations are produced in silico, they can suggest physical mechanisms that are out of reach in a real macroscopic experiment, both in vitro and in vivo. As a short introduction to this subject, the basic ideas and methodological issues of molecular simulations are discussed below; for a

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more detailed treatment we refer to general references (88-90) and to a recent review on the applications to biological systems (91). The basic methods used in molecular simulations are Montecarlo (MC) and Molecular Dynamics (MD). Both approaches are based on mechanical models of the materials under study at atomic resolution. In general, the atomic degrees of freedom are consistently modeled by a potential energy function, which either enters the canonical MaxwellBoltzmann distribution (MC) (92) or, by differentiation, produces the forces (see below equation (4.1)) that make the system to evolve in time (MD) (93). MC simulations are equilibrium time-independent methods for statistically sampling the Maxwell-Boltzmann probability distribution of the atomic degrees of freedom over the phase space of the system; the sampling is obtained by using algorithms for the generation of random numbers (88). The MD approach allows not only a statistical sampling of equilibrium properties by averaging over the very long trajectories generated by the dynamics, but also to directly access the dynamical evolution of microscopic quantities to be related to the macroscopic experiments (88). In MD, the forces acting on each of the N atoms that make up the system are derived from a potential energy function U(r1,r2,…,rN), whose parameters are estimated from experiments on model molecular systems or from quantum-chemical calculations (91). From the potential energy function, as it is well known, the force acting on the i-th atom can be computed as:

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Fi(r1,r2,…,rN) = − iU(r1,r2,…,rN)

(4.1)

that is minus the gradient of the potential energy function with respect to the set {ri} of the three Cartesian coordinates of the i-th atom, i  ri (93). Forces are calculated for all the N atoms of the system and then entered into classical Newton‘s equations of motions. These equations are solved, at each time step (typically, femtoseconds), through standard numerical integration algorithms and the generated time evolution of the molecular system can be colorfully visualized through computer graphics interfaces, such as ―visual molecular dynamics‖ (VMD) (94). The general form of the potential energy function is:

U(r1 , r2 ,..., rN )  

1 1 k b (b  b 0 ) 2   k (   0 ) 2   2 bonds 2 angles 1  k (1  cos(n   ))  2 dihedrals

(4.2)

   12   6  q q   ij ij i j   4 ij             rij   Drij   rij  i< j       N

that is used, with minor modifications, in all known force fields for biomolecular simulations. Among the packages used for these simulations, the ―Groningen machine for chemical simulations‖ (GROMACS) is, probably, the most popular (95-97); it is open source software and is available on a website (98). The first two sums of (4.2) are ―harmonic terms‖, characterized by ―elastic‖ constants kb and k and related to the distortions in the set of bond lengths {b} (with respect to the equilibrium bond lengths {b0}) between pairs of atoms, and in

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the set of angles between bonds {} (with respect to the equilibrium values {0}), defined by triplets of atoms. The two sums are over labels bs and s, defined for each pair and triplet of bonded atoms. The third term is a sum over labels , dihedral angles defined for each quadruplet of bonded atoms, and takes into account local torsional energies; k are torsional constants and n and  are tunable parameters for each dihedral. The fourth term takes into account interactions between pairs of non-contiguous, non-bonded atoms, which are modeled, for each pair of atoms labeled by i and j, by the Lennard-Jones potential (parameterized by energy barriers ij and equilibrium positions ij) (92) and by the electric potential (parameterized by the partial charges qi and qj) (90). In this last term rij is the distance between atom i and atom j and D is the dielectric constant. All the terms in equation (4.2) can be expressed as functions of the atomic Cartesian coordinates (90). MD simulations of lipid membranes under the effect of an electric field and realized though the GROMACS suite (95-98) have been described in a number of papers (99-108). One MD research, in particular, created interesting animations that give a vivid representation of molecular simulations applied to electropermeabilization (109, 110). Most MD simulations have been focusing on the kinetics of pore formation, which is a basic theme in the study of the electroporation induction step. The investigations of Böckmann et al. (107) and Ziegler and Vernier (108) can be seen as representative of the status of the art in this field, together with the work of Tieleman (109), and are particularly interesting because the main limitations and source of artifacts are clearly defined by the authors. The picture emerging from this research is that of a pore that is initiated close to a head group defect (fluctuation) in which a cluster of polar heads points inwardly, with respect to the zero field average conformation. At the location of these defects, induced on the outer side of the membrane by the external electric field, water molecules start to penetrate the lipid bilayer, eventually forming hydrogen bonded chains of molecules. The phenomenon is dynamically driven by the electric field, which tends to orient the dipoles of both water and head groups. The time scale on which the pore is formed is around 200 picoseconds. At later times the pore becomes stabilized and presents an inner hydrophilic interface. If the field is removed the pore gradually collapses and resealing is eventually observed. It is worth mentioning that Böckmann and collaborators introduced a method for connecting the pore formation kinetics in MD simulations with kinetic experimental data, which is a major achievement (107). Ziegler and Vernier‘s main contribution is the operational definition of a ―minimum porating field‖, a key quantity that can also be usefully confronted with experimental data (108). It is remarkable the crossconsistency of these two papers, as well as their focus on the issue of the relationships between simulations and experimental data. An important theme also studied by Ziegler and Vernier is the cell membrane response to the application of very short and high intensity pulses (108). In the general setting of an electroporation experiment, microsecond, kilovoltper-meter pulses are used, which create pores that allow the migration of charged and large molecules across the membrane (104) (see also the third section and the subsection ―Molecular uptake‖ inside the fifth section). However, some recent oncological applications of electroporation have been using nanosecond pulses with promising results (111, 112). The application of nanosecond, megavolt-per-meter pulses in the simulations is particularly interesting, since it is based on electric fields with higher power but lower energy, which can possibly be used for effective and less invasive treatments. Therefore, the paper of Ziegler

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and Vernier, in which four lipid membrane systems are compared, is groundbreaking in the electroporation modeling (108). As mentioned in the second section of this chapter, the electropermeabilization process can be viewed as the sequence of five events: induction, expansion, stabilization, resealing, and memory effects (32). It would be important to have molecular clues about all of them, thus entailing time scales from microseconds to hours. Nonetheless MD, despite the increasing power of computers, is still limited to the simulation of a restricted portion of a membrane bilayer, sufficient to accommodate just one pore and for times not exceeding, ordinarily, a few dozens of nanoseconds (91). Therefore, MD falls largely short with respect to its goal, and the related investigations can focus only on the in silico description of the first two steps and with a small spatial target. In order to overcome these limitations, it will probably be necessary to develop coarse grained models, based on self-consistent effective solvent models and on ―super-atoms‖, whose dynamics is governed by specific force fields that generalize at lower resolution the usual force fields (i.e., those embedded in the GROMACS package). On the other hand, despite the undeniable progress made, a major concern in using coarse grained force fields is still in their limited universality and portability across different molecular systems (113). MC methods have not been particularly used in the field of electroporation, since MD is more suitable for the generation of a microscopic view (109, 110). Notably, the potential of this simulative approach was recently underscored in two articles by Kotulska et al. and Kubica (114, 115). In these papers, the ―induction step‖ or pre-pore membrane excitation in the electroporation process is studied by using the very simple lattice model of a dipalmitoylphosphatidylcholine (DPPC) bilayer, with highly stylized interactions. These studies were able to confirm that the pore ignition is due to a tilt of the hydrophilic polar heads, as seen also in the more detailed MD simulations, and that the model membrane starts to change its organization if the applied electric field exceeds 107 V/m, corresponding to a transmembrane potential of approximately 50 mV. When the field is increased above a critical value of 0.5108 V/m, corresponding to a potential of about 250 mV, the reorientation of the polar heads is abrupt, leading to a model of violent pore formation: the value of this critical field is consistent with the experimental findings. Remarkably, in these types of models there are no explicit dipolar water molecules taken into account. However, in MD simulations the coupling between the electric fields and the electric dipoles of both water molecules and polar heads is the key mechanism of the early stages of pore formation, as quoted above. In our opinion, the importance of MC studies is not in proposing detailed molecular descriptions, as in the case of MD, but instead in searching for universal generic mechanisms of pore formation, much in the spirit of statistical mechanics. Merging together MC and MD approaches could eventually lead to a robust physical understanding of the electroporation process, but there is probably a long way still to go.

V. THE IN VIVO SETTING The mathematical-physical models presented in the second and third section, which are based on idealized situations, are more suitable to describe the in vitro electroporation process, where less variables are involved, rather than the cascade of events in vivo.

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Nonetheless, these models can also be seen as starting points or templates of comprehensive theoretical descriptions, which take into account specific biomedical information and experimental results collected in animals and humans. Rephrasing what was written by Somiari et al. in a more extensive way (48), the investigator‘s effort should be addressed to optimize all the parameters at best of the present knowledge in order to maximize the molecule delivery and to minimize irreversible cell damage in vivo. As explained in the third section, also the ECT is not an exception to this criterion. Proper mathematical models may significantly help the development of more effective, convenient, safe, and reproducible methods and protocols for the purpose of molecule (e.g. ions, dyes, drugs, DNA) delivery to a variety of tissues, in clinical as well as in basic research. Therefore, in this section we analyze some major and inter-related issues that are critical for modelers of in vivo electroporation: i) multiple electrical-instrumental options; ii) geometrical and biophysical properties of cells and their milieu; iii) heat generated by the electric field; iv) physiological dynamics and reactivity; v) molecular uptake; vi) adverse effects. Although some of these points play an important role also in the in vitro setting, they are all discussed here, since the need for their optimization reaches its climax in vivo. Results reported in this section are mostly referred to the investigation of mammal species and, specifically, to rodents, pets and human patients, both because the electroporation literature about them outweighs the published works about other species and because their tissues, organs, apparatuses, and organ systems rank among the highest in terms of complexity across living organisms, thus representing a serious challenge for modelers (27, 116-119). Electrical-instrumental options. New techniques, machines, devices, and protocols are constantly in development for the many applications of electroporation, thanks to the dedication of bioengineers and experimental scientists. An exhaustive discussion about them and the related literature would therefore deserve a separate essay. Instead, in this subsection we will examine some of the main technical variables and instrumental options that have been driving the development of electroporation. The key issues to which we will restrict our analysis are: i) electrode size, shape and composition; ii) electrical field strength; iii) pulse duration; iv) pulse shape; v) total number of applied pulses; vi) pulse frequency (26, 73, 75, 120-124 and references therein). The electrode size by itself plays an important role in electroporation, because it decisively contributes to defining the volume where the electric field is induced. Nevertheless, a more complex issue lies in the combined effect of electrode size and shape. Plate electrodes were the first used in ECT, but have been raising substantial doubts about their capability to reach the deepest tumor regions and, more broadly speaking, about warranting a homogeneous performance to electroporation applications in vivo (120, 125). Additionally, they are not suited for every anatomical location, in particular when space is constrained and internal organs are treated (120). Even among externally reachable anatomical sites, plate electrodes are very efficient in treating general cutaneous and subcutaneous targets, but may have difficulties in accessing a number of areas, such as caudal oral cavity, rectum, eyes, etc. (125). The six-needle array electrode, which is a well known alternative (120, 125-127), is mechanically advantageous for intraoperative purposes (e.g. fibrosarcomas) (125) and small tumors, particularly in sensitive areas (127), but the clinical optimization for its field intensity is not simple, as shown by the mathematical work of Dev et al. (128). Davalos et al. (129) investigated which parameters are the most significant for reducing tissue heating (see the dedicated subsection ―Electric field and generated heat‖

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below) and showed that the geometry (i.e., diameter and spacing) of the electrode with two needles is a prominent factor in determining the thermal distribution. The plot of the allowable pulse lengths for which every part of the tissue is exposed to a temperature lower than 50 °C, which shows that pulses can be much longer for plate electrodes rather than for needle electrodes, is indeed quite remarkable (129). In the same paper, Davalos et al. noted that the critical total pulse length (= (number of pulses)·(time durations)) of needle electrodes is short enough to allow, in typical electroporation conditions, an irreversible damage to the tissue surrounding the electrodes, in agreement with the results of Miklavcic et al. (130). However, it was also suggested that spontaneous heat dissipation through the blood flow, the treated tissue, etc. may help improve the electrode performance (129). Besides the two types of needle electrodes mentioned above, many other versions have been used in experiments and clinical applications. These electrodes can be different from each other because they may or may not have a central needle, diversely manage the pulse administration, have a variable number of needles, etc. (120, 125). Interestingly, electrodes of completely alternative design have been recently proposed (e.g. laparoscopic pinch and unipolar plate) (125), but a complete evaluation of their effectiveness is not yet available. A third factor, in addition to size and shape, which has to be considered when modeling the role played by the electrodes, is their chemical composition (26). For instance, concern has been raised about aluminum electrodes, which would be able to dramatically change the pH due to cathodic electrolysis processes, especially under a strong electric treatment (131) and/or long pulses (26). Research in vitro showed that, for short pulses (100 microseconds), the percentage of permeable cells rises in a sigmoidal way when the field strength increases, although this is only effective for delivering small molecules. Instead, for long pulses (5 milliseconds) both small molecules and macromolecules cross the cell membrane (62, 73). It is helpful to discuss electrical field strength and pulse duration together also in vivo because, according to Gehl (26), short pulses (in the order of tenths of milliseconds) are efficiently administered with a high field strength (in the order of kV·cm-1) (132), while long pulses (in the order of tens of milliseconds) can be proficiently coupled with a low field strength (in the order of tenths of kV·cm-1) (133). Indeed, there is empirical evidence that the effects produced by pulses that have short duration and high voltage-to-distance ratio (i.e., the ratio between the applied voltage and the distance between the electrodes) or, instead, long duration and low voltage-todistance ratio are similar, in that both conditions generate approximately the same amount of heat (48). An indirect confirmation of this has been proposed in some investigations (134, 135), which showed that the transfection efficiency was roughly proportional to the amount of heat generated, whenever the investigators worked below the threshold of irreversible cell damage. According to the in vitro acquired knowledge, the combination favored for producing the best results for the specific case of DNA delivery is pulses with low field intensity and long duration (i.e., milliseconds) (73, 136, 137). In fact, this combination preserves the cell viability but still allows an efficient DNA transduction. However, a study in vivo reported that the best transfection performances were reached when the scheme of short pulses and high values of the voltage-to-distance ratio were used instead (138). There is evidence that the pulse duration is dependent on the kind of application, so that, for instance, electro-gene therapy requires longer pulses, in general, than ECT (139). Specifically, pulse durations ranging from 100 microseconds to 50 milliseconds have all been successfully employed for gene transduction (140), while pulses generally not longer than 1 millisecond (141), and often equal to or lower than 100 microseconds (16, 23, 24, 125, 142, 143) have

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been effective for ECT, in agreement with the already mentioned existence of different mechanisms of molecular transport (see below the subsection entitled ―Molecular uptake”). The most common types of waves have been, since the pioneering years, the square, which has a defined intensity and duration (73) and maintains the voltage throughout the life of the pulse (144), and the exponentially decaying (145). Takahashi et al. showed that in human leukemia cell lines the square wave outperforms the exponentially decaying (146), which, in turn, is considered more efficient for skin electroporation (147, 148). These data suggest that the optimality of this physical variable is fairly context-dependent. More recently, other waves have been proposed for electroporation, in the attempt to achieve higher efficiency and greater adaptability to the in vivo setting. Among them, deserving of being mentioned are the exponentially enhanced pulse (EEP), which starts at a very low voltage and is gradually increased up to a pre-determined maximum voltage (144), and bipolar oscillating pulses (149), such as the bipolar square pulse, which gives higher membrane permeabilization levels than unipolar pulses (150), and the rectangular-like biphasic pulse (149) administered in bursts (i.e., pulse trains with short interpulse intervals) (151) for reducing the electroporation morbidity (see also below the discussion about pulse frequencies) (152). The EEP has been successfully used for delivering plasmid DNA (144), while the bursts of rectangular-like biphasic pulses were originally optimized for ECT (151, 152). Interestingly, bipolar oscillating pulses are supposed to be more conservative of the pH and, more generally, of the physicochemical properties of the medium (131, 153). Finally, we wish to point out, as representative of modeling studies on this subject (122 and references therein), the work of Kotnik et al., who tested the in vitro efficiency (in terms of permeabilization, cell death and molecular uptake) of different unipolar rectangular/trapezoidal and sine-modulated pulses as well as bipolar rectangular, sinusoidal, and triangular pulses. They reported that the time during which the voltage-to-distance ratio exceeds a critical threshold is the factor that most likely determines the efficiency of electropermeabilization, independently of the pulse shape (122). According to experiments in vitro, the more pulses there are in a sequence, the greater is the obtained permeabilization, but this positive correlation is lost for macromolecules like DNA if the pulse is not long enough (namely, for durations below the millisecond time range). Additionally, it was described that keeping the cumulative pulse duration (i.e., the total pulse length as defined above) constant the number of permeabilized cells is approximately unchanged. Instead, the total number of delivered molecules is increased when the highest number of pulses is applied, thus evidencing an interesting difference between these two measures of electroporation efficiency (73). Another study in vitro showed that increasing the pulse number amplifies the slope of the curve that describes the molecular uptake as a function of the field strength, so that, for a fixed value of the electric field, more molecules per cell are delivered when more pulses are administered (154). A recent in vivo investigation in muscle tissue reported that a sequence of four low field strength pulses (80 V·cm-1) applied after a high field strength pulse (600 V·cm-1) warrants the highest efficiency of electrotransfection, when compared with sequences that have the same cumulative pulse duration and that are made by more (eight) or less (one) total pulses (155). These results confirm the need to keep into account general and case-specific criteria in the optimization of the total number of pulses applied. The development of a model that would estimate the probability of cell populations to be porated, as well as the number and size of pores formed,

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while keeping into account the number of electric pulses administered and their length, would likely help to better frame the above illustrated experimental results. Frequency plays a role in electroporation at least in terms of repetition frequency for trains of pulses and in terms of frequency of the cycle of the waveform. In experiments of gene transfer in vivo, where the optimality as for field intensity, pulse length and number of pulses was already reached, a considerable gain of efficiency was obtained by using square waves with higher pulse repetition frequencies, in the specific range 0-2 Hertz (Hz) (133). In order to assess if any reduction of the side effects associated with such low repetition frequencies is possible (see also the subsection entitled ―Clinical adverse events‖), Pucihar et al. compared very high (in the order of kHz) and standard repetition frequencies (in the order of Hz) in in vitro experiments (156). Briefly, their paper shows that the molecular uptake efficiencies obtained in the range comprised between these two extremes are reasonably similar, thus suggesting the possibility to use high repetition frequencies also in in vivo applications (156), and in agreement with the approach previously followed by Daskalov et al., who used repetition frequencies of 1 kHz in ECT (151). According to Chang et al., alternating current (AC) has a better preservation of the cell viability and yields higher efficiency in electroporation applications with respect to DC (157); it is therefore worthwhile to theoretically analyze the effect of frequency changes of the AC waveform. This situation is described by the Schwan equation for AC fields (158, 159), which, using the formulation (for pure dielectric membranes (see the next subsection)) of Marszalek et al. (160), states:

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ΔΨE = 1.5∙r∙E∙cosθ/[1 + (ωτ)2]1/2.

(5.1)

In (5.1), ΔΨE is the transmembrane potential of AC fields, 1.5 is a constant value corresponding to the factor k already discussed in equation (2.1), r is the (outer) cell radius, E is the applied field strength in volts per centimeter, θ is the angle between the field line and a normal from the center of the spherical cell to a point of interest on the plasmalemma, and ω = 2∙π∙f. The variable f is the waveform frequency of the applied AC field, while τ = r∙Cmemb∙(ρint + ρext/2). Cmemb, ρint, and ρext are, respectively, the (specific) capacitance of the membrane in F·cm-2 (see also the second section), the resistivity of the cytoplasmic fluid, and that of the external medium, both in Ω∙cm (160). The Schwan equation suggests that ΔΨE becomes lower when the AC waveform frequency is increased; remarkably, this result is in agreement with in vitro experimental results, which show that, above 100 kHz, the cell permeabilization decreases with the AC field frequency (124). In the last years researchers have indeed been considering AC fields also in the in vivo setting: the positive results of Liu et al., who used alternating current sine-waves (ACSWs) with a waveform frequency of 60 Hz and a field strength of 20 V·cm-1 for electrotransfection of skeletal muscle of mice (161) will hopefully warrant further investigations concerning the applications of this type of electric fields. Cell and tissue properties. Equations (2.1), (2.4) and (5.1), which describe the potential of the lipid bilayer when an electric field E is applied, indicate that reaching the permeabilization threshold directly depends on the cell radius r. Specifically, as already mentioned in the second section, the larger is r, the more sensitive the cell is to an applied electric field (51). When cells have large variations in size, like it happens, for example, for Ehrlich ascites cells, a good level of permeabilization can hardly be obtained (26), probably because it is difficult to choose a field intensity E, which is appropriate for the full range of r

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values of the cell population treated. However, even more puzzling is dealing with the cell shape factor, except for the case of a perfectly symmetric sphere or any close approximations. A more general shape that has been studied for this issue is the spheroidal model, i.e., a shape with two equal semiaxes and a third one, called the symmetry semiaxis, which can be longer or shorther than the other two. From equation (2.4), which is referred to the steady state of the electric field exposure, it is possible to calculate (for homogeneity of notation with other published formulas) the membrane potential in any point, when the membrane is a pure dielectric (λmemb = 0  g = 1), as ΔΨE = 1.5∙E·r∙cosθ (see the variable definitions in the second section). A compact formula from Valic et al. (162), which has been applied to arbitrarily oriented ellipsoidal cells with a non-conductive membrane, is:

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 E 

 1  ri E i  . i x, y, z 1  L i 



(5.2)

Assuming that the origin of the coordinate system is in the ellipsoid center, ΔΨE is the transmembrane potential induced by E, ri are the three components of the vector of the point P(x, y, z) on the membrane surface, Ei are the three components of E, and Li are three ―depolarizing factors‖ in the x, y, and z directions, which depend only on the geometrical properties of the ellipsoid (162). Notably, Lx + Ly + Lz = 1 and, for spheres, Lx = Ly = Lz = 1/3, for symmetry reasons. Spheroids are special ellipsoids whose modeling is particularly important: indeed oblate spheroids, which have the symmetry semiaxis shorter than the other two, mimic discoidal cells, while prolate spheroids, which have the symmetry semiaxis longer than the other two, resemble ―cigar-shaped‖ cells. Therefore, cases such as discoidal erythrocytes on one side and elongated skeletal muscle cells, cells of simple columnar epithelium, and retina photoreceptor cells on the other can all be reasonably well described using the spheroid approximation (117, 163, 164). There is a considerable list of papers dedicated to the subject, whose results contribute to mathematically describe what happens in spheroidal cells (162, 164, 165), and even offer important clues about the general ellipsoidal shape (166). Here, for the sake of brevity, we refer to an article by Kotnik and Miklavcic, who derived the analytical description of the transmembrane voltage induced by an electric field both on prolate and oblate spheroidal cells using spherical coordinates (167). Their work shows that the maximum values of the induced transmembrane voltage are reached only in a small region of the membrane in very prolate cells, and in the majority of the membrane in very oblate cells. The case of irregularly shaped cells has been numerically analyzed by Pucihar et al. (50), who coupled microscopic imaging for the cell shape assessment with numerical modeling. This group reported a very good agreement between numerical results and analytical solutions for standard cases, such as isolated spherical cells and tilted oblate spheroidal cells, thus giving a first validation of the method. Then, the authors observed a relatively good similarity between experimental and numerically calculated values of the transmembrane potentials of irregularly shaped cells. Finally, it was shown that their numerical method is quite sensitive to cell shape variations, since the replacement of irregular shapes with more regular ones, such as hemiellipsoids, generates results having some relevant differences. This last point further confirms how critical is the exact assessment of the induced potential difference (i.e., ΔΨE) of complexly shaped

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membranes (48, 50, 168), such as those of macrophages, dendritic cells and fibroblasts, which have protrusions, branched projections, etc. in their external surface (117, 163). Going back to the general model, we observe that, since the electric potential difference of the plasmalemma relies on the cell orientation with respect to the direction of the electric field (according to the definition of θ in (2.1), (2.4), and (5.1)), describing ΔΨE at each point of the cellular system may be very challenging, unless the level of cell size variation and asymmetry is fairly limited. This observation is particularly important when planning electropermeabilization in vivo, because in many tissues the relative and global cell orientation is much more stable than in vitro, due to the mechanical linkage to the surrounding cells and to the extracellular matrix (48) via occluding, communicating, and anchoring junctions (163). Additionally, it has been proposed that cell junctions may be relevant for electroporation in vivo (169). This hypothesis received support by the works of Fear and Stuchly (170-172), who analyzed the relationship between gap junctions and transmembrane potential induced by an electric field. Their research offers preliminary clues concerning the effect of AC and DC fields on chains or clusters of cells. Notably, because the induced transmembrane potential for a specific electric field depends on the cell size (see equations (2.4) and (5.1)), it is larger for cells connected by gap junctions than for single cells; the potential also grows with the gap conductivity, particularly beyond conductivity levels around 10-5 siemens per meter (S/m) (171). Limiting the analysis to DC fields and AC fields whose frequencies are considerably lower than the relaxation frequency (i.e., ―the frequency at which the magnitude of the transmembrane potential is 3 decibel (dB) below the DC value‖) it was shown that: i) small groups of cells connected by gap-junctions can be modeled by single cells with similar shape and proper size (i.e., their equivalent cells); ii) gaps influence the potential in the interior parts of cell chains and clusters (171). Interestingly, the relaxation frequency becomes lower when larger cell configurations, smaller gaps, and lower gap conductivities are considered (172). The choice of the appropriate mathematical model can be critical: for instance, when the gap resistivity is higher the current flow in the interiors of the cell configuration becomes increasingly complex and cannot be directly represented by simple models (170). Importantly, the above results were obtained by an applied field set at 1 V/m (170-172) or with a maximum magnitude of 1 V/m (172), even though the authors state that ―results scale linearly with applied field strength‖ (172). Overall, further theoretical investigations and experimental validation are necessary for fully evaluating the relevance of these findings to the issues discussed in this section, due to the computational and modeling limitations of the above studies and to the very large difference between their standard electric fields and those used in electroporation (see the second section). Any theoretical description of electroporation that does not include proteins in the bilayer is partial and simplified, since they represent up to 60% of cell membranes and are acted upon by the applied electric field (28). Notably, a MD study suggests that pores may not be formed close to peptide channels, thus supporting a protein role in the variability associated with pore opening and density (102). This represents further complexity, in addition to the well known physical observations of Weaver and Chizmadzhev regarding: i) a local tendency of newly created pores to stay away from already existing ones; ii) a local migration of pores from each other; iii) a global movement of pores towards the cell poles (corresponding to the positions where θ, as described in equation (2.1), equals 0 and π) (40). Moreover, depending on the experimental conditions and type of analysis, the following information may be relevant for

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evaluating the membrane protein effects: i) the intramembrane electric field has a powerful allosteric action on proteins of the plasmalemma (28); ii) it has been reported the association between membrane pores and channels in denatured Na+/K+ ATPase, when using strong electric fields (28, 173); iii) after the exposure to an intense electric shock, it was observed a reduction of the conductance of both Na+ and K+ channels as well as a reduced ionic selectivity of the K+ channels against Na+ ions, which gave a depolarization of the membrane resting potential (28, 174, 175). According to Tsong, the gating potentials of protein channels are much lower than the breakdown potential of the lipid bilayer, and this would induce at first a voltage-sensitive protein channel opening (time scale: microseconds). These channels are subject to be irreversibly denatured by Joule heating (time scale: milliseconds) and excised (time scale: minutes) and their gating action may not be sufficient, in general, to prevent the growth of the electric potential that leads to the cell poration (176). Importantly, it is known that altering the integrity of microtubules and actin filaments accelerates the resealing process (see also the second section), thus suggesting that the cytoskeleton contributes to the stability of electro-induced pores (177). More recently, it was found that electroporation directly disrupts cytoskeleton structures in endothelial cells and fibroblasts, although some debate does exist about the level of involvement of actin filaments (178, 179). Therefore, electroporation can be seen, as for the cytoskeleton involvement, as a negative feedback process, where the higher is the disorganization of microtubules and microfilaments induced by the electric field the faster is the resealing, which leads to a reduced molecular delivery. Altogether, in order to raise the resemblance between theoretical quantitative description and observed cell system, both in the early and late phases of electroporation, protein and cytoskeleton events could usefully be taken into account. One possible way to deal with the impressive intra- and inter- cellular heterogeneity sketched above could be using mathematical models inspired by statistical mechanics, which allow finding critical global descriptors even for extremely mixed populations. An evident difference between electroporation in vivo and in vitro is that the first one is generally performed with sparse and/or limited extracellular fluid, while in the second one, which happens in cuvettes, there is often abundant extracellular fluid. It is therefore worthy to discuss below the results of some investigations focused on the issue of cell density in electroporation applications. One study, based on an in vitro system of multicellular spheroids, which simulates some of the in vivo tissue complexity, evidenced that the uptake of molecules in cells after electropermeabilization is lower and more heterogeneous than in isolated cells. This effect was attributed to multiple factors, including non-uniform cell size (the size of interior cells was up to 30% smaller than cells in the periphery), slow diffusion from outside to inside, and heterogeneous electric field strength (180). An investigation performed onto dense cell suspensions reported that increasing the cell density dramatically reduces the fraction of cells permeabilized (181). Both the outcomes have potentially negative implications for the electroporation efficiency in vivo. Another research proposed that as the quantity of extracellular liquid decreases, a fixed electrical field will yield an increase in transmembrane potential across the cytoplasmic membrane due to a higher resistance of the current pathways around the cell (121, 182). Nonetheless, recent theoretical investigations showed that the transmembrane voltage progressively decreases when cells are closer to each other, like it happens in tissues (181, 183, 184). Furthermore, the extracellular fluid is important for the transport of cytoplasmic material out of the cell, and it was suggested that cell death from electroporation is more likely in vitro than in vivo, probably because the loss

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of intracellular molecules is larger when there is an abundant extracellular volume, like for cells in suspension (26, 121). Based on the scheme proposed by Tsong, this outcome can be directly attributed to osmotic unbalance (176). Moving to a more macroscopic point of view, it is well known that tissues and organs are structures that are not easily modeled (116-119). Their heterogeneity as for shapes, sizes, histological compositions and structures, physical states (e.g. liquid and solid), densities, electrical features, etc. is formidable (117-119, 163), and interferes with any attempt to create models of the electric field effects at this scale. According to Dean et al., we should look at living bodies as ―composite volume conductors‖ made by a number of spatially distributed tissues with differing electrical properties (185). A classic example concerning the importance of this diversity comes from emergency medicine of electric insults: in fact, it was determined that the nervous tissue has the lowest electrical resistance, followed by blood vessels, muscle, skin, tendon, fat, and bone. Therefore, skin burns are due to its high electric resistance and nerve tissue is often damaged by electric shocks (186). Interestingly, electrical conduction within biological tissues is dependent on ion concentration and mobility, rather than on electrons, and, in turn, ion mobility is temperature-dependent (185). Bound charges, such as membrane electrical bilayers and polar molecules determine complex tissue dielectric properties (185). From the specific point of view of electroporation, it was observed that the cell-to-cell mutual electrical shielding induces a decrease in the amplitude of the cellular transmembrane voltage (181) and that, in general, the spatial distribution of ΔΨE is much less easily described when cells are not isolated, since equations (2.1)-(2.4) cannot be directly applied (181). Moreover, the conductivity changes over time due to the ongoing electroporation, which are already important at the cell level, become particularly critical for tissues and, even more, organs, thus reducing the capability of modelers to make quantitative assessments point by point and moment by moment in the target system (28, 181, 185). Since a description of all possible biological scenarios, anatomical locations, and techniques for dealing with the macroscopic heterogeneity of electroporation applications is well beyond the scope of this section, we will refer here only to the specific case of the electric fielddependent drug delivery to and through the skin. Indeed, it is known that if the voltage of the applied pulses across the outermost layer of the epidermis, i.e., the stratum corneum, is greater than a threshold value that, according to Hui (187), can be set at roughly 75-100 V, and whose precise assessment depends on the external temperature (188), there is a localized electroporation. In this peculiar type of tissue poration, microchannels are created through the stratum corneum, which is made by 6-15 layers of flattened, dead cells surrounded by lamellae of 6-10 lipid bilayers (187, 188). Due to the major role that heat and temperature variations play in skin poration and to the prominence of the related modeling in the published literature, this process will be theoretically examined in the next subsection. Electric field and generated heat. Gehl et al. described a quantitative method to calculate the electric field strength across tissues as a function of the voltage-to-distance ratio and with respect to the electrode configuration (121). Briefly, in their model, the potential in a specific point is calculated as the mean value of the potentials in the surrounding points, while assuming that the medium is homogeneous. Additionally, the authors inferred the potentials on the boundary based on an equivalence relation between electric charge and potential. Finally, the electric field distribution was obtained by performing a discrete differentiation. In a compact analytical way, the Laplace equation has been proficiently used to estimate the electrical potential distribution induced in a tissue by electroporation (75):

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·(σΦ) = 0.

21

(5.3)

In (5.3), Φ is the electric potential, σ is the tissue electrical conductivity, and  is the gradient symbol (used with the same meaning also below); this equation is implicitly referred to the electric field E as well, since E = − Φ (189). The electrical boundary condition of the tissue that is in contact with the first electrode equals the applied voltage. The electrical boundary condition at the interface between the other electrode and the tissue is zero. Finally, the boundaries where the analyzed domain is not in contact with neither of the two electrodes are treated as electrically insulative (129). Solving the Laplace equation also enables one to calculate the associated Joule heating (p), ―the heat generation rate per unit volume‖ in the presence of an electric field (75): p = σ|Φ|2.

(5.4)

The Pennes bioheat equation, which accounts both for metabolism and blood flow, is the most popular equation used to solve problems about heat transfer in the body (190). Notably, the tissue heating resulting from electropermeabilization procedures can be calculated (75) by adding the Joule heating term of (5.4) to the Pennes equation. Consequently, a modified version of the Pennes bioheat equation has been obtained:

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·(kT) + wbcb(Ta − T) + q''' + p = ρcp∂T/∂t

(5.5)

where k is the tissue thermal conductivity, T is the temperature, wb is the blood perfusion, cb is the heat capacity of the blood, Ta is the arterial temperature, q''' is the metabolic heat generation, ρ is the tissue density, cp is the heat capacity of the tissue, and p is defined as above (75). Equation (5.5) is a very valuable tool for modelers. An example of the applicability of (5.5) was given by Maor et al., who, in the context of IRE studies performed on rat arteries, numerically solved a variant of this equation and found the temperature distribution in the analyzed domain (191). Then, moving from this result, a model of thermal insult was also developed. This theoretical description uses the damage integral (192-194), which, in its general form, is: 

 ( )   Ae [ E / RT ] dt

(5.6)

0

where Ω is an indicator of thermal injury, τ is the moment when the damage is assessed, A measures the frequency of molecular collisions, e is the Euler‘s number, E is an energy barrier for molecular denaturation, R is the gas constant, T is the absolute temperature, t is the time, and it is assumed that the damage starts when t = 0. Additionally, in this model of IRE thermal damage, T = T(t). Notably, equation (5.6) is based on Arrhenius‘ law (195). A study in vitro proved that electrotransfection is boosted by cooling the cells at the time of electropermeabilization (196). In vivo, instead, it was reported that the temperature increase in the treatment of skeletal muscle cells is modest, when using a standard electroporation setting (197, 198). However, in the view of Edd and Davalos (75), the

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bioelectrical properties of areas exposed to electric fields locally change due to cell permeabilization as well as even minor heating of the tissues (199-201). Several investigations have been showing that thermal damage can be significant in the skin and that heat-induced voltage drops may occur in the stratum corneum (see also the subsection ―Cell and tissue properties‖) (187, 202-208). Below, as paradigmatic of theoretical descriptions of this subject, we discuss the model of skin electroporation dependent on pulsed electric fields, based on a MATLAB (a.k.a. Matrix Laboratory, The MathWorks Inc., Natick, MA, USA) (209) simulation package, which was recently proposed by Pliquett et al. (188). Briefly, this model describes a specific process, consisting in the formation of pores between adjacent corneocytes and having the heat actively involved in the cascade of events, which is conveniently split into five phases: i) the stratum corneum is charged near the electrode until the transepidermal potential difference is large enough to create new aqueous pathways into it; ii) the pulsed fields drive a high current density through the electropore to generate Joule heating; iii) the temperature rise at the pore perimeter increases the probability of further local electroporation; iv) the heat-generated wave of further electroporation propagates until the pore surface becomes so large that the current density drops, thus making the generated heat insufficient to reach the phase transition temperature of the plasma membrane sphingolipids; v) after the electric field action ends, the stratum corneum cools down and there is a partial recovery of the electrical skin resistance. Altogether, the theoretical analysis suggests that mechanisms of tissue electroporation different from the ordinary cell electroporation, including local heat production that depends on (203), influences (208) or directly participates to (188) the porating process, are involved in the in vivo electropermeabilization of cells in the skin. Therefore, this analysis exemplifies how, moving to a more macroscopic level, it is possible to observe a switch from a phenomenon (e.g. the cell membrane poration) to an articulated process (e.g. the tissue poration) that may require further modeling. Future experimental and theoretical investigations on the above mentioned thermic processes will likely help to test the correctness of the different models proposed (188, 202-208) and drive the development of clinical applications. Physiological mechanisms. As it is well known, in the in vivo setting there are complex and inter-related physiological dynamics as well as reactivity to stimuli and traumas of the whole organism that play no role in vitro (119, 210, 211). Here we will discuss some macroscopic factors that should be typically considered for the purposes of integrated modeling: the pro-inflammatory action, the immunological system response and the blood flow modifications. Despite their importance for the optimization of the molecular delivery, these mechanisms are not easily described in a quantitative way, in particular because they involve many issues that go beyond the electropermeabilization framework stricto sensu. A possible strategy to cope with their complexity could be to build mathematical models, which simulate their effects by ―plugging‖ generalization term(s) into simplified and well characterized equations. Cases of inflammatory cell infiltration leading to an increase of the electroporation performance have been reported (e.g. DNA vaccines) (212, 213). However, some articles describe a limited contribution of this phenomenon (214, 215), thus suggesting that its influence is context-specific. Theoretically speaking, it is possible to hypothesize a negative interference between electroporation-induced leukocyte infiltration (212) in the treated tissue and diffusion process of the administered molecules through the cell membranes, in particular in elaborate and multi-stage protocols. Nevertheless, to the best of our knowledge, no

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investigation is available about the timing of the acute inflammation events triggered by electric pulses and their possible overlapping with the electroporation phases. Many research groups have been using electroporation for the transduction of DNA and RNA into lymphocytes and antigen-presenting cells (216-223). In this framework, electrovaccination protocols, whose aim is to elicit an antigen-dependent immune reaction, have been developed (224, 225). Importantly, however, the immune system can be activated and/or play a significant biological role also when the primary targets of electropermeabilization are cells not belonging to this system and no strong antigenic signal is directly elicited. A broad-spectrum immunogenic effect of electroporation is indeed supported by relevant findings: i) the pulse administration by itself can recruit cells involved in antigen presentation and trigger the immune response (226); ii) there is an anti-tumor synergism between ECT and cytokines, which is effective both locally and systemically (26, 227-230). It has been proposed that the immune system response triggered by electroporation could be dependent on the release of antigens from the cytosol and/or on changes in the plasmalemma of treated cells (26). This last point has recently received support by the observation that cells exposed to nanosecond pulsed electric fields externalize phosphatidylserine molecules, which is enough to induce their phagocytic clearance (231, 232). Overall, the published research shows an amplified and/or altered response to electric pulses in oncology applications and gene therapy, which could potentially be modeled if more quantitative data about the immune system response mechanisms were available. In a number of investigations, blood flow modifications were also described. It was suggested that the characteristic hypoperfusion that appears in the pulsed areas is induced by the constriction of the resistance vessels surrounding the electroporated area and by the combined effect of interstitial edema and reduced intravascular pressure, which are due to the endothelial electropermeabilization (230, 233-235). These events are important when any drug uptake is involved, because of the consequent vascular lock (i.e., longer drug retention in electroporated tissues). However, according to a numerical analysis of Edd and Davalos (75), blood vessels behave like a highly conductive core that is surrounded by a shell of lowconductivity tissue (i.e., the endothelium). The high-conductivity cores would primarily lead to increased current flow and higher Joule heating; the subsequent thermal damage spreading, when involving the endothelium of large blood vessels and other cavities, would indeed cause the vascular lock. Part of the vascular effect are the perfusion delays, which can be divided into short (mostly around 1-2 minutes) and long (up to 30 minutes) term, according to Gehl et al. (26, 235). The first effect was described as a sympathetically mediated Raynaud-like phenomenon (236) and associated with reversible permeabilization, while the second would signal irreversible permeabilization or slow resealing kinetics (235). Specifically, it was found that there is a direct relation between electric field strength and length of the perfusion delay, with a threshold generally lower than or equal to 1.2 kV/cm below which the delay is of the short term type, for pulses between 10 microseconds and 20 milliseconds. Notably, different pulse durations give different curves and critical thresholds of perfusion delay. Based on these curves, when long pulses (in particular, above 500-1000 microseconds) are used, the electric field strength range that generates reversible permeabilization becomes smaller (235). Therefore, because long pulses support a better electrophoresis, but reduce the range of electric field strength that generates reversible permeabilization, it is evident that a careful optimization is necessary (26, 235), in particular when trying to transduce DNA (133). Recently, Sersa et al., with the support of a simple physical model, demonstrated that the

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vascular effect synergizes with the bleomycin action in ECT of mice with experimentally induced sarcomas. Specifically, this scientific team showed that the already described first phase of vascular disrupting action is followed by a drug-induced second wave (24 hours after treatment), which is dependent on endothelial cell swelling and apoptosis (237). The entire vascular effect would deserve, in our opinion, a dedicated mathematical modeling, by means, for instance, of delay differential equations (DDE) or delay partial differential equations (DPDE), in the perspective to optimize permeabilization triggering and tissue-specific control of the vascular-related effects. Molecular uptake. The pore creation induced by the applied electric field directly benefits the delivery of small molecules into cells. Indeed, transient pores allow or boost the movement of solutes through the cell membrane by simple diffusion (see equations (2.7) and (2.8)), which follows the concentration gradient, even when the electric field application is interrupted (26, 32, 61, 238). The availability of pores whose size is larger than the molecular size is a major limiting factor of this cross-membrane transport (63). Small molecules are expected to diffuse across the porated membrane for a time longer than the electric pulse(s), while it was described that the macromolecular delivery does not benefit from post-pulse free diffusion (239). The molecular uptake potentially rests also on other mechanisms, such as: i) electrophoresis, ii) electro-osmosis, iii) transport dependent on the transmembrane potential, and iv) induced endocytosis. Specifically, a number of articles reported that, during the pulse administration, the electrophoretic force allows the migration of DNA, which is a polyanionic macromolecule, toward the positive electrode with a higher efficiency than diffusing neutral molecules, both in vitro and in vivo (72, 78, 240, 241). However, several issues that challenge the importance of this mechanism have been raised by Huang and co-workers (242). The electro-osmotic hypothesis, whose relevance is debated (243-245), explains the molecular transport through the cell membrane in terms of hydrodynamic flows (56, 246). It was suggested that electro-osmosis occurs when the electric field is parallel to the negatively charged cell surface, because negative charges cause mobile positive ions to accumulate locally, thus inducing a movement of fluid, which exerts a force on the protruding portion of molecules (247). Another mechanism under investigation has been the post-pulse molecular transport, through residual electropores, dependent on the transmembrane potential (238). Indeed, according to Weaver (245), the resting transmembrane voltage (see the introduction), even when it is small, potentially contributes to the observed asymmetric cell responses to electroporation fields (243) as well as to a prolonged delivery of ions and charged molecules after the electric field intensity goes back to zero (245). Other research showed that induced endocytosis (248) also plays an important role when applying electroporation protocols (249250). Notably, even though macromolecules (defined, in this context, as chemical compounds having a molecular weight > 4 kilodalton (kDa)) are subject to direct uptake only if they are in the extracellular fluid before administering the electric field, proteins and other large molecules introduced after the electric pulses can still enter the cell, thanks to mechanisms of endocytosis induced by electroporation (29, 249, 250). Since at least some of the endocytotic phenomena are non-specific (250), the enhanced cell uptake potentially involves many different solutes, independently of their main physiological and/or electropermeabilizationrelated mechanisms of transmembrane transport. In the specific case of DNA transfection, which is much less effective in vivo than in vitro (132, 251, 252), we observe that models focused on gene expression efficiency rather than molecular uptake need to deal with some

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extra variables, such as the intracellular content of adenosine-5'-triphosphate (ATP). In fact, according to Rols et al., ATP plays an important role in the translocation of plasmid DNA across nuclear membranes and in DNA expression (81). Importantly, it was reported by Delteil et al. that the presence of serum negatively affects cationic lipid-mediated gene transfer but not cell electroporation and gene transfer to the nucleus in vitro; this research group also suggested that other factors are therefore more likely responsible of the lower transfection levels obtained in vivo (253). Molecular shape, size, and electric charge, as well as other chemical-physical properties, critically affect the delivery efficiency of electropermabilization protocols (40, 47, 123). The size and shape factors (245) are fairly straightforward when considering that the largest molecular dimension needs to be smaller than the pore size (see also the third section). Additionally, the so called ―foot-in-the-door‖ hypothesis states that an enhanced and extended uptake of small molecules is possible if a long macromolecule (e.g. plasmid DNA, large dextran, polymer) temporarily stays with a fraction of its length inside a pore and inhibits the pore resealing process (47, 245, 254). The Born energy (i.e., the energy associated with the field of ions or charged molecules) measures the energy cost for the insertion and delayed transit of an electrically charged molecular entity across the pore; therefore, it accounts for the low conduction of small pores and is an interfering factor for the molecular uptake (245). The classical calculation of this physical quantity for charged molecules moving across membrane pores was proposed by Parsegian (255):

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Ep = e2/(2·εp·a) + P(εhc/εp)·[e2/(εhc·b)].

(5.7)

In this formula and below, Ep is the energy of the molecule inside the pore, e is the total charge magnitude, εp is the pore dielectric constant, εhc is the hydrocarbon (i.e., the membrane) dielectric constant, εw is the water dielectric constant, a is the molecule radius, b is the pore radius, and P(εhc/εp) is a function numerically solved in the original paper (see below) (255). The first addend of (5.7) is the ―bulk term‖ and is due to the direct interaction between molecule and pore, while the second addend is the ―induced energy‖ and depends on the charge induced at the boundary between pore and membrane. The pore is supposed to be cylindrical and the formula can be applied only when b 400 Hz), which additionally reduce the force of the generated muscle contractions (119, 156, 262, 263). Also for the purpose of decreasing this type of electroporative toxicity, a new experimental approach, which uses AC and low field strength, has been developed in vivo (161). Interestingly, it was noticed that in clinical settings not carefully pre-evaluated, the local heat generation may be massive, so that pain becomes temporarily unbearable for the patients (129). Actual negative side effects, such as osteomyelitis, dysphagia, fistulas and wound breakdown were described in an ECT study on electroporation of head and neck neoplasms (123, 264). After a first paper describing necrotic areas in experiments of DNA transduction of skeletal muscle (265), another investigation in a similar setting confirmed this outcome and also showed histological evidence of significant inflammation as well as an increase in the blood levels of creatine phosphokinase (CPK), which indicates myofiber lysis (266). The issue of muscle damage was addressed by Durieux et al. (267), who reported that, when using square waves, the major causes of this outcome are the actual DNA delivery to the pool of cells and the following DNA expression; these results are in agreement with the observations of Hartikka et al. (266). They also showed that lowering the electric field intensities and the cumulative pulse lengths mostly helps to reduce the observed damage (267). A list of other agents potentially involved in muscle damage due to DNA electrotransfer, which deserve further investigation, can be found in a review of McMahon and Wells (268 and references therein). Notably, due to the relative novelty of electroporation techniques, physicians and veterinarians have been constantly working for achieving a good control of unpleasant sensations, undesired side effects and pain associated with electroporation-based treatments (24, 125, 269, 270). Altogether, the available literature clearly shows the importance of both direct and indirect negative outcomes of electropermeabilization in vivo and their dependence on different (electrical and biomedical) factors and contexts. Therefore, defining algorithms (like, for instance, those used in

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statistical decision theory (271)) that help to prevent the range of adverse effects by singling out cases and protocols with the highest associated risk, would be a relevant achievement for modelers.

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CONCLUSION Electroporation applications have been rapidly growing in the last years, as it is witnessed, for example, by these facts: i) electrotransfection is becoming more and more popular in biomedical laboratories, thanks to its capability to deal also with ―difficult-totransfect cells‖ (272); ii) electroporation protocols are in development or under consideration also for large animals (273-277); iii) original biotechnologies depending on the application of electric fields are constantly proposed by the scientific community (278-280); iv) scientists are working to develop gene electrotransfer protocols in vivo and clinical trials based on these types of applications are in progress (281-283); v) ECT has already been used in about 40 cancer centers in Europe and in the USA for palliation, cytoreduction before surgical resection, and direct tumor treatment (283). However, researchers have recently been commenting on how limited still is the understanding of the electropermabilization events (29, 32), despite many dedicated papers from a biological, computational, mathematical, medical, and physical perspective. It is our opinion that this lack of knowledge, from the modeling standpoint, is not equally distributed. In fact, investigators have been mathematically and physically studying this phenomenon for many years (32, 40, 63, 162), with results particularly suitable for in vitro applications and, recently, also the microscopic description of pore formation and evolution based on atomistic simulations seems to be opening a route of great interpretative power (107-109). On the other hand, there is still a limited availability of quantitative models for coping with the amazing complexity of the in vivo setting and for leading translational researchers who wish to design the best protocols for their patients (26, 29, 48, 127). Specifically, it would be important to come out with precise assessments concerning which of the many variables examined play major roles for the different biomedical applications, in order to properly integrate these variables into new and more comprehensive mathematical models. Even though the factors discussed in our last section have a great generality and broad scope, it is evident that most of them need to be evaluated in the specific context of the experimental and clinical goals set by the investigators and that the study of other factors may be necessary to fully accomplish this purpose. Hopefully, our overview and this book as a whole will stimulate theoretical and experimental scientists to further join their forces and inspire new investigations of the many aspects of this fascinating subject both in vitro and in vivo, including the most challenging ones.

ACKNOWLEDGMENTS The authors are grateful to Tammy L. Moser for the careful review of this chapter.

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[252] Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol. 1998 Feb;16(2):168-171. [253] Delteil C, Teissié J, Rols MP. Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells. Biochim Biophys Acta. 2000 Aug 25;1467(2):362-368. [254] Polk C, Postow E. (Eds) Handbook of biological effects of electromagnetic fields. CRC Press 1995 Dec 21. Boca Raton, FL, USA. 2nd Edition. [255] Parsegian A. Energy of an ion crossing a low dielectric membrane: solutions to four relevant electrostatic problems. Nature. 1969 Mar 1;221(5183):844-846. [256] Gehl J, Skovsgaard T, Mir LM. Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs. Anticancer Drugs. 1998 Apr;9(4):319-325. [257] Jaroszeski MJ, Dang V, Pottinger C, Hickey J, Gilbert R, Heller R. Toxicity of anticancer agents mediated by electroporation in vitro. Anticancer Drugs. 2000 Mar;11(3):201-208. [258] Rols MP, Bachaud JM, Giraud P, Chevreau C, Roché H, Teissié J. Electrochemotherapy of cutaneous metastases in malignant melanoma. Melanoma Res. 2000 Oct;10(5):468-474. [259] Rodríguez-Cuevas S, Barroso-Bravo S, Almanza-Estrada J, Cristóbal-Martínez L, González-Rodríguez E. Electrochemotherapy in primary and metastatic skin tumors: phase II trial using intralesional bleomycin. Arch Med Res. 2001 Jul-Aug;32(4):273276. [260] Vernhes MC, Cabanes PA, Teissie J. Chinese hamster ovary cells sensitivity to localized electrical stresses. Bioelectrochem Bioenerg. 1999 Feb;48(1):17-25. [261] Zupanic A, Ribaric S, Miklavcic D. Increasing the repetition frequency of electric pulse delivery reduces unpleasant sensations that occur in electrochemotherapy. Neoplasma. 2007;54(3):246-250. [262] Simmons RM. (Ed) Muscular contraction. Cambridge University Press 1992 Jun 26. Cambridge. 1st Edition. [263] Solaro RJ, Moss R. (Eds) Molecular control mechanisms in striated muscle contraction (Advances in muscle research). Kluwer Academic Publishers. Dordrecht, The Netherlands. 2002 Aug 31. 1st Edition. [264] Allegretti JP, Panje WR. Electroporation therapy for head and neck cancer including carotid artery involvement. Laryngoscope. 2001 Jan;111(1):52-56. [265] Mathiesen I. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 1999 Apr;6(4):508-514. [266] Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M, Nishioka WK, Wheeler CJ, Manthorp M, Sawdey M. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188. Mol Ther. 2001 Nov;4(5):407-415. [267] Durieux AC, Bonnefoy R, Busso T, Freyssenet D. In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage. J Gene Med. 2004 Jul;6(7):809-816. [268] McMahon JM, Wells DJ. Electroporation for gene transfer to skeletal muscles: current status. BioDrugs. 2004;18(3):155-165.

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[269] Tjelle TE, Salte R, Mathiesen I, Kjeken R. A novel electroporation device for gene delivery in large animals and humans. Vaccine. 2006 May 22;24(21):4667-70. Epub 2005 Sep 1. [270] Wallace M, Evans B, Woods S, Mogg R, Zhang L, Finnefrock AC, Rabussay D, Fons M, Mallee J, Mehrotra D, Schödel F, Musey L. Tolerability of two sequential electroporation treatments using MedPulser DNA delivery system (DDS) in healthy adults. Mol Ther. 2009 May;17(5):922-928. Epub 2009 Mar 10. [271] Berger JO. Statistical decision theory and Bayesian analysis. Springer-Verlag 1993 Mar 25. New York. 2nd Edition. [272] Jordan ET, Collins M, Terefe J, Ugozzoli L, Rubio T. Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biomol Tech. 2008 Dec;19(5):328-334. [273] Babiuk S, Baca-Estrada ME, Foldvari M, Storms M, Rabussay D, Widera G, Babiuk LA. Electroporation improves the efficacy of DNA vaccines in large animals. Vaccine. 2002 Sep 10;20(27-28):3399-3408. [274] Scheerlinck JP, Karlis J, Tjelle TE, Presidente PJ, Mathiesen I, Newton SE. In vivo electroporation improves immune responses to DNA vaccination in sheep. Vaccine. 2004 Apr 16;22(13-14):1820-1825. [275] Cemazar M, Tamzali Y, Sersa G, Tozon N, Mir LM, Miklavcic D, Lowe R, Teissie J. Electrochemotherapy in veterinary oncology. J Vet Intern Med. 2008 JulAug;22(4):826-831. [276] Brown PA, Bodles-Brakhop A, Draghia-Akli R. Plasmid growth hormone releasing hormone therapy in healthy and laminitis-afflicted horses-evaluation and pilot study. J Gene Med. 2008 May;10(5):564-574. [277] Dharmapuri S, Peruzzi D, Mennuni C, Calvaruso F, Giampaoli S, Barbato G, Kandimalla ER, Agrawal S, Scarselli E, Mesiti G, Ciliberto G, La Monica N, Aurisicchio L. Coadministration of telomerase genetic vaccine and a novel TLR9 agonist in nonhuman primates. Mol Ther. 2009 Oct; 17(10):1804-1813. Epub 2009 Jul 21. Erratum in: Mol Ther 2010 Feb;18(2):447. [278] Yuan TF. Electroporation: an arsenal of application. Cytotechnology. 2007 Jun;54(2):71-76. Epub 2007 Jun 16. [279] Watson D, Sleator RD, Hill C, Gahan CG. Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract. BMC Microbiol. 2008 Oct 9;8:176. [280] Chen X, Fang H, Rao Z, Shen W, Zhuge B, Wang Z, Zhuge J. An efficient genetic transformation method for glycerol producer Candida glycerinogenes. Microbiol Res. 2008;163(5):531-537. Epub 2008 Jul 2. [281] Cemazar M, Golzio M, Sersa G, Rols MP, Teissié J. Electrically-assisted nucleic acids delivery to tissues in vivo: where do we stand? Curr Pharm Des. 2006;12(29):38173825. [282] Heller LC, Heller R. In vivo electroporation for gene therapy. Hum Gene Ther. 2006 Sep;17(9):890-897. [283] Sersa G, Cemazar M, Snoj M. Electrochemotherapy of tumours. Curr Oncol. 2009 Mar;16(2):34-35.

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Chapter 2

TECHNICAL ASPECTS OF THE ELECTROCHEMOTHERAPY Ivan Dotsinsky, Nicolay Mudrov and Tsvetan Mudrov

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Center of Biomedical Engineering, Bulgarian Academy of Sciences, Sofia, Bulgaria

The electrochemotherapy is based on electroporation of the cell membrane and simultaneous administration of cytotoxic drugs. Usually, the poration is achieved by applying short high-voltage electric pulses. The generated electric field acts selectively on the cells while leaving the connective tissue unaffected. There are some possibilities of defining the distribution of the electric field on the area to be treated. Advantage of the technique could be found in completing the treatment in one session usually on an out-patient basis with minimum side-effects. In recent years, the method of electroporation has become also a powerful tool for other cell manipulations.

ELECTROPORATION. SHORT DESCRIPTION OF THE PHENOMENON. TRANSMEMBRANE VOLTAGE INDUCED BY ELECTRIC FIELD ON ISOLATED CELL Electroporation is associated with the creation of aqueous pathways (electropores) in the cell membrane as a result of applied short intensive electric field. This phenomenon allows molecules, ions, and water to pass from one side of the membrane to the other. The electroporation is reversible if the membrane returns into the normal state after the end of the field exposure, which is possible when its parameters have been appropriately selected. Otherwise, the pores can not be resealed. The process becomes irreversible that leads to the cell death. The electroporation was found to be a practical way to place drugs or other molecules such as gene constructs into cells. The transmembrane voltage induced on spherical cells was analytically described firstly for the spherical shape. Schwan (1957) assumed the membrane as nonconductive despite its

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low conductivity compared to the intra- and extracellular environment and derived the equation

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Δø =

3 ER cos  2

where Δø is the induced transmembrane voltage in a point of the cell, E is the applied electric field, R is the cell radius, and υ is the polar angle between the field direction and the radius drawn through the point. Evidently, the maximum voltage is induced at the points where the electric field is perpendicular to the membrane, i.e. at the angles 00 and 1800, called poles. The Schwan‘s equation describes the static state established several μs after the onset of the DC electric field. Regardless of the above mentioned limitations, this simplified but fundamental equation continues to be used in most of the investigations. Later on, Kotnik et al (1997) extended the equation by adding the electric conductivities of the cytoplasm, the cell membrane and the external medium. They also introduced the membrane thickness but assumed the conductivities and the thickness do not change. Meanwhile, numerical assessments of transmembrane voltage in case of spheroidal cells appeared (Klee and Plonsey, 1976). Some authors claimed that an analytical description of such cell shape, even if possible, will no befit the reality due to the nonuniform membrane thickness, which is unrealistic but inevitable in spheroidal geometry. Kotnik and Miclavcic (2000) showed that for all spheroidal cells, the membrane thickness is irrelevant to the induced transmembrane voltage under the assumption of a nonconductive membrane. They reported an analytical description of the voltage induced on prolate and oblate spheroidal cells and analyzed its variation in cases of constant membrane surface area and constant cell volume. Gimsa and Wachner (2001) proposed an analytic equation of the transmembrane voltage Δø induced by a homogeneous AC field on arbitrarily oriented cells of the general ellipsoidal shape. The equation is an extension of the early derived formula of Fricke (1953), which expresses the DC steady-state transmembrane voltage Δø for a cell of the general ellipsoidal shape with negligible membrane conductance and a highly polarizable cytoplasm. Δø =

1 aE 1  na

Here a, and na stand for the semiaxis oriented in field direction and the depolarizing factor along this semiaxis, respectively. Depending on the axial ratio of the ellipsoid, the depolarizing factor can take values varying between 0 and 1. For the spherical shape, Fricke‘s equation can be reduced to the well-known expression, Δø = 1.5RE The improved analytic equation of Gimsa and Wachner (2001) uses a special finite element model to describe the dependence of Δø on: i) field frequency, ii) cell size and shape, iii) membrane capacitance, iv) conductivities of cytoplasm, membrane and external medium, v) location of the membrane site under consideration, and vi) orientation of the cell with

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respect to the field. Further on, the authors found that Δø can be unambiguously defined for non-spherical cells, provided that the membrane thickness is thin in comparison to the cell dimensions. The induced transmembrane voltage is asymmetric because of the resting transmembrane voltage, which is inherently present in cells (Kotnik and Miclavcic, 2001). Normally, its absolute value is higher at the pole facing the positive electrode, because of the negative potential of the cell interior with respect to the cell exterior. As a result, the membrane electropermeabilization obtained by unipolar pulses is necessarily asymmetric. This asymmetry can be counterbalanced by applying a symmetrical bipolar pulse. Thus, the impact of the first unipolar pulse is opposed to that of a second pulse of the same duration and amplitude, but with the reversed polarity. Although most investigators agree that transient hydrophilic pores are responsible for the transport of both small and macromolecules through the membrane, the mechanism of reaching such behavior is not yet fully understood (Saulis and Satkauskas, 2004). Still, the common accepted interpretation of the membrane permeabilization includes the following states: i) initial hydrophobic, characterized by the original orientation of the lipids; ii) creation of initial hydrophilic pores under strong electric field; iii) reorientation of the lipids adjacent to the aqueous inside a pore in a manner that their hydrophilic heads are facing the pore, while the hydrophobic tails are hidden inside the membrane (Weaver and Chizmandzhev, 1996). The appearance of initial hydrophilic electropores is favored in several studies as a reasonable explanation of the observed subsequent larger pore distribution within the entire membrane (Freeman et al, 1994). However, Chang and Reese (1990) consider that it is not clear whether discrete physical pores can be really formed in the electropermeabilized membrane since such electropores have never been seen by electromicroscopy, except for some artefacts that do not prove the assertion (Mir et al, 1995; Weaver and Chizmandzhev, 1996). They doubt that a particular opening is the true electropore opening. An alternative description of the process considers a cross-fractured neck of elongated membrane evagination, which may have a smaller opening at its apex. Some authors suggested that the "'electropores" could be membrane "nano-scale defects" (Rubinsky, 2007), "cracks," or "crater-like structure". The discovered by the video microscopy rapid changes of cell shape and diameters suggest that there has been a fast exchange of intracellular and extracellular material after the electroporation. Shrinkage was observed, which might be caused by a rapid escape of cellular content, presumably hemoglobin molecules. The outflow of hemoglobin apparently lasted only for a few seconds, after that the cells stopped shrinking. Later on, the cells gradually swelled. The membrane pores resealed and became too small to allow hemoglobin molecules to enter while water can still pass through. Another possible interpretation pays attention to the increases of fluctuations in the lipid bilayer as a cause of the appearance of large hydrophilic pores (Lopez et al, 1998). Davalos et al (2004) used experimental data for numerical simulations to demonstrate that electrical impedance tomography (EIT) can produce an image of the electroporated area. They suggest that the EIT may support the electrochemotherapy in introducing the therapeutic molecules into cells in tissue at predetermined areas of the body.

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TECHNIQUES FOR INDUCING THE ELECTRIC FIELD Electropermeabilization typically uses high voltage pulses of μs to ms duration to generate the necessary electric field for opening pores in the cell membrane (Rodamporn et al. 2007). Optimum electroporation parameters vary depending upon the cell type and purpose. Electric field strengths of 1000V/cm and 100 μs pulses are applied for drug delivery and low voltage but longer pulses, such as 200 V/cm, 20-50 ms are used for gene therapy. The short field exposure must be considered as a complex stress applied on the cell assembly (Vernhes et al, 1099). In vitro experiments are usually carried out by charging a capacitor and then discharging it through the cell suspension. This capacitance charge-discharge has some disadvantages due to the difficulty of maintaining stable pulse duration independently of the load impedance (Daskalov et al, 1999). Besides the parameters of the electric pulses, the efficiency of electropermeabilization in vitro depends on the molecular composition of the membrane and the osmotic pressure (Kotnik et al, 2001). Chang (1989) suggested that electroporation or electrofusion can be induced not only by applying electric pulses of high-intensity but also using an additional oscillating electric field. While the DC field relies solely on the dielectric breakdown of the cell membrane, the oscillating field can produce a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of DC field and oscillating field is expected to enhance the efficiency of cell poration and fusion. The oscillating frequency of the field, which was used with the study, varied from a few kHz to 1 MHz. The electric pulse width was selected from 100 μs to 2 ms depending on the cell type used for fusion or gene transfection, respectively. The peak amplitude of the oscillating field was changed from 0.5 to 5 kV/cm. Both single and train pulses were generated. The obtained electric field was applied in the case of electroporation whereas a second electric field was needed for successful electrofusion.

ELECTROPORATION IN CELL SUSPENSION AND TISSUES In vitro experiments are only a rough model for studies under in vivo conditions. In the case of electrochemotherapy, such experiments usually result in evolution of some basic principles dealing with drug uptake and cell survival (Lebar et al, 1998). Usually the electroporation begins at the caps around the poles (Miclavcic and Kotnik, 2004). For stronger fields, the caps area gets larger. The obtained asymmetry may be compensated to some extent by using of bipolar electrodes. In general, the Schwan‘s equation is not valid for cells in suspension. This is due to the non-homogeneity of the field outside a given cell that is distorted by the presence of other cells in the suspension. The larger the volume occupied by the cells, the lower the accuracy of the predicted transmembrane voltage. Briefly, it depends both on the geometrical and electrical cell properties and on the cell density in the suspension. The cells in tissues are far from the spherical shape. They are not homogenous and have different electric properties. When they are electroporated, the activated areas redistribute the

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field, which becomes higher around the non-porated regions. This dynamic state turns back to rearrangement of the electric field and vice versa.

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ELECTROCHEMOTHERAPY. ELECTROGENETRANSFECTION The phenomenon electroporation (electropermeabilization) was discovered first in vitro. The last two decades, new techniques were intensively developed for in vivo drug delivery into the cancer cells (electrochemotherapy) and for gene transfection (Miclavcic et al, 1998; Mahmood et al, 2008). The mechanisms of molecule transport across the membrane are: the diffusion supported by the difference in molecule concentration; the eletrophoresis driven by the electric field; the osmotic pressure difference around the membrane (Miclavcic and Kotnik, 2004). The diffusion is the main component of the small molecule transport while for the macromolecules such as deoxyribonucleic acid (DNA) the electrophoretic forces significantly improve the uptake into the cells (Rols and Teissie, 1998). These differences reflect on the duration chosen from tens of μs through several ms for the electric pulses applied in both cases. The common used cytotoxic chemotherapeutics are bleomycin, which is equally effective intravenously or intratumorally and cisplatin with more expressed intratumoral impact. They permeate very slowly the plasma membrane (Miclavcic and Kotnik, 2004) but the electric pulse delivery at the time, when the drug reaches the highest concentration, increases the transport through the membrane. This is the technique that has the main influence on the improved electrochemotherapy. Still, secondary mechanisms as the decrease of the tumor blood flow contribute to the effectiveness of the electrochemotherapy. Kranjc et al (2005) found the application of electric pulses to the tumors induces profound but transient reduction of tumor oxygenation. Another way to enhance the bleomycin internalization consists of combination with photochemical treatment. Berg et al (2005) reported a synergistic delay in tumor growth after photochemical delivery of bleomycin, whereas no complete responses were observed with the bleomycin alone. Usually, the electropermeabilization is applied for drug transport to cutaneous tumors (Mir et al, 1995). A derived method is the transdermal electroporation applying the electric pulses across the dermal layer (Ciobanu el al, 2007). Lately, the reversible alternation of the cell membrane is used for DNA introducing aimed to genetical modification. While electroporation has reached clinical trials for in vivo delivery of chemotherapeutic agents to cancers, the application of this techniques for plasmid DNA transfer is still at preclinical studies, although some of then are promising for the possibility to tailor gene therapies to individual diseases (Heller and Lucas, 2000). When a DNA injection into the muscle is combined with electroporation, the gene expression is increased by two or three orders of magnitude. Sukharev et al (1992) described the effect of DNA interaction with membrane electropores and provided additional evidences for the key role of DNA electrophoresis in cell electrotransfection. They found that the longer the DNA fragment, the greater must be the increase in permeability. The use of a two-pulse technique allowed the authors to separate the

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two effects provided by a pulsed electric field: membrane electroporation and DNA electrophoresis. A first pulse of 6 kV/cm and 10 μs created high number of pores, whereas the transfection remained low. The second pulse of much lower strength of 0.2 kV/cm, but with substantially higher duration of 10 ms, did not cause poration and transfection by itself but enhanced the transfection by about one order of magnitude. The process rose monotonously with the increase of the second pulse duration. The authors estimated the electropore lifetime by varying the delay between the two pulses. Miller et al (2005) found that the irreversible electroporation (IRE) was studied only as an undesirable effect, which has to be prevented during electrochemotherapy and gene therapy. They suggested the possible use of IRE for inducing a Joule heating thermal ablation, which is distinct from the usually applied techniques of the thermal domain and may result in complete cancer cell ablation. Rubinsky (2007) confirmed that the irreversible electroporation affects only the membrane of living cells. The last three decades the applications of reversible electroporation were become dominated and IRE was studied only to define the upper limit of parameters that induce reversible electroporation. Lately, IRE is beginning to emerge as an important medical technology in its own right. While ignored by medicine, it has been used in the food industry for sterilization and preprocessing of food. It seems that some of the medical applications, which engage thermal effects of electrical fields in tissue ablation, also produce electroporation. Question rose whether IRE can be used to produce substantial volumes of tissue ablation in vivo with negligible thermal effects. Some authors show the use of irreversible electroporation in treatment of the prostate.

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OPTIMIZATION OF THE PULSE PARAMETERS Many authors have studied the influence of the pulse parameters on the electropermeabilization level. Their optimization is aimed to find these minimal electrical conditions that lead to a significant antitumor effect. Usually, the field intensity (the amplitude), the number and the duration of different shapes of pulses have been in the focus of the investigations (Chang, 1989; Rols and Teissie, 1990 and 1998; Tekle et al, 1991; Tovar and Tung, 1991; Sukharev et al, 1992; Wolf et al, 1994; Mir et al, 1995; Tomov, 1995; Sersa et al, 1996; Lebar et al, 1998; Daskalov et al, 1999; Vernhes et al, 1999; Somiary et al, 2000; Kotnik et al, 2001; Lebar et al, 2002; Pucihar et al, 2002; Davalos et al, 2004; Giardino et al, 2006; Chao Cheng-wei et al, 2006; Ciobanu el al, 2007; Mahmood et al, 2008; Yu Zhou et al, 2008). The electropermeabilization occurs only at a threshold value of the transmembral potential (Rols and Teissie, 1990; Sukharev et al, 1992; Wolf, 1994; Rols and Teissie, 1998). Above this threshold the quantity of the porated cells increase following approximately an ascendant sigmoidal law while the percentage of surviving cells decrease by the same but descendant law. Lebar et al (1998) examined the in vivo effect of the applied electric energy on the cell survival and the membrane permeabilization. The highest percentage of stained living cells was observed with four long pulses of 200 μs repeated with 0.5 Hz frequency at 200 V. However, both poration and survival varied significantly between various types of cells maybe due to the different size and membrane structure.

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The life time of the induced permeabilization disappears progressively after the application of the electric field (Rols and Teissie, 1990). Several theoretical models have been proposed to explain the phenomenon of the permeabilization. One considers the membrane as homogenous fluid, which breaks when the electroinduced compressive force is not balanced. Another model is based on the existence of natural defects in the membrane, which grow in size under the applied field. The pore size depends on the intensity of the applied electric field but also on the cell size and density (Giardino et al, 2006). The electrophoresis plays an important role in case of macromolecule transport and sufficiently prolonged pulses are necessary for an adequate uptake (Sukharev et al, 1992; Wolf, 1994; Rols and Teissie, 1998). Research of Mir (1990) has shown that the induction of electropores is affected by three major factors. First, cell-to-cell biological variability causes some cells to be more sensitive to electroporation than other. Second, electropores can be induced if the product of the pulse amplitude and the pulse duration is above a lower limit threshold. Third, the number of pores and the effective pore diameter increase with higher values of the product. The threshold is understood to be largely dependent on a fourth factor, the reciprocal of cell size. It is related to the upper threshold of the field strength that porate such large area of the cell that cannot be repaired by any spontaneous or biological process. An additional important consideration is that during the electroporation pulse, the electric field causes electric current to flow through the cell suspension or tissue. Biologically relevant buffers for cells, bathing media, and fluid in extra-cellular space in tissues contain ionic species at concentrations high enough to cause electric currents to flow. These currents can lead to dramatic heating which is biologically unacceptable. Principles of physics suggest that the early part of exponentially decayed pulses does most of the membrane porating but the late part continues to heat the medium. Miller et al (2005) carried out experiments with pulses of various amplitudes. The pulse duration was selected to produce in all cases comparable temperatures that did not exceed 50°C. Pulses leading to strength of 2500, 2000, 1500, 1000, and 500 V/cm were applied for 1, 1.5, 3, 6, and 24 ms, respectively. The results showed the use of multiple pulses as more effective for cancer cell ablation than the delivering of the same energy in one single pulse. The authors found that a field of 1500 V/cm generated by sets of ten pulses of 300 μs duration and 100 ms pause between them can produce complete cancer cell ablation. The perturbed areas of the cell membrane rapidly decreases after the end of the electric pulse generation (Mir et al, 1995). However, some permeable structures remain present for relatively long periods. Their lifetime depend on the number of pulses applied. Ciobanu el al (2007) experimented on B16F10 cell line and studied the influence of the pulse duration on the drug internalization at field strength of 2.35 kV/cm, which was found to assure sufficiently high level of poration together with cell viability. One or two pulses were applied. The optimal pulse duration is specified in the range of 100 through 200 μs since the porated cells number increased very rapidly and reached a plateau after 100 μs, while the viable cells percentage dropped after 200 μs. Mahmood et al (2008) communicated that the results of their investigations showed a significant and linear increase in the DNA transfection efficiency when the number of pulses was increased up to five with pause between them approximately 15 s at constant field strength of 20 kV/cm and pulse duration of about 10 ms.

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The critical pulse amplitude leading to electropermeabilization becomes lower if the pulse number and/or the duration increase (Rols and Teissie, 1990; Rols and Teissie, 1998). At the same time the molecular uptake enhances. The pulse duration is shown to be crucial for the penetration of macromolecules that is known to be much more complex than the simple diffusion of small molecules through the permeabilized plasma membrane. Cumulative effects are observed when repeated pulses are applied. Vernhes et al (1999) investigated the stress effects of field strength, pulse duration and number of pulses with respect to the Joule energy. The authors found that the loss in cell viability was not related to the energy delivered to the system. At a given energy, a strong field during short cumulated pulse affected more the viability than when used a weak field associated with a long cumulated pulsation. At another field strength and for a given cumulated pulse duration, an accumulation of short pulses was also observed to be very damaging for cells. A control by the delay between the pulses suggested a memory effect. Tomov (1995) studied the porating action of exponential and rectangular electric pulses. The quantitative dependence of the percentage of porated cells on the amplitude and duration of the pulse was derived, assuming that the probability of poration is a function of the square of the membrane potential. Experimental studies supported that: i) the percentage of porated ceils is related to the increase of the pulse amplitude and duration, and ii) poration occurs when the pulse amplitude exceeds a threshold value. Chang (1989) used radio frequency sinusoidal waves from several kHz to 1 MHz for modulation of rectangular pulse and found this protocol is more efficient in both cell fusion and cell poration. Electropermeabilization is very often performed using bursts of rectangular pulses. Their typical durations are in the range from hundreds of μs to tens of ms, while the intervals between the pulses vary from several ms to several s (Rols and Teissie, 1998; Sukharev et al, 1992). Lebar et al (2002) examined how the inter-pulse interval of a train of rectangular pulses influences the electroporation of some bilayer lipid membranes. They used 100 μs pulse duration and found the threshold voltage decreased linearly with the logarithm of the interpulse interval. Threshold dropped to that of a single pulse with an interval of 1 μs. When the interval exceeded 1 μs, the influence on the response of each pulse of the train declined towards that of the previous one. The results suggested a lower electroporation threshold of the membranes with burst of pulses with less than 1 ms inter-pulse interval. Pucihar et al (2002) investigated the effect of the pulse repetition frequency on the uptake into electropermeabilized cells in vitro from the point of view of applications in electrochemotherapy. They suggested prospects for efficient use of burst of pulses in clinical electrochemotherapy. Kotnik et al (2001) compared the efficiency of three sequences of rectangular pulses consisting of: i) eight 1 ms unipolar pulses; ii) eight 1-ms symmetrical bipolar; iii) four 2 ms symmetrical bipolar. All sequences were delivered in intervals of 1 s. The conclusion is that the concentrations of ions released by bipolar pulses are in order of magnitude lower than those released by unipolar pulses of the same amplitude and duration. The authors found that the development of bipolar pulse generators is another step toward the establishment of electropermeabilization as a standard tool in biology and medicine.

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Bipolar pulses may be applied by alternating the polarity of consecutive unipolar pulses (Vernhes et al, 1999). Sersa et al (1996) reported for cyclic reorientation of electrodes supplied by burst of unipolar pulses. Vernhes et al (1999) varied the frequency and the interpulse duration keeping the duration and the number of pulses constant. They studied the effect of repetition frequency from 0.5 to 100 Hz and found high permeabilization and survival of the cells can be obtained even at 100 Hz. Pucihar et al (2002) investigated this effect at higher frequencies. The results reported confirm the increase of the uptake of Lucifer Yellow with increasing the pulse amplitude to a certain threshold, after that the uptake becomes smaller as a result of irreversible cell permeabilization. Similar process was observed with a peak of uptake with increasing the frequency up to 10 Hz while no significant differences were obtained between 1 Hz, 1 kHz and 2.5 kHz, which required higher amplitudes for achieving the same uptake. Daskalov et al (1999) reported on the field strength, the pulse width and the number of pulses in a sequence, which were implemented in instruments, built and clinically experimented by them. The rectangular shape was accepted to be more efficient for in vivo electropermeabilization. The stimulating instrument is transformer-coupled to the patient. Since short rectangular pulses of about 100 μs with a voltage up to 1250 V have usually smoothed leading and failing edges in the range of 30 through 60 μs, a toroidal core transformer was built allowing the generation of practically rectangular shape. The authors considered that the lack of detail on the tissue impedance impede the assessment of the properties of different pulse shapes. Because the mechanism of electroporation is not well understood, the development of protocols for a particular application has usually been achieved empirically by adjusting the pulse parameters (Mir, 1990). Daskalov et al (1999) constituted a logical protocol for electrochemotherapy as follows. The stimuli were a succession of 8 exponential or rectangular pulses of 100 μs duration at 1 s intervals in monophasic mode. In the biphasic mode, only rectangular pulses of 50+50 μs width were used in two sub-modes: i) with 1 s interval or ii) as a burst of 8 pulses spaced at 1 ms with a total duration of 7.1 ms. The pulse amplitude was selected in the range of 750 to 1250 V, depending on the tumor size. The electrical field strength varied form 330 to 1250 V/cm. The application of 8 pulses as a single burst was well accepted by the patients, compared to the succession of 8 separate stimuli. The use of biphasic stimuli was found logical as they apply the electric field partially in two opposite polarities to the transmembrane potential of the cells in the tissue. Thus, the biphasic stimuli might have an enhanced action on larger quantity of cells. Some of the results obtained by Daskalov et al (1999) suggested that lower intensities might be effective. They brought up also the question how to quantify the intensity, as the analogy with the defibrillation leads to substitute energy for voltage as a dose factor. The electrochemotherapy is accompanied with unpleasant sensations because of the contraction of muscles around the electrodes. These sensations are repeated with every pulse of a train of pulses if the frequency of repetition is low. They can be smoothed down to only one if each pause between the pulses is shorter than the duration of a tetanic contraction since the nerve axon cannot be excited for the refractory period (Daskalov et al, 1999; Pucihar et al, 2002). Daskalov et al (1999) drew attention also to the better tolerability of the biphasic pulses.

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The critical parameters governing the electroporation efficiency, as predicted by both theory and experimental analysis, must be optimized for each tissue in order to maximize the gene delivery while minimizing the irreversible cell damage (Somiary et al, 2000). Very general recommendations for the choice of pulse parameters may be suggested as pulse amplitude in the range of 200 to 2000 V/cm and pulse duration from hundreds of μs trough several ms (Miclavcic and Kotnik, 2004). Bipolar pulses should be preferred since they demonstrate lower poration threshold, higher uptake and better viability. However, the optimal parameter values strongly depend on the cell type, the molecule to be introduced and some specific conditions of the therapy. For the moment the electrochemotherapy is mainly applicable for treatment of tumor nodules located at cutaneous or subcutaneous tissues but some appropriate technologies for electrical pulse delivering to deep-seeded tumors seem to be promising (Giardino et al, 2006). Electroporation has been proved to enhance the efficacy of intramuscular delivery of DNA. However, this technique causes pain and discomfort to the patient, especially with higher voltages, usually from 100 to 1200 V/cm for electroporation in animals. Yu Zhou et al (2008) studied the effect of DNA vaccination of mice at lower voltages in the range of 5-10 V. The results proved that a low-voltage electroporation can induce immunity and protect the mice effectively. Tekle et al (1991) examined and compared the transfection efficiency on NIH 3T3 cells. They found that the efficiency of DNA transfection in vitro is higher with bipolar 60 kHz square wave of 400 μs duration compared to a unipolar wave with the same parameters. The results indicated that besides the role of the unipolar pulse shape, the resting membrane potential may be also responsible for the asymmetrical cell permeabilization. Pavselj and Miclavcic (2008) reported models of subcutaneous tumor during electrochemotherapy and of skin during gene electrotransfer. They used commercially available software based on the finite element method and found such approach will be useful for evaluation in advance of different pulse parameters or electrode geometries in parallel with the usually carried out in vitro and in vivo experiments.

ELECTRODES The electric field distribution is strongly depending on the electrode shape and the geometry of their location around the tumor (Miclavcic and Kotnik, 2004). Several designs of electrodes are used such as parallel plates, parallel wires, needle arrays, monopolar configuration but the resulting impedance is not considered (Daskalov et al, 1999). Two parallel plates of a distance in the range of 1-4 mm are typically used for electroporation of cells in suspension. The best materials for electrode design are platinum and stainless steel but they are very often implemented by aluminum since this metal is cheaper (Miclavcic and Kotnik, 2004). However, such design has the disadvantages to introduce significant voltage drop and metal ions at the electrode-to-solution interface. The plate electrodes are also preferred for electroporation in vivo. Sometimes, they are mounted on a caliper to ensure variable interelectrode distance, which is suitable for electrochemotherapy of cutaneous and subcutaneous tumors. The implementation of needle electrodes allows sophisticated field

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distribution by introducing of more complex pulse generation consisting of sequentially activating of electrode pairs, thus devising the tumor volume in smaller fractions. Many tumors regrew after the drug uptake in these areas where the intensity of the electric field was below a threshold (Miclavcic et al, 1998). Therefore, the influence of the electrode geometry on the field distribution continues to be studied in detail. The authors reported three-dimensional finite element model to calculate values of electric field for different electrode sets. The results demonstrated that the model is reliable and can be very useful in search for electrodes system that may better cover the tumors with sufficiently high electric field thus making the electrochemotherapy more efficient.

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GENERATORS Some authors (Chang, 1989; Tekle et al, 1991; Kotnik et al, 2001) found in the past years that the commercially available electroporators offered not sufficiently flexible choice of pulse parameters for the specific needs. Therefore, the results of Chang (1989) and Tekle et al (1991) were based on mutual dependence between number and duration of the pulses in a train because of the used signals from common pulse generators. Rodamporn et al (2007) reported a designed, developed and tested low cost programmable electroporation system for biological applications. It can generate electric fields of 100 to 1000V/cm by programmable pulse duration from 10 μs to 20 ms using a standard commercial electroporation cuvette. Recently at the market are available commercialized systems, e.g. ECM 830 made by BTX Harvard, allowing the control of voltage levels, duty cycles and pulse durations Apparatus can generate voltage ranged from 30 V to 3 kV and pulse duration from 10 μs to 600 μs (http://www.btxonline.com/products/electroporation/Default.asp). No detail is available on the type of the instrument output. Chao Cheng-wei et al (2006) reported for lever tumor implanted in rats, necrotized by means of a BK-92A device for electrochemotherapy. The parameters used were voltage of 6 V and current of 3 mA for about 10 min. Platinum positive electrode with 0.7 mm in diameter was inserted into the tumor centre. Three other widely spaced negative electrodes are located peripherally. The distance between the positive and a negative electrode was about 5 mm.

RISK OF FIBRILLATION Recently, several authors paid attention that during defibrillation and cardioversion the heart cells are exposed to potential gradients, which increase the transmembrane potential. If it is sufficiently high, pathological increases in cell permeability can occur (Al-Khadra et al, 2000). Tovar and Tung (1991) experimented with isolated frog heart cells and found that this is happened with monophasic and biphasic pulses of about 1 V and 0.2-0.4 ms duration. Khadra et al (2000) used optical mapping techniques for study the electrical activity in coronary-perfused rabbit hearts during electric shocks from 50 to 500 V. They found the electroporation was observing through the transient depolarization, the reduction of action potential amplitude and the dV/dt upstroke. The electroporation was localized to a small

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epicardial area near the electrode, as well as throughout the entire endocardium. No arrhythmia in association with electroporation was observed. The authors hypothesized the electroporation may be actively involved in extinguishing the ongoing fibrillatory activity and/or blocking the reinitiate of a new arrhythmia. Electrochemotherapy of tumors located close to the heart could lead to adverse effects, especially if electroporation pulses were delivered during the vulnerable period of the heart or some types of arrhythmias are present. Mali et al (2008) examined the influence of electroporation pulses on heart functioning. They found no pathological morphological changes in the electrocardiogram but observed transient RR interval decrease after the treatment. Although no adverse effects due to electroporation have been reported so far, the probability for complications could increase in cases of internal tumors, in tumor ablation by irreversible electroporation and with pulses of longer durations. Therefore, the authors introduced pulse generation synchronized with the QRS complex of the electrocardiogram. It is known that the most appropriate time for such intervention is before the onset of the vulnerable period of the ventricles, since it may be prolonged, e.g. after premature heart beats. Evidently, a pulses generation immediately after the QRS detection is the most reasonable. The time reserve for safe delivery after the detection and before the onset of the vulnerable period is approximately 60 ms. Finally, the authors concluded that a synchronized electroporation would increase the patient safety in cases of anatomical locations presently not accessible to existing electroporation devices and electrodes. Maor et al (2007) presented a study on the long term effects of IRE treatment of cancer tumors on large blood vessels. Sequences of ten IRE pulses with 3.8 kV/cm and 100 μs duration were applied with a frequency of 10 Hz to the carotid artery in six rats. The tissue conductivity was measured during the procedure. The results showed a predicted increase, which has been used to control the experiment. All the animals survived the procedure and showed no side effects. Histology performed 28 days after revealed that the connective matrix of the blood vessels remained intact and the number of vascular smooth muscle cells in the arterial wall decreased with no evidence of aneurysm, thrombus formation or necrosis. The authors concluded that during treatment of cancer near large blood vessels, the IRE damages only the cell membrane and no other types of molecules of the tissue. The large blood vessels showed at most an ablation of cells. Therefore, the IRE may have possible applications to treatment of pathological processes such stenosis and for attenuating atherosclerotic processes in clinical important locations as coronary, carotid and renal arteries. Music et al (1992) give a warning of the risk of fibrillation when electroporation pulses are applied to tumors located close to the heart muscle, specifically if the pulses are delivered during the vulnerable period of the heart or coincide with period of arrhythmias that may lowered the fibrillation threshold. The defibrillation shocks, which are aimed at resynchronizing the electrical activity in the heart, may induce pores in cellular membranes resulting in transient or permanent electrical and mechanical dysfunction of the heart (Nikovski et al, 2005). Seok Chan Kim (2008) maintains that a strong internal defibrillation shock may damage the heart via electroporation expressed as disruption of cell membranes. He inserted an implantable defibrillator in the right ventricle and discovered the electroporation by assessing the uptake of membrane-impermeant propidium iodide through electropores into the ventricular myocardium.

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Sersa et al (2008) carried out histological evaluation and physiological measurements of tumors based on prediction of a mathematical model to confirm that electroporation and electrotherapy of tumors have a vascular disruption action. Takashi Ashihara et al (2001) reported a computer simulation study aimed to examine whether the electroporation contributes to the mechanism of ventricular defibrillation. The conclusion was that the electroporation mechanism may play an important role in electrical defibrillation.

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OUR OWN STUDIES AND INSTRUMENT DESIGN We developed and implemented in the medical practice a family of electroporators based on the initial investigations of Daskalov el al (1999). The last of them, called CHEMOPULSE II, is a portable instrument with small dimensions of 250х190х85 mm and weight of 4 kg. The equipment is built around a toroidal transformer. It generates optimized bursts of biphasic rectangular pulses that increase the hydrophilic pores in the membrane and as a result – the drug uptake into the cells. The pulses are 8 with duration of 50+50 μs and pause between them of 1 ms. This short sequence is accepted by the patients as a single contraction of the muscles near located to the tumor. The burst of pulses is optimized during continuous clinical tests. Therefore, the operation with the instruments is considerably simplified. The operator has to select gradually the pulse amplitude from 100 through 2200 V, but special means against involuntary provoked high voltage shock are taken to protect the patient. A microcontroller circuit reduces the initial (default) pulse voltage up to 100 V. The family of electroporators are currently operating in the National Oncology Institute of Sofia and the Regina Elena Cancer Institute – Rome where many investigations and routine surgeries have been carried out. The results obtained were published in series of papers by Spugnini et al (2003 through 2009) that addressed the different needs for intraoperative and postoperative electrochemotherapy in terms of electrical parameters as well as the development of customized electrodes for the different body districts.

REFERENCES Al-Khadra A, Nikolski V, Efimov IR (2000): ‗The Role of electroporation in defibrillation‘, Circulation Research 87, 797-804 Berg K, Dietze A, Kaalhus O, Hogset A (2005): ‗Site-specific drug delivery by photochemical internalization enhances the antitumor effect of bleomycin‘, Clinical Cancer Research, 11, 8476-8485. Chang DC (1989): ‗Cell poration and cell fusion using an oscillating electric field‘, Biophysical Journal, 56, 641-652. Chang DC, Reese TS (1990): ‗Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy‘, Biophysical Journal 58, 1-12.

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Chao Cheng-wei, Tian Lian-ming, Wang Pei-jun, Zuo Chang-jing, Zhang Huo-jun (2006): ‗Electrochemotherapy for rat implanted liver tumor‘, Chinese Medical Journal, 119(8), 696-700. Ciobanu F, Radu M, Moisescu M, Surleac M, Bajenaru L, Savopol T, Covacs E (2007): ‗Electroporation of malignant cells for enhanced uptake of therapeutic drugs‘, Romanian Journal of Biophysics, 17(3), 211-217. Daskalov I, Mudrov N, Peycheva E (1999): ‗Exploring new instrumentation parameters for electrochemotherapy‘, IEEE Engineering in Medicine and Biology, 18, 62-66. Davalos RV, Otten DM, Mir LM, Rubinsky B (2004): ‗Electrical impedance tomography for imaging tissue electroporation‘, IEEE Transactions on Biomedical Engineering, 51 (5), 761 – 767, doi: 10.1109/TBME.2004.824148. Fricke H (1953): ‗The electric permittivity of a dilute suspension of membrane- covered ellipsoids‘, Journal of Applied Physics, 24, 644–646. Freeman SA, Wang MA, Weaver JC (1994): ‗Theory of electroporation of planar bilayer membranes: predictions of the aqueous area, change in capacitance, and pore-pore separation‘, Biophysical Journal, 67, 42–56. Giardino R, Fini M, Bonazzi V, Cadossi R, Nicolini A, Carpi A (2006): ‗Electrochemotherapy a novel approach to the treatment of metastatic nodules on the skin and subcutaneous tissues‘, Medicine and Pharmacotherapy, 60, 458- 462. Gimsa J, Wachner D (2001): ‗Analytical description of the transmembrane voltage induced on arbitrary oriented ellipsoidal and cylindrical cells‘, Biophysical Journal, 81, 18881896. Heller L, Lucas ML (2000): ‗Delivery of plasmid DNA by in vivo electroporation‘, Gene Therapy and Molecular Biology, 5, 55-60. Klee M, Plonsey R (1976): ‗Stimulation of spheroidal cells—the role of cell shape‘, IEEE Transition on Biomedical Engineering, 23, 347–354. Kotnik T, Bobanovic F, Miklavcic D (1997): ‗Sensitivity of transmembrane voltage induced by applied electric fields—a theoretical analysis‘, Bioelectrochemistry and Bioenergetics, 43, 285–291. Kotnik T, Miclavcic D (2000): ‗Analytical description of transmembrane voltage induced by electric fields on spheroidal cells‘, Biophysical Journal, 79, 670-679. Kotnik T, Mir LM, Flisar K, Puc M, Miclavcic D (2001): ‗Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part I. Increased efficiency of permeabilization‘, Bioelectrochemistry, 54, 83-90. Kranjc S, Cemazar M, Grosel A, Sentjurc M, Sersa G (2005): ‗Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice‘, BMC Cancer, 5, 5 pages, doi:10.1186/147-2407-5-115. Lebar AM, Kopitar NA, Ihan A, Sersa G, Miclavcic D (1998): ‗Significance of treatment energy in cell electropermeabilization‘, Electro- and magnetobiology, 17, 255-262. Lebar AM, Troiano GC, Tung L, Miklavcic D (2002): ‗Inter-pulse interval between rectangular voltage pulses affects electroporation threshold of artificial lipid bilayers‘, IEEE Transactions on NanoBioscience, 3(1), 116-120. Lopez A, Rols MP, Teissie J (1998): ‗31P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells‘, Biochemistry, 27(4), 1222–1228.

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Mahmood T, Tamkina Z, Naqvi SMS (2008): ‗Multiple pulses improve electroporation efficiency in Agrobacterium tumefaciens‘, Electronic Journal of Biotechnology, 11 (1), 4 pages, http://www.ejbiotechnology.info/content/vol11/issue1/full/1/ Mali B, Jarm T, Corovic S, Snezna M, Paulin-Kosir, Cemazar M, Sersa G, Miclavcic D (2008): ‗The effect of electroporation pulses on functioning of the heart‘, Medical and Biological Engineering and Computing, 46, 745-757. Maor E, Ivorra A, Leor J, Rubinsky B (2007): ‗The effect of irreversible electoporation on blood vessels‘, Technology in cancer research and treatment, 6(4), 1-6. Miclavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G (1998): ‗The importance of electric field distribution for effective in vivo electroporation of tissues‘, Biophysical Journal, 74, 2152-2158. Miclavcic D, Kotnik T (2004): ‗Electroporation of electrochemotherapy and gene therapy‘, In: Bioelectromagnetic Meidicine, Eds. Paul J Kosch, Marko Markov, Taylor and Francis. Miller L, Leor J, Rubinsky B (2005):‘Cancer cells ablation with irreversible elelectroporation‘, Technology in cancer research and treatment, 4(6), 1-7. Mir LM, Orlowski S, Belehradek J, Teissie J, Rols M-P, Sersa G, Miclavcic D, Gilbert R, Heller R (1995): ‗Biomedical applications of electric pulses with special emphasis on antitumor electrochemotherapy‘, Bioelectrochemistry and Bioenergetics, 38, 203-207. Music B, Jarm T, Jager F, Miklavcic D (1992): ‗Algorithm for synchronization of electroporation pulse delivery with electrocardiogram‘, American Journal of Physiology Heart and Circulatory Physiology, 263 (4), H1128-H1136, doi: 10.2495/BIO030371. Nikovski VP, Effimov IR (2005): ‗Electroporation of the heart‘, Technology in cancer research and treatment, 4(6), S147-S154. Pavselj N, Miclavcic D (2008): ‗Numerical modeling in electroporation-based biomedical applications‘, Radiology and oncology, 42(3), 159-168. Pucihar G, Mir LM, Miclavcic D (2002): ‗The effect of pulse repetition frequency on the uptake into electropermeabilized cells in vitro with possible applications in electrochemotherapy‘, Bioelectrochemistry, 57, 167-172. Rodamporn S, Beeby SP, Harris NR, Brown AD, Chad JE (2007): ‗Design and construction of a programmable electroporation system for biological applications‘, Proceeding of the ThaiBME, 234-238. Rols M-P, Teissie J (1998): ‗Electropermeabilization of mammalian cells to macromolecules: Control by pulse duration‘, Biophysical Journal, 75, 1415-1423. Rols M-P, Teissie J (1990): ‗Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon‘, Biophysical Journal, 58, 1089-1098. Rubinsky (2007): ‗Irreversible Electroporation in Medicine‘, Technology in Cancer Research and Treatment 6 (4), 255-259 Saulis G, Satkauskas S (2004): ‗Electroporation in biological membranes‘, Veterinarija ir zootechnika, 26(48),82-88. Seok Chan Kim (2008): ‗Electroporation by strong interval defibrillation shock in intact structurally normal and chronically infracted rabbit hearts‘, MSc degree, Case western reserve university, pages 49. http://www.ohiolink.edu/etd/send-pdf.cgi/Kim%20Seok%20Chan.pdf?acc_num=case 1196122659

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Sersa G, Cemazar M, Semrov D, Miklavcic D (1996): ‗Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice‘, Bioelectrochemistry and Bioenergetics, 39, 61–66. Sersa G, Jarm T, Kotnik T, Coer A, Podkrajsek M, Sentjurc M, Miclavcic D, Kadivec M, Kranjc S, Secerov A, Cemazar M (2008): ‗Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma‘, British Journal of Cancer 98(2), 388-398. Somiary S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, Malone RW (2000): ‗Theory and in vivo application of electroporative gene delivery‘, Molecular Therapy, 2, 178-187. Spugnini EP, Porrello A (2003): ‗Potentiation of chemotherapy in companion animals with spontaneous large neoplasms by application of biphasic electric pulses‘, Journal of Experimental and Clinical Cancer Research, 22(4), 571-580. Spugnini EP, Citro G, Porrello A (2005): ‗Rational design of new electrodes for electrochemotherapy‘, Journal of Experimental and Clinical Cancer Research, 24(2), 245-254. Spugnini EP, Vincenzi B, Baldi F, Citro G, Baldi A (2006): ‗Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors‘, Anticancer Research, 26(6B), 4585-4589. Spugnini EP, Vincenzi B, Citro G, Santini D, Dotsinsky I, Mudrov N, Montesarchio V, Laieta MT, Esposito V, Baldi A (2007): ‗Adjuvant electrochemotherapy for the treatment of incompletely excised spontaneous canine sarcomas‘, In Vivo, 21(5), 819-822. Spugnini EP, Citro G, Mellone P, Dotsinsky I, Mudrov N, Baldi A (2007): ‗Electrochemotherapy for localized lymphoma: a preliminary study in companion animals‘, Journal of Experimental and Clinical Cancer Research, 26(3), 343-346. Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C, Citro G, Porrello A (2007): ‗Intraoperative versus postoperative electrochemotherapy in high grade soft tissue sarcomas: a preliminary study in a spontaneous feline model‘, Cancer Chemotherapy and Pharmacology, 59(3), 375-381. Spugnini EP, Vincenzi B, Citro G, Tonini G, Dotsinsky I, Mudrov N, Baldi A (2009): ‗Electrochemotherapy for the treatment of squamous cell carcinoma in cats: a preliminary report‘, Veterinary Journal, 179(1), 117-120. Sukharev SI, Klenchin VA, Serov SM, Chernomordik LV, Chizmandzhev YA (1992): ‗Electroporation and electrophoretic DNA transfer into cells‘, The effect of DNA interaction with electropores‘, Biophysical Journal, 58, 1320-1327. Schwan HP (1957): ‗Electrical properties of tissue and cell suspensions‘, Advances in biological and medical physics, 5, 147–209. Takashi Ashihara, Takenori Yao, Tsunetoyo Namba, Makoto Ito, Takanori Ikeda, Ayaka Kawase, Sunao Toda, Toru Suzuki, Masashi Inagaki, Masaru Sugimachi, Masahiko Kinoshita, Kazuo Nakazawa (2001): ‗Electroporation in a Model of Cardiac Defibrillation‘, J. Cardiovascular Electrophysiology, 12, 1393–1403, doi: 10.1046/j.15408167.2001.01393.x.

Tekle E, Astumian RD, Chock PB (1991): ‗Electroporation by using bipolar oscillating electric field: An improved method for DNA transfection of NIH 3T4 cells‘, Proc. Natl. Acad. Sci. USA 88, Biochemestry, 88, 4230-4234.

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Tomov TC (1995): ‗Quantitative dependence of electroporation on the pulse parameters‘, Bioelectrochemistry and Bioenergetics, 37, 101-107. Tovar O, Tung L (1991): ‗Electroporation of cardiac cell membranes with monophasic or biphasic rectangular pulses‘, Pacing and clinical electrophysiology, 14(11), 1887-1892. Vernhes M-C, Cabanes P-A, Teissie J (1999): ‗Chinese hamster ovary cells sensitivity to localized electrical stresses‘, Bioelectrochemistry and Bioenergetics, 48, 171-257. Weaver JC, Chizmandzhev YA (1996): ‗Theory of electroporation: A review‘, Bioelectrochemistry and Bioenergetics, 41, 135-160. Wolf H, Rols M-P, Boldt E, Neumann E, Teissie J (1994): ‗Control by pulse parameters of electric field-mediated gene transfer in mammalian cells‘, Biophysical Journal, 66, 524531. Yu Zhou, Fang Fang, Jianjun Chen, Huadong Wang, Haiyan Chang, Zhongdong Yang, Ze Chen (2008): ‗Electroporation at Low Voltages Enables DNA Vaccine to Provide Protection against a Lethal H5N1 Avian Influenza Virus Challenge in Mice‘, Intervirology, 51, 241-246, doi: 10.1159/000156483.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 3

NON-THERMAL IRREVERSIBLE ELECTROPORATION FOR TISSUE ABLATION Paulo A. Garcia, Robert E. Neal II and Rafael V. Davalos School of Biomedical Engineering and Sciences Virginia Tech – Wake Forest University Blacksburg, VA 24061, U.S.

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ABSTRACT Non-thermal irreversible electroporation (IRE) is a promising new technique for the ablation of undesirable tissues, particularly tumors and arrhythmogenic regions in the heart (Davalos, Otten et al. 2002; Davalos, Mir et al. 2005; Edd, Horowitz et al. 2006; Al-Sakere, André et al. 2007; Al-Sakere, Bernat et al. 2007; Edd and Davalos 2007; Onik, Mikus et al. 2007; Rubinsky 2007). The procedure involves placing electrodes around the targeted tissue and delivering a series of low energy (intense but short) electric pulses. These pulses induce irrecoverable structural changes in the cell membranes of the targeted tissue, ultimately leading to cell death. IRE is a form of molecular surgery since it only affects a single molecular component of the treated volume, the cell membrane. In addition, the procedure is minimally invasive, requires only a few minutes for administration, promotes an immune response, supports rapid lesion resolution, and may be monitored in real-time with ultrasound (Al-Sakere, Bernat et al. 2007; Lee, Loh et al. 2007; Maor, Ivorra et al. 2007; Onik, Mikus et al. 2007). IRE has the ability to create complete and predictable cell ablation with sharp transition between normal and necrotic tissue, while preserving important components of the tissue such as the extracellular matrix, major blood vessels, myelin sheaths, and nerves. This chapter introduces IRE, then describes the relevant issues to consider and how to account for them when planning IRE therapies.

INTRODUCTION Non-thermal irreversible electroporation (IRE) is a new surgical technique to ablate undesirable tissue (Davalos, Mir et al. 2005). The technique is easy to apply, can be

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monitored and controlled, is not affected by local blood flow, and does not require the use of adjuvant drugs. The minimally invasive procedure involves placing needle electrodes into or around the targeted area to deliver a series of short and intense electric pulses that induce structural changes in cell membranes that promote cell death. Electroporation, which results in an increase in the permeability of the cell membrane, is initiated by exposing cells or tissues to electric pulses (Weaver and Chizmadzhev 1996; Weaver 2003). As a function of the induced transmembrane potential (the electric potential difference across the plasma membrane), the electroporation pulse can either: have no effect on the cell membrane, reversibly permeabilize the cell membrane, after which the cells can survive (reversible electroporation), or permeabilize the cell membrane in a manner that leads to cell death (irreversible electroporation), presumably through a loss of homeostasis if not from other superimposed damage modes. This increase in transmembrane potential is dependent on a variety of conditions such as tissue type, cell size, and pulse parameters including pulse shape, duration, number, and repetition rate. However, for a specific tissue type and set of pulse conditions, the primary parameter determining the extent of electroporation is the electric field to which the tissue is exposed (Edd and Davalos 2007). Recently, IRE was shown to be an effective method to treat tumors through studies with aggressive cutaneous mouse sarcoma tumors in vivo in preclinical mouse models (Al-Sakere, André et al. 2007). The electrical pulses were delivered through two plate electrodes placed across the tumors. Complete regression was achieved in 12 out of 13 treated tumors when eighty 100 µs pulses were delivered at a repetition rate of 1 pulse every 3.3 seconds using an applied electric field of 2500 V/cm (Figure 1). Histology verified that ablation occurred as a direct result of irreversible membrane permeabilization (Al-Sakere, André et al. 2007). These results were achieved with a single treatment that lasted less than five minutes.

Figure 1. Tumor volume (mm3) within nude mice after IRE treatment as a function of days after treatment. A: No treatment – tumors continue to grow. B: Eight 1000 µs pulses at 0.03 Hz deterred growth tumors and 4/13 tumors completely regressed. C: Eighty 100 µs pulses at 0.3 Hz deterred growth and 12/13 tumors completely regressed. Parameters were characterized by the same total energized (8 ms) and treatment durations (267 s). Adapted from (Al-Sakere, André et al. 2007).

The goal of this chapter is to introduce readers to the field of non-thermal IRE for tissue ablation, with particular application to cancer therapy, and to supply readers with the tools and understanding necessary to design appropriate treatment protocols. To this end, after providing a historical perspective, we present the fundamental theory that determines how electric field and temperature distributions will result from a chosen electrode configuration, pulse characteristics, and the electrical and thermal properties of the tissue.

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IRREVERSIBLE ELECTROPORATION FOR NON-THERMAL TISSUE ABLATION In many medical procedures, such as the treatment of benign or malignant tumors, it is important to ablate undesirable tissue in a controlled and focused way. Over the years, several minimally invasive methods have been developed to selectively destroy specific areas of undesirable tissues as an alternative to resection surgery. Cryosurgery, for example, is a low temperature thermal technique in which tissue is frozen on contact with a cryogen-cooled probe (Rubinsky 2000; Davalos, Mir et al. 2005). The area affected by low temperature therapies can be easily monitored through imaging. However, the probes are large and difficult to use, there is a discrepancy between the visualized cooled regions, and the the outcome is affected by blood flow (the ―cold sink‖ effect). Nonselective chemical ablation uses agents, such as ethanol, to cause the tissue ablation (Shiina, Tagawa et al. 1993; Davalos, Mir et al. 2005). This therapy is easy to apply, but the affected area cannot be controlled because of local blood flow transport of the chemical species. Focused ultrasound uses highintensity ultrasound beams to heat the undesirable tissue to coagulation (Lynn, Zwemer et al. 1942; Foster, Bihrle et al. 1993; Davalos, Mir et al. 2005). Radiofrequency ablation (RF) is a technique in which an active electrode is introduced into the undesirable area to heat the tissue to coagulation (Organ 1976; Davalos, Mir et al. 2005). Interstitial laser coagulation is yet another thermal technique in which tumors are slowly heated to temperatures exceeding the threshold of protein denaturation using low power lasers (Bown 1983; Davalos, Mir et al. 2005). High temperature thermal therapies have the advantage of ease of application. The disadvantage is that the extent of the treated area is difficult to control because blood circulation has a strong local effect on the temperature field that develops in the tissue. In addition, damage to regions outside the target area, such as the blood vessels, extracellular matrix, and other vital physiologic structures is inevitable. Consequently, when destroyed, the possible regeneration of the tissue might take months to years, and scarring is unavoidable. Davalos, Mir and Rubinsky postulated that IRE could be induced in vivo to destroy substantial volumes of targeted tissue prior to the onset of thermal damage (Davalos, Mir et al. 2005). It had been shown on cells in vitro that IRE is an effective means to kill mammalian cells, including cancer cells (Pinero, Lopez-Baena et al. 1997; Krassowska, Nanda et al. 2003). However, if IRE could not ablate a significant amount of tissue prior to the onset of thermal damage, there would be no benefit to using IRE over thermal ablation techniques since it would act in superposition. Alternatively, there would be tremendous advantages in using IRE if it could non-thermally kill the targeted area while sparing major blood vessels, connective tissue, nerves, and the surrounding tissue. The integrity of these structures is vital for the healing of the tissue after surgery. Their hypothesis that IRE could be used as an independent modality for tissue ablation was confirmed in small animal models in the liver (Edd, Horowitz et al. 2006), and on tumors (Al-Sakere, André et al. 2007), as well as in large animal models in the liver (Rubinsky, Onik et al. 2007), the prostate (Onik, Mikus et al. 2007), and the heart (Lavee, Onik et al. 2007). These in vivo studies yielded a wealth of information pertaining to the additional benefits of IRE ablation procedures as described below. An original in vivo study conducted by Edd et al. used a single 20-ms-long square wave pulse of 1000 V/cm (chosen to have no significant thermal effect) applied across rat livers

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using two plate electrodes three hours before sacrificing the animals and performing histology on the liver (Edd, Horowitz et al. 2006). It was found that the livers experienced microvascular occlusion while large vessel architecture was preserved as well as a strong demarcation between regions that were unaffected, and those that experienced IRE induced cell death. The preservation of vasculature was more thoroughly investigated by Maor et al. (Maor, Ivorra et al. 2007), where ten 100 µs pulses of 3800 V/cm at a frequency of 10 pulses per second were administered across the carotid artery of six rats 28 days before histology. This study found a large decrease in the number of vascular smooth muscle cells without evidence of aneurysm, thrombus formation, or necrosis. In addition to promoting the efficacy of performing IRE procedures on regions near major blood vessels and the potential application of IRE to treat pathological processes involving excessive proliferation of vascular smooth muscle cells, such as restenosis, an increase in conductivity of the tissue during treatment was found, resulting from the increased permeability of the cell membranes. In a study on swine liver from (Lee, Loh et al. 2007), a total of 11 lesions were created using monopolar and bipolar electrodes with ninety 100 µs pulses ranging from 1000 to 1667 V/cm. This study showed complete hepatic cell death without structural destruction as well as hypoechoic properties to the ablated regions during procedure administration, allowing realtime procedure monitoring using ultrasound. A preclinical study on the implications of IRE for the ablation of prostate tissue (both cancerous and regions exhibiting prostatitis) was performed by (Onik, Mikus et al. 2007), where one or four bipolar or monopolar electrodes were placed in the prostates of six beagle dogs before administering eighty pulses of 100 µs ranging from 1000 to 3000 V/cm. This study found IRE lesions with a narrow transition between complete necrosis and unaffected tissue, with complete destruction of the IRE lesion and resolution within two weeks, as observed by marked shrinkage. The shrinking regions resulting from the procedure show strong adaptability of IRE protocols for the treatment of pathologies involving swollen tissues and organs. This study also examined the effects on sensitive peripheral structures such as the urethra, blood vessels, nerves, and rectum that have experienced problems with thermal techniques and found all to be unaffected by the IRE treatment application. The preservation of the microvasculature experienced in this study also raises the question for the possibility of normal tissue regeneration within the ablated regions. A unique observation in the IRE lesions from (Onik, Mikus et al. 2007) was evidence of an immunologic reaction, prompting the possibility of a tumor specific immunological reaction that may be promoted by IRE, further enhancing treatment outcome. This also shows the potential for destruction of micro-metastasis in affected lymph nodes, possibly reducing the risk for recurrence. Furthermore, a study was performed on the immunologic response by (Al-Sakere, Bernat et al. 2007) on implanted tumors from a mouse sarcoma cell line. They used IRE parameters previously found in (Al-Sakere, André et al. 2007) to yield complete tumor regression (plate electrodes with four trains of 16 pulses 100 µs in length at 2500V/cm with a 90° electrode rotation between trains) on mice sacrificed 1-72 hours after treatment. Immunohistochemistry was performed, and it was determined that IRE from this procedure did not require an immunological response to produce ablation, suggesting the efficacy of using IRE on immunodepressed patients. These IRE animal experiments verified the many beneficial effects resulting from this special mode of non-thermal cell ablation. With major structures such as the extracellular

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matrix, major blood vessels, and myelin sheaths preserved, there is extremely rapid lesion resolution with healthy tissue (Rubinsky, Onik et al. 2007), preventing scar formation and promoting a beneficial immune response (Rubinsky, Onik et al. 2007). Preventing scar formation is especially important because it allows the determination of treatment success or failure through imaging, something not possible when using thermal techniques (Sickles and Herzog 1980; Onik, Mikus et al. 2007). This method also allows treatment in the heart (Lavee, Onik et al. 2007) and blood vessels (Maor, Ivorra et al. 2007) without the danger of coagulation in the blood stream and subsequent emboli.

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HISTORY It is difficult to discern when IRE was first observed. The literature suggests that the initial studies could have been as early as the 18th century (Nollet 1754; Biedermann 1898; Fuller 1898; Rockwell 1903; Frankenhaueuser and Widen 1956). However, it was not until 1967 that Sale and Hamilton demonstrated the non-thermal lethal effect of high electrical fields on organisms (Hamilton and Sale 1967; Sale and Hamilton 1967; Sale and Hamilton 1968; Rubinsky 2007). They concluded that the damage to the cell membrane occurs when the transmembrane potentialsof around 1 V are reached. This result (threshold) is based on the theoretical potential of an insulating sphere in a conducting medium in an analysis that has become a classic in the field of electroporation (Hamilton and Sale 1967; Sale and Hamilton 1967; Sale and Hamilton 1968; Rubinsky 2007). For decades, IRE has been studied extensively within in vitro cellular systems, in particular the food industry for sterilization and preprocessing of food (Doevenspeck 1961; Toepfl, Mathy et al. 2006). IRE has also been considered an effective means to destroy both gram positive and gram negative bacteria and amoebae with regards to water decontamination for biofouling control (Schoenbach, Peterkin et al. 1997; Rowan, MacGregor et al. 2000; Joshi and Schoenbach 2002; Vernhes, Benichou et al. 2002). Another context in which IRE has been studied is in the delayed cell damage in highvoltage accidents (Lee and Kolodney 1987; Lee 2005) and the post-electric-shock arrhythmias during defibrillation (Jones, Proskauer et al. 1980). Lee et al. showed that electrical injury is attributed to thermal damage as well as IRE in superposition (Lee and Kolodney 1987; Lee 2005). It had also been observed in medical applications involving thermal ablation using electrical fields that the electrosurgical tools also induce electroporation and that coagulation may be due to electrofusion of the membranes (Belov 1978). IRE is currently being studied as part of a family of non-thermal methods to ablate tissue with electrical pulses, which includes electrochemotherapy (ECT) (Mir, Orlowski et al. 1991; Mir, Glass et al. 1998; Mir 2001; Marty, Sersa et al. 2006; Mir, Gehl et al. 2006; Al-Sakere, André et al. 2007) and supra-poration (Beebe, White et al. 2003; Deng, Schoenbach et al. 2003; Gowrishankar and Weaver 2006; Al-Sakere, André et al. 2007). ECT is a relatively new minimally invasive tissue ablation technique that employs reversible electroporation pulses to facilitate the penetration \of non-permeant or low-permeant drugs, such as bleomycin or cisplatin, into cells. A major advantage of ECT is that it selectively kills only rapidly dividingcells, such as tumor cells. In tissue ablation, ECT is a safe and highly efficient

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method to introduce non-permeable cancer drugs into malignant cells and is currently used to treat cutaneous and subcutaneous tumors in humans (Mir, Orlowski et al. 1991; Belehradek, Domenge et al. 1993; Mir, Glass et al. 1998; Gothelf, Mir et al. 2003; Sersa, Cemazar et al. 2003; Al-Sakere, André et al. 2007). However, this method requires the combination of chemical agents with an electric field, which IRE does not. Supra-poration is achieved by means of nanosecond electrical pulses in the tens of nanoseconds range and field strengths of 40-80 kV/cm (Beebe, White et al. 2003; Deng, Schoenbach et al. 2003; Al-Sakere, André et al. 2007). In supra-poration, cell death is not a consequence of the irreversible cell membrane permeabilization as in IRE, but the probable result of Ca2+ ions released inside the cells (Beebe, White et al. 2003; Al-Sakere, André et al. 2007). R. Nuccitelli et al. (Nuccitelli, Pliquett et al. 2006) described antitumor effects in mice using this technique. The new techniques based on the non-thermal delivery of electric pulses, namely ECT, IRE and supra-poration, have inherent advantages and disadvantages for tissue ablation. It is quite likely that each will find appropriate uses in modern medicine, separately or in combination.

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THEORY OF IRREVERSIBLE ELECTROPORATION The natural transmembrane potential is on the order of 70 mV in healthy cells. If the potential drop across the membrane is made to exceed approximately 1 V by the action of an applied electric field, structural rearrangement of the lipid bilayer occurs, creating permanent aqueous pathways or pores for ions and macromolecules to pass through, i.e. electroporation (Sale and Hamilton 1967). The typical formula to approximate the induced transmembrane potential (Vm) resulting from an applied electric field for a cell in suspension is:



Vm  rEa cos( )  1  f f s 



2 0.5

(1)

where λ is the shape factor of the cell (1.5 for spherical cells), r is the radius of the cell, Ea is the applied electric field,  is the angle between electric field and the vector from the cell center to any point on its surface, fs is approximately equal to the frequency where the beta dielectric dispersion occurs (below which the cell membrane charge is in step with the electric field) and f is the frequency of the assumed sinusoidal Ea (Lee, Zhang et al. 2000). This results from the simplifying model of a cell as a resistor (intra- and extra-cellular pathresistance) in series with a capacitor (membrane capacitance). For most cases, the transient terms can be neglected because the electroporation pulse (100 µs - 50 ms) is much larger than the membrane charging time (about 1 µs for spherical cells about 10 μm in diameter) (Weaver 2000). Despite studies, relatively little is known about the mechanism by which IRE causes cell death. There have been numerous experimental studies on cell viability following the delivery of an electric pulse (Hulsheger and Niemann 1980; Gabriel and Teissie 1995; Lubicki and Jarayam 1997; Krassowska, Nanda et al. 2003). Yet, there are disagreements within the literature. There is an ongoing debate whether IRE-induced cell death is caused by: (1) cell membrane rupture (Weaver 1995; Weaver 1995), (2) excessive leakage through pores

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(Hoffman 1989; Weaver 1995; Weaver 1995), or (3) thermal damage to cells (Kekez, Savic et al. 1996). The three proposed mechanisms have different scaling laws that relate the strength (E) and duration (d) of the threshold electric pulse for cell death. In particular, for rupture: ln(d) ~ 1/E2; for leakage: d ~ 1/E; and for thermal damage: d ~ 1/E2 (Krassowska and Filev 2007). Some studies find a correlation between cell death and the total energy delivered by the pulse (Okino, Tomie et al. 1992; Kekez, Savic et al. 1996), while others do not (Schoenbach, Peterkin et al. 1997; Vernhes, Cabanes et al. 1999); and yet others correlate cell death with the total pulse charge (Krassowska, Nanda et al. 2003). Despite the varying mechanism theories, we present guidelines for applying IRE to treat pathologic tissues and planning medical treatments.

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NUMERICAL MODELING FOR TREATMENT PLANNING In tissue, there are a number of conditions that determine the extent of electroporation, such as tissue type and temperature, as well as a number of pulse parameters, including duration, number, shape, and repetition rate. However, for a given set of conditions, the primary parameter affecting the degree of electroporation is the local electric field strength (Miklavcic, Beravs et al. 1998; Davalos, Otten et al. 2002). Therefore, in order to design protocols for an IRE procedure, the electric field distribution, which is dependent on the procedure‘s specific electrode-tissue geometry, pulse amplitude, and tissue conductivity distribution, must be determined. Furthermore, to verify that a specific protocol does not induce thermal effects, the temperature distribution can be calculated from the electric field distribution, the electric pulse parameters, and tissue electrical and thermal properties. Knowledge of the electric field and temperature distribution enables surgeons and researchers to reliably predict the results of an IRE procedure. This insight enables surgeons to plan and optimize the electrode geometry and voltage parameters for varying types of tissue and heterogeneities to:  Ensure treatment of the entire region, especially when multiple applications are necessary  Minimize applied voltages in order to reduce charge delivered  Visualize where potential thermal damage may occur to surrounding tissues  Reduce treatment time, invasiveness, and number of procedures  Superimpose medical images to plan treatment of the appropriate region

Models: To illustrate how the IRE treated area/volume depends on the electrode configuration and applied voltage, two electrode types are analyzed as examples, as depicted in Figure 2. Case A shows single 2-mm diameter bipolar electrode, and case B shows two 1-mm diameter monopolar electrodes, separated by a distance of 10 mm. The red regions are energized and the black ones are set to ground.

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Figure 2. Example models. Case A: One 2-mm diameter bipolar electrode with conducting regions separated by a distance of 10 mm. Case B: Two 1-mm diameter monopolar electrodes separated by a distance of 10 mm. The red regions are energized and the black ones are set to ground.

Electric Field Distribution The methods used to generate the electric field and temperature distributions in tissue are similar to the ones described by Edd and Davalos (Edd and Davalos 2007). The electric field distribution associated with the electric pulse is given by solving the Laplace equation: (2) where σ is the electrical conductivity of the tissue and is the electrical potential (Edd and Davalos 2007). Boundary conditions most often include surfaces where electric potential is specified, as in the case of a source or sink electrode, or surfaces that are electrically insulating, as on the free surfaces of the tissue, for example. The electrical boundary condition along the tissue that is in contact with the energized electrode is . The electrical boundary condition at the interface of the other electrode is boundaries are treated as electrically insulating:

. The remaining

(3)

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The models are fully defined and readily solvable using a numerical method once an appropriate set of boundary conditions and the properties of the tissue are defined. The computations were performed with a commercial finite element package (FEMLab, Comsol AS, Stockholm, Sweden). The analyzed domain extends far enough from the area of interest (i.e. the area near the electrodes) that the electrically and thermally insulating boundaries at the edges of the domain do not significantly influence the results in the treatment zone. The models for the two treatment relevant electrode geometries outlined above may be seen in Figure 3. A voltage of 2000 V was placed on the energized electrode, and the resulting electric field distribution in tissue with an electric conductivity of σ = 0.2 S·m-1 has been mapped out in the three Cartesian planes (y-z, z-x, and x-y). Based on these models, a general distribution pattern can be seen. It is important to remember that this is a visualization of how the field strength disperses, and that the values of the field seen in the legend will vary with the voltage applied to the energized electrode. Thus, applying 4000 V or 1000 V to the energized electrode will result in identical electric field contours of significantly larger and smaller strengths, respectively. It is also important to note that the two needle array of Case B has a larger exposed surface area than the bipolar electrode of Case A, contributing to its larger volume of high electric field regions.

Figure 3. Electric field distribution for the bipolar electrode (A-C) of Case A and the two monopolar electrodes (D-F) of Case B. Images A and D show a cross-section in the y-z plane, B and E in the z-x plane, and C and F in the x-y plane (looking into the electrodes).

From the images in Figure 3, it can be clearly seen that the strongest electric fields will occur directly beside the electrodes as two distinct ellipses that will connect at the center, forming a ―peanut‖ shape, before expanding into an ellipse. For the bipolar electrode of Case A, the greatest fields occur in concentric rings (Figure 3C) expanding out from the electrode, indicating that inserting this electrode directly into the targeted tissue would yield the most symmetric results. The monopolar needle array has the greatest fields immediately around the conducting surfaces and then expanding inwards between them, suggesting this arrangement would work better when placed slightly on opposing outer regions of the targeted tissue.

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Joule Heating Joule heating refers to the heat generation rate per unit volume caused by an electric field. As described in (Davalos, Rubinsky et al. 2003), the joule heating source term is evaluated by solving the Laplace equation for the potential distribution associated with an electrical pulse. The associated joule heating rate per unit volume, q , from an electric field, is the square of the local electric field magnitude,   , times the electrical conductivity of the tissue:

q    

2

(4)

A convenient equation to estimate the increase in temperature (ΔT) of homogeneous tissue for the parallel plate configuration from the joule heating is:

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T 

 2   t , c p

(5)

where Δt is the total duration of the pulses, ρ is density and cp is specific heat of the tissue (Krassowska, Nanda et al. 2003). This equation assumes no heat dissipation between the pulses, and no fringe effects at the electrode edge. Furthermore, this equation assumes that the biological properties are uniform and the contributions from blood flow, metabolic heat generation, and electrode heat dissipation are negligible. To account for these other effects, the Pennes bioheat equation is often used to assess tissue heating associated with thermally relevant procedures, because it accounts for the dynamic processes that occur in tissues, such as blood perfusion and metabolism. Blood perfusion is an effective way to dissipate (take away) heat contrary to metabolic processes which generate heat in the tissue. Modifying this equation to include the joule heating term gives the equation the following form:

  (kT )  wb cb (T  Ta )  q '''     c p 2

T t

(6)

where k is the thermal conductivity of the tissue, T is the temperature above the arterial temperature (Ta = 37°C), wb is the blood perfusion per unit volume, cb is the heat capacity of the blood, q’’’ is the metabolic heat generation, ρ is the tissue density, and cp is the heat capacity of the tissue. However, it has been suggested that these factors have a negligible contribution to the overall temperature distribution as compared with joule heating (Davalos, Rubinsky et al. 2003). Therefore, we have neglected the blood perfusion and metabolic heat generation terms in our models. Several thermal boundary conditions can be employed to study the heat exchange between the electrodes and the tissue (Davalos, Rubinsky et al. 2003; Becker and Kuznetsov 2006; Becker and Kuznetsov 2007). In these models, the electrodes were considered as infinite fins,

as described in (Davalos, Rubinsky et al. 2003), which

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dissipate heat from the tissue through the electrodes to the environment. However, in other studies, the boundaries are taken to be adiabatic to predict the maximum temperature rise in the tissue: (7)

COMMON PHYSICAL PROPERTIES: For all the models, we used the physical properties of homogeneous liver tissue to provide insight, but the properties can be easily adapted for other tissues. The values of the liver tissue heat capacity (cp = 4 kJ·kg-1K-1), electrical conductivity ( = 0.2 S·m-1), thermal conductivity (k = 0.5 W·m-1K-1), and density (ρ = 1000 kg∙m-3) used in the models are taken from the literature (Swarup, Stuchly et al. 1991; Deng and Liu 2001; Davalos and Rubinsky 2008). The tissue temperature is assumed to be initially the same as the physiological temperature (37 oC).

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Temperature Distribution: An additional comparison of numerical models explored between the two electrode geometries depicted in Figure 2 may be seen in Figure 4 using the joule heating term to observe changes in temperature. The images represent the electric field and temperature distributions of the two geometries at time t = 50 µs, during the application of a single IRE pulse. From Figure 4, it can be seen that large volumes of tissue may be treated with IRErelevant electric fields (A) and (C); maintaining the same shape of distribution observed in Figure 3. Parts (B) and (D) shows the thermal effects and where they are most prevalent, which is at the edges of the energized surfaces. It should be noted that, although the thermal effects have been depicted to help visualize their distribution, the maximum temperature found at the end of the 50 µs pulse was only 43°C. This temperature is well below the range of thermal lesioning or scarring, typically taken to be 50°C (Diller 1992). Therefore, this figure, demonstrates the significantly large volumes of tissue that may be treated by IRE without the occurrence of any considerable thermal damage. This is in accord with previously published studies (Davalos, Otten et al. 2002; Davalos, Mir et al. 2005; Edd, Horowitz et al. 2006; Al-Sakere, André et al. 2007; Onik, Mikus et al. 2007). Higher energy applications (multiple pulses or longer pulses) will increase the temperature change. However, for most clinical application purposes, the volume of tissue undergoing thermal damage will typically never exceed 5% of the volume of tissue ablated by IRE. Further thermal damage assessment is possible by using the Pennes bioheat equation, calculating the equivalent thermal dose from an IRE treatment, or by assessing the thermal effects using the thermal damage equation.

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Figure 4. Electric field and temperature distributions for the bipolar (A,B) and monopolar electrodes (C,D). Parts A and C depict the electric field for IRE-relevant ranges from 650 to 1000 V/cm while B and D show the temperature region from 37°C to 43°C.

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Equivalent Thermal Dose For procedures involving time varying temperatures, thermal damage can be assessed by calculating the amount of time it would take to equivalently damage the tissue as if it was held at a constant temperature, typically 43°C (Sapareto and Dewey 1984; Becker and Kuznetsov 2006; Al-Sakere, André et al. 2007; Becker and Kuznetsov 2007; Edd and Davalos 2007; Davalos and Rubinsky 2008). The following expression is the duration necessary to hold the tissue at 43°C to result in an equivalent thermal dose:

t43 

where

t  final

R

( 43Tt )

t

(8)

t 0

Tt is the average temperature during Δt with R  0.25 when Tt  43C and R  0.5

when Tt  43C (Sapareto and Dewey 1984; Damianou, Hynynen et al. 1993). Figure 5 shows the equivalent thermal dose curves for the two monopolar electrode configuration described in Case B of Figure 2. The thermal dose was calculated for an eighty pulse (50 µs pulse length) IRE treatment at a frequency of 1 pulse per second using 1500, 2000 and 2500 V as the input voltage. Thermal doses were calculated along the electrodeelectrode axis extending 10 mm to the left and right from the middle of the electrodes, as shown at the bottom of Figure 5. The highest thermal doses occurred at the electrode tissue interface because these locations experienced the maximum electric fields. However, at 2 mm from the interface the thermal dose decreases by a factor of 10 due to heat diffusion to areas

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of lower temperature. Increasing the voltage results in more joule heating, as described in Equation 5. The 500 V intervals examined exhibited thermal doses that increased by roughly an order of magnitude each. Nevertheless, the thermal doses remained well below a typical thermal damage threshold of t43 = 120 min that has been found for common soft tissues (Damianou, Hynynen et al. 1993; Ho, Ju et al. 2007). This shows that the temperature increase generated by the entire IRE procedure is not responsible for the tissue death.

Figure 5. Thermal dose curve along the electrode-electrode axis for eighty (50 µs) pulses at a frequency of 1 Hz for 1500, 2000 and 2500 V.

Thermal Damage Equation: An additional assessment of thermal effects is thermal damage. Since thermal damage is a function of temperature and duration at elevated temperatures, the negligible heating associated with these case studies is emphasized by the fact that an electroporation pulse is typically a very small fraction of a second long (Al-Sakere, André et al. 2007; Becker and Kuznetsov 2007; Edd and Davalos 2007). One of the distinguishing features of IRE is that it does not induce thermal damage (Tropea and Lee 1992; Davalos, Mir et al. 2005; Lee and Despa 2005). The thermal damage can be calculated to assess whether a particular set of voltage parameters will induce thermal effects in addition to IRE. Thermal damage occurs when cells or tissues are exposed to a temperature higher than the physiological temperature for an extended period of time. If the period of exposure is long, thermal damage can occur at

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temperatures as low as 42C. However, 50oC is generally chosen as the target temperature (Diller 1992). Thermal damage, Ω, is quantified by the Arrhenius type equation:

     e  Ea / RT dt

(9)

where δ is the frequency factor, Ea is the activation energy, R is the universal gas constant, and T is the temperature (Henriques and Moritz 1947; Diller 1992; Rylander, Feng et al. 2005; Feng, Tinsley Oden et al. 2008).

SPECIAL CONSIDERATIONS

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Heterogeneous Tissue: In an IRE treatment, if the conductivity distribution in the targeted region is homogenous, the results in Figure 6 can be applied directly to estimate the size of the treated region as a function of electrode geometry and applied voltage. Exposing the entire tissue to the electric field magnitude necessary to achieve cell death is important. However, there can be factors that would make the targeted domain heterogeneous, such as the presence of large blood vessels, multiple tissue types, or tissues with anisotropic properties, such as muscle. Three electric field contour levels (500, 750 and 1000 V/cm) are used to illustrate the effects of heterogeneity in the electric field distribution. Under these circumstances, the reader would need to understand these guidelines when making their own model to match their specific procedure. Figures 6 - 8 shows the tissue treated with a 2000 V (50 µs pulse length) IRE pulse using the same dimensions as Case B in Figure 2. It is important to note that different tissues may have different electric field thresholds to cause IRE. Figure 7 shows the electric field distribution for an IRE procedure in which the electrical conductivity of the surrounding tissue (σ = 0.1 S·m-1) is half the magnitude of the treated tissue (σ = 0.2 S·m-1). The same electric field contour levels (500, 750 and 1000 V/cm) that were used in the homogeneous tissue discussion were used in this analysis. Having two different electrical conductivities affects the electric field distribution, so knowledge of the physical properties of the tissue is important for more accurate predictions. The treated area by IRE is increased when the surrounding tissue to the region of interest has a smaller electrical conductivity. For example, an electric field of 750 V/cm covers the entire region of interest but was not sufficient to treat the homogeneous tissue. This scenario can occur in mammary tumors in which the fat surrounding the tissue has lower electrical conductivity than the tumor itself. Figure 8 shows the electric field distribution for an IRE procedure in which the electrical conductivity of the surrounding tissue (σ = 0.4 S·m-1) is twice the magnitude of the treated tissue (σ = 0.2 S·m-1). The same electric field contour levels (500, 750 and 1000 V/cm) that were used in the two previous examples were used in this analysis. The treated area by IRE is reduced when the surrounding tissue to the region of interest has a larger electrical conductivity. For example, an electric field of 500 V/cm is now required to cover the entire region of interest which is lower than was needed to treat the homogeneous tissue. These results are to give insight to the reader about the influence of the electrical conductivity in the electric field distribution in heterogeneous tissue. For more information on these effects, other

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studies of electric fields on heterogeneous tissue can be found in (Miklavcic, Beravs et al. 1998; Edd and Davalos 2007; Esser, Smith et al. 2007).

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Figure 6. Electric field [V/cm] distribution using a 2000 V (50 µs) IRE pulse in homogeneous tissue . The black line outlines the area to be treated with IRE.

Figure 7. Electric field [V/cm] distribution using a 2000 V (50 µs) IRE pulse in heterogeneous tissue. The black line outlines the area to be treated with IRE . Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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Figure 8. Electric field [V/cm] distribution using a 2000 V (50 µs) IRE pulse in heterogeneous tissue. The black line outlines the area to be treated with IRE .

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Dynamic Properties During Electroporation Researchers have shown that there is a change in tissue conductivity during and after pulsing, as a result of electroporation (Bhatt, Gaylor et al. 1990; Davalos, Otten et al. 2002; Pavlin and Miklavcic 2003; Davalos, Otten et al. 2004; Miklavcic, Sel et al. 2004). The electrical conductivity of tissue during electroporation increases as a result of electroporation (Lee, Zhang et al. 2000; Pavlin and Miklavcic 2003; Davalos, Otten et al. 2004; Miklavcic, Sel et al. 2004). These changes can be readily incorporated into the reader‘s numerical models (Davalos, Otten et al. 2002; Davalos, Otten et al. 2004). Since the conductivity changes during IRE, it provides an active means for the physician to monitor the procedure by measuring the change in current. This also allows imaging of the irreversibly electroporated tissue with electrical impedance tomography to verify treatment success (Davalos, Otten et al. 2002; Davalos, Otten et al. 2004; Lee, Loh et al. 2007).

Temperature Dependent Properties If it is necessary to take into consideration the thermal effects from a treatment, then other tissue properties such as the mass density, heat capacity and thermal conductivity are needed. If these properties cannot be directly measured, the properties of the tissue can be

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taken from the literature, for example from (Duck 1990). It should be noted that the thermal and electrical conductivities of biological tissues are dependent on temperature and their dependence can be found in literature and incorporated into the models if necessary. Since IRE produces negligible heating, the change in conductivity is not usually significant. For example, thermal and electrical conductivities increase by about 0.25% and 1.5% per degree Celsius rise in temperature, respectively, in liver (Duck 1990).

CONCLUSION

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This chapter introduced the field of non-thermal IRE and its potential to ablate large tissue volumes. The advantages of IRE over other focal ablation techniques lay within its ability to ablate tissue through a non-thermal mechanism. This method preserves the extracellular matrix, nerves, major blood vessels, and other sensitive tissues, enhancing treatment outcome. Furthermore, the ablation area can be predicted using numerical modeling for accurate treatment planning, and application of the procedure can be monitored in realtime using ultrasound. This ablation of the targeted areas exhibits rapid lesion creation and resolution, prompting the repopulation of the region with healthy cells. Though treatment success is not dependent upon the immune system, a tumor specific immune response capable of helping to destroy any residual micro-metastases occurs, decreasing the chances of recurrence. These aspects, in conjunction with short treatment times and the minimally invasive nature of treatment administration, show strong potential for using IRE as an effective tissue ablation modality for the improved treatment of many localized tissue pathologies. This chapter then focused on applying the basic principles involved in IRE therapies to facilitate accurate treatment planning for clinical therapies.

REFERENCES Al-Sakere, B., F. André, et al. (2007). "Tumor Ablation with Irreversible Electroporation." PLoS ONE 2(11): e1135:1-8. Al-Sakere, B., C. Bernat, et al. (2007). "A study of the immunological response to tumor ablation with irreversible electroporation." Technology in Cancer Research and Treatement 6: 301-306. Becker, S. M. and A. V. Kuznetsov (2006). "Numerical modeling of in vivo plate electroporation thermal dose assessment." Journal of Biomechanical Engineering 128(1): 76-84. Becker, S. M. and A. V. Kuznetsov (2007). "Thermal Damage Reduction Associated with in Vivo Skin Electroporation: A Numerical Investigation Justifying Aggressive PreCooling." International Journal of Heat and Mass Transfer 50: 105-116. Beebe, S. J., J. White, et al. (2003). "Diverse effects of nanosecond pulsed electric fields on cells and tissues." DNA Cell Biol 22(12): 785-96. Belehradek, M., C. Domenge, et al. (1993). "Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial." Cancer 72(12): 3694-700.

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Belov, S. V. (1978). "Effects of high-frequency current parameters on tissue coagulation." Biomedical Engineering 12: 209-211. Bhatt, D. L., D. C. Gaylor, et al. (1990). "Rhabdomyolysis due to pulses electric fields." Plast. Reconstr. Surg. 86(1): 1-11. Biedermann, W. (1898). Electrophysiology. London, Macmillan. Bown, S. G. (1983). "Phototherapy in tumors." World J Surg 7(6): 700-9. Damianou, C., K. Hynynen, et al. (1993). "Application of the thermal dose concept for predicting the necrosed tissue volume during ultrasound surgery." Ultrasonics Symposium 2: 1199-1202. Davalos, R. V., L. M. Mir, et al. (2005). "Tissue ablation with irreversible electroporation." Annals of Biomedical Engineering 33(2): 223-231. Davalos, R. V., D. M. Otten, et al. (2004). "Electrical impedance tomography for imaging tissue electroporation." IEEE Transactions on Biomedical Engineering 51(5): 761-767. Davalos, R. V., D. M. Otten, et al. (2002). "A feasibility study for electrical impedance tomography as a means to monitor tissue electroporation for molecular medicine." IEEE Transactions on Biomedical Engineering 49(4): 400-403. Davalos, R. V. and B. Rubinsky (2008). "Temperature considerations during irreversible electroporation." International Journal of Heat and Mass Transfer 51(23-24): 5617-5622. Davalos, R. V., B. Rubinsky, et al. (2003). "Theoretical analysis of the thermal effects during in vivo tissue electroporation." Bioelectrochemistry 61(1-2): 99-107. Deng, J., K. H. Schoenbach, et al. (2003). "The effects of intense submicrosecond electrical pulses on cells." Biophys. J .84(4): 2709-14. Deng, Z. S. and J. Liu (2001). "Blood perfusion-based model for characterizing the temperature fluctuations in living tissue." Phys A STAT Mech Appl 300: 521-530. Diller, K. R. (1992). Modeling of bioheat transfer processes at high and low temperatures. Bioengineering heat transfer. Y. I. Choi. Boston, Academic Press, Inc. 32: 157-357. Doevenspeck, H. (1961). "Influencing cells and cell walls by electrostatic impulses." Fleishwirtshaft 13: 986-987. Duck, F. A. (1990). Physical Properties of Tissues: A Comprehensive Reference Book. San Diego, Academic Press. Edd, J., L. Horowitz, et al. (2006). "In vivo results of a new focal tissue ablation technique: irreversible electroporation." IEEE Transactions on Biomedical Engineering 53(7): 14091415. Edd, J. F. and R. V. Davalos (2007). "Mathematical modeling of irreversible electroporation for treatment planning." Technology in Cancer Research and Treatment 6: 275-286. Esser, A. T., K. C. Smith, et al. (2007). "Towards solid tumor treatment by irreversible electroporation: intrinsic redistribution of fields and currents in tissue." Technol Cancer Res Treat 6(4): 261-74. Feng, Y., J. Tinsley Oden, et al. (2008). "A two-state cell damage model under hyperthermic conditions: theory and in vitro experiments." J. Biomech. Eng .130(4): 041016. Foster, R. S., R. Bihrle, et al. (1993). "High-intensity focused ultrasound in the treatment of prostatic disease." Eur Urol 23 Suppl 1: 29-33. Frankenhaueuser, B. and L. Widen (1956). "Anode break excitation in desheated frog nerve." J. Physiol. 131: 243-247. Fuller, G. W. (1898). Report on the investigations into the purification of the Ohio river water at Louisville Kentucky. New York, D. Van Nostrand Company.

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Gabriel, B. and J. Teissie (1995). "Control by electrical parameters of short- and long-term cell death resulting from electropermeabilization of Chinese hamster ovary cells." Biochim. Biophy.s Acta 1266(2): 171-8. Gothelf, A., L. M. Mir, et al. (2003). "Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation." Cancer Treat Rev 29(5): 371-87. Gowrishankar, T. R. and J. C. Weaver (2006). "Electrical behavior and pore accumulation in a multicellular model for conventional and supra-electroporation." Biochem Biophys Res Commun 349(2): 643-53. Hamilton, W. A. and A. J. Sale (1967). "Effects of high electric fields on microorganisms. 2. Mechanism of action of the lethal effect." Biochimica et Biophysica Acta (BBA) 163: 3743. Henriques, F. C. and A. R. Moritz (1947). "Studies in thermal injuries: the predictability and the significance of thermally induced rate processes leading to irreversible epidermal damage." Arch. Pathol. 43: 489-502. Ho, C.-S., K.-C. Ju, et al. (2007). "Thermal therapy for breast tumors by using a cylindrical ultrasound phased array with multifocus pattern scanning: a preliminary numerical study." Physics in Medicine and Biology 52: 4585-4599. Hoffman, G. A. (1989). "Cells in electric field. Physical and practical electronic aspects of electro cell fusion and electroporation." Electroporation and Electrofusion in Cell Biology: 389-407. Hulsheger, H. and E. G. Niemann (1980). "Lethal effects of high-voltage pulses on E. coli K12." Radiat Environ Biophys 18(4): 281-8. Jones, J. L., C. C. Proskauer, et al. (1980). "Ultrastructural injury to chick myocardial cells in vitro following "electric countershock"." Circ Res 46(3): 387-94. Joshi, R. P. and K. H. Schoenbach (2002). "Mechanism for membrane electroporation irreversibility under high-intensity, ultrashort electrical pulse conditions." Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 66(5 Pt 1): 052901. Kekez, M. M., P. Savic, et al. (1996). "Contribution to the biophysics of the lethal effects of electric field on microorganisms." Biochim Biophys Acta 1278(1): 79-88. Krassowska, W. and P. D. Filev (2007). "Modeling electroporation in a single cell." Biophys. J. 92(2): 404-17. Krassowska, W., G. S. Nanda, et al. (2003). "Viability of cancer cells exposed to pulsed electric fields: the role of pulse charge." Ann. Biomed. Eng. 31(1): 80-90. Lavee, J., G. Onik, et al. (2007). "A Novel Nonthermal Energy Source for Surgical Epicardial Atrial Ablation: Irreversible Electroporation." The Heart Surgery Forum 10(2): E162167. Lee, E. W., C. T. Loh, et al. (2007). "Imaging guided percutaneous irreversible electroporation: ultrasound and immunological correlation." Technology in Cancer Research and Treatement 6(4): 287-294. Lee, R. C. (2005). "Cell Injury by Electric Forces." Annals of the New York Academy of Sciences 1066: 85-91. Lee, R. C. and F. Despa (2005). Distinguishing Electroporation from Thermal Injuries in Electrical Shock by MR Imaging. Engineering in Medicine and Biology 27th Annual Conference, Shanghai, China, IEEE. Lee, R. C. and M. S. Kolodney (1987). "Electrical injury mechanisms: Electrical breakdown of cell membranes." Plast Reconstr Surg 80(5): 672-9.

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Lee, R. C., D. Zhang, et al. (2000). Biophysical Injury Mechanisms in Electrical Shock Trauma. Ann.. Rev.Biomed. Eng. M. L. Yarmish, K. R. Diller and M. Toner. Palo Alto, Annual Review Press. 2: 477-509. Lubicki, P. and S. Jarayam (1997). "High voltage pulse application for the destruction of the Gram-negative bacterium." Bioelectrochem Bioenerg 43: 135-141. Lynn, J. G., R. L. Zwemer, et al. (1942). "A new method for the generation and use of focused ultrasound in experimental biology." J. Gen. Physiol. 26(2): 179-193. Maor, E., A. Ivorra, et al. (2007). "The effect of irreversible electroporation on blood vessels." Technology in Cancer Research and Treatement 6(4): 307-312. Marty, M., G. Sersa, et al. (2006). "Electrochemotherapy - an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of the ESOPE (European Standard Operating Procedures of Electrochemotherapy) study." Eur. J. Cancer Supplements 4(11): 3-13. Miklavcic, D., K. Beravs, et al. (1998). "The importance of electric field distribution for effective in vivo electroporation of tissues." Biophysical Journal 74(5): 2152-2158. Miklavcic, D., D. Sel, et al. (2004). Sequential Finite Element Model of Tissue Electropermeabilisation. Proceedings of the 26th Annual International Conference of the IEEE EMBS, San Francisco, CA. Mir, L. M. (2001). "Therapeutic perspectives of in vivo cell electropermeabilization." Bioelectrochemistry 53(1): 1-10. Mir, L. M., J. Gehl, et al. (2006). "Standard Operating Procedures of the Electrochemotherapy." Eur. J .Cancer Supplements 4(11): 14-25. Mir, L. M., L. F. Glass, et al. (1998). "Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy." Br. J. Cancer 77(12): 2336-42. Mir, L. M., S. Orlowski, et al. (1991). "Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses." Eur J Cancer 27(1): 68-72. Nollet, J. A. (1754). Recherches sur les causes particulieres des phenomenes electriques. Paris, Chez H.L. Guering and L.F. Delatour. Nuccitelli, R., U. Pliquett, et al. (2006). "Nanosecond pulsed electric fields cause melanomas to self-destruct." Biochem Biophys Res Commun 343(2): 351-60. Okino, M., H. Tomie, et al. (1992). "Optimal electric conditions in electrical impulse chemotherapy." Jpn. J. Cancer Res. 83(10): 1095-101. Onik, G., P. Mikus, et al. (2007). "Irreversible electroporation: implications for prostate ablation." Technol Cancer Res Treat 6(4): 295-300. Organ, L. W. (1976). "Electrophysiologic principles of radiofrequency lesion making." Appl Neurophysiol 39(2): 69-76. Pavlin, M. and D. Miklavcic (2003). "Effective Conductivity of a Suspension of Permeabilized Cells: A Theoretical Analysis." Biophysical Journal 85: 719–729. Pinero, J., M. Lopez-Baena, et al. (1997). "Apoptotic and necrotic cell death are both induced by electroporation in HL60 human promyeloid leukaemia cells." Apoptosis 2(3): 330-6. Rockwell, A. D., Ed. (1903). The medical and surgical uses of electricity: including the Xray, Finsen light, vibratory therapeutics, and high-frequency currents. New York, E.B. Treat and Company. Rowan, N. J., S. J. MacGregor, et al. (2000). "Pulsed electric field inactivation of diarrhoeagenic Bacillus cereus through irreversible electroporation." Lett Appl Microbiol 31(2): 110-4.

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Rubinsky, B. (2000). "Cryosurgery." Annual Review of Biomedical Engineering 2(1): 157187. Rubinsky, B. (2007). "Irreversible Electroporation in Medicine." Technology in Cancer Research and Treatement 6(4): 255-260. Rubinsky, B., G. Onik, et al. (2007). "Irreversible electroporation: A new ablation modality – clinical implications." Technology in Cancer Research and Treatment 6(1): 37-48. Rylander, M. N., Y. Feng, et al. (2005). "Optimizing HSP Expression in Prostate Cancer Laser Therapy Through Predictive Computational Models." Journal of Biomedical Optics 11(4): 04111131-16. Sale, A. J. and W. A. Hamilton (1967). "Effects of high electric fields on micro-organisms. 1. Killing of bacteria and yeasts." Biochimica et Biophysica Acta 148: 781-788. Sale, A. J. and W. A. Hamilton (1968). "Effects of high electric fields on micro-organisms. 3. Lysis of erythrocytes and protoplasts." Biochim Biophys Acta 163(1): 37-43. Sapareto, S. and W. Dewey (1984). "Thermal dose determination in cancer therapy." Int. J. radiation oncology Biol. Phys. 10: 787-800. Schoenbach, K. H., F. E. Peterkin, et al. (1997). "The effect of pulsed fields on biological cells: Experiments and applications." IEEE Trans Biomed Eng 25: 284-292. Sersa, G., M. Cemazar, et al. (2003). "Electrochemotherapy: advantages and drawbacks in treatment of cancer patients." Cancer Therapy 1: 133-42. Shiina, S., K. Tagawa, et al. (1993). "Percutaneous ethanol injection therapy for hepatocellular carcinoma: results in 146 patients." AJR Am J Roentgenol 160(5): 1023-8. Sickles, E. and K. Herzog (1980). "Intramammary scar tissue: a mimic of the mammographic appearance of carcinoma." Am. J. Roentgenol. 135(2): 349-352. Swarup, A., S. Stuchly, et al. (1991). "Dielectric properties of mouse MCA1 fibrosarcoma at different stages of development." Bioelectromagnetics 12: 1-8. Toepfl, S., A. Mathy, et al. (2006). "Review: Potential of high hydrostatic pressure and pulsed electric field for energy efficient and environmentally friendly food processing." Food Review International 22: 405-423. Tropea, B. I. and R. C. Lee (1992). "Thermal Injury Kinetics in Electrical Trauma." J. Biomech. Engr. 114: 241-250. Vernhes, M. C., A. Benichou, et al. (2002). "Elimination of free-living amoebae in fresh water with pulsed electric fields." Water Res. 36(14): 3429-38. Vernhes, M. C., P. A. Cabanes, et al. (1999). "Chinese hamster ovary cells sensitivity to localized electrical stresses." Bioelectrochem Bioenerg 48(1): 17-25. Weaver, J. C. (1995). "Electroporation in cells and tissues: A biophysical phenomenon due to electromagnetic fields." Radio Sci 30: 205-221. Weaver, J. C. (1995). "Electroporation theory. Concepts and mechanisms." Methods Mol. Biol .48: 3-28. Weaver, J. C. (2000). "Electroporation of cells and tissues." IEEE Transactions on Plasma Science 28(1): 24-33. Weaver, J. C. (2003). "Electroporation of Biological Membranes from Multicellular to Nano Scales." IEEE Transactions on Dielectrics and Electrical Insulation 10(5): 754-768. Weaver, J. C. and Y. A. Chizmadzhev (1996). "Theory of electroporation: a review." Bioelectrochem. Bioenerg. 41: 135-60.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 4

MECHANISMS OF MICROORGANISM INACTIVATION BY PULSED ELECTRIC FIELDS Gintautas Saulis* Department of Biology, Faculty of Nature Sciences, Vytautas Magnus University, Kaunas, Lithuania

ABSTRACT

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To implement pulsed electric fields (PEF) for killing of microorganisms as a routine food processing and preservation technology, better understanding of the actual mechanisms that govern microbial inactivation by this technique is required. Also, the parameters of the electric treatment assuring the best yield of microbial inactivation have to be determined. Here, with the purpose of creating the model capable to predict the kinetics of microorganism inactivation, the kinetics of cell death induced by PEF has been analyzed taking into account the following stages of this process: 1) pore formation due to membrane electroporation; 2) increasing of the membrane permeability during the PEF treatment and 3) processes taking place after PEF treatment (leakage of intracellular compounds, pore shrinkage and disappearance, etc.). Using the set of chosen parameters, theoretical relationships between the external electric field strength required to porate cell, Ep, and the length of an exponential pulse, i, was studied. It has been obtained that the cell poration time depends on the pulse intensity: the shorter the pulse length, the higher the field strength has to be. This dependence is more pronounced for short pulses (i 10 m) the electric field strength required for the poration of cell membranes increases significantly slower with the decreasing pulse length. Then, the kinetics of pore disappearance was analysed - the theoretical dependence of the fraction of completely resealed cells on the post-pulse incubation time was calculated. All dependences obtained theoretically are compared with the experimental relationships obtained for various cell lines. Good agreement between theory and experiments has been obtained.

*

Address for correspondence: Gintautas Saulis, Department of Biology, Faculty of Nature Sciences, Vytautas Magnus University, 58 K. Donelaičio str., Kaunas, 44248, Lithuania Tel: +370-612-82773, Fax: +370-37406572, Email: [email protected].

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Keywords: Electroporation, hydrophilic pore, pore formation, pore disappearance

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INTRODUCTION The use of pulsed electric fields (PEF) for killing of microorganisms is a promising new non-thermal food processing technology. Till now, a variety of microorganism species have been successfully inactivated by PEF in various aqueous solutions, such as milk, orange or apple juice, whole egg, pea soup and various buffer solutions [1-3]. Synergism with other preservation methods has also been confirmed [4-7]. To implement this method, first, better understanding of the way how microorganisms die due to the action of pulsed electric fields is required. For practical purposes, the parameters of electric treatment assuring the best yield of microbial inactivation have to be determined. This should be based on development of the models that would allow predicting microbial inactivation. Unfortunately, the models currently used for this purpose are, in most part, purely empirical and based on simple approximations of the experimentally obtained dependences by some of the well-known mathematical equations, such as a sigmoid curve, Bigelow, Weibull frequency distribution functions and other ones [8-11]. Meanwhile, the models describing the kinetics of the microorganism inactivation, should account the actual mechanisms that govern microbial inactivation by PEF [12]. It is known that the primary cause of inactivation of microorganisms leading to the loss of cell membrane functionality and leakage of intracellular contents is electroporation of the cell membrane occurring due to the exposure of cells by pulsed electric field [13-16]. Here, the complete process of the microorganism inactivation by PEF has been analyzed. This process consists of the following stages: 1) pore formation due to membrane electroporation; 2) increasing of the membrane permeability due to the increase in the number and/or sizes of pores during the PEF treatment and 3) processes taking place after PEF treatment (leakage of intracellular compounds, pore shrinkage and disappearance, etc.). Theoretical dependences describing the kinetics of each of these processes have been presented and compared with the experiments.

THEORY The cell electroporation as well as cell killing (inactivation of microorganisms) by PEF treatment should be regarded as an "all-or-nothing events" [12, 17, 18]. Quantitatively, this can be depicted by two curves describing the dependence of the fraction of electroporated and killed (inactivated) cells on the duration of an electric pulse (as shown in Figure 1) or an electric field strength [12]. In the case of small pores, there will be some difference between the fraction of electroporated (dashed line) and dead cells (solid line) Fp - Fd as shown in Figure 1 [12]. The process of microorganism inactivation consists of the following main stages [12]: 1) Initial stage (with the duration from nanoseconds to milliseconds): creation of pores when an electric pulse is applied ('electroporation' curves in Figure 1);

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2) Stage of evolution of the pore population: change in the number of pores and their sizes during an electric treatment; 3) Post-treatment stage: cell death (complete inactivation) ('inactivation' curve in Figure 1) or returning of the cell to its initial viable state due to pore resealing. In the latter case, the damage of the cells, induced by pulsed electric fields, is sub-lethal.

Figure 1. The schematic representation of the dependences of the fraction of electroporated and dead (inactivated) cells on the electric pulse duration. In the majority cases, there is some difference between the fraction of electroporated and dead cells Fp - Fd. The area between these two curves represents the cells which were electroporated but retained their viability due to pore resealing – the damage of these cells, induced by pulsed electric fields, was sub-lethal.

The dependence of the fraction of electroporated cells, Fp, on the parameters of an electric treatment can be written as [17]:



Fp E0 , i   1  exp  k f ( E0 ) i



(1)

where kf(E0) is the rate of pore formation in a cell, E0 is the electric field strength, and i is the duration of the electric treatment. For a spherical cell, kf(E0) can be calculated from [17]:

k f ( E0 ) 

 W f (0)  1 ( w /  m  1) 2 2 a 2 exp  r* )(1.5E0 a y   0 ) 2 ]dy (2)   exp[( Cm al 2k BT  k BT  1

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where  is the frequency of lateral fluctuations of lipid molecules, a is the cell radius, al is the area per lipid molecule, kB is Boltzmann's constant, T is the absolute temperature, Wf(0) is the energy barrier to pore formation at m=0, r* is the pore radius corresponding to the top of this barrier, 0 is the resting potential, m and w are the relative permittivities of the membrane and the water inside the pore, Cm is the specific capacity of the membrane. From equations (1) and (2), it follows that increasing the amplitude of electric pulses is much more effective that lengthening the treatment time, which is consistent with the experimental data [19, 20]. Using equations (1) and (2), one can obtain theoretical relationships between the parameters of the electric treatment resulting in cell electroporation ('electroporation' curves in Figure 1) for any type of an electric treatment. The relationship between the external electric field strength required to porate cell, Ep, and the time constant of an exponential pulse, i, calculated by using the set of chosen parameters is shown in Figure 2. For the calculations the following set of parameters (the "standard cell") was used:  = 1011 s-1, al = 0.6 nm2, Wf(0) = 45 kT, r* = 0.3 nm [21, 22], a = 3.5 m, Cm = 1 F/cm2 w = 81, m = 2, T = 295 K, 0 = 25 mV, and m = 0.3 s. It is seen from Figure 2 that the shorter the pulse length, the higher the field strength should be to electroporate the cell. This dependence is more pronounced for short pulses.

Figure 2. Theoretical dependence of the electric field strength necessary for electroporation, E0.5, and corresponding transmembrane potential m on the time constant of an exponential electric pulse calculated according to Eqns. (1) and (2).

The stage of evolution of the pore population (the change in the number of pores and their sizes) during an electric treatment is especially important for achieving microorganism inactivation because often small initial pores do not lead to the cell death (see Comparison Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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with Experiments and Discussion). It is considered that short pulses often fail to cause irreversible damage because there is no enough time for pores to grow and expand beyond a critical threshold radius [23]. The dynamics of the pore population can be estimated on the basis of Smoluchowski equation [24]:

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nr , t  / t  D p

 2 n(r , t ) D p  W  n(r , t ) D p   2W        nr , t  k BT  r  r k BT  r 2  r 2

(3)

where n(r,t) is the pore density function, Dp – pore diffusion constant, W – pore energy, and r – pore radius. Theoretical modeling carried out on the basis of Eq. (3), shows that while electric pulse is on, the pore population evolves by increasing both the number and sizes of pores [25-27]. To create larger pores longer pulses are needed [28, 29]. After the end of an electric treatment, the cell or microorganism can either die or retain its viability because of the capability of pores to reseal [30]. The process of pore resealing consists of a few stages of the quick (microseconds–milliseconds, minutes) reduction of pore size until the value of about 0.5 nm and the stage of the slow (tens of minutes-hours) complete pore closure [30]. Due to this, the membrane barrier function for larger molecules (sucrose, proteins, enzymes) is restored within a few minutes at 37 oC while complete resealing can take a few hours [30]. Pore resealing is important for practical applications of PEF for microorganism inactivation as it effects the number of molecules exchanged between the intracellular fluid and the surrounding medium which influences the cell viability and the likelihood of its death [12, 23]. By changing the conditions, at which cells or microorganisms are kept after PEF treatment, it is possible either to retain cell viability or facilitate cell death, depending on the particular needs. So, it is necessary to know how long the pores remain open. Because the last stage of pore resealing – complete closure of small pores – is much slower than the first ones (reducing the pore size), the kinetics of pore disappearance in a cell after an electric pulse has been terminated can be analyzed as a one-step process [31]. This allows describing the dependence of the fraction of the cells the pores in which have completely disappeared on the post-pulse incubation time, Fr(t), as [31]: 

Fr t   (1  Firr )   Pn (0)1  exp( k r t )

(4)

n 1

where n is the number of pores created in the cell membrane during an electric pulse, kr is the rate of pore resealing, Firr is the fraction of the cells that have been damaged irreversibly, and Pn(0) is the probability that there are n pores in a cell just after the pulse.

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COMPARISON WITH EXPERIMENTS AND DISCUSSION

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For cell electroporation, electric pulses of variuos durations are used – starting from as short as a few nanoseconds [32-34] to several [35-37] or several tens [38-40] of milliseconds. Usually, the duration of electric pulses in within the range of microseconds [41-43]. Experimental data show that, in the case of short electric pulses, 'threshold' electroporation of cells and liposomes involves pores with radii of about 0.3 nm, that is, just large enough to let small ions, such as K+, Rb+, Tl+ or Na+ (radii of hydrated ions are 0.17– 0.33 nm [44-46]), through but still impermeable to slightly larger molecules such as mannitol, sucrose or propidium iodide (radii ~0.4–0.5 nm) [35, 47-50]. The leakage of intracellular substances important for cell viability (ATP, proteins, enzymes) is limited through small pores. Due to this, in the majority cases, there will be some difference between the fraction of electroporated and dead cells Fp - Fd as shown in Figure 1 [12], especially when short electric pulses (in the range of tens of microseconds or shorter) are used. The 'electroporation' curve can be obtained by measuring the loss of intracellular K+ [51]. The 'death or inactivation' curve is usually obtained by the viability tests, e.g. by cells clonogenic [51, 52] or the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays [53]. An example of the curves describing the dependence of the fraction of electroporated and killed cells on the amplitude of a square-wave electric pulse with the duration of 2 ms, obtained for rat glioma C6 cells, are shown in Figure 3 (Saulis and Saule, unpublished data). It can be seen from this figure, that the difference between the fraction of electroporated and dead cells Fp – Fd can be as high as 70 %.

Figure 3. The dependences of electroporated and dead cells on the amplitude of a square-wave electric pulse with the duration of 2 ms obtained for rat glioma C6 cells.

From equations (1) and (2), it follows that increasing the amplitude of electric pulses is much more effective that lengthening the treatment time, which is consistent with the Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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experimental data [19, 20]. The shape of the dependence shown in Figure 2 is similar to the dependence of the fraction of electroporated human erythrocytes on the duration of an electric pulse obtained experimentally [42]. As it was mentioned in section Theory, calculations carried out on the basis of Eq. (3), show that during an electric pulse, the pore population evolves by increasing both the number and sizes of pores [25-27]. To create larger pores longer pulses are needed [28, 29]. Figure 4 shows the dependences of the fractions of human erythrocytes the membrane of which has become permeable to mannitol and sucrose. The erythrocytes were exposed to an electric pulse of various field strengths (E0 = 2–6 kV/cm). It can be seen from this figure that pores permeable to mannitol appeared at electric field intensities higher than 2.25–2.5 kV/cm, and that with increasing pulse intensity there is an increase in the number of cells permeable to mannitol or sucrose. Figure 4 also indicates that the pores, the presence of which is sufficient for a human erythrocyte to be regarded as porated, are small. For example, the exposure of the cells to a 3 kV/cm electric pulse leads to the poration of almost 100 % of the cells, but in only 30 % of the cells the pores through which mannitol can enter the cell were created. The pores permeable to sucrose only appeared in 5.5 % of the cells. The radius of the sucrose molecule is about 4.4–5.2 Å [47, 54, 55]. Thus, it can be concluded that the average radius of pores created by the short (time constant about 22 s) exponential electric pulse with the amplitude between 2 and 6 kV/cm is in the range of 0.2–0.5 nm [30, 56].

Figure 4. Dependences of the fraction of electroporated (permeable to K+ ions) human erythrocytes and the cells with pores through which mannitol or sucrose can enter the cell on the electric pulse amplitude. Cells were subjected to an electric pulse ( = 20 s) at varying field strengths at 20 °C.

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Figure 5. Dependences of the fraction of electroporated mouse hepatoma MH-A22 cells and cells permeable to bleomycin on the amplitude of a square-wave electric field pulse with the duration of 2 ms. (Figure 5 from Saulis et al. [51], reproduced with the kind permission of Elsevier).

The dependences of the fraction of electroporated cells (permeable to potassium ions, the hydrated radius of which is 0.16–0.22 nm [45]) and cells permeable to bleomycin (radius about 0.8 nm) on the pulse intensity obtained for mouse hepatoma MH-22A cells exposed to a single square-wave electric pulse with the duration of 2 ms are shown in Figure 5 [51]. It can be seen from this figure, that the curves showing the dependence of the fraction of the cells that have become permeable to bleomycin are close to the ones showing the release of intracellular potassium ions. This means that, irrespective of the amplitude of an electric pulse, in all cells that were electroporated the pores larger than the size of the molecules of bleomycin were created [51]. So, experimental studies obtained in different laboratories on diferent cells confirm the conclusions obtained in theoretical studies [25-27] that inreasing the amplitude and/or the duration of the electric pulse, increases the number and sizes of pores [28]. Usually, to create larger pores longer pulses are needed [28, 51, 57-59]. Experimental points in Figure 6 show the dependences of the fraction of Chinese hamster ovary cells, the membrane of which has restored its impermeability to trypan blue, on the time elapsed after the pulse. The data were taken from the paper published by Rols et al. (1990) [60]. Electroporation conditions were 10 square-wave pulses, i = 100 s,  = 1 Hz, E0 = 1.5 (filled circles), and 1.8 (open triangles) kV/cm, and resealing was monitored at T = 21 oC. Solid lines in Figure 6 are the theoretical distribution functions calculated from Eq. (4) [31].

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Equation (4) shows that increasing the time of incubation at elevated temperature increases the fraction of resealed cells, which is consistent with experimental data [31]. This means that the time necessary for the resealing varies from cell to cell, and thus the process of pore disappearance after electroporation is fundamentally stochastic [31]. So, the PEF killing efficiency should be higher for lowered post- PEF treatment temperature. Some observations indicate that this might be the case. When cells were post-incubated at lower temperature, the killing rate of Saccharomyces cerevisiae was higher [61].

Figure 6. Experimental points show the dependence of the fraction of Chinese hamster ovary cells, the membrane of which has restored its impermeability to trypan blue, on the time passed after the pulse. 10 square-wave pulses, i = 100 s,  = 1 Hz, E0 = 1.5 kV/cm (curve 1), and 1.8 kV/cm (curve 2). Resealing was monitored at T = 21 oC. Solid curves are the theoretical curves plotted according Eq. (4). (Figure from Saulis [31], reproduced with the kind permission of Biophysical Society).

An analysis of the process of microorganism inactivation by PEF presented here shows that it is a multi-step process in which pore formation due to electroporation is just the first step. Besides pore formation, it also includes the phase of increasing of the membrane permeability during the PEF treatment and several processes taking place after it (leakage of intracellular compounds, pore shrinkage and disappearance, etc.). The latter processes can last for up to a few hours [30]. Stochastic nature of the processes of pore formation and resealing governs wide distribution of electroporation and inactivation times among individual cells even in the case of a homogenous cell population and uniform exposure by PEF of all cells [17, 31]. The

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author hopes that the analysis presented here is helpful in the further development of more general models describing the kinetics of microorganism inactivation.

ACKNOWLEDGMENT This work was in part supported by grant T-39/09 from the Lithuanian State Science and Studies Foundation.

REFERENCES

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[12] Saulis, G. and P. C. Wouters (2007) Probable mechanism of microorganism inactivation by pulsed electric fields. In Food Preservation by Pulsed Electric Fields: From Research to Application. H. L. M. Lelieveld, S. Notermans and S. W. H. De Haan (Eds.), Woodhead Publishing Limited, Cambridge, pp. 138-155. [13] Wouters, P. C., Bos, A. P., and Ueckert, J. (2001) Membrane permeabilization in relation to inactivation kinetics of Lactobacillus species due to pulsed electric fields. Appl. Environ. Microbiol., 67, 3092-3101. [14] Tsong, T. Y. (1991) Electroporation of cell membranes. Biophys. J., 60, 297-306. [15] Kekez, M. M., Savic, P., and Johnson, B. F. (1996) Contribution to the biophysics of the lethal effects of electric field on microorganisms. Biochim. Biophys. Acta, 1278, 7988. [16] Sale, A. J. H. and Hamilton, W. A. (1967) Effects of high electric fields on microorganisms. I. Killing of bacteria and yeasts. Biochim. Biophys. Acta, 148, 781788. [17] Saulis, G. and Venslauskas, M. S. (1993) Cell electroporation. Part 1. Theoretical simulation of the process of pore formation in the cell. Bioelectrochem. Bioenerg., 32, 221-235. [18] Simpson, R. K., Whittington, R., Earnshaw, R. G., and Russell, N. J. (1999) Pulsed high electric field causes 'all or nothing' membrane damage in Listeria monocytogenes and Salmonella typhimurium, but membrane H+-ATPase is not the primary target. Int. J. Food Microbiol., 48, 1-10. [19] Hulsheger, H., Potel, J., and Niemann, E. G. (1981) Killing of bacteria with electric pulses of high field strength. Radiat. Environ. Biophys., 20, 53-65. [20] Qin, B., Zhang, Q., Barbosa-Canovas, G. V., Swanson, B. G., and Pedrow, P. D. (1995) Pulsed electric field treatment chamber design for liquid food pasteurization using a finite element method. Trans. ASAE, 38, 557-565. [21] Saulis, G. and Venslauskas, M. S. (1993) Cell electroporation. Part 2. Experimental measurements of the kinetics of pore formation in human erythrocytes. Bioelectrochem. Bioenerg., 32, 237-248. [22] Glaser, R. W., Leikin, S. L., Chernomordik, L. V., Pastushenko, V. F., and Sokirko, A. I. (1988) Reversible electrical breakdown of lipid bilayers: formation and evolution of pores. Biochim. Biophys. Acta, 940, 275-287. [23] Joshi, R. P. and Schoenbach, K. H. (2000) Electroporation dynamics in biological cells subjected to ultrafast electrical pulses: a numerical simulation study. Phys. Rev. E., 62, 1025-1033. [24] Pastushenko, V. F., Chizmadzhev, Yu. A., and Arakelyan, V. B. (1979) Electric breakdown of bilayer lipid membranes. II. Calculation of the membrane lifetime in the steady-state approximation. Bioelectrochem. Bioenerg., 6, 53-62. [25] Weaver, J. C. and A. Barnett (1992) Progress toward a theoretical model for electroporation mechanism: membrane electrical behaviour and molecular transport. In Guide to Electroporation and Electrofusion. D. C. Chang, B. M. Chassy, J. A. Saunders and A. E. Sowers (Eds.), Academic Press, Inc., New York, pp. 91-117. [26] Joshi, R. P., Hu, Q., and Schoenbach, K. H. (2004) Modeling studies of cell response to ultrashort, high-intensity electric fields-Implications for intracellular manipulation. IEEE Trans. Plasma Sc., 32, 1677-1686.

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[27] Krassowska, W. and Filev, P. D. (2007) Modeling electroporation in a single cell. Biophys. J., 92, 404-417. [28] Gowrishankar, T. R., Esser, A. T., Vasilkoski, Z., Smith, K. C., and Weaver, J. C. (2006) Microdosimetry for conventional and supra-electroporation in cells with organelles. Biochem. Biophys. Res. Commun., 341, 1266-1276. [29] Schelly, Z. A. (2002) Transient electro-optics of organized assemblies. Colloid. Surface. A, 209, 305-314. [30] Saulis, G., Venslauskas, M. S., and Naktinis, J. (1991) Kinetics of pore resealing in cell membrane after electroporation. Bioelectrochem. Bioenerg., 26, 1-13. [31] Saulis, G. (1997) Pore disappearance in a cell after electroporation: theoretical simulation and comparison with experiments. Biophys. J., 73, 1299-1309. [32] Garon, E. B., Sawcer, D., Vernier, P. T., Tang, T., Sun, Y., Marcu, L., Gundersen, M. A., and Koeffler, H. P. (2007) In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies. Int. J. Cancer, 121, 675-682. [33] Beebe, S. J., Fox, P. M., Rec, L. J., Willis, E. L., and Schoenbach, K. H. (2003) Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. FASEB J., 17, 1493-1495. [34] Schoenbach, K. H., A. Abou-Ghazala, T. Vithoulkas, R. W. Alden, R. Turner, and S. Beebe (1997) The effect of pulsed electrical fields on biological cells. In Proceedings of the 11th IEEE International Pulsed Power Conference. C. Cooperstein and I. Vitkovitsky (Eds.), Baltimore, MD, pp. 73-78. [35] Saulis, G. and Praneviciute, R. (2005) Determination of cell electroporation in small volume samples by using a mini potassium-selective electrode. Anal. Biochem., 345, 340-342. [36] Clapper, D. L. and Lee, H. C. (1985) Inositol trisphosphate induces calcium release from nonmitochondrial stores in sea urchin egg homogenates. J. Biol. Chem., 260, 13947-13954. [37] Muller, F., Ivics, Z., Erdelyi, F., Papp, T., Varadi, L., Horvath, L., and Maclean, N. (1992) Introducing foreign genes into fish eggs with electroporated sperm as a carrier. Mol. Mar. Biol. Biotechnol., 1, 276-281. [38] Kang, J. H., Toita, R., Niidome, T., and Katayama, Y. (2008) Effective delivery of DNA into tumor cells and tissues by electroporation of polymer-DNA complex. Cancer Lett., 265, 281-288. [39] Blair-Parks, K., Weston, B. C., and Dean, D. A. (2002) High-level gene transfer to the cornea using electroporation. J. Gene Med., 4, 92-100. [40] Reschke, D. K., Frazier, M. E., and Mallavia, L. P. (1990) Transformation of Rochalimaea quintana, a member of the family Rickettsiaceae. J. Bacteriol., 172, 51305134. [41] Mussauer, H., Sukhorukov, V. L., Haase, A., and Zimmermann, U. (1999) Resistivity of red blood cells against high-intensity, short-duration electric field pulses induced by chelating agents. J. Membr. Biol., 170, 121-133. [42] Kinosita, K. and Tsong, T. Y. (1977) Voltage-induced pore formation and hemolysis of human erythrocytes. Biochim. Biophys. Acta, 471, 227-242.

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[43] Riemann, F., Zimmermann, U., and Pilwat, G. (1975) Release and uptake of haemoglobin and ions in red blood cells induced by dielectric breakdown. Biochim. Biophys. Acta, 394, 449-462. [44] Cosgrove, R. F. and Fairbrother, J. E. (1977) Bioassay method for polyene antibiotics based on the measurement of rubidium efflux from rubidium-loaded yeast cells. Antimicrob. Agents Chemother., 11, 31-33. [45] Hille, B. (1975) Ionic selectivity of Na and K channels of nerve membranes. In Membranes: A Series of Advances. G. Eisenman (Ed.), Marcel Dekker, Inc., New York, pp. 255-323. [46] Moore, W. J. (1972) Physical Chemistry. Englewood Cliffs, NJ, Prentice-Hall. [47] Kinosita, K. and Tsong, T. Y. (1977) Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature, 268, 438-441. [48] Pakhomov, A. G., Bowman, A. M., Ibey, B. L., Andre, F. M., Pakhomova, O. N., and Schoenbach, K. H. (2009) Lipid nanopores can form a stable, ion channel-like conduction pathway in cell membrane. Biochem. Biophys. Res. Commun., 385, 181186. [49] Pakhomov, A. G., Shevin, R., White, J. A., Kolb, J. F., Pakhomova, O. N., Joshi, R. P., and Schoenbach, K. H. (2007) Membrane permeabilization and cell damage by ultrashort electric field shocks. Arch. Biochem. Biophys., 465, 109-118. [50] El-Mashak, E. M. and Tsong, T. Y. (1985) Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles. Biochemistry, 24, 2884-2888. [51] Saulis, G., Satkauskas, S., and Praneviciute, R. (2007) Determination of cell electroporation from the release of intracellular potassium ions. Anal. Biochem., 360, 273-281. [52] Freshney, I. R. (2005) Culture of Animal Cells. A Manual of Basic Technique, John Wiley and Sons, Inc. [53] Kotnik, T., Macek, L., Miklavcic, D., and Mir, L. M. (2000) Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent. Biotechniques, 28, 921-926. [54] Renkin, E. M. (1954) Filtration, diffusion, and molecular sieving through porous cellulose membranes. J. Gen. Physiol., 38, 225-243. [55] Schultz, S. G. and Solomon, A. K. (1961) Determination of the effective hydrodynamic radii of small molecules by viscometry. J. Gen. Physiol., 44, 1189-1199. [56] Saulis, G. (2005) The loading of human erythrocytes with small molecules by electroporation. Cell. Mol. Biol. Lett., 10, 23-35. [57] Sowers, A. E. and Lieber, M. R. (1986) Electropore diameters, lifetimes, numbers, and locations in individual erythrocyte ghosts. FEBS Lett., 205, 179-184. [58] He, H., Chang, D. C., and Lee, Y.-K. (2007) Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells. Bioelectrochemistry, 70, 363-368. [59] Saulis, G. (1999) Cell electroporation: estimation of the number of pores and their sizes. Biomed. Sci. Instrum., 35, 291-296. [60] Rols, M. P., Dahhou, F., Mishra, K. P., and Teissie, J. (1990) Control of electric field induced cell membrane permeabilization by membrane order. Biochemistry, 29, 29602966.

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[61] Gaskova, D., Sigler, K., Janderova, B., and Plasek, J. (1996) Effect of high-voltage electric pulses on yeast cells: Factors influencing the killing efficiency. Bioelectrochem. Bioenerg., 39, 195-202.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 5

ELECTROCHEMICAL PROCESSES OCCURING DURING CELL ELECTROMANIPULATION PROCEDURES Gintautas Saulis* Biophysical Research Group, Department of Biology, Faculty of Nature Sciences, Vytautas Magnus University, Kaunas, LT-44246, Lithuania

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ABSTRACT During cell electromanipulation procedures, a cell suspension is exposed to a highvoltage electric pulse and a strong electric current passes through the solution. At each electrode-solution interface, electrochemical reactions occur that transfer electrons either to or from the electrode. In this chapter, theoretical background of the electrochemical processes taking place during cell electroporation procedures has been provided. These may include the evolution of gas, the separation of substances, the dissolution of the electrode or the appearance of new substances in the solution. The processes of electrolysis lead to the changes of the temperature, pH, and the chemical composition of the experimental medium. The consequences of these electrochemical processes, both primary and secondary ones, which may be important for optimization of cell electromanipulation procedures, have been discussed. When using high-voltage electrical pulses for electroporation of cells and tissues, scientists must keep in mind that the products generated due to electrochemical reactions can influence the biochemical processes taking part in their experimental systems.

Keywords: Cell electromanipulation, electroporation, pH shift, electrolysis reactions, electrodes, Aluminium, stainless-steel, hypochloric acid

*

Address for correspondence: Gintautas Saulis, Department of Biology, Faculty of Nature Sciences, Vytautas Magnus University, 58 K. Donelaičio str., Kaunas, 44248, Lithuania, Tel: +370-612-82773, Fax: +370-37406572, Email: [email protected].

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INTRODUCTION Pulses of strong electric field (from 1 to 300 kV/cm) are commonly utilized for cell electromanipulation in vitro (electroporation, electrofusion, electrotransformation, etc.) [1-3]. Exposure of cell suspension to such strong electric fields results in a temporary increase of cell membrane conductivity and permeability [1, 2]. These changes in a membrane barrier function are attributed to the formation of transient aqueous pores in the cell membrane [1-4]. However, an electrically induced cell membrane perturbation is not the only consequence of the exposure of cell suspension to a strong electric field. When an electric current passes through the aqueous solution, it causes heating (Joule heating) and at the same time various chemical reactions occur at the surface between the solution and the electrodes (electrolysis). These may include the evolution of gas, the separation of substances, the dissolution of the electrode or the appearance of new substances in the solution [5]. The processes of electrolysis lead to the changes of the temperature, pH, and the chemical composition of the experimental medium. For example, the release of Al3+ from aluminum electrodes [6-10], Cu2+ from copper electrodes [11] and Fe2+/Fe3+ from stainless steel electrodes has been observed [8, 11, 12]. Diverse electrode materials are used in commercially available and home-made electrodes with the most popular ones being stainless steel [8, 11-17], aluminum [6, 8, 9, 14, 18, 19], and platinum [14, 20, 21]. Scientists must keep in mind that the electrodes they use may be involved in the biochemical processes taking part in their experimental systems. When the processes of electrolysis are not taken into consideration, it is not only difficult to improve the experimental procedures, but this can also lead to misinterpretation of the results obtained as, for example, was the case when Al+3 ions released from the electrodes stimulated the conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3 [6]. As a contrary example, a work of Tada et al. [22] can be mentioned. In this study, by replacing chloride ions with organic acids in electroporation buffers, the authors avoided the formation of the Cl2 gas at the inert gold-plated stainless steel anode and, as a result, obtained higher survival rates and transformation frequencies of rice protoplasts [22]. It must be stressed that the electrolysis reactions go along with the permeabilization of the cell membrane. Therefore any uncontrollable changes of the physicochemical properties of a solution during procedures of cell electromanipulations are especially undesirable, as various solutes may enter the cells through the pores created by strong electric fields [4, 13]. Studying the processes of electrolysis occurring during cell electroporation procedures became especially important when electroporation was recently started to be used in vivo for electrochemotherapy [23], transdermal drug delivery [24], gene therapy [25], as well as nonthermal pasteurization of liquid foods [26, 27] as the interaction between electrode materials and tissues or food products during electric treatment should be minimized [27]. Here, electrochemical processes, which can take place during the treatment by highvoltage electrical pulses used in various electroporation experiments, and the consequences of these processes have been reviewed.

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THEORETICAL BACKGROUND When, during cell electromanipulation procedures, a cell suspension is exposed to an electric pulse, a strong electric current passes through the solution. At each electrode-solution interface, the electrochemical reactions occur, that transfer electrons either to or from the electrode, thereby allowing charge to flow completely throughout the circuit consisting of the high-voltage pulse generator and the chamber with a solution. Several primary electrochemical half-reactions can take place at each electrode-solution interface. Which one of these reactions occurs under particular conditions, depends on the relative ease of other competing reactions. Let us shortly analyze them.

Primary Cathodic Half-Reactions There are two possible cathodic half-reactions that can occur when an electric current passes through a water solution: i) the reduction of metal cations. For example,

K  (aq)  e   K ( solid)

(1a)

ii) the reduction of water molecules (or hydrogen ions in acidic solutions)

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2 H 2 O  2e   H 2 ( gas)  2OH  (aq)

(1b)

The species actually reduced is the one that has the most favourable reduction potential. Solutions which are generally utilized in cell electromanipulation experiments contain mainly the salts of sodium, potassium, calcium and magnesium. Because Na+, K+, Ca2+, and Mg2+ ions are more difficult to reduce than water [5], in these solutions the water reduction reaction (Eq. (1)) takes place at the cathode.

Primary Anodic Half-Reactions There are three possible anodic half-reactions that can occur when an electric current passes through a water solution: i) the oxidation of water molecules

2 H 2 O  O2 ( gas)  4 H  (aq)  4e 

(2a)

ii) the oxidation of the anion of the solute. For example,

2Cl  (aq)  Cl 2 ( gas)  2e  Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

(2b)

Gintautas Saulis

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iii) the oxidation of the metal of the electrode (in the case of a non-inert anode):

M ( solid)  M n (aq)  ne 

(2c)

For example,

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2 Al ( solid)  2 Al 3 (aq)  6e 

(2d)

Whether or not any of these reactions occurs depends on the relative ease of the other two possible anodic reactions and the anode potential [5]. When a non-inert metal electrode is used, for example, an electrode made from stainless steel or aluminum, usually, the most favourable half-reaction is the last one (Eq. (2c)). That is, in such a case, the release of the metal ions from the electrode into the solution occurs [6, 8, 11, 12, 15-17]. However, when the anode potential is sufficiently high, several reactions can proceed simultaneously, with the different intensity. For example, besides the dissolution of the noninert anode, an oxygen evolution [28] or a direct oxidation of Cl− ions, which are usually present in the solutions used in electroporation experiments, can occur [29], as shown in Eqs. (2a) and (2b). In the case of an inert anode (e.g., platinum), the only two competing reactions are the first and the second one (Eq. (2b)). Which of these reactions, Eq. (2a) or Eq. (2b), would occur depends on the particular conditions. The solutions used for electroporation-mediated cell transformation or other electroporation experiments usually contain high concentrations of chloride ions. The standard-state potentials for oxidation of water and chlorine ions are close to each other (Eoox = -1.23 V for water and -1.36 V for Cl- ions) [5, 30]. High concentrations of NaCl, which usually is the case in the electroporation experiments, decreases the potential required to oxidize the Cl- ions. Hence, when an inert anode is utilized, in many cases, we find the oxidation of Cl- and the production of Cl2 at the anode [22].

Secondary Chemical Reactions Besides the primary electrochemical reactions – the reactions which occur at the electrode-solution interfaces during an electric pulse, – secondary chemical reactions often occur in the solution. There might be several secondary reactions depending on a variety parameters, such as the pH of the the solution, its composition and the electrode material. As the examples of the secondary chemical reactions, the following processes can be mentioned: 



building of complexes/coagulants by the metal ions released from the electrodes, e.g. Fe3+, Al3+ or Cu2+, with the molecules present in the solution and the adsorption of soluble or colloidal substances on these complexes/coagulants [31]; dissolution of the aluminium cathode due to a chemical attack by hydroxyl ions generated as as result of the reduction of water molecules (see Eq. (1b)), especially, at high pH values [32]:

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2 Al  6 H 2 O  2OH   2 Al (OH ) 4  3H 2 

103

(3)

formation of the hypochloric acid (HClO), which occurs due to the reaction of water with chlorine (Cl2) produced at the anode [30]:

Cl 2  H 2 O  HClO  H   Cl 

(4)

Products of any of these secondary reactions can also make influence on the processes taking place during electroporation experiments.

CONSEQUENCES OF ELECTROCHEMICAL PROCESSES As it has been shown in the previous section of this chapter, a variety of primary and secondary chemical reactions might occur due to the action of high-voltage electric pulses. Let us discuss the possible consequences of these electrochemical processes.

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Consequences of Cathodic Processes Most cations present in the solutions, which are generally utilized in cell electromanipulation procedures, such as Na+, K+, Ca2+, and Mg2+, are more difficult to reduce than water [5]. Therefore, in these solutions the water reduction reaction takes place at the cathode (Eq. (1b)). Two products are generated during this water reduction half-reaction, namely, HO- ions and hydrogen gas. Evolution of any of these products is undesirable in electroporation experiments. First, the evolution of OH- ions changes the pH of a solution at the cathode. The change of the pH of a solution occuring due to the exposure of an electrolyte solution to an electric pulse, has been noticed many times [18, 33-38]. The shift in pH when using iontophoresis for transdermal drug delivery was reported by Kari [38] and Meyer et al. [37]. Pillai et al. [39] observed the shift of the pH of a donor solution of up to 4-5 pH units during ionthophoresis. Large changes in pH in the vicinity of electrodes (the acidification around the anode and alkalinisation at the cathode) were also supposed to be the determining factors in the destruction of tissues in electrochemical treatment of tumors [40, 41]. A more detailed analysis of this effect was carried out recently [7, 10]. It has been shown, that the increase in the pH value of electroporation solution of a whole chamber volume, caused by the application of electric field pulses, commonly used in cell electromanipulation procedures, can exceed 1-2 pH units (Figure 1) [7, 10]. In addition, the aluminum cathode gave approximately two-fold higher pH [10], most likely due to secondary chemical reactions.

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Figure 1. The change of the pH of a medium as a function of the intensity of electrotreatment for the sodium chloride solution (139 – 149 mM NaCl) buffered with various amounts of phosphates: ( ● ) 5.6, ( □ ) 11.2, and ( ▲ ) 15 mM of Na2HPO4/NaH2PO4. The error bars represent standard deviation of the mean. Dotted line shows the dependence of the solution pH on the amount of the HO- ions added (139 mM NaCl solution containing 15 mM Na2HPO4/NaH2PO4 (pH 7.4) was titrated with 100 mM NaOH). Both electrodes, cathode and anode, made of stainless steel were utilized. (Figure from Saulis et al. [10], reproduced with the kind permission of Elsevier).

Another product of a water reduction half-reaction described by Eq (1b) is hydrogen gas. The production of hydrogen gas at the cathode leads to the formation of gas bubbles. These bubbles reduce the dielectric strength of a solution and limit the maximum intensity of electric pulse that can be applied to the cell suspension [42, 43].

Consequences of Anodic Processes During each of the anodic half-reactions the products, which can be undesirable are produced. These include: (1) the production of H+ at the anode (Eq. 2a); (2) the production of O2 gas (Eq. (2a); (2) the production of Cl2 gas (Eq. (2b)); and (4) the release of the metal ions from the electrode (Eq. 2c).

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Diverse electrode materials are used in commercially available and home-made electrodes with the most popular ones being stainless steel [8, 11-17], aluminum [6, 8, 9, 14, 18, 19], and platinum [14, 20, 21]. Other materials used include gold [14, 22], carbon [44], nickel and NiCr [19, 45], silver [14], brass [46], copper [11], tungsten [47], and other ones. When a non-inert metal electrode is used, in most cases, the release of the metal ions from the electrode into the solution should occur, according to Eq. (2c). The release of Al3+ from the aluminum anode [6, 8-10], Cu2+ from the copper anode [11] as well as Fe2+/Fe3+, chromium and manganese ions from the stainless steel anode has been observed during cell electroporation experiments (Figure 2) [8, 11, 12, 15, 17, 48].

Figure 2. Iron concentration in the electroporation cuvette, which is equiped with stainless-steel electrode plates, after the electric discharge of a high-voltage capacitor. 0.8 ml of KHBS buffer was subjected to the electric pulse under constant electric field of 660 V/cm (A) or constant capacitance of 1500 F (B). (Figure from Stapulionis [11], reproduced with the kind permission of Elsevier)

Several effects caused by the metal ions released from the electrodes have been observed during electroporation experiments. In one of the first reports of such effects, the stimulation of the conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3 by Al+3 ions released from the electrodes was noticed [6]. The reduction of the cell viability by the iron ions released from the stainless steel anode has also been reported (Figure 3) [8]. So, the scientists must keep in mind that the

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electrodes, which are used in their electroporation experiments, may be involved in the biochemical processes taking part in their experimental systems.

Figure 3. Cell survival (mean±S.D.) without (○) and with electropermeabilization (●) as the function of Fe2+/Fe3+ concentration in the suspension. The cells were incubated for 1 h at room temperature. Electropermeabilization was performed at the beginning of the incubation using a train of eight unipolar rectangular pulses, each of 100 μs duration and 240 V amplitude (1200 V/cm voltage-to-distance ratio), delivered in 1 s intervals. (Figure from Kotnik et al. [8], reproduced with the kind permission of Elsevier)

One more consequence of this process is the complexation of the metal ions released from the electrodes with the molecules present in the solution [11, 31, 49], which might also affect the efficiency of various procedures in which the treatment with high-voltage electric pulses is used. By the way, the aluminum electrodes caused about two times greater precipitation of macromolecules (DNA, RNA and proteins) than stainless steel electrodes [11].

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A)

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B)

Figure 4. Typical three-dimensional AFM images of the surface of the stainless-steel anode: (A) polished prior to the exposure by high-voltage electric pulses and (B) after the exposure to 120 exponential pulses with the duration of about 20 μs (dissolution charge Qdiss = 0.24 As/cm2). Scanning area 30 x 30 μm2; z range: (A) 157 and (B) 410 nm. (Figure from Saulis et al. [50], reproduced with the kind permission of Elsevier).

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In addition, the dissolution of the anode material can cause the increase of the roughness of the electrode surface. Recently, the changes of the surface topography of stainless-steel and aluminium electrodes occurring due to the action of electric pulses, which are commonly utilized in cell electroporation procedures, have been studied by using atomic force microscopy. After the treatment of the chambers filled with 154 mM NaCl solution by a series of short (20–40 s), high-voltage (4 kV) pulses with the total dissolution charge of 0.20–0.26 As/cm2, the roughness of the surface of the electrodes has increased, depending on the total amount of the electric charge that has passed through the unit area of the electrode. Up to a three–fold increase of the surface roughness of the stainless-steel and aluminium anodes was observed due to the dissolution of the anode material (Figure 4) [50, 51]. Therefore, the use of high-voltage electric pulses leads to the increase of the inhomogeneity of the electric field at the electrode, which leads to the non-equal treatment of each cell and facilitates the occurrence of the electric breakdown of the liquid samples. When an inert anode is utilized, in many cases, two anodic half-reactions can take place the oxidation of water molecules (Eq. (2a)) and the oxidation of the anion of the solute (Eq. (2b)). When the anode potential is sufficiently high, these reactions can proceed simultaneously [28, 29]. The production of H+ changes the solution pH at the anode - it becomes more acidic. This might affect the cell viability or various biochemical processes. For example, the pH increase of initially acidic solution containing ionic metallic species (e.g. Cu2+, Zn2+, Ni2+, Cr3+, etc.) may induce their coprecipitation in the form of their corresponding hydroxides [28]. Because, usually, the main purpose of electromanipulation procedures is the enhancement of the membrane permeability, the exposure of the cell suspension to a strong electric pulse can cause the change of the pH of not only extracellular solution but intracellular medium as well. It is therefore quite possible that the change of pH, in some cases, might be one of the factors causing cell death [36]. The drastic acidification around the anode (as well as alkalinisation at the cathode) was supposed to be the determining factor in the destruction of tissues in electrochemical treatment of tumors [40, 41]. If an oxidation of Cl− ions, which are usually present in the solutions used in electroporation experiments, occurs [29], Cl2 gas is produced at the anode [22]. The chlorine is a strong oxidant that can oxidize some organic compounds and promote electrode reactions. It has been shown that the Cl2 gas produced at the anode can be toxic to some cells - replacing chloride ions with organic acids in electroporation buffers increased survival rates and transformation frequencies of rice protoplasts [22]. The production of either O2 (Eq. (2a)) or Cl2 (Eq. (2b)) gas causes intensive bubling of the solution. This limits the intensity of an electric pulse which can be applied to the sample as increases the risk of arcing, that is, the dielectric breakdown of the liquid samples, which is observed as a spark [42, 43]. Arcing can lead to dramatic effects from just ejection of the sample from the cuvette [52] or the reduction of the transfection efficiency [53] to irreversible damage of electroporation cuvettes, power supplies or electroporators [54, 55].

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Consequences of Secondary Chemical Reactions

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It is supposed that aluminium cathode is attacked by hydroxyl ions generated during water reduction half-reaction (see Eqs. (1b) and (3)) [32]. This leads to a variety of secondary chemical reactions with different effects. For example, when studying the changes of the pH value of the electroporation solution, caused by the application of high-voltage electric pulses, the aluminum cathode gave approximately two-fold higher pH, most likely, due to secondary chemical reactions [10]. These secondary reactions might be responsible for anomalous heating of a solution in electroporation experiments with aluminum electrodes observed by Pliquett et al. [18]. Recently, a substantial release of the aluminium ions from the aluminium cathode caused by the application of electric field pulses, commonly used in cell electroporation experiments was observed [7, 10]. The release of aluminum ions not only from the anode but also from the cathode, can also explain the fact, that aluminum electrodes caused almost exactly two times greater precipitation of macromolecules (DNA, RNA and proteins) than stainless steel electrodes, reported by Stapulionis [11]. Chlorine generated during the primary anodic half-reaction (Eq (2b)) can participate in the secondary reactions as chlorine is a strong oxidizing agent [30]. Hulsheger and Niemann have shown that when solutions containing chloride (Cl2) compounds were treated by electric pulses, hypochloric acid (HClO) was produced as a result of a reaction of chlorine with water (see Eq. (4)) [33]. The concentration of HClO, which is more bactericidal than Cl2 gas, depended on the amount of chlorine in the solution and the pH. These authors suggested that this hypochloric acid contributed to inactivation of bacteria Escherichia coli by pulsed electric fields [33].

CONCLUSION When using high-voltage electrical pulses for electroporation of cells and tissues, scientists must keep in mind that, besides an electrically induced cell membrane permeabilization, various primary electrochemical and secondary chemical reactions occur at the surfaces between the solution and the electrodes as well as in the solution and the products generated due to these reactions can influence the biochemical processes taking part in their experimental systems.

ACKNOWLEDGMENT This work was supported in part by grants No. 409 and B-08020 from the Lithuanian State Science and Studies Foundation.

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[9]

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[17] Roodenburg, B., Morren, J., Berg, H. E. I., and de Haan, S. W. H. (2005) Metal release in a stainless steel pulsed electric field (PEF) system. Part II. The treatment of orange juice; related to legislation and treatment chamber lifetime. Innovative Food Sc. Emerg. Technol., 6, 337-345. [18] Pliquett, U., Gift, E. A., and Weaver, J. C. (1996) Determination of the electric field and anomalous heating caused by exponential pulses with aluminum electrodes in electroporation experiments. Bioelectrochem. Bioenerg., 39, 39-53. [19] Haritou, M., Yova, D., Koutsouris, D., and Loukas, S. (1998) Loading of intact rabbit erythrocytes with fluorophores and the enzyme pronase by means of electroporation. Clin. Hemorheol. Microcirc., 19, 205-217. [20] Riemann, F., Zimmermann, U., and Pilwat, G. (1975) Release and uptake of haemoglobin and ions in red blood cells induced by dielectric breakdown. Biochim. Biophys. Acta, 394, 449-462. [21] Marszalek, P., Liu, D.-S., and Tsong, T. Y. (1990) Schwan equation and transmembrane potential induced by alternating electric field. Biophys. J., 58, 10531058. [22] Tada, Y., Sakamoto, M., and Fujimura, T. (1990) Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions. Theor. Appl. Genet., 80, 475-480. [23] Mir, L. M., Orlowski, S., Belehradek, J., and Paoletti, C. (1991) Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. Eur. J. Cancer, 27, 68-72. [24] Prausnitz, M. R., Bose, V. G., Langer, R., and Weaver, J. C. (1993) Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery. Proc. Natl. Acad. Sci. U. S. A., 90, 10504-10508. [25] Jaroszeski, M. J., Heller, R., and Gilbert, R. (Editors) (2000) Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery. Totowa, NJ, Humana Press Inc. [26] Qin, B. L., Pothakamury, U. R., Barbosa-Canovas, G. V., and Swanson, B. G. (1996) Nonthermal pasteurization of liquid foods using high-intensity pulsed electric fields. Crit. Rev. Food Sc. Nutr., 36, 603-627. [27] Knorr, D., Ade-Omowaye, B. I. O., and Heinz, V. (2002) Nutritional improvement of plant foods by non-thermal processing. P. Nutr. Soc., 61, 311-318. [28] Mouedhen, G., Feki, M., Wery, M. D. P., and Ayedi, H. F. (2008) Behavior of aluminum electrodes in electrocoagulation process. J. Hazard. Mater., 150, 124-135. [29] Vlyssides, A. G., Karlis, P. K., and Zorpas, A. A. (1999) Electrochemical oxidation of noncyanide stripperswastes. Environ. Int., 25, 663-670. [30] Holtzclaw, H. F., Jr. and Robinson, W. R. (1988) General Chemistry. Lexington, D. C. Heath and Company. [31] Mouedhen, G., Feki, M., De Petris Wery, M., and Ayedi, H. F. (2007) Behaviour of aluminum electrodes in electrocoagulation process. J. Hazard Mater., 150, 124-135. [32] Picard, T., Cathalifaud-Feuillade, G., Mazet, M., and Vandensteendam, C. (2000) Cathodic dissolution in the electrocoagulation process using aluminium electrodes. J. Environ. Monit., 2, 77-80. [33] Hülsheger, H. and Niemann, E. G. (1980) Lethal effect of high-voltage pulses on E. coli K12. Radiat. Environ. Biophys., 18, 281-288.

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[34] Miklavcic, D., Sersa, G., Kryzanowski, M., Novakovic, S., Bobanovic, F., Golouh, R., and Vodovnik, L. (1993) Tumor treatment by direct electric current - tumor temperature and pH, electrode material and configuration. Bioelectrochem. Bioenerg., 30, 209-220. [35] Prausnitz, M. R., Lau, B. S., Milano, C. D., Conner, S., Langer, R., and Weaver, J. C. (1993) A quantitative study of electroporation showing a plateau in net molecular transport. Biophys. J., 65, 414-422. [36] Potter, H. (1988) Electroporation in biology: methods, applications, and instrumentation. Anal. Biochem., 174, 361-373. [37] Meyer, B. R., Katzeff, H. L., Eschbach, J., Trimmer, J. S., Zacharias, S. B., Rosen, S., and Sibalis, D. (1989) Transdermal delivery of insulin to albino rats using electrical current. Am. J. Med. Sci., 297, 321-325. [38] Kari, B. (1986) Control of blood glucose levels in alloxandiabetic rats by iontophoresis of insulin. Diabetes, 35, 217-221. [39] Pillai, O., Kumar, N., Dey, C. S., Borkute, S., Nagalingam, S., and Panchagnula, R. (2003) Transdermal iontophoresis of insulin: Part 1. A study on the issues associated with the use of platinum electrodes on rat skin. J. Pharm. Pharmacol., 55, 1505-1513. [40] Nordenstrom, B. E. W. (1994) Survey of mechanisms in electrochemical treatment (ECT) of cancer. Eur. J. Surg., 574, 93-109. [41] Berendson, J. and Olsson, J. M. (1998) Bioelectrochemical aspects of the treatment of tissue with direct current. Electro and Magnetobiology, 17, 1-16. [42] Zhang, Q., Monsalve-Gonzalez, A., Barbosa-Canovas, G. V., and Swanson, B. G. (1994) Inactivation of E. coli and S. cerevisiae by pulsed electric fields under controlled temperature conditions. Trans. ASAE, 37, 581-587. [43] Zhang, Q., Barbosa-Canovas, G. V., and Swanson, B. G. (1995) Engineering aspects of pulsed electric field pasteurization. J. Food Eng., 25, 261-281. [44] Sale, A. J. H. and Hamilton, W. A. (1967) Effects of high electric fields on microorganisms. I. Killing of bacteria and yeasts. Biochim. Biophys. Acta, 148, 781788. [45] Zhelev, D. V., Dimitrov, D. S., and Tsoneva, I. (1988) Electrical breakdown of protoplast membranes under different osmotic pressures. Bioelectrochem Bioenerg, 19, 217-225. [46] Obermeyer, G. and Weisenseel, M. H. (1995) Introduction of impermeable molecules into pollen grains by electroporation. Protoplasma, 187, 132-137. [47] Kustermann, S., Schmid, S., Biehlmaier, O., and Kohler, K. (2008) Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina. Cell Tissue Res., 332, 195-209. [48] Roodenburg, B., Morren, J., Berg, H. E. I., and de Haan, S. W. H. (2005) Metal release in a stainless steel pulsed electric field (PEF) system. Part I. Effect of different pulse shapes; theory and experimental method. Innovative Food Sc. Emerg. Technol., 6, 327336. [49] Kobya, M., Can, O. T., and Bayramoglu, M. (2003) Treatment of textile wastewaters by electrocoagulation using iron and aluminum electrodes. J. Hazard. Mater., B100, 163178. [50] Saulis, G., Rodaite-Riseviciene, R., and Snitka, V. (2007) Increase of the roughness of the stainless-steel anode surface due to the exposure to high-voltage electric pulses as revealed by atomic force microscopy. Bioelectrochemistry, 70, 519-523.

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[51] Rodaite-Riseviciene, R., Saulis, G., and Snitka, V. (2009) Changes of the electrode surface roughness induced by high-voltage electric pulses as revealed by AFM. Acta Phys. Pol. A, 115, 1095-1097. [52] Lambert, H., Pankov, R., Gauthier, J., and Hancock, R. (1990) Electroporationmediated uptake of proteins into mammalian cells. Biochem. Cell Biol., 68, 729-734. [53] Ward, L. J. H. and Jarvis, A. W. (1991) Rapid electroporation-mediated plasmid transfer between Lactococcus lactis and Escherichia coli without the need for plasmid preparation. Lett. Appl. Microbiol., 13, 278-280. [54] Friesenegger, A., Fiedler, S., Devriese, L. A., and Wirth, R. (1991) Genetic transformation of various species of Enterococcus by electroporation. FEMS Microbiol. Lett., 63, 323-327. [55] Potter, H. (1993) Application of electroporation in recombinant DNA technology. Methods Enzymol., 217, 461-478.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 6

ULTRASTRUCTURAL MODIFICATIONS INDUCED BY “ELECTROPORATION” Agnese Molinari1, Giuseppe Arancia1and Enrico P. Spugnini2 1

Department of Technology and Health, Italian National Institute of Health, Rome, Italy 2 SAFU Department, Regina Elena Cancer Institute, Rome, Italy

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ABSTRACT Despite the large bulk of literature on electroporation, there is a lack of knowledge about the cellular mechanism(s) and subcellular components controlling this phenomenon. In this chapter, we describe a number of ultrastructural alterations occurring in the tissues and in the cellular membranes after exposure to high voltage pulses. Specifically, experimental and ultrastructural data coming from literature body on transdermal drug delivery are briefly discussed. Moreover, alterations following the exposure of orthotopic melanomas and red blood cells to trains of biphasic pulses are reported. To look insight the intimate mechanism(s) controlling electroporation, several imaging techniques such as light and electron microscopy, electrochemical imaging, confocal microscopy have been employed. Freeze-fracture analysis allowed in more than one case to evidence alterations of cell membranes and defects in the dynamic assembly of lipids and proteins. Such modifications could be the hallmarks of a reduction of lipidprotein cohesion and of changes in lipid orientation inside cell membranes, as postulated in several mathematical models applied to electroporation.

Keywords: electrochemotherapy, nude mouse, erythrocytes, stratum corneum, light and electron microscopy, freeze-fracturing, thin sectioning

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INTRODUCTION Electroporation induces structural changes in the membrane phase, making lipid and lipid-protein membranes porous and permeable in a transient and mostly reversible way. It is a technique used to facilitate the uptake of macromolecules such as DNA (direct electroporative gene transfer), as well as to introduce chemotherapeutics into cancer tissues (Fromm et al., 1985; Gehl, 2003; Mir et al., 1988; Neuman et al., 1982; Orlowski et al., 1988; Poddevin et al., 1991). As demonstrated by a number of in vitro studies, the application of high voltage exponentially-decaying electric pulses (electroporation, electroinjection) to cells in suspension results in cross-membrane flows of material, or even in cell fusion if cells are adjacent (Kinosita and Tsong, 1977; Sencia et al., 1979; Zimmermann and Scheurich, 1981). Due to its properties, electroporation proved to be effective at enhancing the in vitro cytotoxicity of anticancer molecules (electrochemotherapy, ECT) (Gehl, 2003; Orlowski 1988; Poddevin et al., 1991; Salford et al., 1993) by driving drugs into exposed cells, mostly by varying the diffusion coefficient. The major obstacle to the penetration of anticancer compounds into the cells is, in fact, represented by their cell membranes. As it is well known, anticancer drugs cross the cell membrane by diffusion (e.g. cisplatin) (O‘Dwyer et al., 1997) or by means of facilitated transport through carrier proteins (e.g. methotrexate) (De Vita et al., 1995). Remarkably, ECT has been also studied in vivo and in clinical trials. It is potentially useful for treating patients with metastatic tumors, such as melanoma, and even selected primary tumors, such as head and neck squamous cell carcinomas and basal cell carcinoma. Although several chemotherapeutic agents have been tested with electroporation therapy, bleomycin and cisplatin are the two most widely used (Byrne and Thompson, 2006). Of interest, studies on electrochemotherapy performed on dogs with spontaneous neoplasms by using trains of biphasic pulses, evidenced a greater sensibility of tumor cells to permeabilizing wave trains when compared with normal surrounding tissues (Spugnini et al., 2006a, b). As far as the clinical application of ECT is concerned, the combined administration of antitumor compounds and permeabilizing electric pulses proved to be a very promising approach in veterinary oncology, wherein is becoming a primary treatment (Spugnini and Porrello, 2003; Spugnini et al., 2009), whereas in humans is still limited to cutaneous neoplasms in order to improve transd et al ermal drug delivery (Prausnitz et al., 1993). Despite the large bulk of publications on electropermeabilization, there are no ultrastuctural studies that succeeded in visualizing and quantifying the phenomenon of ―pore‖ or ―permeabilizing defects‖ formation within minutes after the electric induction, i.e., in the main biophysical transient phase of in vivo electroporation. Even if the electroporation is an efficacious and elegant way to introduce chosen molecules into the cells, the molecular mechanisms supporting the induction of ―permeabilizing defects‖ in membrane assemblies remain poorly understood. Recently, on the basis of data acquired by the scientific community, the concept of ―acqueous pore‖ (transient and long lasting electropore) as previously modeled (Neumann et al., 1999) has been revisited (Teissie et al., 2005), thus giving a remarkable contribution to defining a model, which chronologically describes the ultrastructural modifications. Despite

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the large body of literature on ECT, very few ultrastructural studies have been performed to elucidate the mechanisms of membrane electropermeabilization and to understand the overall ultrastructural modifications. In the present chapter, the results obtained in preclinical studies on two experimental models (skin and melanoma xenografts), by using mainly morphological and ultrastructural methods, are described and discussed. In our opinion, these results, besides to demonstrate the validity of the ultrastructural analysis in gaining insight into the subcellular and molecular mechanisms of electropermeabilization, might supply useful information for the optimization of ECT and let us hope an its rapid and valuable application against human tumors.

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ELECTROPORATION FOR TRANSDERMAL DRUG DELIVERY Transdermic administration by electroporation has been developed over recent years for applying drugs in a variety of pathological processes, and it is regarded as a viable alternative to traditional ways (oral, intramuscular, parenteral) of drug administration. It has the potential advantages over other methods of delivery in terms of convenience, non-invasiveness, and reduction of drug degradation. The use of electroporation for transdermal drug delivery was suggested by Prausnitz et al. (1993) who demonstrated that electroporation of skin is feasible. In their pioneer study they suggested that electroporation occurs in the intercellular lipid bilayers of the stratum corneum by a mechanism involving transient structural changes. Electroporation has enabled to treat a variety of pathologies by administering several drugs through the skin, including the transdermal delivery of liposomal formulations (Badkar et al., 1999). The mechanism of transdermal drug transport by electroporation has been widely reviewed (Prausnitz, 1996; Denet et al., 2004). Electrically, the skin can be modelled as a resistor and capacitor in parallel with most of the resistance residing in the stratum corneum (Yamamoto, 1977). Thus, if the skin is exposed to an electric pulse most of the pulse voltage would fall across its uppermost stratum, the stratum corneum, making it the site of electroporation. The stratum corneum represents the major barrier to molecular transport of the skin. It consists of several layers of flattened, enucleated, keratin-filled corneocytes surrounded by lamellae of lipid bilayers on average. The lipid bilayers consist primarily of cholesterol, free fatty acids, sphingolipids, and ceramides, most of which have saturated fatty acids. The electrical breakdown associated with dramatic increase in transport has been observed for transdermal voltages of 30-100 V (100 – 1500 V applied voltages), which well correspond to the range of voltages used for electroporation in cells, i.e. 0.3 – 1.0 V per bilayer (Denet et al., 2004). In order to characterize the localization of molecular and ionic transport across skin subjected to high voltage pulses, several studies have been performed, by applying imaging approaches based on real time video fluorescence microscopy, electrochemical deposition imaging, ‗gel localized microscopy‘ (GLM) and confocal microscopy (Pliquett et al., 1996, 1998; Prausnitz et al., 1996). The transdermal-transport by high voltage pulses has been shown to occur through highly ‗local transport regions‘ (LTRs) of the stratum corneum. Pliquett et al. (1998) proposed a

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hypothetical structure of a LTR, contained within a ‗local dissipation region‘ (LDR). Proposed LTR model is basically represented by a cylindrical region, with a thickness of the stratum corneum (10 to 20 m) and a radius (10 to 100 m) strongly dependent on ‗high voltage‘ pulse duration. Also the LDR size depends on pulse length: all data obtained by Pliquett et al. (1998) demonstrate that it is larger than LTR size. Moreover, the pulse voltage seems less important for LTR size than for LTR number. The small ion transport seems to be concentrated within LDR; the molecular transport is even more localized, occurring within the smaller LTR. Imaging of LTRs in real time fluorescence microscopy leads to the hypothesis that for large high voltage (700 to 1500 V across electrodes) pulses the entire corneocytes (fully fluorescent) are involved in the transport. In contrast, long pulses at medium high voltage (50 to 400 V across electrodes) suggest that the molecular transport occurs around the corneocytes. The examinations of electrochemical images based on AgCl deposits on an Ag anode, located behind the specimen, provided the visualization of the resistivity distribution over the stratum corneum. LTRs are surrounded by a diffuse ring of low resistivity (LDR) that is larger than the central low resistivity region of LTR. Finally, GLM demonstrated that the molecular transport occurs mainly at the center of a LTR. The application of high voltage pulses to the human stratum corneum may cause multilamellar electroporation followed by localized heating. The heating occurs within the acqueous pathway created by the local electrophoretic driving force and the thermal effects seem to be responsible for the expansion of LTR/LDR (Pliquett et al., 1998). Using time-resolved freeze-fracture electron microscopy, Gallo et al. (1999, 2002) observed the development of local disruption areas of the lipid lamellae in the stratum corneum, in the form of vesicle formation. In their studies Gallo and colleagues conducted electrical and ultrastructural measurements to also examine the temperature dependence of the pulse-induced permeabilization of the stratum corneum (Gallo et al., 2002). They observed that resistance of the stratum corneum was reduced manyfold during the applied pulse. The extent of this reduction increased with the increase of pulse voltage until reaching a threshold value, above which the resistance reduction was less dependent on the pulse voltage. In addition, they demonstrated that the stratum corneum was more susceptible to electropermeabilization at high temperature (the threshold voltage being lower). Timeresolved freeze fracture electron microscopy revealed numerous aggregates of lipid vesicles in all samples pulsed above the threshold voltage: the aggregates persisted long after the electric resistance was recovered. Moreover, freeze fracture studies also indicated that the temperature of the stratum corneum influences the vesicle formation and their kinetics in pulsed samples. At both cold (4°C) and room temperature (25°C), multi or unilamellar vesicles of various sizes (0.1–3 m) were found to aggregate in the lamellar lipid region or confined hydrated areas of the stratum corneum. These aggregates varied from 1 m to 50 m across. Aggregates of vesicles persisted long after the electric resistance was recovered. Some similarities were found by Gallo et al. (1999) between the aggregates and the LTRs: both appeared shortly after the pulse and lasted minutes to hours afterwards. Aggregate sizes varied from 1 to 30 m in diameter, somewhat smaller than LTR, ranging from 40 to 80 m in diameter. The difference in size could be due to the different imaging apparatus employed: in the case of aggregates the freeze-fracturing at TEM resolution; in the case of LTR a fluorescent imaging apparatus which detected the diffusion of the fluorescent dye.

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However, the relationship between the transient permeability of the skin and the formation of the vesicle aggregates remains to be clarified. Other ultrastructural studies have been made in order to demonstrate how the ―pores‖, which allow the entrance through the skin, are induced by the electroporation. As above described, the transport of substances through the skin as a result of electroporation might occur by means of a double pathway (intercellular and/or transcellular), the contribution of each depending on the electromagnetic flow (Pliquett et al., 1996, 1998). Molecular transport would be totally transcellular in the case of short, high voltage pulses; mostly intercellular at lower voltages and/or longer pulses. Ortega et al. (2006) in an elegant ultrastructural study analyzed the way in which macromolecules (i.e. India ink in a suspension of soy lectin liposomes) pass through the stratum corneum and epidermidis, by means of electroporation by electromagnetic waves (pulsed-modulated sinusoidal hectometric waves over the zone for 20 min). Light and electron microscopy observations performed on semithin and ultrathin sections, respectively, allowed to demonstrate that transport was both through the intercellular spaces, or ―pores‖, between the hair follicle cells, and via the transcellular pathway, as suggested by the ultrastructural evidence of particles in the corneous layers, in epidermics keratinocytes and in the papillar dermis immediately below. In addition, these observations showed that the application of electromagnetic waves did not produce morphological and ultrastructural alterations in the treated zones, confirming the efficacy of electroporation by electromagnetic waves for administering substances through the skin and transfollicle, in the absence of side effects. The efficacy and no invasive nature of electroporation for the transdermic administration of macromolecules were thus reinforced.

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ELECTROPORATION ON MELANOMA XENOGRAFTS In order to get insights about modifications induced in vivo by electroporation, we investigated the major transient phase after delivery of trains of biphasic pulses (Daskalov et al., 1999; Spugnini and Porrello, 2003) in a mouse melanoma xenograft system (Spugnini et al., 2007). The electroporation was followed by ultrathin sectioning and freeze-fracture electron microscopy. Overall, consistency of these results was assessed through a comparison with electroporative effects obtained in cat red blood cells. The ultrastructural results are here discussed taking into account the current knowledge about the mechanism(s) of cell membrane electropermeabilization and proposing, whenever it is possible, links with the theoretical background. Our observations performed by TEM on ultrathin sectioned tumors, implanted in mice and in vivo electroporated, revealed alterations of cell membrane ultrastructure. Embedded and thin sectioned control tumors showed a fine preserved structure characterized by cells with dense cytoplasm and regular nuclei containing dispersed chromatin and prominent nucleoli (Figure 1 a). At higher magnification, numerous vesicles were often observed in the narrow space between adjacent cells (Figure 1 c). These vesicles, shedding from the plasma membrane, contained granular electron dense material. In the cytoplasm organelles such as mitochondria and Golgi apparatus displayed the typical morphology with parallel cristae in the former, and flattened membrane-bound sacs in the latter (Figure 1 e).

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In tumors explanted five minutes after treatment with electric pulses at 1250 V/cm, the tissue architecture appeared almost unaltered: melanoma cells were closely connected and displayed a dense cytoplasm and regular-shaped nuclei (Figure 1 b). However, at higher magnification, extracellular vesicles displayed fragmented membranes and their inner content released in the intercellular space (Figure 1 d). In the same way, the intracellular membranes underwent detectable changes following electric treatment: the evident dilation of the mitochondrial cristae and stacked membranes of Golgi apparatus are shown in Figure 1 f.

Figure 1. TEM observations of ultrathin sections of control melanoma xenograft before (a, c and e) and after treatment with electric pulses at 1250 V/cm (b, d and f). At low magnification the tumor tissue shows regular organization and structure (a). At higher magnification, numerous vesicles are detectable in the intercellular space (c) and the cytoplasm contains well preserved organelles (e). After the treatment, the general architecture appears to be quite unaltered when compared to control samples (b). On the contrary, vesicles in the extracellular space show evident membrane alterations with release of their content (d). Also intracytoplasmic organelles display significant structural alterations (f) .

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When the electroporation treatment was performed with an electric field of 2450 V/cm, more pronounced alterations could be observed: the intercellular spaces resulted to be enlarged, many cells showed indentation of the nuclear envelope (Figure 2 a) and swollen cytoplasmic organelles (Figure 2 b).

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Figure 2. TEM observations of tumor tissue treated with electric pulses at 2450 V/cm. (a) At low magnification the intercellular spaces appear to be enlarged and many nuclei show indented profile. (b) At higher magnification the alterations of the cytoplasmic organelles induced by the intense electric pulses are well evident.

The disruption of the vesicle membranes released in the intercellular space, observed in electroporated samples, suggested that one of the mechanisms of tumor destruction induced by electroporation can be mediated by the interruption of the intercellular flow of vesicles. This issue is very relevant, because many tumor cells shed specialized membrane vesicles (exosomes) that carry messengers and mediators (Andreola et al., 2002) and recent reports demonstrated the important role played by exosomes on the tumor growth (Liu et al., 2006). On the other hand, also the intracellular membranes (endoplasmic reticulum, mitochondrial cristae and Golgi stacks) showed to be affected: these alterations could be ascribed to a variation of membrane permeability induced by electroporation (Maccarrone et al., 1995). The increase of the electric field to 2450 V/cm induced deeper alteration of the general tissue architecture of melanoma xenograft with the total loss of intercellular vesicles, the enlargement of the intercellular gaps and the swelling of the intracellular organelles. All these alterations were suggestive of a cytotoxic damage elicited by the electric field (SalomskaitèDavalgienè et al., 2002). The possible modifications of the molecular organization, occurring in the plasma membrane after electric pulses treatment, were also analyzed by means of the freeze-fracture technique (FF). Since in quickly frozen biological samples the fracture preferentially runs along the inner hydrophobic plane of membranes, the observation by TEM of fracture surface replicas allows the visualization of both protoplasmic (PF: lipid leaflet adjacent to the cytoplasm) and exoplasmic (EF: external lipid leaflet) fracture faces. The proteins present in both PF and EF appear as small spherical particles, with a diameter ranging approximately from 8 to 10 nm (IMPs: intramembrane particles), dispersed in the smooth lipid matrix. FF analysis performed on tissue samples before and after electroporation clearly showed modifications of the molecular organization of the plasma membrane (proteins and lipids),

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thus suggesting that electroporation in vivo is more complex than previously hypothesized. Figures 3 a and 3 b show portions of the PF and EF, respectively, of the plasma membrane of freeze-fractured untreated tumor cells. These samples are cognate of embedded and thin sectioned melanoma tumors grown in nude mice, above described (Figure 2).

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Figure 3. Freeze-fractured control melanoma cells. In both protoplasmic (a) and exoplasmic (b) fracture faces of the plasma membrane the protein intramembrane particles appear to be randomly distributed on a smooth lipid matrix.

In control tissue IMPs appeared to be randomly distributed in the lipid layers of the plasma membrane, thus showing the common pattern of cellular membranes out of highly specialized regions (e.g. tight junctions, nuclear envelope, mitochondrial membranes). Similarly to many other cell types (erythrocytes, lymphocytes, etc.), there was a higher IMP density in the PF (Figure 3 a) than in the EF (Figure 3 b) of the plasma membrane of melanoma cells. After treatment at 1250 V/cm for five minutes some slight but appreciable ultrastructural changes were revealed. Figure 4 a shows the fracture faces of the plasma membrane of two adjacent tumor cells. On both EF (upper membrane) and PF (lower membrane) is evident an ultrastructural shift from randomly distributed to cluster aggregated IMPs. In addition, numerous small rounded areas with rough structure were often detectable (arrows in Figure 4 a). This appearance suggests that areas with rough structure are protein aggregates that span the two lipid layers of the plasma membrane and, therefore, are exposed by the fracture on both EF and PF. The observation at higher magnification allowed a better visualization of the IMP clusters (Figure 4 c: PF; Figure 4 e: EF) which are much more visible on the PF (arrows in Figure 4 c) where the intramembrane proteins are more numerous. When the electric treatment was performed at 2450 V/cm, IMPs redistribution was even more evident on both PF (Figure 4 b) and EF (Figure 4 d). Also the number of areas with rough structures was noticeably increased and, in some membranes, they appeared to be quite large and with a complex shape (Figure 4 f). These results indicate the occurrence of a remarkable alteration in the physico-chemical properties of lipid bilayer and, consequently, in the protein-lipid interaction mechanisms.

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It appears that electroporative uptake of DNA or drugs is anticipated by field-induced structural changes in the membrane phase, comprising transient, yet long-lived permeation sites, pathways, channels or pores (Neumann et al., 1999).

Figure 4. Freeze-fractured melanoma cells after treatment at 1250 V/cm (a, c, e) and at 2450 V/cm for 5 min (b, d, f). (a) In both protoplasmic (lower membrane in the panel) and exoplasmic (upper membrane in the panel) fracture faces of the plasma membrane numerous roundish areas with rough structure are detectable (arrows). Moreover, after electric field exposure, IMPs are no more randomly distributed but aggregated in large clusters (arrows) in both PF (c) and EF (e). Under treatment at 2450 V/cm, the IMPs redistribution and the formation of areas with rough structure appear to be even more evident on both PF (b) and EF (d). In some plasma membranes such rough areas assume large size and complex shape (f).

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The special structural order of a long-lived, potential permeation site was initially modeled as follows: the basic concept was that the energy brought by the external field to the membrane could support the formation of regular ―toroidal pores‖ in the lipid bilayer. The size of these pores was linked to a balance between line tension of the pore, membrane tension and induced transmembrane potential. Creation of these pores would be linked to a transition of hydrophobic pores (packing fluctuations of the lipid bilayer) to the hydrophilic pores lined by the lipid polar heads, to minimize the hydrophobic contact with water. The stationary pores kept open by the electrically induced transmembrane field undergo size limitations (≤ 1 nm diameter) in order to prevent discharging of the membrane interface by ion conduction. However, additional lateral tension due to long-lasting Maxwell stress on cells may lead to further pore enlargements (Neumann et al., 1999). Nevertheless, this model based on a symmetrical lipid bilayer is quite simplistic, because it does not take into account several problems concerning the complexity of cell membranes, where proteins and glycoproteins coexist with highly mobile lipids. Indeed, in a biological cell membrane, phospholipids undergo hop diffusion in compartmentalized cell membranes. Such compartmentalization depends on various transmembrane proteins anchored to the actin-based membrane skeleton meshwork that acts as rows of pickets that temporarily confine phospholipids (Fujiwara et al., 2002). More recently, a sophisticated view of the different steps involved in membrane electropermeabilization has been proposed (Teissie et al., 2005): this new model comes from previous ones (Teissie and Rols, 1994; Schmeer et al., 2004) and uses data dealing with a direct field effect on membrane proteins (Rols and Teissie, 1998). The ―four-state model‖ proposed by Teissie et al. accounts for different state transitions of lipids that under the electric field pass from resting state (L1) to three transition states: the orientation of the polar heads of lipids changes (L2), the cohesion of their assembly is affected for a short time (L3), and the assembly is stressed by the field for a long time interval (L4). Also proteins change their resting state (P1) to a state where they are affected by the loss of cohesion of the lipid assembly (P2) to a condition where the field is directly stressing the polypeptide on a relatively long term (several hours) (P3). States P1L1, P1L2 are supposed to be poorly ―leaky‖ and so-called ―closed‖, while all the other states (P1L3, P2L2, P2L3) are highly ―leaky‖. Following this theory, macromolecules can cross directly the membrane only in the state P3L4. As stated by the authors, these molecular descriptions are highly speculative and need further experimental validations. The high mobility of lipids in a membrane and our experimental data seem to support the hypothesis of ―defects in the dynamic assembly‖ of lipids and proteins more than ―aqueous pores‖ induced by the electropermeabilization. This hypothesis is based on an analogy of properties between lipids at the thermal phase transition and electropermeabilized membranes: dynamic mismatches between lipid domains, or between lipids and transmembrane protein segments, could explain the transfer of polar species both at thermal phase transition and under electric field in the absence of regular channels (Cruizero-Hansson and Mouritsen, 1988; el-Mashak and Tsong, 1985). Movements across membranes would take place in ―fluctuating defects‖, with polar compounds that do not move together with structural pores but are dragged coherently along a ―zigzag‖ line, following the reptation mechanism of polymer transport (Ginsburg and Stein, 1987; Lieb and Stein, 1986). The loss of cohesion induced by electropermeabilization should allow such reptation between the lipid chains or lipid and protein molecules.

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It was suggested that because of their small size there are few evidence, if not at all, for visible electropores. The large ―pore-like crater‖ structures or ―volcano funnels‖ of 50 nm to 0.1 m diameter, observed in electroporated red blood cells (Chang and Reese, 1990), at the present time are seen as misleading experimental artifacts, most probably resulting from the enlargement of smaller primary pores by osmotic or hydrostatic pressure. No such defects were ever observed under iso-osmolar conditions or in other cell models. The ―areas with rough structure‖, and the clustering of IMPs observed in the plasma membrane of tumor cells after treatment with 1250 V/cm, could be hallmarks of alterations of lipid and protein cohesion of the assembly and/or change of lipid orientation postulated in the above enunciated ―four-state model theory‖. Such modifications of the cell membranes were dose-dependent: in fact, when the electric treatment was performed at 2450V/cm, IMPs lateral redistribution was more evident. These ultrastructural findings were confirmed by observations carried out on electroporated red blood cells. In order to verify if the effects induced by the electric pulses on the molecular organization of melanoma cell membranes were peculiar for this cell type or, rather, they were representative of a typical response of cells to electroporation, a different membrane model was taken into consideration. In particular, red cells from the peripheral blood of cat were analyzed by freeze-fracturing and scanning electron microscopy after the in vitro exposure to the electric pulses. The plasma membrane of control erythrocytes, observed by TEM after freeze-fracturing, showed its typical molecular architecture. IMPs, randomly distributed on both PF and EF (Figures 5 a and 5 c, respectively), appeared much higher in density on the PF than on the EF, as above described for melanoma tumor cells and as previously reported for human erythrocytes in a number of papers.

Figure 5. Freeze-fracturing of cat erythrocytes, before (a, c) and after (b, d) in vitro treatment with 400 V/cm electric pulses. In both protoplasmic (a) and exoplasmic (c) fracture faces of the plasma membrane of untreated red cells, IMPs appear to be randomly distributed on a smooth lipid matrix. The exposure to the electric field induced the appearance of numerous granular aggregates protruding fromboth PF (b) and EF (d) of the plasma membrane.

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Despite the different models and treatment conditions, the electric pulses induced ultrastructural alterations similar to those observed in melanoma cells. In fact, after 400 V/cm electric pulses, numerous granular aggregates protruding from the fracture faces could be observed on both PF (Figure 5 b) and EF (Figure 5 d). Also in this experimental model the structural changes resulted to be dose-dependent: in fact, after exposure to 800 V/cm electric pulses, the peculiar areas with rough structures increased in size (Figure 6 a: PF; Figure 6 b: EF).

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Figure 6. Cat erythrocytes, freeze-fractured after in vitro treatment with 800 V/cm electric pulses. The peculiar structural changes of the plasma membrane observed after 400 V/cm treatment (Figure 8) are much more evident when red cells are treated with 800 V/cm electric field. (a): PF; (b): EF.

The alterations of red cell membranes treated with electric field and freeze-fractured were similar to those observed in melanoma cells and were clearly different from ―volcano-like‖ pores. Also in this case, roughly structured areas support the hypothesis of ―defects in the dynamic assembly‖ of lipids and proteins rather than the ―acqueous pores‖ scenario. In addition, their ramified morphology might indicate that membrane domains are suitable for the ―zigzag‖ alignment of polar compounds (Ginsburg and Stein, 1987; Lieb and Stein, 1986). Additionally, electropermeabilization induced the alteration of the discocytic shape of red blood cells. Alterations of the molecular organization of the whole plasma membrane and, in particular, of the connections between the membrane proteins and the underlying cytoskeletal proteins can strongly influence the cell shape. Thus, cat erythrocytes were also observed by scanning electron microscopy, before and after treatment with 400 V/cm electric field. While untreated red cells showed their typical discocytic shape (Figure 7 a), most erythrocytes exposed to the electric field underwent evident morphological changes (Figure 7 b), some of them assuming a star-like shape (Figure 7 c). This outcome gives an indirect proof of the relevant alterations occurring in plasma membranes of electroporated red blood cells as well. The first consequence of the interaction of an electric field with a lipid vesicle is its deformation from a quasi-spherical shape to an ellipsoidal one. Experimentally, shape deformation was observed only in lipid vesicles and never reported in cells, except when repetitive pulses were applied (Chang and Reese, 1990; Popov et al., 1991; Riske and Dimova, 2005; Tekle et al., 2001). In particular, an intense alternating current electric field can induce formation of cell protrusions. Membrane-applied force is sufficient to produce native-like cell protrusions: actin microfilaments can be organized into bundles directly under the action of membrane-applied force even in conditions where activity of the cytoskeleton is

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inhibited (Popov et al., 1991). On the other hand, it has been reported that electroporation transforms discocytic erythrocytes into echinocytes (Henszen et al., 1997). Such shape transition seems to be determined by passive transmembrane redistribution of phospholipids (Schwarz et al., 1999). These findings support the hypothesis that ―rough areas‖ observed by us in membranes of echinocytic erythrocytes after electroporation could be representative of ―defects in the dynamic assembly of lipids and proteins‖, which are likely associated with passive transmembrane redistribution of phospholipids.

Figure 7. Control and electric field treated erythrocytes observed by SEM. (a) Untreated red cells show their typical discocytic shape. (b) Most treated erythrocytes undergo evident morphological changes. (c) Some of the treated cells show a star-like shape.

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CONCLUSION Electroporation is now seen as a method to enhance the efficiency of drug delivery in several pathologies. The data obtained by in vitro and in vivo studies strongly suggest that it represents a promising alternative as a no invasive delivery of macromolecules for transdermal and topical drug delivery (Denet et al., 2004). In addition, the results obtained in orthotopic mouse melanoma xenograft model supply useful information for the optimization of ECT and let us hope its rapid and valuable application against human tumors (Spugnini et al., 2009). The ultrastructural alterations observed in our models were suggestive of electric fieldinduced defects in the dynamic assembly of lipids and proteins rather than of the formation of ―hydrophilic toroidal pores‖ and were consistent with ultrastructural observations on electroporated red blood cells. Due to the short lag time chosen in all experiments (five minutes after electroporation), the observed ultrastructural effects are likely indicative of patterns present when all major electroporation effects already act, and the cell is going to begin a relatively slow process of resealing. In biophysical terms, and based on the ultrastructural evidence described in this chapter, we hypothesize that the membrane flux of molecules in the electroporation setting, quantitatively described by an equation derived from Fick‘s laws for diffusion (Teissie et al., 2005), is potentially affected both at the level of permeability coefficient and of membrane curvature variation. Notably, this last value depends on the locally measured cell radius and is therefore importantly influenced by the extra-cellular vesicle release here described. All together, the ultrastructural data herein reported, even if not exhaustive at all, seem to confirm that the application of an electric field can induce important changes in the molecular

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organization of the cell membranes that can be exploited to improve the entry of drugs, thus enhancing their therapeutic effects.

ACKNOWLEDGMENT We thank Dr. Marisa Colone and Mr. Giuseppe Formisano for their invaluable technical assistance.

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REFERENCES Andreola, G; Rivoltini, L; Castelli, C; Huber, V; Perego, P; Deho, P; Squarcina, P; Accornero, P; Lozupone, F; Lugini, L; Stringaro, A; Molinari, A; Arancia, G; Gentile, M; Parmiani, G; Fais, S. Induction of lymphocyte apoptosis by tumor cell secretion of FasLbearing microvesicles. J. Exp. Med., 2002, 195, 1303–1316. Badkar, AV; Betageri, GV; Hofmann, GA; Banga, AK. Enhancement of transdermal iontophoretic delivery of a liposomal formulation of colchicines electroporation. Drug Deliv., 1999, 6, 111-115. Byrne, C.M. and Thompson, J.F. (2006). Role of electrochemotherapy in the treatment of metastatic melanoma and other metastatic and primary skin tumors. Expert. Rev. Anticancer Ther., 6, 671-678. Chang, D.C. and Reese, T.S. (1990). Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J., 58, 1-12. Cruzeiro-Hansson, L. and Mouritsen, O.G. (1988). Passive ion permeability of lipid membranes modelled via lipid-domain interfacial area. Biochim. Biophys. Acta, 944, 6372. Daskalov, I; Mudrov, N; Peycheva, E. Exploring new instrumentation parameters for electrochemotherapy. Attacking tumors with bursts of biphasic pulses instead of single pulses. IEEE Eng. Med. Biol. Mag., 1999, 18, 62-66. Denet, A-R; Vanbever, R; Préat, V. Skin electroporation for transdermal and topical delivery. Adv. Drug Deliv. Rev., 2004, 56, 659-674. DeVita, VT; Hellman, S; Rosenberg, SA (Eds). Biologic therapy of cancer. 2nd Edition. Philadelphia: Philadelphia JB Lippincott; 1995. El-Mashak, E.M. and Tsong, T.Y. (1985). Ion selectivity of temperature-induced and electric field induced pores in dipalmitoylphosphatidylcholine vesicles. Biochemistry, 24, 28842888. Fromm, M; Taylor, LP; Walbot, V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Nat. Acad. Sci. USA, 1985, 82, 5824-5828. Fujiwara, T; Ritchie, K; Murakoshi, H; Jacobson, K; Kusumi, A. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol., 2002, 157, 1071-1081. Gallo, SA; Sen, A; Hensen, ML; Hui, SW. Time-dependent ultrastructural changes to porcine stratum corneum following an electric pulse Biophys. J., 1999, 76, 2824–2832.

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Gallo, SA; Sen, A; Hensen, ML; Hui, SW. Temperature-dependent electrical and ultrastructural characterizations of porcine skin upon electroporation. Biophys. J., 2002, 82, 109–119. Gehl, J. (2003). Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol. Scand., 177, 437-447. Ginsburg, H. and Stein, W.D. (1987). Biophysical analysis of novel transport pathways induced in red blood cell membranes. J. Membr. Biol., 96, 1-10. Henszen, MM; Weske, M; Schwarz, S; Haest, CW; Deuticke, B. Electric field pulses induce reversible shape transformation of human erythrocytes. Mol. Membr. Biol., 1997, 14, 195-204. Kinosita, K. Jr and Tsong, TT (1977). Hemolysis of human erythrocytes by transient electric field. Proc. Natl. Acad. Sci. USA, 74, 1923-1927. Lieb, W.R. and Stein, W.D. (1986). Non-stokesian nature of transverse diffusion within human red cell membranes. J. Membr. Biol., 92, 111-119. Liu, C; Yu, S; Zinn, K; Wang, J; Zhang, L; Jia, Y; Kappes, JC; Barnes, S; Kimberly, RP; Grizzle, WE; Zhang, HG. Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function. J. Immunol., 2006, 176, 1375-1385. Maccarrone, M; Bladergroen, MR; Rosato, N; Finazzi Agrò, AF. Role of lipid peroxidation in electroporation-induced cell permeability. Biochem. Biophys. Res. Commun., 1995, 209, 417-425. Mir, LM; Banoun, H; Paoletti, C. Introduction of definite amounts of nonpermanent molecules into living cells after electropermeabilization: direct access to the cytosol. Exp. Cell Res., 1988, 175, 15-25. Neumann, E; Kakorin, S; Toensing, K. Fundamentals of electroporative delivery of drugs and genes. Bioelectrochem. Bioenerg., 1999, 48, 3-16. Neumann, E; Schaefer-Ridder, M; Wang, Y; Hofschneider, PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J., 1982, 1, 841-845. O‘Dwyer, PJ; Johnson, SW; Hamilton, TC. Cisplatin and its analogues. In: De Vita VT, Editor. Principle and practice of oncology. Cancer. 5th Edition. Philadelphia: Lippincott, pp. 418-431, 1997. Orlowski, S; Belehradek, J Jr; Paoletti, C; Mir, LM. Transient electropermeabilization of cells in culture. Increase of cytotoxicity of anticancer drugs. Biochem. Pharmacol., 1988, 37, 4727-4733. Ortega, VV; Martínez, AF; Gascón, JY; Sánchez, NA; Baños, MA; Rubiales, FC. Transdermal transport of India ink by electromagnetic electroporation in Guinea pigs: an ultrastructural study. Ultrastruct. Pathol., 2006, 30, 65-74. Pliquett, UF; Vanbever, R; Preat, V; Weaver, JC. Local transport regions (LTRs) in human stratum corneum due to long and short ‗high voltage‘ pulses. Bioelectrochem. Bioenerg,, 1998, 47, 151-161. Pliquett, UF; Zewert, TE; Chen, T; Langer, R; Weaver, JC. Imaging of fluorescent molecule and small ion transport through human stratum corneum during high voltage pulsing: localized transport regions are involved. Biophys. Chem., 1996, 58, 185-204. Poddevin, B; Orlowski, S; Belehradek, J Jr; Mir, LM. Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. Biochem. Pharmacol., 1991, 42, S67-75. Popov, SV; Svitkina, TM; Margolis, LB; Tsong TY. Mechanism of cell protrusion formation in electrical field: the role of actin. Biochim. Biophys. Acta, 1991, 1066, 151-158.

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Prausnitz, M.R. (1996). The effect of current applied to the skin: a review for transdermal drug delivery. Adv. Drug Deliv, Rev., 18, 395-425. Prausnitz, MR; Bose, VG; Langer, R; Weaver, JC. Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery. Proc. Natl. Acad. Sci. USA, 1993, 90, 10504-10508. Prausnitz, MR; Gimm, JA; Guy, RH; Langer, R; Weaver, JC; Cullander, C. Imaging regions of transport across human stratum corneum during high-voltage and low-voltage exposures. J. Pharm. Sci., 1996, 85, 1363-1370. Riske, K.A. and Dimova, R. (2005). Electro-deformation and poration of giant vesicles viewed with high temporal resolution. Biophys. J., 88, 1143–1155. Rols, M.P. and Teissié J. (1998). Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys. J., 75, 1415-1423. Salford, LG; Persson, BR; Brun, A; Ceberg, CP; Kongstad, PC; Mir, LM. A new brain tumor therapy combining bleomycin with in vivo electropermeabilization. Biochem. Biophys. Res. Commun., 1993, 194, 938-943. Salomskaitè-Davalgienè, S; Venslauskas, MS; Pauziené, N. Histological analysis of electrochemotherapy influence in Lewis lung carcinoma. Medicina, 2002, 38, 540-544. Schmeer, M; Seipp, T; Pliquett, U; Kakorin, S; Neumann E. Mechanism for the conductivity changes caused by membrane electroporation of CHO cell pellets. Phys. Chem. Chem. Phys., 2004, 6, 5564-5574. Schwarz, S; Deuticke, B; Haest, CW. Passive transmembrane redistributions of phospholipids as a determinant of erythrocyte shape change. Studies on electroporated cells. Mol. Membr. Biol., 1999, 16, 247-255. Sencia, M; Takeda, J; Abe, S; Nakamura, T. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol., 1979, 20, 1441-1443. Spugnini, EP; Arancia, G; Porrello, A; Colone, M; Formisano, G; Stringaro, A; Citro, G; Molinari, A. Ultrastructural modifications of cell membranes induced by "electroporation" on melanoma xenografts. Microsc. Res. Tech., 2007, 70, 1041-1050. Spugnini, EP; Citro, G; Baldi, A. Adjuvant electrochemotherapy in veterinary patients: a model for the planning of future therapies in humans. J. Exp. Clin. Cancer Res., 2009, 28, 114-119. Spugnini, EP; Dragonetti, E; Vincenzi, B; Onori, N; Citro, G; Baldi, A. Pulse-mediated chemotherapy enhances local control and survival in a spontaneous canine model of primary mucosal melanoma. Melanoma Res., 2006a, 16, 23-27. Spugnini, E.P. and Porrello, A. Potentiation of chemotherapy in companion animals with spontaneous large neoplasms by application of biphasic electric pulses. J. Exp. Clin. Cancer Res., 2003, 22, 571-580. Spugnini, EP; Vincenzi, B; Baldi, F; Citro, G; Baldi, A. Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors. Anticancer Res., 2006b, 26, 4585-4589. Tekle, E; Astumian, RD; Friauf, WA; Chock, PB. Asymmetric pore distribution and loss of membrane lipid in electroporated DOPC vesicles. Biophys. J., 2001, 81, 960-968. Teissie, J; Golzio, M; Rols, MP. Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of?) knowledge. Biochim. Biophys. Acta, 2005, 1724, 270-280.

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Teissie, J and Rols, MP. Manipulation of cell cytoskeleton affects the lifetime of cell membrane electopermeabilization. Ann. N.Y. Acad. Sci., 1994, 720, 98-110. Tsong, TY and Kinosita, K Jr. Use of voltage pulses for the pore opening and drug loading, and the subsequent resealing of red blood cells. Bibl. Haematol., 1985, 51, 108-114. Yamamoto, T. Electrical properties of the epidermal stratum corneum. Med. Biol. Eng., 1976, 14, 151-158. Zimmermann, U and Scheurich, P. High frequency fusion of plant protoplasts by electric fields. Planta, 1981, 151, 26-32.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 7

ELECTROPORATION IN BACTERIA Maria Papagianni Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece

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ABSTRACT Application of rDNA technology to manipulate the genome of bacteria requires suitable and highly efficient transformation systems. There are many approaches available, with the most popular technique, because of its practical interest, that of electroporation. In electrotransformation, electric pulses are applied to a mixture of bacteria and plasmids in an aqueous solution. The methodology is simple and includes five steps: preparation of electrocompetent cells, mixture of cells and DNA, application of an electric field of high intensity, incubation of the mixture to ensure expression and selection of transformants. Since its first application, around twenty years ago, the method has been proved to be highly efficient, easy to use, and suitable for both Grampositive and -negative bacteria. Today, an impressive number of protocols on various species can be found in literature and several manufacturers provide reliable equipment and protocols adapted to it. A number of parameters are known to affect the process of electrotransformation of bacteria. The method is highly species-dependent and the most important among many other parameters related to the characteristics of the applied electric field, are its strength and the duration of the pulse. In spite of the widespread application of the method in molecular biology and biotechnology, the molecular processes that support the introduction of macromolecules into the bacterial cell remain rather unknown. This chapter will focus on the application of the method in bacteria and the parameters that influence the efficiency of transformation. The current progress and methodology aspects of the technique will be critically discussed.

INTRODUCTION Efficient transfer of foreign DNA in the genome of cells is a problem that can cause restrictions of the power of modern biotechnology and cell biology. The phospholipid bilayer

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arrangement of the plasma membrane has a hydrophobic exterior and interior and therefore, polar molecules, including DNA, are not able to freely pass through it. In 1982, Neumann and co-workers introduced a method based on bioelectrochemistry, the electrotransformation method, that has been proved to be a highly efficient technique (Purves et al., 2003). Electrotransformation is achieved through electroporation of cells, a mechanical method that introduces temporarily a large electric pulse that disturbs the phospholipid bilayer, allowing polar molecules like DNA to pass into the treated cell. Following a quick shock, in the appropriate application of the method, the membrane spontaneously reassembles after disturbance, with the cell remaining intact. Several methods other than electroporation are used to internalize otherwise membraneimpermeant molecules into cells. Such methods, used widely in cell biology, include microinjection, microprecipitates, liposomes, and biological vectors (Alberts et al., 2002). Microinjection, although widely used, demands that each cell be injected individually. The method therefore, is possible to be applied at most only a few hundred cells at a time. Introduction of large molecules into cells with the liposomes method is to cause membranous vesicles that contain the molecules of interest to fuse with the plasma membrane. To introduce new genes into the nucleus, gold particles coated with DNA are shot into cells at high velocity. Electroporation, compared to these methods, has both advantages and disadvantages. Its main advantage is its effectiveness with nearly all cell types (Nickoloff, 1995; Eynard and Teissie, 2000). Another advantage of equal importance is that it allows large populations of cells to be permealized simultaneously. Also, the amount of DNA required is smaller than for other methods (Withers, 1995). Elecroporation has been shown to be successful not only with cells in suspension but with intact tissues as well, with important applications of the method in agrobiotechnology and medicine. In 1993, Klöti and co-workers reported on the successful gene transfer into intact scutellum cells by electroporating zygotic wheat embryos without any special pretreatment. In medicine, electroporation is gaining recently increased attention as a technology to enhance clinical chemotherapy and gene therapy of tissues. Electroporation of tissues has been demonstrated for applications such as targeted delivery of chemotherapeutics to tumors, efficient gene transfection of cells in vivo, and increased skin permeability for transdermal drug delivery (Jaroszeski et al., 1999, 2000; Prausnitz, 1999; Mir, 2001; Canatella et al., 2004). Disadvantages of the method of electroporation include cell damage in case of application of pulses of wrong length or intensity (Weaver, 1995), and the non specific import or export of molecules from the cell that may cause ion imbalances and consequently cell death (Weaver, 1995). Electroporation is now routinely used for transformation of intact mammalian, bacterial, yeast and plant cells (protoplasts and intact cells). This chapter will focus on the application of the method in bacteria. The current progress and methodology of the technique will be critically reviewed. Since its first description, the method has been proved to be a highly efficient and easy to use technique for both Gram-positive and -negative bacteria. The methodology is simple and includes five steps: preparation of competent cells, mixture of cells and DNA, application of an electric field of high intensity, incubation of the mixture to ensure expression, and finally a selection assay to separate the transformants. Today, a large number of electroporation protocols are available for a large number of bacterial species. Electroporation apparatus (electropulsers) are provided by several manufacturers and in many

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cases also protocols. In spite of the widespread application of the method in cell biology and biotechnology, the molecular processes that support the introduction of macromolecules into the bacterial cell remain largely uknown. A number of parameters however, are known to affect the process of electrotransformation. In most cases, adaptation of the protocols through trial and error and experience are the most effective tools to obtain increased yields of transformants.

KEY STEPS PRIOR TO ELECTROPORATION

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Preparation of Electrocompetent Cells Various treatments have been applied to bacterial cells prior to electroporation in order to increase transformation efficiencies. According to Eynard and Teissie (2000), each treatment that increases the plasmid interfacial concentration leads to increased transformation rates. The efficiency of transformation is also influenced by the cell envelope quality, yet the mechanisms remaining unknown. The efficiency of electrotransformation is strongly correlated to the level of electropermealization (Sixou et al., 1991). Since the physical barrier of the cell wall has to be weakened enough in order that an adequate amount of DNA will enter the cell, pre-treatment with various chemicals that increase permeability, and subsequently the rate of transformation, has often been proposed. Chemical treatment prior to electroporation tends to be common practice today with bacteria. Initial applications of electroporation for the transformation of both Gram-positive and -negative bacterial species, achieved limited transformation efficiencies. Various chemicals are proposed today in electrotransformation protocols, while their mechanism of action is not always known. CaCl2, for example, is used successfully with Escherichia coli (Brown, TA, 2000) and what makes the treatment successful remains unknown. For Lactococcus lactis, pre-treatment with lysozyme has been proposed by Powel et al. (1988), threonine, by Van der Lelie et al. (1989) and Dornan and Collins (1999), glycine, by Holo and Nes (1988) and Le Bourgeois et al. (2000). Papagianni et al. (2007) pretreated L. lactis cells with lithium acetate (LiAc) and dithiothreitol (DTT), agents that had never been used before with L. lactis or other bacteria, and observed a tremendous improvement in transformation efficiency without affecting their survival rate. Pretreatment of Clostridium perfrigens with lysostaphin provided the highest transformation efficiencies (3.0 x 105 transformants per microgram DNA for Cl. perfrigens strain 13) as reported by Scott and Rood (1989). Lysostaphin treatment however, was omitted in the work of Jiraskova et al. (2005) while a simpler and more rapid protocol was proposed for Cl. perfrigens with increased transformation rates. Chemical treatment of cells prior to electroporation aim to reduce the strength of the physical barrier of the cell wall. Bacterial cell walls, in both Gram-positive and -negative bacteria, gain much of their strength from a rigid layer of peptidoglycan. Proteins and crosslinked polysaccharides contribute to the rigidity within individual layers of the polymer (Tsien et al., 1978). Approximately 40 layers of peptidoglycan are found in cell walls of Gram-positive bacteria, accounting for approximately 50% of the total cell wall thickness. Gram-negative bacteria generally contain a maximum of 2-3 layers of peptidoglycan that

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accounts for only a 5-10% of the total cell wall thickness (Beveridge, 1981). As a result of these structural differences, and while the Gram-negative bacteria contain an additional outer membrane, the cell wall of Gram-positive species is generally thicker and more resistant to physical stress (Shepard and Gilmore, 1995). Powel and co-workers (1988) found that transformation efficiencies of the Gram-positive L. lactis increased when treated with lysozyme prior to electroporation, indicating that the intact cell wall and the glycocalyx provided a physical barrier to electroporation and uptake of plasmid DNA. Effective lysozyme treatment requires mild treatment with minimal amounts of the enzyme. Extensive treatment leads to damage of cells and even lysis upon application of the electroporation pulse and extents the time required before the cells could be placed in hypotonic selective growth media (Powel et al., 1988; Shepard and Gilmore, 1995). Lysozyme is used widely as cell-weakening agent in molecular biology and biotechnology. Cell susceptibility to treatment with lysozyme varies among species. Apart from cell wall hydrolyzing enzymes, amino acids, e.g. glycine and theonine have also been used. Glycine is commonly used as a cell-weakening agent prior to electroporation of Gram-positive bacteria (Dunny et al., 1991). Glycine has been applied with L. lactis (Holo and Nes, 1988; Le Bourgeois et al., 2000), Pediococcus spp. (Caldwell et al., 1996), Lactobacillus spp. (Bhowmik and Steele, 1993; Mason et al., 2005), Leuconostoc spp. (David et al., 1989) and other lactic acid bacteria (Luchansky et al., 1988), Enterococcus spp. (GruzRodz and Gilmore, 1990; Shepard and Gilmore, 1995), Streptococcus spp. (Buckley et al., 1999), Mycobacterium spp. (Lee et al., 2002) and others. The action of glycine is through replacement of alanine and impediment of synthesis and assembly of cell wall (Hammes et al., 1973; Buckley et al., 1999). Cells treated with glycine are able to regenerate functional cell walls within hours. Addition of glycine however, may have deleterious effects to exponentially grown cultures whose synthesis of cell walls must occur rapidly (Dunny et al., 1991). To overcome glycine toxicity, glycine was added to S. salivarius cultures during early exponential phase in the work of Buckley et al. (1999) and this is generally followed in protocols with other microorganisms. The only study in which glycine (5%) was added during late exponential phase cultures was that of Stepanov and co-workers (1990) in a procedure of preparation of cryotransformable Bacillus anthracis cells. Glycine concentration may vary in various protocols from 1.5% (Lee et al., 2000) to 5% (Buckley et al., 1999) and must be used in osmotically stabilized media to enhance the stability of the weakened cells. In the protocol developed for electroporation of L. lactis subsp. cremoris by Holo and Nes (1989), the cells were cultured in the presence of high concentrations of glycine in media osmotically stabilized with 0.5 M sucrose. The protocol followed by Holo and Nes resulted in high transformation efficiencies in L. lactis subsp. cremoris, a strain that previously had not been transformed following treatment with lysozyme. Also, no cell lysis was observed upon application of the electric pulse. Thus, the use of glycine in osmotically stabilized media allowed for balance between the need for membrane access by DNA and sufficient viability following electroporation. Apart from glycine, other amino acids used in electroporation protocols have been theonine and cysteine. Theonine was used as a racemic mixture of the two L-and D- forms with L. lactis in the works of McIntyre and Harlander (1989b) and Dornan and Collins (1990), and enhanced electrotransformation efficiency. It has also been used with Actinomyces spp. in the work of Yeung and Kozelsky (1994) and strains of B. subtilis by McDonald and co-workers (1995). According to the last work, the action of theronine is not

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through a direct incorporation in the peptidoglycan structure but by an inhibition of the diaminopimelic acid (DAP) incorporation in peptidoglycan. Cysteine was used in electrotransformation of Cl. thermocellum by Tyurinand co-workers (2004). For electrotransformation of Gram-negative bacteria, the protocols suggested for E. coli are usually adopted with modifications, and therefore cells are usually treated with CaCl2 as a cell wall weakening agent (Wirth et al., 1989). CaCl2 has also been used with lactococcal strains in the works of Lillehaug et al. (1977) and Kobayashi et al. (2002). Instead of CaCl2, MgCl2 has also been used with L. lactis (Corneau et al., 2001). MgCl2 was used in electrotransformation of Lb. plantarum by Alegre et al. (2004) and with B. subtilis and B. licheniformis by Xue et al. (1999). The transformation efficiency of L. lactis, treated with various combinations of LiAc and DTT before electroporation was examined by Papagianni and co-workers (2007). Pretreatment with these chemicals was applied for the first time in bacteria. Electrotransformation efficiencies of up to 105 transformants per g DNA have been reported in the literature for L. lactis LM0230. Following treatment with LiAc and DTT before electroporation, in early log phase cells, increased transformation efficiency to 225 ± 52.5 x 107 transformants per g DNA, while untreated cells or treated with LiAc alone transformation efficiency approximated 1.2 ± 0.5 x 105 transformants per g DNA. For mycobacteria and other mycolic-acid-containing bacteria, e.g. Corynobacterium glutamicum, a treatment with isonicotinic acid hydrazide (INH) (an inhibitor of mycolic acid synthesis) is used (Hermans et al., 1990; Haynes and Britz, 1990). For these species Tween 80 is also suggested for enhancing the electrotransformation efficiency (Haynes and Britz, 1989), as it prevents cell-clumping and changes the composition of mycolic acid in cutaneous corynobacteria.

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Growth Stage and Concentration of Cells Although electroporation has been successfully demonstrated for a large number of strains, transformation efficiencies and optimum electroporation conditions appear to vary rather widely. Successful electroporation is highly dependant in a number of parameters. Growth phase at the time of harvesting the cells and cell density have both significant influence on transformation efficiency. The period from early to mid-log phase is regarded as the period of great electrocompetence for most cells. 2-5-fold increased transformation efficiencies have been reported with cells from mid-log phase compared to those obtained with late exponential phase cells (Calvin and Hanawalt, 1988). McIntyre and Harlander (1989a) studied the effects of the growth stage and cell concentration on transformation efficiency in L. lactis. Washed cell suspensions at optical densities of OD600 0.2, 0.45, 0.7, and 1.2, corresponding to early-log, mid-log, late-log, and stationary phases, respectively, were diluted to make cell concentrations of 1010, 109, 108 and 107 CFU/ml, and electroporated. The results showed that transformation efficiencies were dramatically reduced when cells were diluted prior to electroporation or when early- or latelog phase cells were used. Other investigators however, have recommended the use of earlyor mid-log phase cells and have utilized cell concentrations in the range of 2 x 108 to 5 x 109 CFU/ml (Chassy and Flickinger, 1987; Miller et al., 1988; Powel et al., 1988).

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Growth Medium Composition and Growth Temperature For some species, the growth medium and the growth temperature have been shown to influence the transformation efficiency. McIntyre and Harlander (1989b) reported results on the electrotransformation of L. lactis that clearly indicate that a chemically defined growth medium, such as FMC or RPMI 1646, with the addition of 0.24% DL-theonine, improves the efficiency of electroporation. Complex media appeared to create less favorable microenvironments around cells with regard to electro pulsing. Improved transformation efficiencies were reported for Staphylococcus aureus (Schenk and Laddaga, 1992) by using yeast extract in the growth medium rather than the conventional SOC medium. The same effect was observed also for E. coli C by Taketo (1989). Growth temperature is another parameter that influences the outcome of the process of electroporation. Chuang et al. (1995) reported that growth of E. coli at 18oC, instead of the conventional 37oC, increased the transformation efficiency. Generally, classical growth temperatures are not regarded as optimal growth temperatures for the preparation of electrocompetent cells (Glenn et al., 1992; Eynard and Teissie, 2000).

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Size, Form, and Concentration of DNA Transformation efficiency is markedly affected by the concentration of DNA in the electroporation medium. Increased transformation efficiencies have been achieved through increased DNA concentrations (Dower et al., 1988). There is a logarithmic relationship between the total number of electrotransformants and DNA concentration (Dower et al., 1988; Rittich and Spanova, 1996). McIntyre and Harlander (1989a) found a linear relationship between the number of electrotransformants (nt) and DNA concentration (c) at low DNA concentrations. It follows from this fact that at low DNA concentrations, the logarithmic relationship can be approximated by a linear one. However, above the saturation concentration (csat) the number of transformants remains constant. Rittich and Spanova (1996) developed a simple mathematical model for the relationship between the number of transformed cells and the concentration of DNA using published data for many different microorganisms. The model assumes adsorption of DNA molecules on the surface of cells and penetration through the cell wall. The limits and the validity of the application of the model for different bacterial strains electroporated by plasmid DNA, were verified using regression analysis. Simple equations were derived for the relationship between the number of transformed cells and the concentration of DNA which allow calculation of the concentration cmax at which the number of electrotransformed cells is maximum (ntmax):

1 1 x   nt nt max nt max c where, Kx is the DNA-receptor dissociation constant.

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(Eqn. 1)

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For low concentrations of DNA, where c Carboplatin [Nanda et al., 1998]. EPT (ECT) of human epidermoid carcinoma of larynx (HEp-2) xenografted subcutaneously in nude mice resulted in complete regression of 83% of the treated mice 67 days after treatment [Nanda et al., 1998]. A substantial increased drug uptake in tumours has been demonstrated for bleomycin and cisplatin: the accumulation of these two drugs in tumours was increased two to fourfold, compared to tumours without electroporation [Belehradek et al., 1994; Cemazar et al., 1998]. Thus these drugs have been identified as potential candidates for electrochemotherapy of cancer patients [Mir, 2006].

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Clinical studies performed in patients with different cancer histotypes achieved high rates of local tumor control [Mir et al., 1991a; Glass et al., 1997; Heller et al., 1998; Kubota et al., 1998]. In other clinical trials a high rate of local tumor disappearance was achieved in patients with basal cell carcinoma, malignant melanoma, head and neck squamous cell carcinoma, and breast adenocarcinoma tumors treated by high voltage electrochemotherapy with bleomycin [Belehradek et al., 1993; Domenge et al., 1996; Panie et al., 1998; Hofmann et al., 1999; Rodriguez-Cuevas et al., 2001; Bloom and Goldfarb, 2005; Campana et al., 2008]. The prerequisite for effective electrochemotherapy is a sufficient drug concentration and distribution within the tumour, as well as an adequate electric field distribution [for review see also Dev and Hofmann, 1994; Hofmann et al., 1999a; Sersa et al., 2008]. Currently, Electrochemotherapy is mainly used as a palliative treatment of cutaneous and subcutaneous tumour nodules, mostly single or multiple cutaneous metastases of melanoma, and proved to be a highly efficient and safe approach [Heller et al., 1996; Mir et al., 1998; Sersa et al., 2000; Sersa, 2006; Byrne and Thompson, 2006], and long-term remission–up to several years–can be obtained [Sersa et al., 2000; Byrne et al., 2005].

1.4.3. Electrotherapy by High Frequency Electric Fields Application of low-intensity alternating electric fields in the frequency range of 100-300 kHz, delivered by means of insulated electrodes, was shown to possess inhibitory effect on the growth rate of a variety of human and rodent tumor cell lines in vitro and malignant tumors in animals. Thus, complete proliferation arrest was achieved at intensities of 1.4 and 2.25 V/cm in melanoma and glioma cells, respectively [Kirson et al., 2004]. In vivo treatment of tumors in mice resulted in significant slowing of tumor growth and extensive destruction of tumor cells within 3-6 days [Kirson et al., 2004; Kirson et al., 2007]. These findings led to the initiation of a pilot clinical trial of the effects of this treatment in 10 patients with recurrent glioblastoma (GBM). Median time to disease progression in these patients was more than double the reported medians of historical control patients [Kirson 2007]. Using this treatment, Salzberg and coworkers reported a temporal regression of 3 out of 6 treated tumors in patients with progressive cancer, whereas the other patients showed partial responses or progressive disease [Salzberg et al., 2008]. Exposure of cell and animals to a much higher electromagnetic frequency in the range of the millimetric wave radiation revealed to affect cancer. The effects of low power millimetric wave radiation on the growth of tumor and healthy cells were examined by employing a wide-band frequency range between 53.57-78.33 GHz with a radiation density power of 2.7 x 10-17 watt/Hz [Chidichimo et al., 2002]. Following one hour of radiation treatment given every other day to three tumoral human stable cell lines produced a noticeable inhibition of the cellular growth. The analogous treatment given to two healthy cell lines gave weak growth stimulation. A scanning electron microscopy study of MCF-7-and K562 -irradiated cells revealed that the treatment induced profound morphological changes of the membrane [Chidichimo et al., 2002]. In-vivo study on the effect of millimeter electromagnetic waves at a frequency of 42.2 GHz (possessing incident power density of 36.5±5 mW/cm2) demonstrated that cyclophosphamide caused a marked enhancement in tumor metastases (fivefold), which was significantly reduced when cyclophosphamide-treated animals were irradiated [Logani et al., 2006]. Millimeter waves also increased NK cell activity suppressed by cyclophosphamide, suggesting that a reduction in tumor metastasis by the radiation is mediated through activation of NK cells [Logani et al., 2006].

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1.4.4. Electro-Hyperthermia An additional use of electric energy for the treatment of cancer was proposed as a new branch of hyperthermia. This approach called extracellular hyperthermia or electrohyperthermia heats up the targeted tissue by means of electricity [for review see Fiorentini and Szasz, 2006].

2. ANTI–TUMORAL EFFECTS OF LOW ELECTRIC FIELD CANCER TREATMENT-ENHANCED CHEMOTHERAPY (LEFCT-EC) We have shown previously that exposure of cells to non-permeabilizing unipolar-pulsed low electric fields (LEF) led to an efficient enhanced uptake of molecules and macromolecules via an endocytic-like process. We have made use of this new phenomenon to enhance the efficiency of incorporation of chemotherapeutic agents into tumor cells in-vivo. In our studies we examined the anti-tumor effectiveness of treatment based on exposure of solid tumors in mice to low electric fields in the absence (LEFCT) or presence of chemotherapeutic agents (LEFCT-EC). The effect was studied in several experimental models of mouse malignant tumors such as melanoma (B16F10), breast carcinoma (DA3), squamous cell carcinoma (SQ2), prostate carcinoma (TRAMP-C1), and colon carcinoma (CT-26). The studies addressed two major issues:

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I.What is the effect of LEFCT-EC on primary tumor eradication and metastatic progression of experimental metastatic tumors? II.What is the involvement of immunological components in the arrest of metastatic growth achieved by LEFCT-EC?

2.1. Experimental Design And Methods: 2.1.1. Experimental Metastatic Tumors We used experimental models of mouse tumors that develop spontaneous metastases and are suitable for evaluating the clinical relevance of this therapeutic approach. In these model systems we evaluated the efficacy of LEFCT-EC on tumor metastases, tumor growth and survival, and explored the optimal conditions of this procedure. The following tumor cells were used in the studies: 1. Highly metastatic and weakly immunogenic clone of the tumor cell line B16-F10.9 melanoma. Metastases in the lung appear after removal of the primary tumor. 2. The DA-3 mammary adenocarcinoma is weakly immunogenic, and metastases in the lung appear in the presence of the primary tumor. 3. Metastatic clone of squamous cell carcinoma - SQ2 cell line. 4. Transgenic adenocarcinoma of mouse prostate cell line (TRAMP C-1). 5. The metastatic line of colon carcinoma CT-26, metastases mainly in the lung, but also in the liver, in the presence of the primary tumor.

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2.1.2. Chemotherapy The chemotherapeutic drugs used in this study were: 5-Fluorouracil (5-FU), BCNU (1,3bis(2-Chloroethyl)-1-nitrosurea), Taxol, cisplatinum, doxorubicin, and bleomycin. In most of the experiments the drugs were injected into the tumor, and in several experiments into the tail vein or into the peritoneum.

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2.1.3. Low Electric Field Cancer Therapy Protocol Mice are subjected to a single treatment, once the tumor reaches the size where initiation of metastasis takes place. Typically a volume not exceeding 100 μl solution of the chemotherapeutic agents was injected. The exposure to electric field was carried out 2 minutes after intratumoral injection of the chemotherapeutic agents or 10 minutes after intravenous or intraperitoneal administration of the appropriate chemotherapy. To expose tumors to electric fields, we used stainless steel electrode needles (Karlsbader insect pins No 0; BioQuip Products, Rancho Dominquez, CA), soldered at their brassy ends with thin isolated copper wires. The electrodes were arranged with the cathode needle penetrating the middle of tumor while three to four anode needles were inserted percutaneously into the perimeter of the tumor. The needles were connected to an electric pulse generator (Grass S48 Stimulator). The typical electric parameters used were: field strength, 40 V/cm; repetition frequency, 500 Hz; and pulse width, 180 μs. The mice were exposed to the electric stimulus for 12 min. Mice treated with either electric fields (LEFCT) alone or with electric fields and chemotherapy (LEFCT-EC) were anesthetized 10 min before the treatment. The following experimental groups were used: a) Non-treated tumor bearing mice (TB). b) Tumor bearing mice treated with an intratumoral, intravenous or intraperitoneal injection of the chemotherapeutic drug. c) Tumor bearing mice treated with low electric field (LEFCT). d) Tumor bearing mice treated with the chemotherapeutic drug and low electric field, (LEFCT-EC). The parameters tested: a) Primary tumor reduction. b) Survival time. c) Rate of cure. The statistical significance (p 0.1). Although increasing the pulse duration and number of pulses using caliper electrodes enhanced gene transfer, no significant difference in gene transfer was noted with consistent or alternating polarity. If electrophoretic force contributed to DNA electrotransfer, lower gene expression would occur with alternating polarity.[21] Other laboratories have proposed that two components are required for optimal gene electrotransfer: cell permeabilization with high-voltage pulses and electrophoretic forces on DNA with low-voltage pulses.[24, 26] It has been observed that one high-voltage pulse followed by four low-voltage pulses resulted in 10-fold higher gene expression than one highvoltage pulse followed by one low-voltage pulse when the lag time between pulses was set from 1 to 300 s. Due to this result, it was suggested that low-voltage pulses are important in producing an electrophoretic force.2 Our laboratory replicated the results with our device using pulses of alternating polarity. However, no difference in gene transfer efficiency was observed when low-voltage pulses were delivered with consistent or alternating polarity. Also, enhancement of gene expression indicates low voltage pulses may induce permeabilization. To verify this hypothesis, we injected mice with Evans Blue dye followed by six low-voltage pulses at 80 V/cm for 99 ms. Myocyte uptake of Evans Blue dye was detected 1 day after dye injection in mice with a caliper electrode given low-voltage pulses with consistent or alternating polarity and in mice given pulses under standard conditions. Muscles without electroporation had no accumulation of Evans Blue dye, suggesting lowvoltage pulses can induce membrane permeabilization.[21] Additional experiments have confirmed electrophoresis is not involved in electrogene transfer. Klenchin and colleagues suggested the efficacy of gene transfer in vitro decreased significantly when the electrophoretic mobility is diminished by increasing medium viscosity or when Mg2+ is added to the medium.[27]

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Figure 3. The pulses with consistent (A) and alternating (B) polarity.[21]

However, levels of luciferase expression were not affected by increasing concentrations of Ficoll or Mg2+ following electroporation with consistent or alternating polarity pulses. Another experiment involved injecting DNA (10 μg/μl) between the sheath covering the muscle known as the epimysium and the muscle fiber of the quadriceps immediately followed by six pulses. The DNA may only move either towards the muscle or epimysium given the presumed electrophoretic force with direction dependent on the pulse polarity. If the electrophoretic force is present, the efficiency of electrogene transfer should be enhanced when the muscle is in contact with the anode of the caliper electrode and the epimysium is in contact with the cathode during unidirectional electroporation. When polarity is reversed, the efficiency of electrogene transfer would considerably decrease as DNA is driven away from cells. More than a 100-fold enhancement in gene expression was detected for both polarity groups in comparison to a control group without electroporation. [21]

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Figure 4. Luciferase expression after DNA electro-transfer into the quadriceps with the pulses of consistent or alternating polarity. Electro-gene transfer was done with either caliper or syringe electrodes delivering short (20 μs) or long (20 ms) pulses.[21]

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THE FIELD STRENGTH FOR ELECTROGENE TRANSFER CAN BE FURTHER REDUCED USING SINE-WAVE CURRENT PULSES Although electrogene transfer is a useful tool for enhancing gene expression in various organs, toxicity due to high field strengths of conventional direct current square waves must be minimized for routine use in treating human disease states. Previously, all commercial electropulsators were designed to convey direct current sine square-wave pulses.[24-26] Direct current square-wave pulses require high field strengths which often result in irreversible tissue damage.[19, 28, 29] Human volunteer studies of direct current square-wave electroporation in skin30 and muscle[31] also have reported increased pain sensation. The recent conclusion that electrophoresis does not affect electrogene transfer led us to investigate the use of alternating current. Similar to earlier findings,[21] the alternating current sine-wave pulses had no net electrophoretic forces yet resulted in higher electrogene transfer efficiency. Efficient gene transfer may be safely achieved with pulses of alternating current sine waves with a frequency of 60 Hz. Compared to conventional direct current square wave pulses, the field strength could be decreased to as low as 20 V/cm and electrogene transfer increased greater than 10 fold with less toxicity (Figure 5). Initially, the effect of field strength was examined with alternating current sine-wave and direct current square-wave pulses. When field strength varied from 10 to 30 V/cm with a fixed pulse strength of 600 ms, all groups tested with alternating current sine-wave pulses had a significant improvement in gene expression (p < 0.01) resulting in a 10 to 20 fold increase in luciferase expression. The current through needle electrodes with alternating current sinewave pulses was 11 to 13 mA as measured with an Extech digital thermometer.

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Figure 5. Electrogene transfer into quadriceps of mice with alternating current (AC) sine-wave and direct current (DC) square-wave pulses. The muscle in the control was transferred with a caliper electrode with direct current square-wave (6 pulses, 200 V/cm, 20 ms pulse duration).[22]

Levels of luciferase expression increased with an increase in alternating current sinewave pulse duration with a maximum expression at 600 ms. A pulse duration of 800 ms or higher resulted in decreased gene expression, perhaps due to increased tissue damage. Contrarily, gene expression did not increase with longer pulse duration using direct current square-wave pulses; a significant difference in luciferase expression was examined between the alternating current and direct current groups. In addition, enhanced gene expression was observed by live animal imaging for luciferase expression and fluorescence imaging for green fluorescent protein expression.[22] Alternating current sine-waves should generate 70.7% of the heat generated by squarewaves as calculated with integral of the wave functions to reduce muscle damage and possibly increase transfection efficiency.[32] By observing hematoxylin-and-eosin-stained transverse sections of stained muscle, larger areas of muscle necrosis were noted in direct current groups. Alternating current sine-wave pulses had more than a 10 fold increase in electrogene transfer efficiency with less damage than direct current square-waves. Total creatinine kinase levels as a marker for skeletal muscle damage were also collected from mice (n = 5) 2 hours after electrogene transfer. Alternating current sine-wave pulses had an average of 37% less creatinine kinase than direct current pulses, confirming histological observations.[22] Alternating current sine-wave gene transfer is further characterized by DNA dose, gene expression kinetics, and transfer to various tissues. Significant levels of luciferase expression in muscle could be detected by as little as 1 μg DNA and reached a plateau when injected with 5 μg DNA. Gene expression could be detected as early as 6 hours after injection of luciferase DNA to at least 80 days with peak levels of approximately 900 ng luciferase at day

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7. Peak levels could be regained in the muscle by a second electrogene transfer in the same muscle at day 80.[22] Alternating current sine-wave pulses can be used to efficiently transfer naked DNA into additional tissues besides muscle. Gene expression was detected 6 hours in liver and skin or 20 hours in HPV16E7+ tumor after alternating current sine-wave or direct current pulse electrogene transfer. An 8 fold increase in gene expression in the liver and 10 fold increase in skin and tumor occurred in tissues treated with alternating current sine-wave pulses in comparison to direct current square-wave pulses. Alternating current sine-waves were also used to transfer plasmid DNA to the skin of neonatal mice as gene therapy protocols for the induction of immune tolerance are used in the neonate population. Luciferase expression was detected 4 hours after electrogene transfer with a peak level at 14 hours. Normal behavior of the neonatal mice following electrogene transfer indicates that the protocol was both safe and effective.[22] The observed safety of alternating current sine-wave gene transfer allowed for the treatment of mice with hemophilia B (R333Q and FIXKO) with electrogene transfer of the hFIX (human protein IX) plasmid. Peak levels of circulating hFIX levels were reached at day 5 with 83 ng/ml in R333Q mice and 64 ng/ml in FIXKO mice. Therapeutic levels of hFIX 60 ng/ml lasted at least 40 days in R333Q mice while measured levels of hFIX were less than 20 ng/ml at day 13 in FIXKO mice. An inhibitory antibody response to hFIX occurs in FIXKO mice after gene transfer but not in R333Q mice, perhaps explaining the difference in kinetics between the two mouse models. The clotting activity in the mouse models following electrogene transfer corresponded with levels of hFIX in the blood. In comparison, human IX protein levels and clotting activity remained undetected in both mouse models when treated with direct current square-waves.[22] The mechanism behind electrogene transfer using alternating current sine-wave pulses at low voltages is not clear. Currently, electrogene transfer is performed by commercially available electropulsators that generate direct current square-wave pulses with a frequency of 1 Hz.24-26 We used alternating current sine-wave pulses with a frequency of 60 Hz, which alternated the polarity three times during a 50 ms pulse, but a direct current square-wave (1 Hz) does not alternate during that time (Figure 5). The polarity of alternating current sinewave per pulse alternates 36 times when the pulse duration increases to 600 ms, resulting in an increase of gene transfer efficiency. Therefore, we hypothesize that both bipolar current and the frequency may play important roles in destabilizing the cell membrane for DNA entry. Another advantage of using alternating current sine-waves is its induction of lower damage than direct current square-wave pulses. It has been estimated that the heat produced by alternating current is only 70.7% of the heat produced by direct current. Presumably, production of less heat by alternating current is one of mechanisms for this reduction in the amount of damage.[22]

CONCLUSION Several recent advances have occurred in electrogene transfer. The syringe electrode allowed gene transfer at much lower threshold values due to a design based on the principles of field strength distribution.[20] Previous needle electrodes have been devised; however, the

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needles were not dually used for injection.[25] With conventional electrodes, the field strength rapidly disperses with increasing distance between the injection site and electrode. This dissipation necessitates a field strength higher than the threshold value, leading to tissue damage. The syringe electrode alleviates this dilemma by delivering an electric field directly to the placement site for injected DNA. In addition, the electric field is strongest within a cylindrical area surrounding the syringe electrode. Consequently, gene transfer may be achieved at a lower field strength and with lower potential for muscle damage. Thus, the syringe electrode may be useful for clinical applications of gene therapy.[20] In earlier in vitro studies, electrophoresis was suspected to be an important factor for efficient DNA transfer due to the effect of an electric field on polyanionic DNA. An electrophoretic force is involved in in vitro DNA transfer due to an electrically conductive cell culture medium.[27] However, the query was whether or not electrophoresis could drive DNA across the cellular membrane, especially in vivo. Data from the above series of experiments indicate that electrogene transfer in vivo is not facilitated by DNA electrophoresis. Rather, efficient gene transfer by electroporation occurs due to cell membrane permeabilization and passive DNA diffusion.[21] In order for electrogene transfer to be utilized in humans, the field strength must be reduced to decrease toxicity while maintaining efficiency. The use of alternating current sinewaves offers an attractive alternative to direct current square-waves, as seen in the treatment of mice with hemophilia B.[22] Although safety guidelines for human gene therapy are lacking, the voltage of 36 V is a guideline for electrical products approved to be safe for humans[.33, 34] Also, the maximum amperage that allows a person to release his or her hand from the current source (15 mA for a 70 kg man at 60 Hz) should be considered in electrogene transfer.[35] Efficient in vivo electrogene transfer using alternating current sinewave pulses may be achieved with a field strength of 20 V, current between 11 and 13 mA, and less damage than traditional direct current square-wave pulses at 1 Hz. However, conditions have been examined where direct current pulses were not observed to cause serious tissue damage, although gene transfer using alternating current sine-wave pulses may be more advantageous in a clinical setting due to lower field strengths. This new technique of alternating current sine-wave electrogene transfer may be useful in production of therapeutic circulating proteins, DNA vaccines, and antibody therapy. Hopefully, new electropulsators will be developed to treat human diseases and clarify the mechanisms involved in alternating current sine-wave electrogene transfer.[22]

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Electrogenetherapy: Electrogene Transfer Using Low Field Strength [4]

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Somiari, S.; Glasspool-Malone, J.; Drabick, J. J.; Gilbert, R. A.; Heller, R.; Jaroszeski, M. J.; Malone, R. W., Theory and in vivo application of electroporative gene delivery. Molecular Therapy 2000, 2, 178-187. Bloquel, C.; Fabre, E.; Bureau, M. F.; Scherman, D., Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications. Journal of Gene Medicine 2004, 6, S11-S23. Heller, L.; Jaroszeski, M. J.; Coppola, D.; Pottinger, C.; Gilbert, R.; Heller, R., Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo. Gene Therapy 2000, 7, 826-829. Heller, R.; Jaroszeski, M.; Atkin, A.; Moradpour, D.; Gilbert, R.; Wands, J.; Nicolau, C., In vivo gene electroinjection and expression in rat liver. FEBS Letters 1996, 389, 225-228. Suzuki, T.; Shin, B. C.; Fujikura, K.; Matsuzaki, T.; Takata, K., Direct gene transfer into rat liver cells by in vivo electroporation. FEBS Letters 1998, 425, 436-440. Rizzuto, G.; Cappelletti, M.; Mennuni, C.; Wiznerowicz, M.; DeMartis, A.; Maione, D.; Ciliberto, G.; La Monica, N.; Fattori, E., Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure. Human Gene Therapy 2000, 11, 1891-1900. Muramatsu, T.; Shibata, O.; Ryoki, S.; Ohmori, Y.; Okumura, J., Foreign gene expression in the mouse testis by localized in vivo gene transfer. Biochemical and Biophysical Research Communications 1997, 233, 45-49. Rols, M. P.; Delteil, C.; Golzio, M.; Dumond, P.; Cros, S.; Teissie, J., In vivo electrically mediated protein and gene transfer in murine melanoma. Nature Biotechnology 1998, 16, 168-171. Byrnes, C. K.; Malone, R. W.; Akhter, N.; Nass, P. H.; Wetterwald, A.; Cecchini, M. G.; Duncan, M. D.; Harmon, J. W., Electroporation enhances transfection efficiency in murine cutaneous wounds. Wound Repair and Regeneration 2004, 12, 397-403. Dean, D. A.; Machado-Aranda, D.; Blair-Parks, K.; Yeldandi, A. V.; Young, J. L., Electroporation as a method for high-level nonviral gene transfer to the lung. Gene Therapy 2003, 10, 1608-1615. Aihara, H.; Miyazaki, J., Gene transfer into muscle by electroporation in vivo. Nature Biotechnology 1998, 16, 867-870. MacColl, G. S.; Goldspink, G.; Bouloux, P. M. G., Using skeletal muscle as an artificial endocrine tissue. Journal of Endocrinology 1999, 162, 1-9. Danko, I.; Fritz, J. D.; Jiao, S. S.; Hogan, K.; Latendresse, J. S.; Wolff, J. A., Pharmacological enhancement of in-vivo foreign gene-expression in muscle. Gene Therapy 1994, 1, 114-121. Lu, Q. L.; Bou-Gharios, G.; Partridge, T. A., Non-viral gene delivery in skeletal muscle: a protein factory. Gene Therapy 2003, 10, 131-142. Bigey, P.; Bureau, M. F.; Scherman, D., In vivo plasmid DNA electrotransfer. Current Opinion in Biotechnology 2002, 13, 443-447. Muramatsu, T.; Nakamura, A.; Park, H. M., In vivo electroporation: a powerful and convenient means of nonviral gene transfer to tissues of living animals (Review). International Journal of Molecular Medicine 1998, 1, 55-62. Liu, F.; Huang, L., A syringe electrode device for simultaneous injection of DNA and electrotransfer. Molecular Therapy 2002, 5, 323-328.

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Feng Liu, Amber Frick, and Jue Wang

[21] Liu, F.; Heston, S.; Shollenberger, L. M.; Sun, B.; Mickle, M.; Lovell, M.; Huang, L., Mechanism of in vivo DNA transport into cells by electroporation: electrophoresis across the plasma membrane may not be involved. Journal of Gene Medicine 2006, 8, 353-361. [22] Liu, F.; Sag, D.; Wang, J.; Shollenberger, L. M.; Niu, F. L.; Yuan, X.; Li, S. D.; Thompson, M.; Monahan, P., Sine-wave current for efficient and safe in vivo gene transfer. Molecular Therapy 2007, 15, 1842-1847. [23] Neumann, E.; Kakorin, S.; Toensing, K., Fundamentals of electroporative delivery of drugs and genes. Bioelectrochemistry and Bioenergetics 1999, 48, 3-16. [24] Satkauskas, S.; Bureau, M. F.; Puc, M.; Mahfoudi, A.; Scherman, D.; Miklavcic, D.; Mir, L. M., Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis. Molecular Therapy 2002, 5, 133140. [25] Miklavcic, D.; Semrov, D.; Mekid, H.; Mir, L. M., A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy. Biochimica Et Biophysica Acta-General Subjects 2000, 1523, 73-83. [26] Bureau, M. F.; Gehl, J.; Deleuze, V.; Mir, L. M.; Scherman, D., Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochimica Et Biophysica Acta-General Subjects 2000, 1474, 353-359. [27] Klenchin, V. A.; Sukharev, S. I.; Serov, S. M.; Chernomordik, L. V.; Chizmadzhev, Y. A., Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis. Biophysical Journal 1991, 60, 804-811. [28] Durieux, A. C.; Bonnefoy, R.; Busso, T.; Freyssenet, D., In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage. Journal of Gene Medicine 2004, 6, 809-816. [29] McMahon, J. M.; Wells, D. J., Electroporation for gene transfer to skeletal muscles current status. Biodrugs 2004, 18, 155-165. [30] Wallace, M. S.; Ridgeway, B.; Jun, E.; Schulteis, G.; Rabussay, D.; Zhang, L., Topical delivery of lidocaine in healthy volunteers by electroporation, electroincorporation. or iontophoresis: an evaluation of skin anesthesia. Regional Anesthesia and Pain Medicine 2001, 26, 229-238. [31] Tjelle, T. E.; Salte, R.; Mathiesen, I.; Kjeken, R., A novel electroporation device for gene delivery in large animals and humans. Vaccine 2006, 24, 4667-4670. [32] Nielsen, K. G.; Nielsen, O.; Thomsen, H. K., Device and methods for the measurement of energy-transfer in experiments involving thermal and electrical injuries of skin. Forensic Science International 1981, 17, 203-209. [33] Dalziel, C. F.; Lee, W. R., Lethal electric currents. IEEE Spectrum 1969, 6, 44-50. [34] Lee, R. H., Electrical Safety in Industrial Plants. IEEE Spectrum 1971, 8, (6), 51-57. [35] Lyster, T.; Jorgenson, D.; Morgan, C., The safe use of automated external defibrillators in a wet environment. Prehospital Emergency Care 2003, 7, 307-311.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 11

ELECTROPORATION – TREATING MICE OR MEN? Angela M. Bodles-Brakhop and Ruxandra Draghia-Akli* VGX Pharmaceuticals, Inc., The Woodlands, Texas 77381, U.S.

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ABSTRACT Electroporation is a novel strategy that may provide opportunities for therapeutic and prophylactic treatments for diseases for which a cure is yet available. The recent interest in using plasmid DNA as a gene therapy tool has resulted in the improvement and optimization of physical methods of delivery, in particular in vivo electroporation. Electroporation increases transfer of DNA vaccines or therapeutic plasmids to the skin, muscle or tumor resulting in higher levels of expression and clinical benefits. Numerous preclinical studies have shown that electroporation can be successfully used in many species facilitating transition of this technology to humans. The first gene therapy product delivered by electroporation has been approved for use in farm pigs. With the advent of human clinical trials examining the use of electroporation the results are greatly awaited.

Keywords: electroporation, gene therapy, plasmid

INTRODUCTION The development of novel non-viral vaccines and therapeutic plasmids for the prevention or treatment of diseases has warranted the development of a delivery system that will enable satisfactory expression of the desired molecule. Electroporation (EP) is a technique by which cell membranes are made permeable by rapid electrical pulses allowing the entry of macromolecules into cells and tissues [1]. The delivery of square waves to targeted tissues is either based on constant-voltage or constant-current concepts. Since the first report *

Address all correspondence and requests for reprints to: Ruxandra Draghia-Akli, M.D., Ph.D. VGX Pharmaceuticals, Inc. 2700 Research Forest Drive, Suite 180, The Woodlands, Texas 77381, USA Telephone: 281-296-7300, ext. 107, Fax: 281-296-7333, E-mail: [email protected].

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demonstrating its use in vitro over 25 years ago [2] the process of EP has developed many applications in vitro and in vivo at the cellular and whole tissue level, including gene transfer in mammalian cells, genetic manipulation in plant cells, genetic transformation of bacteria and yeast, introducing compounds such as drugs or proteins into cells, cell loading, as well as production of hybridoma and human monoclonal antibodies. Although viral transfection systems have been historically superior in efficacy to nonviral methods, their adverse immunological drawbacks [3] have led to the increase in naked plasmid DNA utilization, although for this application there have been issues of low expression. Compared to viral-mediated therapies, plasmid DNA can be easily designed and manipulated to incorporate desired elements, is relatively easier to manufacture, and does not include intrinsic elements that could generate a negative or undesired immune response in the host. For example, optimization of erythropoietin (EPO) by changing the leader sequence and optimizing the gene codon usage resulted in higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in mice, rabbits and cynomolgus monkeys [4]. In order to boost efficacy of delivery of non-viral gene therapy several methods are currently under investigation such as lipid-mediated entry into cells [5], gene gun delivery and jet injection [6], and sonoporation [7]. In this chapter we will concentrate on the physical delivery method of EP and its combination with gene therapy. As mentioned above, the technique of EP results in the transient destabilization of cellular membranes by localized and controlled electric fields, facilitating the entry of foreign molecules into cells and tissues [1]. While some controversy still exists regarding the exact mechanism of cellular entry, most agree that the entry of plasmid DNA occurs through a multistep mechanism involving the interaction of the DNA molecule with the destabilized membrane during the pulse followed by its passage across the membrane [8]. Modeling of EP in a single cell revealed three stages: charging of the cell membrane, creation of pores, and evolution of pore radii with a 1-ms, 40 kV/m pulse resulting in approximately 341,000 pores [9]. To enable entry of plasmid DNA into cells, the pores should have sufficiently large radii (>10 nm), remain open long enough for the DNA to enter the cell (milliseconds), and should not cause membrane rupture [10]. Recently, different EP protocols comparing low against low and high voltage pulse combinations in dogs were evaluated. In a series of reports, the combination of 1 high voltage pulse (600 V/cm, 100 mus), followed by 4 low voltage pulses (80 V/cm, 100 ms, 1 Hz) yielded a similar efficiency as the standard low voltage pulses and was carried out more quickly and therefore determined to be more suitable for potential use in clinical practice [11]. Furthermore, plasmid DNA administered with EP is more protected from degradation most likely due to its early compartmentalization into the nuclei of the muscle cells compared to simple injection alone [12]. Previous constant-voltage EP techniques did not take into account the resistance of the tissue and often resulted in tissue damage, inflammation, and loss of plasmid expression; while these phenomena are acceptable for vaccine delivery, and may even result in better immune responses in some circumstances [13] plasmid delivery for gene therapy purposes often require long-term expression and optimized, mild EP conditions that preserve the tissue integrity. Therefore, investigation of parameters for DNA administration using adaptive constant-current EP were carried out. Studies in mice and pigs revealed that age- and tissuespecific resistance, pulse pattern, and other variables associated with EP need to be

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individually optimized for each species to achieve maximum plasmid expression [14;15]. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses short-term and long-term transfection efficiency in murine skeletal muscle have all been shown to affect the duration of transgene expression [16]. Furthermore, plasmid injection variables such as concentration, volume and formulation of the DNA vaccine, also need to be optimized to yield high expression and immunogenicity. It has been found that concentrated formulations result in better expression and immunogenicity [17]. These findings, in not only small lab animals but also in larger animals such as pigs, demonstrate that this technology can be translated to large animals and suggests that similar success in humans is achievable. The end of the 20th century was key for the advancement of EP with several papers being published on the subject [18-22], followed by a large number of studies demonstrating the potential of this technology for many applications. The use of animal model studies has been important for the development of EP, gene therapy and DNA vaccination. The combinations of these technologies have enabled successful preclinical studies in many areas of disease prevention and treatment and have provided the foundation for the transition to clinical trial status and beyond. The first plasmid-mediated gene therapy delivered by EP was recently approved for commercialization for the reduction of peri-natal mortality in farm pigs [23]. In this chapter we will focus on EP studies at the preclinical level that have been published in the last year(s), concluding with the clinical trials that are currently underway.

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ANIMAL MODELS IN PRECLINICAL STUDIES There are numerous preclinical studies that have investigated the potential of EP gene therapy and that have lead to current clinical trial status. Table 1 shows the breadth of preclinical studies that have been carried out using EP in the last year; as shown, most are in the area of DNA vaccination. Animals used for these preclinical studies include mice, hamsters, ferrets, rabbits, dogs, pigs, cattle, horses, and non-human primates; some such as the ferrets and non-human primates are more relevant for DNA vaccine therapy studies. Figure 1 shows the percentage of EP preclinical studies grouped by animals within the last year. Importantly, some companion and farm animal studies have also been carried out, using a heterogeneous population that is more representative of a human heterogeneous population than small laboratory animals, enabling the transfer of the technology to a veterinarian setting as well as providing important data for clinical studies [24]. The use of larger animals, including non-human primates and companion animals, provides convincing evidence of the ability to transfer EP to humans. The first studies examining in vivo EP were carried out in mice in 1998 in a murine melanoma tumor model [18] or by IM administration of plasmid DNA [20;25]. Since then numerous studies involving mice have been carried out. In fact, preclinical studies using mice outnumber any other. Most preclinical studies are initially carried out in mice due to cost effectiveness. Important findings are often made in small animals that then lead to larger animal studies including non-human primate before the final transition to clinical studies; nevertheless, too many times results achieved in mouse models do not translate to primates.

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Rodents 64% Multiple species 16% Rhesus macaques 8% Cattle 4%

Dogs 4% Horses 2%

Crustaceans 2%

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Figure 1. Percentage of EP preclinical trials by species within the last year. Rodents, including mice, rats and hamsters, make up the largest group (n = 32). The multiple species group (n = 8) includes research that was carried out in two or more animals such as mice, rabbits or pigs and rhesus macaques. Both gene therapy and plasmid DNA based vaccine preclinical trials are included.

EP seems to bridge this gap and now many EP studies performed in non-human primates yield similar or even better results compared to rodents. Table 1 shows the range of studies using mice for plasmid DNA administered with EP. Studies in larger animals include dogs, horses, and cattle. Companion animal and farm animal studies are not only important for scale up to humans but also provide value information for veterinarian research and future therapies as well as bridging the gap between the two disciplines. Taking care of our four legged friends and improving the quality of life of production animals are increasingly important issues for many people. Of most interest are studies in nonhuman primates due to the critical information that they can provide for numerous human diseases and because they are often the final stepping stone before transition to clinical trial status. While taking into consideration cost and endangered or threatened species limitations, the use of old world monkeys such as macaques that diverged from humans over 15 million years ago in preclinical evaluation are important due the phylogenetic similarities to humans [26]. Some examples of preclinical studies that have utilized a combination of gene therapy and EP are discussed below.

Anemia The causes of anemia are many and the benefits to finding a treatment would be great. Approximately 10 years ago the first report of the correction of anemia in mice after the single injection of as little as 1 μg of plasmid erythropoietin (Epo) DNA followed by EP showed a 100-fold increase in the production and secretion of protein from mouse skeletal muscle [27]. A following study demonstrated the effectiveness of this procedure in rats [28]. Induction of a long-lasting and dose-dependent increase in hematocrit levels was observed in mice and normal and uremic rats by several groups [29-32].

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Table 1. Preclinical studies with EP. Only studies in the last year have been documented. PubMed http://www.ncbi.nlm.nih.gov search terms: DNA vaccine, electroporation, plasmid Title

Animal

DNA plasmid/vaccine

Reference

1

Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles.

mice

plasmid vascular endothelial growth factor

[79]

2

In vivo DNA electrotransfer into muscle.

mice

3

4

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5

Gene electrotransfer into murine skeletal muscle: a systematic analysis of parameters for longterm gene expression. Age-dependent impairment of HIF-1alpha expression in diabetic mice: Correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells. Electroporation of corrective nucleic acids (CNA) in vivo to promote gene correction in dystrophic muscle.

mice

[103]

mice

corrective nucleic acids (CNAs)

[80]

mice

7

Factor IX gene therapy for hemophilia.

mice

8

In vivo DNA electrotransfer into muscle.

mice

10

[16]

mice

6

9

[102]

plasmid gWIZCA5, which encodes a constitutively active form of HIF-1alpha

KGF-1 for wound healing in animal models

Comparison of electrically mediated and liposomecomplexed plasmid DNA delivery to the skin. Parameters for DNA vaccination using adaptive constant-current electroporation in mouse and pig models.

pCAGGS-IL-5 and pCAGGSlacZ DNA plasmid encoding green fluorescent protein (GFP)

mice, rats

mice, pigs

Plasmid Keratinocyte growth factor-1 Plasmid DNA encoding hF.IX pCAGGS-IL-5 and pCAGGSlacZ a plasmid encoding the reporter luciferase SEAP, EGFP, HA or NA expressing plasmids

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[104] [85] [102]

[105]

[17]

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222

Table 1. (Continued) Title

Animal

11

Growth enhancement of shrimp (Litopenaeus schmitti) after transfer of tilapia growth hormone gene.

crustaceans

12

Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor.

rats

13

14

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15

Double-blinded, Placebocontrolled plasmid GHRH trial for cancer-associated anemia in dogs. Plasmid growth hormone releasing hormone therapy in healthy and laminitis-afflicted horses-evaluation and pilot study. Effects of plasmid growth hormone-releasing hormone treatment during heat stress.

DNA plasmid/vaccine pE300tiGH15 plasmid containing the tilapia growth hormone gene (tiGH) cartilage oligomeric matrix protein, COMP-Ang-1 plasmid

Reference

[106]

[107]

dogs

pGHRH

[24]

horses

pGHRH

[49]

cattle

plasmid GHRH

[70]

16

HER2/neu DNA vaccination for breast tumors.

mice

17

DNA immunization using constant-current electroporation affords long-term protection from autochthonous mammary carcinomas in cancer-prone transgenic mice.

mice

18

DNA vaccination for prostate cancer.

mice

19

Protective immunity against neupositive carcinomas elicited by electroporation of plasmids

mice

pCMV-ECDTM, encoding the extracellular and transmembrane region of the HER2/neu anitgen plasmid coding for the extracellular and transmembrane domains of the product of the rat neu(664V-E) oncogene protein prostate-specific antigen (PSA) DNA vaccine plasmids encoding the TM domain

Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

[108]

[109]

[110]

[111]

Electroporation-Treating Mice or Men? Title

Animal

encoding decreasing fragments of rat neu extracellular domain.

20

21 22

23

24

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25

26

27

28

Intramuscular electroporation of a plasmid encoding human plasminogen kringle 5 induces growth inhibition of Lewis lung carcinoma in mice. IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial. Optimisation of intradermal DNA electrotransfer for immunisation. A consensus-hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses. Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine. Humoral immune response after genetic immunization is consistently improved by electroporation. Therapeutic vaccination halts disease progression in BALBneuT mice: the amplitude of elicited immune response is predictive of vaccine efficacy. Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine. Anti-tumor immunity induced by CDR3-based DNA vaccination in a murine B-cell lymphoma model.

mice

mice mice

mice

mice

DNA plasmid/vaccine associated with decreasing fragments of the EC domain encoded by the rat neu oncogene plasmid encoding kringle 5 (K5) domain of human plasminogen (pVAX1-K5) Interleukin-2 (IL2) plasmid (pDNA) pGL3 Luciferase Reporter Vector pCHA5, optimized consensus H5N1 hemagglutinin a marker plasmid DNA (pDNA), pCMV-Luc

223 Reference

[112]

[113] [114]

[115]

[116]

mice

hVEGF165, hFGF-2 and BbKI

[117]

mice

rat ErbB2 antigen

[118]

mice

SARS-CoV N1 and N3 DNA vaccine

[119]

mice

pV(H)CDR3-IL2 and pV(L)CDR3-IL2

[120]

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224

Table 1. (Continued) Title

29

30

31

32

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33

34

Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation. HIV-1 Env vaccine comprised of electroporated DNA and protein co-administered with Talabostat. Markedly enhanced immunogenicity of a Pfs25 DNAbased malaria transmissionblocking vaccine by in vivo electroporation. Electroporation at Low Voltages Enables DNA Vaccine to Provide Protection against a Lethal H5N1 Avian Influenza Virus Challenge in Mice. Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle. Protection abilities of influenza B virus DNA vaccines expressing hemagglutinin, neuraminidase, or both in mice.

Animal

DNA plasmid/vaccine

Reference

mice

HIV type 1 Env DNA vaccine

[121]

mice

HIV-1 Env vaccine

[122]

mice

Pfs25 DNA plasmid

[123]

mice

H5N1 virus hemagglutinin DNA

[124]

cattle

mice

35

Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus.

mice

36

Immunogenicity in mice and rabbits of DNA vaccines expressing woodchuck hepatitis virus antigens.

mice, rabbits

37

Potentiation of an anthrax DNA vaccine with electroporation.

mice, rats, rabbits

38

Mixing of M segment DNA vaccines to Hantaan virus and

hamsters

SeAP-encoding plasmid HBsAg-encoding plasmid plasmid DNA expressing hemagglutinin or neuraminidase plasmid coding for the CHIKCapsid, E1 and E2 codon-optimized DNA vaccine for the woodchuck hepatitis virus surface antigen DNA vaccine encoding anthrax protective antigen pIMS-120 DNA vaccines expressing the M

Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

[95]

[125]

[126]

[94]

[97]

[127]

Electroporation-Treating Mice or Men? Title

Animal

Puumala virus reduces their immunogenicity in hamsters.

39

40

41

42

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43

The relative immunogenicity of DNA vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods. Treatment of SCCVII tumors with systemic chemotherapy and Interleukin-12 gene therapy combination. Systemic IL-12 gene therapy for treating malignancy via intramuscular electroporation. Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs. Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques.

mice, rabbits

DNA plasmid/vaccine segments of Hantaan virus and Puumala virus DNA vaccine expressing a hemagglutinin antigen from an H5N1 influenza virus

225 Reference

[87]

mice

IL-12 plasmid DNA

[128]

mice

IL-12 encoding plasmid DNA

[129]

dogs

Interleukin 12 plasmid DNA

[130]

rhesus macaques

DNA with plasmid-encoded IL-12

[90]

44

Electroporation-mediated HBV DNA vaccination in primate models.

rhesus macaques

45

Heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus DNA antigens.

mice, ferrets and nonhuman primates

Plasmid DNA encoding the HBV preS2-S and an adjuvant plasmid encoding a fused gene of IL-2 and IFNgamma Combination of several consensus influenza antigens, H5 hemagglutinin (pH5HA), N1 neuraminidase (pN1NA), and nucleoprotein antigen (pNP)

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[93]

[86]

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226

Table 1. (Continued) Title

Animal

46

Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques.

pig, rhesus macaques

47

Persistent antibody and T cell responses induced by HIV-1 DNA vaccine delivered by electroporation.

rabbits, macaques

48

Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein in mice and rhesus macaques

49

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50

Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation A therapeutic SIV DNA vaccine elicits T-cell immune responses, but no sustained control of viremia in SIVmac239-infected rhesus macaques.

mice, rhesus macaques

Indian rhesus macaques

SIVmac239/ macaque model

DNA plasmid/vaccine pEGFP-N1 or pSEAP-2, pGag4Y, pEY2E1-B, and WLV104 plasmids DNA encoding HIV-1 env novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein plasmid expressing HIV gag and rhesus macaque IL-15. gp140Env, GagPol, and TatRevNef plasmids

Reference

[88]

[89]

[96]

[91]

[92]

Following this, skin-targeted delivery of rat Epo also resulted in a dose-dependent increase in expression although for a shorter time period [33]. Several other reports suggest that the use of adjuvants such as hyaluronidase [34] and poly-L-glutamate [35] enhances the transfer and expression of genes delivered with EP. In an adenine-induced renal failure rat model Epo-gene transfer by EP increased Epo expression and serum Epo levels, and also increased the hematocrit levels [36]. The administration of an optimized Epo by EP to mice, rabbits and cynomolgus monkeys resulted in higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested [4]. A predefined target dose was achievable with the EP of Epo [37] – an important finding for prescribed amounts to patients. Alternatively, we have shown that correction of cancerassociated anemia is achievable in severely debilitated dogs using plasmid growth hormonereleasing hormone (GHRH) followed EP with many quality of life parameters, such as appetite and activity level of this extremely heterogeneous population also being improved [24]. Importantly, we have shown that plasmid expression can last for a relative long period of time (3-12 months) after one single injection and EP, making repeat administration unnecessary.

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Arthritis There are several types of arthritis and many related conditions that impact the quality of life of countless people. There are medications to alleviate the pain, swelling and stiffness associated with the disease but they have had limited therapeutic effects. Numerous vectors and therapeutic genes have been investigated for the treatment and management of the disease offering new possibilities. Several studies have investigated the use of tumor necrosis factor (TNF) gene therapy in combination with EP for the treatment of arthritis. In an experimental model of rheumatoid arthritis in mice the administration by EP of plasmids encoding three different human TNF-alpha-soluble receptor I variants exerted protective effects [38]. Further studies revealed that local intra-articular administration of the same plasmids by EP decreased joint destruction in the ankles [39]. The onset of collagen-induced arthritis was prevented in mice treated with anti-TNF gene therapy using in vivo EP and the effect lasted for at least 18 days after treatment [40]. In another collagen-induced arthritis model, TNF receptor 2 (dTNFR) constitutively expressed from plasmid DNA, delivered intramuscularly with EP resulted in a therapeutic effect that was dependent on the stage of the disease at the time of treatment [41]. Further studies with a dimeric TNF receptor II plasmid delivered intramuscularly with EP after the onset of arthritis resulted in inhibition of the progression of the disease [42]. Several other groups have published data for therapeutic treatment of arthritis using gene therapy and EP that does not involve TNF. The IM EP of a pro-opiomelanocortin plasmid reduced inflammatory pain in a rat model of rheumatoid arthritis [43]. Administration of tissue inhibitor of metalloproteinases-4 by EP-mediated intramuscular injection of naked DNA using the rat adjuvant-induced arthritis model completely abolished arthritis development [44]. Administration of IL-4 DNA by EP in a murine collagen-induced arthritis model reduced disease onset and severity [45]. Other studies showed that the in vivo EP of plasmid DNA encoding human IL-1Ra reduced the incidence of collagen-induced arthritis [46] while a plasmid expressing IL-10 delivered by EP revealed that IM administration was a potent therapeutic method that significantly inhibited all the clinical and biological features of arthritis [47]. The revelation of the essential role of HSP70 in protecting cells from stressful stimuli led to the plasmid delivery by EP of HSP70 and to a decreased severity of osteoarthritis [48]. In horses afflicted with laminitis and arthritis, we have shown that treatment with plasmid GHRH by EP had a positive impact on the animals health, with correction of the X-ray signs of arthritis, reduction in inflammation, and increased body mass, enabling a return to pasture of treated animals, compared to controls that received standard of care during the 6-months trial [49].

Diabetes Although there have been improvements in insulin preparation and delivery, physiological normal glycemia is still not easily achieved in diabetics. A gene therapy approach may provide the solution and several preclinical studies have investigated this possibility. The ability of skeletal muscle to produce human insulin after EP-enhanced plasmid DNA injection of a furin-cleavable proinsulin cDNA was evaluated in mice and lead to the release of biologically active insulin, with restoration of basal insulin levels, and

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Angela M. Bodles-Brakhop and Ruxandra Draghia-Akli

lowering of fasting blood glucose with increased survival in severe diabetes [50]. In a separate study, it was also shown that a plasmid encoding mouse furin-cleavable preproinsulin II cDNA administered intramuscularly by EP increased plasma (pro)insulin and reduced fasting blood glucose levels for 8 weeks [51]. Another group also showed that intramuscular injection with EP of a naked plasmid DNA encoding human preproinsulin gene achieved effective expression of insulin with decreases in blood glucose levels and increases in the survival of severe diabetic mice [52]. Electrotransfer of the insulin gene into a diabetic mouse model similarly returned their blood glucose to normal levels, with further findings that several proteins in the liver, kidney and serum also returned to normal levels after treatment [53] demonstrating broader beneficial effects of gene therapy. The in vivo application with EP of protein drugs such as glucagon-like peptide 1 (GLP-1), leptin or transforming growth factor beta (TGF-β) restoring glucose homeostasis, promoting islet cell survival and growth or improving wound healing and other complications for type I and II diabetes has been discussed [54]. It has been shown that administration of a GLP-1 analogue via EP-enhanced IM plasmid-based gene transfer, normalized blood glucose levels in type 2 diabetes-prone db/db mice. Furthermore in a type I diabetes mouse model GLP-1 effectively reduced fed blood glucose levels in treated mice and ameliorated diabetes symptoms likely due to significantly enhanced islet beta-cell mass. Improved glucose tolerance and increased circulating insulin and GLP-1 levels were also noted [55]. This suggests that GLP-1 gene therapy with EP may be applicable to diseases where there is either acute or chronic beta-cell injury. Calcitonin gene-related peptide (CGRP) plays an important role in the regulation of T lymphocytes the imbalance of which is an important pathogenic mechanism for insulindependent diabetes mellitus (IDDM). Gene transfer of CGRP via EP in autoimmune diabetic mice resulted in decreased morbidity, ameliorated hyperglycemia and insulin deficiency, and suppressed pro-inflammatory and promoted anti-inflammatory T cells [56]. The production of reactive oxygen species was also inhibited [57]. Peripheral arterial disease, diabetic neuropathies and microangiopathies are serious complications of the diabetic state. The in vivo angiogenic effects of insulin-like growth factor-I (IGF-I) in regenerating diabetic muscle were investigated. Intramuscular administration of plasmid IGF-I delivered by EP amplified angiogenic responses in regenerating muscle and reversed diabetic microangiopathy [58]. Wound healing in a diabetic mouse model was greatly improved with the delivery of keratinocyte growth factor plasmid DNA with EP compared to untreated controls [59]. Furthermore, the therapeutic effect of the administration of hepatocyte growth factor (HGF) plasmid by intramuscular injection combined with electroporation resulted in three-fold increases in endogenous HGF levels compared to untreated controls and an improvement in advanced diabetic nephropathy [60]. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and has neurotrophic and neuroprotective activities and was also investigated as a gene-based therapeutic for diabetic neuropathy. The findings indicated a complete recovery of the sensory deficits, i.e. hypoalgesia, in the diabetic mouse.

Ocular Disease Over 10 years ago the first publication on an EP-mediated gene therapy approach for the treatment of ocular disease demonstrated its potential [61] as an alternative to the painful,

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repeated injections of recombinant protein therapy. Gene expression was observed in the corneal endothelial cells with no apparent cell damage or evidence of inflammation. Since then others have investigated ocular gene transfer by EP, demonstrating that the use of mammalian rather than viral promoters can achieve safe and sustained gene expression [62]. Positive findings were observed when plasmid was injected in combination with EP in three different sites (subretinal, intravitreous, or periocular) with strong expression but no evidence of retinal damage [63]. The EP of TNF-α soluble receptor to the cilliary muscle in rats with endotoxin-induced uveitis resulted in long-lasting gene expression, elevated protein secretion in the aqueous humor and to inhibition of inflammation. Again, no ocular pathology or structural damage was observed [64].

Multiple Sclerosis

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For the long-term treatment of multiple sclerosis (MS), in vivo gene therapy is an attractive alternative to the current repeated injection of recombinant interferon-beta (IFN-β). Experimental autoimmune encephalomyelitis (EAE), an animal model widely used in MS research was used for investigation of a single intramuscular administration of plasmid IFN-β by EP. The achieved therapeutic effect was comparable to the conventional treatment strategy, with a significant inhibition of disease progression [65]. Further studies, compared the administration of bolus protein, with gene-based delivery of IFN- β by IM injection of plasmid DNA followed by EP, and gene-based delivery of IFN-β by IM injection of adenovirus-associated type 1 (AAV1). Comparable long-term induction of IFN-β biomarkers were observed for both gene therapy approaches; an improvement on the short-term effects from bolus protein injection [66]. Together these findings indicate that gene therapy with EP is a viable alternative for long-term IFN-β therapy in MS.

Growth Hormone-Releasing Hormone In aged and cancer-afflicted patients decreased quality of life, cachexia, anemia, anorexia, and decreased activity levels can be experienced all with a negative impact. The growth hormone releasing hormone (GHRH)/ growth hormone (GH)/ insulin-like growth factor-I (IGF-I) axis plays an important role in growth and development and modulation of it with plasmid GHRH has been shown to have beneficial effects and may be used to prevent and or treat the above conditions. As previously discussed we have shown that administration of plasmid GHRH with EP can improve the quality of life of dogs with cancer [24]. Furthermore we have shown that plasmid GHRH can increase weight gain without organomegaly while improving hematological parameters in young healthy dogs [67]. In geriatric and cancer-afflicted dogs similar results were found with increased IGF-I levels, and increased scores for weight, activity level, exercise tolerance, and appetite [68]. Treatment of companion dogs and cats with chronic renal failure with plasmid GHRH via EP also resulted in increased survival, improved quality of life parameters as well as increasing body weight, hematological parameters, and maintenance of kidney function [69]. The above responses in heterogeneous groups of animals that are more representative of human conditions suggest

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that the single administration of plasmid GHRH could also be beneficial to patients with various catabolic conditions. The use of antibiotics and other conventional methods for improving animal welfare are coming under scrutiny; therefore, alternatives that are safe for the public as well as able to enhance the quality of life of the animals are much sought after. Treatment with plasmid GHRH by EP in heat stress cattle resulted in a reduction in mortality with improvements in weight gain, milk production and success of second rate of pregnancy compared to untreated controls [70]. Administration was carried out in calves without the need for anesthesia proving the viability of this method for the vaccination of farm animals. In a large pig study the application of a now commercially available plasmid GHRH to gestating sows resulted in improvements in offspring for three consecutive pregnancies [23]. All of these studies demonstrate that administration of plasmid GHRH with EP warrants further investigation for the food production industry, while providing proof of concept in large studies in large animals for future human studies.

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Other Studies Duchenne muscle dystrophy (DMD) is a progressive and fatal muscle wasting disease caused by the absence of the protein dystrophin [71]. In the early 2000‘s several groups reported the use of EP to deliver dystrophin cDNAs into mice [72-77]. More recently it has been shown that the single electrotransfer-assisted plasmid-based gene transfer of IGF-I hastened functional repair of mouse tibialis anterior muscles after myotoxic injury [78] demonstrating that this approach could potentially be used for treating muscle injuries and skeletal muscle diseases. Others have shown that EP of tibialis anterior female mouse muscles with a plasmid containing the vascular endothelial growth factor (VEGF165) promoted angiogenesis and enhanced myoblast survival after transplantation [79]. The introduction of corrective nucleic acids by in vivo EP has also been shown to potential method for correcting the dystrophin gene (mdx) mutation responsible for muscular dystrophy in the mdx mouse model of human DMD [80]. Other studies on dystrophic muscle involved a pretreatment with a dilute solution of hyaluronidase 2 hours prior to the injection of plasmid DNA and EP [81]. The treatment of other conditions such as peripheral arterial disease [82;83] and hemophilia [84;85] have also benefited from the development of gene therapy and their application by EP showing the scope of conditions that could potentially be treated in humans.

DELIVERY OF DNA VACCINES WITH ELECTROPORATION Many DNA vaccination regimens are delivered by EP, with or without molecular adjuvants (i.e. transgene products that have been shown to enhance or potentiate an immune response). Immunization with DNA vaccines is no different from conventional delivery methods and often fails to induce consistent, robust immune responses, especially in species larger. As a result there are a number of published reports examining EP-mediated DNA vaccination. Investigation of parameters such as target muscle, delay between plasmid delivery and onset of EP pulses and DNA vaccine formulation were carried out in mice and

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pigs revealing that optimization is critical for effectiveness [17]. For protection against pathogenic human and avian influenza virus, the EP of synthetic consensus DNA antigens in mice, ferrets and nonhuman primates induced both protective cellular and humoral immune responses [86]. Furthermore, the efficacy of EP was compared with an alternative delivery method, namely gene gun, for the administration of a DNA vaccine against an influenza virus in mice and rabbits. Overall, both the gene gun and EP methods were more immunogenic than IM injection alone [87]. Studies examining strategies for optimization of delivery of HIV vaccines have been carried out in pigs and rabbits, followed by trials in nonhuman primates [88;89] demonstrating that cellular and humoral immune responses were induced in all animals. Assessment of an IL-12 plasmid-encoded DNA vaccine with optimized HIV gag and env constructs showed that the use of EP to enhance plasmid delivery resulted in dramatically higher cellular as well as humoral responses [90]. These findings were made in comparison with IM injection alone, IM with IL-12, IM injection with EP and indicated that the use of an adjuvant in combination with delivery by EP may overcome previous immunogenicity limitations of DNA based vaccines. Further studies have also shown that improved gene delivery and expression by EP dramatically increases immunogenicity of DNA vaccines. Direct plasmid DNA injection was compared to in vivo IM EP using an optimized HIV gag expression plasmid. The development of cellular immune responses in SIV-infected animals controlling viremia was shown along with an expansion of antigen-specific T cells [91]. Another group demonstrated that there was a similar increase in CD8+ T-cells with the administration of a therapeutic DNA-vaccine during anti-retroviral therapy using EP with or without IL-2 treatment, however, there was not a sustained control of viremia in the SIVmac239-infected rhesus macaques [92]. Therapeutic vaccines for the treatment of chronic HBV infections are also drawing attention. The effect of EP-mediated DNA vaccination with different electro-pulse parameters in rhesus macaques was investigated, revealing that the optimization of EP parameters is important in developing clinical application of DNA vaccination [93]. The investigation of chronic hepatitis B virus infection in mice using a DNA vaccine expressing woodchuck hepatitis virus antigens administered by IM/EP revealed similar findings to IM injection alone but when scaled up to rabbits the IM/EP was much more effective [94]. In cattle, EP of a plasmid encoding a model antigen, hepatitis B surface antigen, resulted in improved cellmediated and humoral immune responses [95]. Other studies have shown that cellular immunity can be induced by a HPV18 DNA vaccine in mice followed by nonhuman primates [96]. Similarly, it was found that EP of an anthrax DNA vaccine in mice, rats and rabbits resulted in enhanced potency over IM injection alone and promoted the rapid induction of broad immune responses [97].

CLINICAL STUDIES The transition of EP to the clinical setting has been progressively making headway. The increased preclinical research involving larger animals and non-human primates utilizing the technology has demonstrated the effectiveness and potential for use in humans as previously discussed in this chapter. As a result there are currently several clinical trials that are

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investigating EP as a delivery method for administration of DNA-based vaccines for a number of diseases as shown in Table 2. The investigation of EP in the administration of bleomycin, an anti-cancer agent that induces DNA strand breaks, demonstrates that EP has many potential applications and is not limited to DNA alone [98-100]. Currently there are three phase I trials investigating EP in the treatment strategy for melanoma. Two of the trials are utilizing intratumoral EP with interleukin based plasmid DNA vaccines (ClinicalTrials.gov Identifier: NCT00223899 and NCT00323206) while the third is an IM administration of a tyrosinase DNA vaccine (ClinicalTrials.gov Identifier: NCT00545987). The remaining five clinical trials involving EP encompass therapies for HIV (ClinicalTrials.gov Identifier: NCT00545987), HPV (ClinicalTrials.gov Identifier: NCT00685412), hepatitis C (ClinicalTrials.gov Identifier: NCT00563173) and prostate cancer (Gene Therapy Clinical Trials Identifier: UK-112) as well as one study examining the effects of EP alone on healthy volunteers (ClinicalTrials.gov Identifier: NCT00721461). Only the latter study has been completed to date; the rest of the trials are either ongoing or recruiting. While the preclinical research leading to the development of these clinical trials has been previously discussed (Bodles-Brakhop et al., Mol Ther, in press), the results of the first phase I trial investigating the dose escalation of plasmid IL-12 with EP (ClinicalTrials.gov Identifier: NCT00323206) that was carried out in patients with metastatic melanoma have now been reported [101]. In this study twenty-four patients were treated with 7 dose levels of plasmid with minimal systemic toxicity and transient pain after EP. It was found that plasmid dose was proportional to increases in IL-12 protein levels and marked tumor necrosis and lymphocytic infiltrate were observed. This report describing the first human trial of EP indicates it is safe, effective, reproducible, and titratable. With the success of this study, we eagerly await the results from the other clinical trials and the progression towards phase II trials and beyond.

CONCLUSION Electroporation has emerged as a compelling new research and medical tool. The numerous preclinical studies underscore the rising interest in the technology with the ultimate desire to produce more effective and efficient vaccines and therapies. There are some areas of the technology that, however, still require fine-tuning such as the optimization of plasmids for enhanced expression, definition of the parameters for EP depending on the target tissue and species, and conveyance of EP to the general public. The optimization and continued development of more efficient plasmid DNA constructs and EP devices will undoubtedly continue to move the field forward. The exact mechanism as to how delivery of plasmid DNA with EP results in increased efficacy and expression is yet to be elucidated but one thought is that EP increases the number of molecules entering the cell and subsequently being expressed [10]. However, in order for the successful transfer of this technology to humans it will be essential to minimize tissue damage (usually associated with discomfort and low tolerability) while maintaining an effective response. Both IM and ID administration of plasmid DNA with EP will require optimization for clinical use.

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Table 2. DNA vaccine clinical trials involving electroporation NCT or GT ID NCT 00223899

NCT 00323206

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NCT 00471133

NCT 00545987

Phase/ Status

Sponsor

I Ongoing

Vical

I Recruiting

H. Lee Moffitt Cancer Center and Research Institute; National Gene Vector Laboratory

I Recruiting

Ichor Medical Systems Incorporated; Memorial SloanKettering Cancer Center

I Recruiting

Rockefeller University, Aaron Diamond AIDS Research Center; Bill and Melinda Gates Foundation; Ichor Medical Systems Incorporated; International AIDS Vaccine Initiative

Condition

Intervention

Primary objective

Metastatic Melanoma

Intratumoral VCL-IM01 (encoding IL-2) with EP

Safety and efficacy, dose escalation

Malignant Melanoma

Intratumoral IL-12pDNA with EP

Toxicity and efficacy, recommended dose for Phase II study, local and systemic response

Melanoma (Skin); Intraocular Melanoma

Biological: Xenogeneic Tyrosinase DNA Vaccine; TriGrid™ delivery system for IM EP

Safety and immunogenicity

HIV Infections

ADVAX DNA-based HIV vaccine TriGrid™ EP delivery system

Safety and immunogenicity

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Angela M. Bodles-Brakhop and Ruxandra Draghia-Akli Table 2. (Continued)

NCT or GT ID

Sponsor

Condition

I Recruiting

VGX Pharmaceuticals Inc.

NCT 00721461

I Completed

Merck; University of California San Diego; Inovio Biomedical Corporation

Healthy

NCT 00563173

I/II Recruiting

Tripep AB, Inovio Biomedical Corporation

Chronic Hepatitis C Virus Infection

I/II Ongoing

Cancer Research UK Oncology Unit Southampton, UK

Prostate cancer

NCT 00685412

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Phase/ Status

UK-112

Papilloma virus Infections

Intervention Biological: VGX-3100 via EP using the CELLECTR A® constant current device IM injection of phosphate buffered saline solution MedPulserT M EP IM administered CHRONVA C-C® in combination with EP Prostate specific membrane antigen (PSMA)/pdo m fusion gene, IM injection with or without EP

Primary objective

Safety and tolerability of escalating doses

Tolerability of the MedPulserTM DNA delivery system

Safety, tolerability and efficacy

---

Websites: ClinicalTrials.gov Gene therapy clinical trials worldwide http://www.wiley.com/legacy/wileychi /genmed/clinical/

It is likely that the advent of skin (ID) EP will dramatically increase patient compliance due to the reduced pain level compared to IM EP, convenience of the site, and the similarity with the tradition route of vaccination. Furthermore, the ability to administer numerous optimized consensus plasmids in a single injection with EP will be an important development for providing broad protection against divergent strains, for example H5N1 influenza [86]. Alternatively the ability to co-administrate plasmids directed to producing various immonogens or transgene products (a new type of ―combination drug‖) at one time will also be an important development for reducing the number of required administrations and lowering cost. The ability to reproduce these results in humans will be critical for the

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advancement of EP. Overall EP is an exciting development and will play an important role in the future application of new class of therapeutics or vaccines for human use.

ACKNOWLEDGMENTS This work was supported by VGX Pharmaceuticals Inc., The Woodlands, TX.

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[46] Jeong JG, Kim JM, Ho SH, Hahn W, Yu SS, Kim S. Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis. J Gene Med 2004 Oct;6(10):1125-33. [47] Khoury M, Bigey P, Louis-Plence P, Noel D, Rhinn H, Scherman D, et al. A comparative study on intra-articular versus systemic gene electrotransfer in experimental arthritis. J Gene Med 2006 Aug;8(8):1027-36. [48] Grossin L, Cournil-Henrionnet C, Pinzano A, Gaborit N, Dumas D, Etienne S, et al. Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J 2006 Jan;20(1):65-75. [49] Brown PA, Bodles-Brakhop A, Draghia-Akli R. Plasmid growth hormone releasing hormone therapy in healthy and laminitis-afflicted horses-evaluation and pilot study. J Gene Med 2008;10(5):564-74. [50] Martinenghi S, Cusella De AG, Biressi S, Amadio S, Bifari F, Roncarolo MG, et al. Human insulin production and amelioration of diabetes in mice by electrotransferenhanced plasmid DNA gene transfer to the skeletal muscle. Gene Ther 2002 Nov;9(21):1429-37. [51] Croze F, Prud'homme GJ. Gene therapy of streptozotocin-induced diabetes by intramuscular delivery of modified preproinsulin genes. J Gene Med 2003 May;5(5):425-37. [52] Wang LY, Sun W, Chen MZ, Wang X. Intramuscular injection of naked plasmid DNA encoding human preproinsulin gene in streptozotocin-diabetes mice results in a significant reduction of blood glucose level. Sheng Li Xue Bao 2003 Dec 25;55(6):6417. [53] Diao WF, Chen WQ, Wu Y, Liu P, Xie XL, Li S, et al. Serum, liver, and kidney proteomic analysis for the alloxan-induced type I diabetic mice after insulin gene transfer of naked plasmid through electroporation. Proteomics 2006 Nov;6(21):583745. [54] Prud'homme GJ, Draghia-Akli R, Wang Q. Plasmid-based gene therapy of diabetes mellitus. Gene Ther 2007 Apr;14(7):553-64. [55] Soltani N, Kumar M, Glinka Y, Prud'homme GJ, Wang Q. In vivo expression of GLP1/IgG-Fc fusion protein enhances beta-cell mass and protects against streptozotocininduced diabetes. Gene Ther 2007 Jun;14(12):981-8. [56] Sun W, Wang L, Zhang Z, Chen M, Wang X. Intramuscular transfer of naked calcitonin gene-related peptide gene prevents autoimmune diabetes induced by multiple low-dose streptozotocin in C57BL mice. Eur J Immunol 2003 Jan;33(1):233-42. [57] She F, Sun W, Mao JM, Wang X. Calcitonin gene-related peptide gene therapy suppresses reactive oxygen species in the pancreas and prevents mice from autoimmune diabetes. Sheng Li Xue Bao 2003 Dec 25;55(6):625-32. [58] Rabinovsky ED, Draghia-Akli R. Insulin-like Growth Factor I Plasmid Therapy Promotes in Vivo Angiogenesis. Mol Ther 2004;9(1):46-54. [59] Marti G, Ferguson M, Wang J, Byrnes C, Dieb R, Qaiser R, et al. Electroporative transfection with KGF-1 DNA improves wound healing in a diabetic mouse model. Gene Ther 2004 Dec;11(24):1780-5. [60] Cruzado JM, Lloberas N, Torras J, Riera M, Fillat C, Herrero-Fresneda I, et al. Regression of advanced diabetic nephropathy by hepatocyte growth factor gene therapy in rats. Diabetes 2004 Apr;53(4):1119-27.

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[61] Oshima Y, Sakamoto T, Yamanaka I, Nishi T, Ishibashi T, Inomata H. Targeted gene transfer to corneal endothelium in vivo by electric pulse. Gene Ther 1998 Oct;5(10):1347-54. [62] Kachi S, Esumi N, Zack DJ, Campochiaro PA. Sustained expression after nonviral ocular gene transfer using mammalian promoters. Gene Ther 2006 May;13(9):798-804. [63] Kachi S, Oshima Y, Esumi N, Kachi M, Rogers B, Zack DJ, et al. Nonviral ocular gene transfer. Gene Ther 2005 May;12(10):843-51. [64] Bloquel C, Bejjani R, Bigey P, Bedioui F, Doat M, BenEzra D, et al. Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNFalpha soluble receptor in uveitis. FASEB J 2006 Feb;20(2):389-91. [65] Jaini R, Hannaman D, Johnson JM, Bernard RM, Altuntas CZ, Delasalas MM, et al. Gene-based intramuscular interferon-beta therapy for experimental autoimmune encephalomyelitis. Mol Ther 2006 Sep;14(3):416-22. [66] Petry H, Cashion L, Szymanski P, Ast O, Orme A, Gross C, et al. Mx1 and IP-10: biomarkers to measure IFN-beta activity in mice following gene-based delivery. J Interferon Cytokine Res 2006 Oct;26(10):699-705. [67] Draghia-Akli R, Hahn KA, King GK, Cummings K, Carpenter RH. Effects Of Plasmid Mediated Growth Hormone Releasing Hormone In Severely Debilitated Dogs With Cancer. Molecular Therapy 2002;6(6):830-6. [68] Tone CM, Cardoza DM, Carpenter RH, Draghia-Akli R. Long-term effects of plasmidmediated growth hormone releasing hormone in dogs. Cancer Gene Ther 2004;11(5):389-96. [69] Brown PA, Bodles-Brakhop AM, Pope MA, Draghia-Akli R. Gene therapy by electroporation for the treatment of chronic renal failure in companion animals. BMC Biotechnol 2009;9(4):doi:10.1186/1472-6750-9-4. [70] Brown PA, Bodles-Brakhop AM, Draghia-Akli R. Effects of Plasmid Growth Hormone Releasing Hormone Treatment During Heat Stress. DNA and Cell Biology 2008;27(11):629-35. [71] Hoffman EP, Brown RH, Jr., Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987 Dec 24;51(6):919-28. [72] Vilquin JT, Kennel PF, Paturneau-Jouas M, Chapdelaine P, Boissel N, Delaere P, et al. Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies. Gene Ther 2001 Jul;8(14):1097-107. [73] Gollins H, McMahon J, Wells KE, Wells DJ. High-efficiency plasmid gene transfer into dystrophic muscle. Gene Ther 2003 Mar;10(6):504-12. [74] Murakami T, Nishi T, Kimura E, Goto T, Maeda Y, Ushio Y, et al. Full-length dystrophin cDNA transfer into skeletal muscle of adult mdx mice by electroporation. Muscle Nerve 2003 Feb;27(2):237-41. [75] Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N, et al. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther 2004 Sep;10(3):447-55. [76] Wong SH, Lowes KN, Quigley AF, Marotta R, Kita M, Byrne E, et al. DNA electroporation in vivo targets mature fibres in dystrophic mdx muscle. Neuromuscul Disord 2005 Oct;15(9-10):630-41.

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[77] Ferrer A, Foster H, Wells KE, Dickson G, Wells DJ. Long-term expression of fulllength human dystrophin in transgenic mdx mice expressing internally deleted human dystrophins. Gene Ther 2004 Jun;11(11):884-93. [78] Schertzer JD, Lynch GS. Comparative evaluation of IGF-I gene transfer and IGF-I protein administration for enhancing skeletal muscle regeneration after injury. Gene Ther 2006 Dec;13(23):1657-64. [79] Bouchentouf M, Benabdallah BF, Bigey P, Yau TM, Scherman D, Tremblay JP. Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles. Gene Ther 2008 Mar;15(6):404-14. [80] Kapsa RM, Wong SH, Quigley AF. Electroporation of corrective nucleic acids (CNA) in vivo to promote gene correction in dystrophic muscle. Methods Mol Biol 2008;423:405-19. [81] Wells KE, McMahon J, Foster H, Ferrer A, Wells DJ. Gene delivery to dystrophic muscle. Methods Mol Biol 2008;423:421-31. [82] Seidler RW, Allgauer S, Ailinger S, Sterner A, Dev N, Rabussay D, et al. In vivo human MCP-1 transfection in porcine arteries by intravascular electroporation. Pharm Res 2005 Oct;22(10):1685-91. [83] Dev NB, Preminger TJ, Hofmann GA, Dev SB. Sustained local delivery of heparin to the rabbit arterial wall with an electroporation catheter. Cathet Cardiovasc Diagn 1998 Nov;45(3):337-45. [84] Liu F, Sag D, Wang J, Shollenberger LM, Niu F, Yuan X, et al. Sine-wave current for efficient and safe in vivo gene transfer. Mol Ther 2007 Oct;15(10):1842-7. [85] Fewell JG. Factor IX gene therapy for hemophilia. Methods Mol Biol 2008;423:375-82. [86] Laddy DJ, Yan J, Kutzler M, Kobasa D, Kobinger GP, Khan AS, et al. Heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus DNA antigens. PLoS ONE 2008;3(6):e2517. [87] Wang S, Zhang C, Zhang L, Li J, Huang Z, Lu S. The relative immunogenicity of DNA vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods. Vaccine 2008 Apr 16;26(17):2100-10. [88] Hirao LA, Wu L, Khan AS, Satishchandran A, Draghia-Akli R, Weiner DB. Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine 2008;26(3):440-8. [89] Cristillo AD, Weiss D, Hudacik L, Restrepo S, Galmin L, Suschak J, et al. Persistent antibody and T cell responses induced by HIV-1 DNA vaccine delivered by electroporation. Biochem Biophys Res Commun 2008;366(1):29-35. [90] Hirao L, Wu L, Khan AS, Hokey D, Yan J, Dai A, et al. Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques. Vaccine 2008;26(25):3112-20. [91] Rosati M, Valentin A, Jalah R, Patel V, von Gegerfelt AS, Bergamaschi C, et al. Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation. Vaccine 2008;26(40):5223-9. [92] zur Megede J, Sanders-Beer B, Silvera P, Golightly D, Bowlsbey A, Hebblewaite D, et al. A therapeutic SIV DNA vaccine elicits T-cell immune responses, but no sustained control of viremia in SIVmac239-infected rhesus macaques. AIDS Res Hum Retroviruses 2008 Aug;24(8):1103-16.

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[93] Zhao YG, Xu Y. Electroporation-mediated HBV DNA vaccination in primate models. In: Li S, editor. Methods Mol Biol. Electroporation Protocols. Preclinical and clinical gene medicine. 423 ed. Totowa, New Jersey 07512: Humana Press; 2008. p. 487-95. [94] Luxembourg A, Hannaman D, Wills K, Bernard R, Tennant BC, Menne S, et al. Immunogenicity in mice and rabbits of DNA vaccines expressing woodchuck hepatitis virus antigens. Vaccine 2008 Jul 29;26(32):4025-33. [95] van Drunen Littel-van den Hurk, Luxembourg A, Ellefsen B, Wilson D, Ubach A, Hannaman D, et al. Electroporation-based DNA transfer enhances gene expression and immune responses to DNA vaccines in cattle. Vaccine 2008 Aug 15;26(43):5503-9. [96] Yan J, Harris K, Khan AS, Draghia-Akli R, Sewell DA, Weiner DB. Cellular immunity induced by a novel HPV18 DNA vaccine encoding an E6/E7 fusion consensus protein in mice and rhesus macaques. Vaccine 2008;26(40):5210-5. [97] Luxembourg A, Hannaman D, Nolan E, Ellefsen B, Nakamura G, Chau L, et al. Potentiation of an anthrax DNA vaccine with electroporation. Vaccine 2008 Sep 19;26(40):5216-22. [98] Belehradek J, Orlowski S, Ramirez LH, Pron G, Poddevin B, Mir LM. Electropermeabilization of cells in tissues assessed by the qualitative and quantitative electroloading of bleomycin. Biochim Biophys Acta 1994 Feb 23;1190(1):155-63. [99] Gothelf A, Mir LM, Gehl J. Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev 2003 Oct;29(5):371-87. [100] Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M. Electrochemotherapy in treatment of tumours. Eur J Surg Oncol 2008 Feb;34(2):232-40. [101] Daud AI, DeConti RC, Andrews S, Urbas P, Riker AL, Sondak VK, et al. Human Trial of In Vivo Electroporation-Mediated Gene Transfer: Safety and Efficacy of Interleukin12 Plasmid Dose Escalation in Metastatic Melanoma. Journal of Clinical Oncology 2008;10.1200/JCO.2007.15.6794. [102] Miyazaki S, Miyazaki J. In vivo DNA electrotransfer into muscle. Dev Growth Differ 2008 Aug;50(6):479-83. [103] Liu L, Marti GP, Wei X, Zhang X, Zhang H, Liu YV, et al. Age-dependent impairment of HIF-1alpha expression in diabetic mice: Correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells. J Cell Physiol 2008 Nov;217(2):319-27. [104] Marti GP, Mohebi P, Liu L, Wang J, Miyashita T, Harmon JW. KGF-1 for wound healing in animal models. Methods Mol Biol 2008;423:383-91. [105] Heller LC, Jaroszeski MJ, Coppola D, Heller R. Comparison of electrically mediated and liposome-complexed plasmid DNA delivery to the skin. Genet Vaccines Ther 2008 Dec 4;6(1):1-16. [106] Arenal A, Pimentel R, Pimentel E, Martin L, Santiesteban D, Franco R, et al. Growth enhancement of shrimp (Litopenaeus schmitti) after transfer of tilapia growth hormone gene. Biotechnol Lett 2008 Jan 18;30(5):845-51. [107] Lee JS, Song SH, Kim JM, Shin IS, Kim KL, Suh YL, et al. Angiopoietin-1 prevents hypertension and target organ damage through its interaction with endothelial Tie2 receptor. Cardiovasc Res 2008 Jun 1;78(3):572-80. [108] Smorlesi A, Papalini F, Pierpaoli S, Provinciali M. HER2/neu DNA vaccination for breast tumors. In: Li S, editor. Methods Mol Biol. Electroporation Protocols. Preclinical

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and clinical gene medicine. 423 ed. Totowa, New Jersey 07512: Humana Press; 2008. p. 473-85. [109] Curcio C, Khan AS, Amici A, Spadaro M, Quaglino E, Cavallo F, et al. DNA immunization using constant-current electroporation affords long-term protection from autochthonous mammary carcinomas in cancer-prone transgenic mice. Cancer Gene Ther 2008;15(2):108-14. [110] Roos AK, King A, Pisa P. DNA vaccination for prostate cancer. In: Li S, editor. Methods Mol Biol. Electroporation Protocols. Preclinical and clinical gene medicine. 423 ed. Totowa, New Jersey, 07512: Humana Press; 2008. p. 463-72. [111] Rolla S, Marchini C, Malinarich S, Quaglino E, Lanzardo S, Montani M, et al. Protective immunity against neu-positive carcinomas elicited by electroporation of plasmids encoding decreasing fragments of rat neu extracellular domain. Hum Gene Ther 2008 Mar;19(3):229-40. [112] Li Y, Han W, Zhang Y, Yuan L, Shi X, Yu Y, et al. Intramuscular electroporation of a plasmid encoding human plasminogen kringle 5 induces growth inhibition of Lewis lung carcinoma in mice. Cancer Biother Radiopharm 2008 Jun;23(3):332-41. [113] Horton HM, Lalor PA, Rolland AP. IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial. Methods Mol Biol 2008;423:361-72. [114] Vandermeulen G, Staes E, Vanderhaeghen ML, Bureau MF, Scherman D, Preat V. Optimisation of intradermal DNA electrotransfer for immunisation. J Control Release 2007 Dec 4;124(1-2):81-7. [115] Chen MW, Cheng TJ, Huang Y, Jan JT, Ma SH, Yu AL, et al. A consensushemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses. Proc Natl Acad Sci U S A 2008;105(36):13538-43. [116] Kanazawa T, Takashima Y, Hirayama S, Okada H. Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine. Int J Pharm 2008 Aug 6;360(12):164-70. [117] Parise CB, Lisboa B, Takeshita D, Sacramento CB, de Moraes JZ, Han SW. Humoral immune response after genetic immunization is consistently improved by electroporation. Vaccine 2008 Jul 23;26(31):3812-7. [118] Cipriani B, Fridman A, Bendtsen C, Dharmapuri S, Mennuni C, Pak I, et al. Therapeutic vaccination halts disease progression in BALB-neuT mice: the amplitude of elicited immune response is predictive of vaccine efficacy. Hum Gene Ther 2008 Jul;19(7):670-80. [119] Dutta NK, Mazumdar K, Lee BH, Baek MW, Kim DJ, Na YR, et al. Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine. Immunol Lett 2008 Jun 15;118(1):65-71. [120] Rinaldi M, Fioretti D, Iurescia S, Signori E, Pierimarchi P, Seripa D, et al. Anti-tumor immunity induced by CDR3-based DNA vaccination in a murine B-cell lymphoma model. Biochem Biophys Res Commun 2008 May 30;370(2):279-84. [121] Liu J, Kjeken R, Mathiesen I, Barouch DH. Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation. J Virol 2008 Jun;82(11):5643-9. [122] Cristillo AD, Galmin L, Restrepo S, Hudacik L, Suschak J, Lewis B, et al. HIV-1 Env vaccine comprised of electroporated DNA and protein co-administered with Talabostat. Biochem Biophys Res Commun 2008;370(1):22-6.

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[123] Leblanc R, Vasquez Y, Hannaman D, Kumar N. Markedly enhanced immunogenicity of a Pfs25 DNA-based malaria transmission-blocking vaccine by in vivo electroporation. Vaccine 2008 Jan 10;26(2):185-92. [124] Zhou Y, Fang F, Chen J, Wang H, Chang H, Yang Z, et al. Electroporation at Low Voltages Enables DNA Vaccine to Provide Protection against a Lethal H5N1 Avian Influenza Virus Challenge in Mice. Intervirology 2008 Sep 24;51(4):241-6. [125] Fang F, Cai XQ, Chang HY, Wang HD, Yang ZD, Chen Z. Protection abilities of influenza B virus DNA vaccines expressing hemagglutinin, neuraminidase, or both in mice. Acta Virol 2008;52(2):107-12. [126] Muthumani K, Lankaraman KM, Laddy DJ, Sundaram SG, Chung CW, Sako E, et al. Immunogenicity of novel consensus-based DNA vaccines against Chikungunya virus. Vaccine 2008 Apr 14;26(40):5128-34. [127] Spik KW, Badger C, Mathiessen I, Tjelle T, Hooper JW, Schmaljohn C. Mixing of M segment DNA vaccines to Hantaan virus and Puumala virus reduces their immunogenicity in hamsters. Vaccine 2008 Sep 19;26(40):5177-81. [128] Torrero M, Li S. Treatment of SCCVII tumors with systemic chemotherapy and Interleukin-12 gene therapy combination. Methods Mol Biol 2008;423:339-49. [129] Zhu S, Li S. Systemic IL-12 gene therapy for treating malignancy via intramuscular electroporation. Methods Mol Biol 2008;423:327-37. [130] Cutrera J, Torrero M, Shiomitsu K, Mauldin N, Li S. Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs. Methods Mol Biol 2008;423:319-25.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 12

ELECTROCHEMOTHERAPY IN VETERINARY MEDICINE, PART I: SOLID TUMORS Enrico P. Spugnini1, Gennaro Citro1 and Alfonso Baldi2 1

2

S.A.F.U. Department, Regina Elena Cancer Institute, Rome, Italy Dept. Biochemistry, Sect. Pathology, Second University of Naples, Naples, Italy

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ABSTRACT The authors review the most important characteristics of electrochemotherapy (ECT) in canine and feline patients affected by solid tumors. In particular, the origins of this technique, and its further developments are described, with particular attention to the evolution of the clinical protocols, the development of novel equipment as well as their translation to human patients. The authors will also report the current state of the art in large animals, in particular in equine.

PRELIMINARY STUDIES AND FIRST PROTOCOLS The first experiment on pets with spontaneous neoplasms were performed in the second part of the ‗90s and continued throughout the early 2000, leading to the development of the first protocols [1-4]. In the first report of ECT (for the treatment of feline sarcoma), Mir and coll. treated 12 cats with relapsing sarcomas with systemic bleomycin coupled with the delivery of a series of 8 single square pulses at the voltage of 1300 V/cm, until tumor coverage was obtained. Three cats also received peritumoral injection of xenogenic CHO living cells (IL 2 secreting). While tumor control seemed not to be achieved by the treatment, the authors claimed an extended survival in 12 cats treated with ECT compared to 11 untreated controls [1]. While this is the first clinical trial on ECT in pets, its design is still much closer to a basic science investigation and its results are of difficult comparison with the clinical studies that followed.

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A few years later, a group of Slovenian investigators tested CDDP based ECT in a small group of dogs (7) and cats (3) with cutaneous neoplasms, adopting again a series of single square pulses and obtaining responses ranging from 1 month to 11 months [2]. A different approach was adopted by Italian investigators that decided to deliver trains of biphasic pulses rather than single pulses; this technique allowed a eight times shorter treatment time and was more proficient in controlling canine and feline tumors [3]. Briefly the tumor‘s bed and the margins for ½ cm in all directions were infiltrated with bleomycin at the concentration of 1.5 mg/ml. Five minutes after the infiltration, trains of 8 biphasic electric pulses lasting 50 + 50 s each, with 1 ms interpulse intervals, were delivered by means of modified caliper electrodes. The treatment was repeated after one or two weeks on the basis of the patients‘ status. In this preliminary phase I/II, 22 companion animals were enrolled with down staged neoplasms, obtaining an overall response rate of 80% and a total of 40% long lasting remissions. This high response rate was probably due to the high degree of permeabilization induced by biphasic pulses (Figure 1). In fact, the neoplastic cells that are randomly arranged, due to the lack of size and shape homogeneity, have different orientations with respect to the field polarity, are also extremely vulnerable to the sudden chance of field orientation induced by this particular ECT technique [3].

Figure 1. The features of a biphasic pulse adopted for in vivo electropermeabilization

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As a consequence of this preliminary investigation, several ad hoc electrodes for biphasic pulses electrochemotherapy were tested in pets, including caliper, modified caliper, needle array, paired needles, laparoscopic pinch electrodes and unipolar electrodes [4]. Figure 2 shows the features of the different electrodes used in veterinary oncology with biphasic pulses based ECT. In particular, electrodes A and D are the electrodes of choice for post operative ECT, while electrodes C and E are preferentially adopted for intraoperative ECT or for the treatment of oral tumors. Electrode G is limited to the eye canthus, while electrode H is used for intra-articular neoplasia.

Figure 2. Different electrodes used for ECT in pet with spontaneous tumors. A,D: calliper and modified calliper electrode; B,C,E,F: needle array electrodes; G: vaccine electrode, H: laparoscopic pinch electrode, F: unipolar electrodes. (From: Spugnini EP, Citro G and Porrello A: Rational design of new electrodes for electrochemotherapy. J Exp Clin Cancer Res 2005; 24: 245-254).

Meanwhile, a group of Slovenian investigators reported a good palliation for the treatment of canine perianal tumors (mostly benign adenomas) with square pulse based ECT [5]. A single preliminary study on horses affected by sarcoids has been published as well [6]. That article reports the successful treatment of 10 sarcoid lesions in three horses.

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ELECTROCHEMOTHERAPY FOR SPECIFIC TUMOR TYPES: PHASE II STUDIES The brilliant results of the first clinical trials on biphasic pulses based ECT led to the investigation of this therapy in cohorts of dogs and cats affected by specific tumor types [719]. The first of these studies was aimed at exploring the potentials of this technique for the control of advanced oral melanoma in dogs [7]. In this report, 10 dogs with malignant melanoma were treated with intralesional bleomycin followed by the application of permeabilizing biphasic electric pulses, rather than with more conventional therapies such as demolitive surgery and hypofrationated radiation therapy. The treatment was well tolerated with a median survival time of 6 months that compares favorably with the data available in the literature (Figure 3).

1,0

0,8

0,6

0,4

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0,2

0,0 0,00

10,00

20,00

30,00

40,00

Survival Time (months)

Figure 3. Kaplan-Meier survival curve of 10 dogs with oral melanoma treated with ECT (Modified from: Spugnini EP, Dragonetti E, Vincenzi B, Onori N, Citro G and Baldi A: Pulse mediated chemotherapy enhances local control and survival in a spontaneous canine model of primary mucosal melanoma. Melanoma Res 2006; 16: 23-27).

Interestingly, four dogs achieved remissions that lasted 16, 17, 31 and 37 months, respectively. The absence of pigmentation at the tumor site in the four long term responders might imply a significant recruitment of the immune system that perhaps contributed to these long lasting remissions. These results were instrumental in planning the treatment of a dog with a large, unresectable, anal melanoma. ECT has been able to successfully palliate the tumor in this patient by restoring anal transit, thus allowing normal defecation for three months [11]. Soft tissue sarcomas (STS) are among the most represented neoplasms in dogs and cats, and pose significant difficulties to clinicians, due to their infiltrative nature and to their

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modest response to chemotherapy. The largest study on electrochemotherapy in companion animals affected by solid cancer [9] was published in 2007. In this paper, 58 cats with incompletely excised STS were randomized to receive intraoperative versus postoperative ECT and compared to a control cohort of 14 cats that received surgery as only therapeutic modality. The authors elected to use ECT in an adjuvant modality because they believed that the treatment could be enhanced by the removal of significant amounts of cancer-associated connective tissue. It has been suggested by studies on electromobility of plasmid DNA in tumor tissues that the tumor collagen content was the major obstacle to the mobility of the construct and this was likely to apply to drug molecules as well [20]. Finally, previous experience with hyaluronidase as an antidote to chemotherapy extravasation in companion animals [21], suggested its use to digest the connective tissue prior to the pulse delivery. This further decreased the amount of ground substance within the infiltrated tissues, thus permitting a more uniform and more selective drug delivery. As in the previous studies on electrochemotherapy, the drug of choice has been bleomycin, followed by trains of biphasic pulses. The results have been extremely good with a median time to recurrence of 12 months for the intraoperative cohort and 19 months for the postoperative group (Figure4 and 5), while the controls had a median time to recurrence of 4 months, as reported in the literature [9].

Figures 4 and 5. Time to recurrence for 19 cats treated with intraoperative ECT and 39 cats treated with postoperative ECT for STS (From: Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C and Porrello A: Intraoperative versus postoperative electrochemotherapy in soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother Pharmacol 2007; 59: 375-381.)

Independent prognostic variables identified for feline STS were tumor size and history of previous treatments with development of drug resistance (Figures 6 and 7). Interestingly, the metastatic rate among these patients was only of 1.7% much lower than the rates reported in the literature (ranging from 10 to 25%), As per canine melanomas, the authors hypothesized that ECT has been able to recruit the immune system (perhaps by uncovering deep tumor antigens), thus resulting in decreased metastatic spread.

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Figure 6. Effect of tumor size on the outcome of cats treated with intraoperative ECT: Tumor < 25 cm Time to recurrence (months) Mean: 19,05; Median: 16,00; Tumor > 25 cm Time to recurrence Mean: 8,00; Median: 5,00 (From: Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C and Porrello A: Intraoperative versus postoperative electrochemotherapy in soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother Pharmacol 2007; 59: 375-381.)

Figure 7. Effect of tumor size and previous treatments on the outcome of cats treated with postoperative ECT (From: Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C and Porrello A: Intraoperative versus postoperative electrochemotherapy in soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother Pharmacol 2007; 59: 375-381.).

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Similar results have been obtained in dogs with STS [9]. In a recent article 22 dogs have been treated with ECT for large mesenchymal neoplasms obtaining a median time to recurrence of 730 days (Figure 8). This study suggests that hemangiopericytomas are extremely responsive to ECT. Figure 9 shows the outcome of a dog with a large hemangiopericytoma that had been previously excised, three times, prior to referral for electrochemotherapy.

Figure 8. Kaplan Meier curve showing the outcome of 22 dogs treated with ECT.

More recently, a case report described the neo-adjuvant role of ECT in the management of a high-grade fibrosarcoma in a Malinois dog [16]. In this case ECT has been used to reduce the tumor volume in order to perform a conservative surgery. The dog then had 1 additional intraoperative ECT session to sterilize the tumor bed from residual disease. This dog has been in remission in excess of 24 months. These studies carry a great significance not only for the novel therapeutic schemes proposed for pets, but also generate a significant amount of preclinical data that can be easily transferred to humans [14, 17]. Another neoplasm that has been investigated as a target for biphasic pulses based electrochemotherapy, is feline sun-induced nasal squamous cell carcinoma (SCC), a neoplasm that is similar to actinic carcinoma in humans [12]. An article published in 2007 describes the outcome of 9 cats with sun induced SCC, reporting a 77% response rate with preservation of the facial architecture (Fig 10). The good results obtained by ECT in cancer pets led to a significant increase of the referrals by veterinarians, resulting, among other things, in the identification and cure of previously unreported neoplasms [8, 13, 22], in particular a thoracic hemangiopericytoma, a ganglioneuroblastoma and a pleomorphic rhabdomyosarcoma in feline patients.

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Figure 9. Outcome of a 4-year-old female syberian husky with a recurring hemangiopericytoma; A: tumor at presentation; B: tumor after two sessions of ECT, note the tumor‘s reduction and the regrowth of hairs; C: The patient at her 4 year recheck, there is no evidence of the neoplasia and the only sign of her local treatment is a circular area of alopecia.

Electrochemotherapy has been also used for the palliation/cure of perianal tumors and anal sac carcinomas either as single therapy or as an adjuvant [15, 18]. Despite the difficulty of treating this difficult district, ECT has been able to achieve a complete remission in 8/8 dogs with perianal adenoma and 3/4 dogs with perianal carcinoma. Only a down-staged patient with carcinoma failed to achieve a complete and lasting remission [15].

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Figure 10. The clinical outcome of two cats with SCC treated with ECT at presentation (A, C) and at follow-up appointments (B, D).

Finally, a dog with an infiltrating anal sac carcinoma that had incomplete surgical resection achieved a remission in excess of 18 months [18]. A recent development of ECT in veterinary oncology is the possible adoption of this therapy in the palliation of metastatic disease. An article published in 2008 describes the successful treatment of a metastatic cascade affecting the cervical lymph nodes of a husky dog with a previously excised submandibular lymph node, by combining the systemic administration of mitoxantrone with the delivery of trains of biphasic pulses [19]. The therapy obtained a complete remission lasting 6 months. This event confirms the results obtained by our group in the treatment of cervical neoplasms in pets, in particular thyroid tumors (Spugnini, unpublished) and suggests a potential significant role of other chemotherapy agents, until now, not investigated in this field. In conclusion, ECT is a safe, inexpensive and efficacious addition to the treatment options available in veterinary oncology and warrants further investigations.

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REFERENCES [1]

[2] [3]

[4] [5] [6] [7]

[8] [9]

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[10]

[11]

[12]

[13]

[14]

[15]

Mir LM, Devauchelle P, Quintin-Colonna F, Delisle F, Doliger S, Fradelizi D, Belehradek J Jr, Orlowski S. First clinical trial of cat soft-tissue sarcomas treatment by electrochemotherapy. Br J Cancer 1997; 76:1617-1622. Tozon N, Sersa G, Cemazar M. Electrochemotherapy: potentiation of local antitumour effectiveness of cisplatin in dogs and cats. Anticancer Res 2001;21:2483-2488. Spugnini EP, Porrello A. Potentiation of chemotherapy in companion animals with spontaneous large neoplasms by application of biphasic electric pulses. J Exp Clin Cancer Res 2003 22:571-580. Spugnini EP, Citro G and Porrello A: Rational design of new electrodes for electrochemotherapy. J Exp Clin Cancer Res 2005; 24: 245-254. Tozon N, Kodre V, Sersa G, Cemazar M. Effective treatment of perianal tumors in dogs with electrochemotherapy. Anticancer Res 2005; 25:839-845. Rols MP, Tamzali Y, Teissié J. Electrochemotherapy of horses. A preliminary clinical report. Bioelectrochemistry 2002; 55:101-105. Spugnini EP, Dragonetti E, Vincenzi B, Onori N, Citro G and Baldi A: Pulse mediated chemotherapy enhances local control and survival in a spontaneous canine model of primary mucosal melanoma. Melanoma Res 2006; 16: 23-27. Baldi A, Spugnini EP. Thoracic haemangiopericytoma in a cat. Vet Rec 2006 159: 598600. Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C and Porrello A: Intraoperative versus postoperative electrochemotherapy in soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother Pharmacol 2007; 59: 375-381. Spugnini EP, Vincenzi B, Citro G, Santini D, Dotsinsky I, Mudrov N, Baldi A. Adjuvant electrochemotherapy for the treatment of incompletely excised spontaneous canine sarcomas. In Vivo 2007; 21: 819-822. Spugnini EP, Filipponi M, Romani L, Dotsinsky I, Mudrov N, Barone A, Rocco E, Laieta MT, Montesarchio V, Cassano R, Citro G, Baldi A. Local control and distant metastasis after electrochemotherapy of a canine anal melanoma. In Vivo 2007; 21: 897-899. Spugnini EP, Vincenzi B, Citro G, Tonini G, Dotsinsky I, Mudrov N, Baldi A. Electrochemotherapy for the treatment of squamous cell carcinoma in cats: A preliminary report. Vet J 2009; 179: 117-120. Spugnini EP, Citro G, Dotsinsky I, Mudrov N, Mellone P, Baldi A. Ganglioneuroblastoma in a cat: a rare neoplasm treated with electrochemotherapy. Vet J 2008; 178: 291-293. Spugnini EP, Baldi F, Mellone P, Feroce F, D'Avino A, Bonetto F, Vincenzi B, Citro G, Baldi A. Patterns of tumor response in canine and feline cancer patients treated with electrochemotherapy: preclinical data for the standardization of this treatment in pets and humans. J Transl Med 2007; 5: 48. Spugnini EP, Dotsinsky I, Mudrov N, Cardosi G, Citro G, D'Avino A, Baldi A. Biphasic pulses enhance bleomycin efficacy in a spontaneous canine perianal tumors model. J Exp Clin Cancer Res 2007; 26: 483-487.

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[16] Spugnini EP, Vincenzi B, Betti G, Cordahi F, Dotsinski I, Mudrov N, Baldi A. Surgery and electrochemotherapy for the treatment of high grade soft tissue sarcoma in a dog. Vet Record 2008; 162: 186-188. [17] Spugnini EP, Citro G, Baldi A. Potential role of electrochemotherapy for the treatment of soft tissue sarcoma: first insights from preclinical studies in animals. J Biochem. Cell Biol 2008; 40: 159-163. [18] Spugnini EP, Dotsinsky I, Mudrov N, Bufalini M, Giannini G, Citro G, Feroce F, Baldi A. Adjuvant electrochemotherapy for incompletely excised anal sac carcinoma in a dog. In Vivo 2008; 22: 47-50. [19] Spugnini EP, Dotsinsky I, Mudrov N, De Luca A, Codini C, Citro G, D‘ Avino A, Baldi A. Successful rescue of a apocrine gland carcinoma metastatic to the cervical lymph nodes by mitoxantrone coupled with trains of permeabilizing electric pulses (electrochemotherapy). In Vivo 2008; 22: 51-54. [20] Zaharoff DA, Barr RC, Li C-Y, Yuan F. Electromobility of plasmid DNA in tumor tissues during electric field-mediated gene delivery. Gene Ther 2002; 9: 1286-1290. [21] Spugnini EP. Use of hyaluronidase for the treatment of extravasation of chemotherapeutic agents in six dogs. J Am Vet Med Assoc 2002; 221: 1437-1440. [22] Spugnini EP, Filipponi M, Romani L, Dotsinsky I, Mudrov N, Citro G, Baldi A. Bilateral pleomorphic rhabdomyosarcoma in a cat treated with electrochemotherapy. J Sm An Pract in press.

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Chapter 13

ELECTROCHEMOTHERAPY IN VETERINARY MEDICINE, PART II: ROUND CELL TUMORS Enrico P. Spugnini1, Alfonso Baldi2 and Gennaro Citro1 1

2

S.A.F.U. Department, Regina Elena Cancer Institute, Rome, Italy Dept. Biochemistry, Sect. Pathology, Second University of Naples, Naples, Italy

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ABSTRACT The authors review the most important characteristics of electrochemotherapy (ECT) in canine and feline patients affected by round cell tumors. In particular, the origins of this technique, and their further developments are described, with particular attention to the evolution of the clinical protocols, the development of novel equipments as well as their translation to human patients.

PRELIMINARY STUDIES AND FIRST PROTOCOLS Round cell tumors, and in particular, mast cell tumors, are frequently reported in veterinary literature [1]. These neoplasms are locally aggressive and can be associated with systemic dissemination when they have a high histological grade. Treatment options include surgery, radiation therapy and systemic therapy. In 2001 Tozon and coll. reported the direct treatment of 3 mast cell tumors (MCT) in two dogs by using series of single square pulses coupled with the intralesional injection of cisplatin [2]. Despite the risk of tumor degranulation, these investigators decided to attack directly the neoplasm with ECT, obtaining a complete response in a dog carrying two nodules and a partial response in the other patient. In 2003 Spugnini and Coll. describe the palliation of a dog with cutaneous lymphoma that harbored multiple lesions, by using intralesional bleomycin associated with the subsequent delivery of trains of biphasic electric pulses [3]. The dog remained in partial remission for 6 months when the owners of the pet elected to discontinue the therapy. In a later study, the same investigators report the treatment of a nasal lymphoma in a cat with ECT

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by using intralesional bleomycin delivered through needle array electrodes. This resulted in a remission in excess of 18 months [4]. Figure 1 shows the above mentioned patient at presentation (A) and at the completion of the therapy (B).

Figure 1. The clinical outcome of a cat with large granular lymphocyte lymphoma treated with ECT.

ELECTROCHEMOTHERAPY FOR SPECIFIC TUMOR TYPES: PHASE II STUDIES The first report of adjuvant electrochemotherapy for the treatment of incompletely excised mast cell tumors [5] was published in 2006. This article described the outcome of 28 dogs with MCT, treated with local injection of the tumors‘ bed, with bleomycin, followed by the application of trains of biphasic pulses, (8 pulses, 1300 V/cm, 50 + 50 µs duration, 1 Hz frequency).

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The overall response rate was 85% with a mean estimated time to recurrence of 52.76 ± 6.5 months (range: 39.99 to 65.54 months, 95% CI). At the time of the writing of the article, the median survival time was not reached (Figure 2).

Figure 2. Kaplan-Meier disease-free survival curve of 28 dogs with cutaneous mastocytoma treated with electrochemotherapy (From: Spugnini EP, Vincenzi B, Baldi F, Citro G and Baldi A: Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors. Anticancer Res 2006; 26: 4585-4589).

Specifically, three dogs died of metastatic disease that developed at the same time of local recurrence; one dog developed multiple cutaneous nodules at different locations and one dog with recurrence was retreated and experienced a total disease-free period of 22 months (Figure 3) The decision of adopting an adjuvant protocol was due to the risk of degranulation of large mast cell tumors treated in this study [1] and on the basis of a previous study that showed the potentials of this strategy [6]. Following the preliminary results obtained in companion animals with lymphoma and based on therapies performed in humans [7], a more thorough investigation has been armed on pets with lymphoma [8]. Indeed in 2007 Peycheva and coll. described the successful outcome of a cohort of 8 patients with stage I mycosis fungoides treated with interferon alpha followed by the application of trains of biphasic pulses [8]. Accordingly to the authors, all the

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29 lymphomatous lesions went into remission and new lesions did not develop over a period of 12 months.

Figure 3. A patient with recurring MCT of the lip; this dog was retreated with surgery and ECT and experienced a remission that lasted 22+ months.

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The aim of this study was the investigation of the potential role of bleomycin-based ECT for the local control of lymphoma in a spontaneous pet model. A total of 6 dogs and cats have been enrolled in this study experiencing complete remissions that lasted from 1 week up to 3 years (Figure 4). The treatment was well tolerated and side effects were limited to transient local inflammation that subsided within 48 hours, without the need of administering any medications.

Figure 4. The clinical outcome of a Yorkshire dog with labial lymphoma treated with ECT, note the complete remission at the end of the therapy.

Of interest, 50% of the patients experience remission in excess of 600 days, without side effects from the therapy. In particular this technique has been able to control lymphoma in difficult districts such as the nose, neck or the retrobulbar cavity (Figure 5).

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.

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Figure 5. A four-year-old male castrated cat treated with ECT for a retrobulbar lymphoma. Note the small areas of alopecia due to the application of needle array electrodes. The patient remained in remission for 635 days.

In 2009, Kodre et al. described the outcome of a group of 9 dogs with 12 mast cell tumors treated with intralesional CDDP coupled with square electric pulses, matched against a group of 16 dogs with 16 lesions treated with surgery alone [9]. The authors reported that they obtaining a response rate for the ECT cohort that compared well with the control group receiving surgery as single treatment. A limit of that study is the lack of grading of MCT, thus limiting the prognostic factors to tumor stage. A very recent communication reported the outcome of neoadjuvant ECT for the treatment of incompletely resected mast cell tumors, adopting local injection of CDDP followed by the application of trains of biphasic pulses [10]. This preliminary communication reported a total of 23 dogs treated with the combination, and an overall 17/23 patients without evidence of disease at different times (Figure 6). The average disease free interval of these dogs was 342 days. Interestingly, none of these dogs had CDDP induced local inflammation and the only side effect was a wound dehiscence in a dog with a large neoplasm. The authors were unable to assess if this should have been ascribed to the ECT or to the damage induced in the surrounding tissues by the neoplasm. A brief report describes the application of ECT as a rescue for drug-resistant canine affected by Transmissible Venereal Tumor (TVT) [11]. TVT is a horizontally transmitted neoplasm of the dog that is passed with coitus and is commonly located in the genitals, the nose, and the perianal area. It is a locally aggressive neoplasm with a low tendency to metastatic spread. Its standard treatment consists in chemotherapy with vincristine, and a rescue protocol with doxorubicine [12]. Electrochemotherapy has been adopted as a rescue

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protocol for three dogs with TVT, that relapsed or was non responsive to vincristine and doxorubicine, obtaining long lasting responses in all three patients (Figure 7).

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Figure 6. Kaplan-Meier disease free curve for 23 dogs with MCT treated with ECT.

Figure 7. A: Close-up image of a male with a preputial TVT, at the time of first evaluation. B: the same patient after one session of ECT, note the tumor shrinkage in excess of 50%. C: the same patient two weeks after the second and last session of ECT: the tumor has been completely replaced by scar tissue. D: the patient‘ prepuce one month after the completion of the two cycles of electrochemotherapy. (Modified from reference 12).

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CONCLUSIONS ECT is a safe, low cost and efficacious addition to the standard therapies for round cell tumors. These cancer cells showed remarkable sensitivity to therapeutical electroporation. This is probably due to their round symmetry and to the absence of extracellular tumor, associated matrix, resulting in optimal permeabilization and increased drug uptake. The possibility to repeat this technique in relapsing tumors and the identification of new drugs, whose efficacy is potentiated by the combination of proper electrical pulses, makes it an appealing alternative to the currently available protocols. Finally, the development of pulsemediated gene therapy shows promising results, that will make electroporation a more effective therapy in the near future.

REFERENCES

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[1]

Thamm DH, Vail DM. Mast cell tumors. In: Withrow SJ, Vail DM (Eds) Small animal clinical oncology, 4th Edition, St Louis (MI), Saunders, pp 402-424. [2] Tozon N, Sersa G, Cemazar M. Electrochemotherapy: potentiation of local antitumour effectiveness of cisplatin in dogs and cats. Anticancer Res. 2001;21:2483-2488. [3] Spugnini EP, Porrello A. Potentiation of chemotherapy in companion animals with spontaneous large neoplasms by application of biphasic electric pulses. J. Exp. Clin. Cancer Res 2003 22:571-580. [4] Spugnini EP, Citro G and Porrello A: Rational design of new electrodes for electrochemotherapy. J. Exp. Clin. Cancer Res. 2005; 24: 245-254. [5] Spugnini EP, Vincenzi B, Baldi F, Citro G and Baldi A: Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors. Anticancer Res. 2006; 26: 4585-4589. [6] Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C and Porrello A: Intraoperative versus postoperative electrochemotherapy in soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother. Pharmacol. 2007; 59: 375-381. [7] Peycheva E, Daskalov I, Tsoneva I. Electrochemotherapy of Mycosis fungoides by interferon-alpha. Bioelectrochemistry 2007; 70: 283-286. [8] Spugnini EP, Citro G, Mellone P, Dotsinsky I, Mudrov N, Baldi A. Electrochemotherapy for localized lymphoma: a preliminary study in companion animals. J. Exp. Clin. Cancer Res. 2007; 26: 343-346. [9] Kodre V, Cemazar M, Pecar J, Sersa G, Cor A, Tozon N. Electrochemotherapy compared to surgery for the treatment of canine mast cell tumors. In Vivo 2009; 23: 5562. [10] Spugnini EP, CitroG, Vincenzi B, Baldi A. 1st Worldvetcancer meeting, February 28thMarch 1st 2008, Copenhagen Denmark, pag 122. [11] Nak D, Nak Y, Cangul IT, Tuna B. A Clinico-pathological study on the effect of vincristine on transmissible venereal tumour in dogs. J. Vet. Med A Physiol .Pathol. Clin. Med. 2005; 52: 366-370.

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[12] Spugnini EP, Dotsinsky I, Mudrov N, Citro G, D'Avino A, Baldi A. Biphasic pulses enhance bleomycin efficacy in a spontaneous canine genital tumor model of chemoresistance: Sticker sarcoma. J. Exp. Clin. Cancer Res. 2008; 27: 58.

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Chapter 14

HISTOPATHOLOGICAL ANALYSIS OF CANINE AND FELINE CANCER TREATED WITH ELECTROCHEMOTHERAPY Alfonso Baldi*1 Feliciano Baldi,1 Pasquale Mellone,1 Alfredo D’Avino,1 Gennaro Citro,2 And Enrico P. Spugnini2 1

Department of Biochemistry, section of Pathology, Second University of Naples, Italy 2 S.A.F.U. Department, Regina Elena Cancer Institute, Rome, Italy

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ABSTRACT Electrochemotherapy (ECT) is a novel anticancer therapy that is currently being evaluated in human and pet cancer patients. ECT associates the administration of an antitumor agent to the delivery of trains of appropriate waveforms. In this chapter we describe the histological features of more than 400 bioptic specimens of tumors coming from a cohort of 215 companion animals with spontaneous tumors enrolled in different phase II ECT trials, over a 10 years period. Patterns of response suggest a dependence on tumor matrix content as well as unmasking of deep antigens, triggering immune mediated tumor eradication. Further studies on this topic are warranted in companion animals as well as humans in order to better define the histopathological response of tumor tissues to ECT treatment.

Keywords: biphasic pulse, cat, dog, electrochemotherapy Human and veterinary oncologists acutely perceive the difficulty of achieving local tumour control in cancer patients affected by various neoplasms. In fact, lack of awareness, inadequate screenings and the sudden onset of rapidly growing cancers often prevent the clinician from curing tumours with surgery alone. Due to these and other reasons, cancer is *

Correspondence to: Alfonso Baldi MD, Associate professor of Pathology, Dept. Biochemistry, Sect. Pathology, Second University of Naples, Via L. Armanni 5, 80138 Naples ITALY phone: +390815666003; fax: +390815569693, email:[email protected].

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considered (with the exception of some benign tumours) a disease that requires multimodality treatment. In humans, such approaches have been developed through multiinstitutional phase II and III trials and usually consist of the association of surgery and radiation therapy (depending on the clinical situation, usually brachytherapy with radiation beam). Chemotherapy is usually combined with the aforementioned treatments in an adjuvant fashion for those cancers with high tendency to metastasize (i.e. breast cancer). In selected cases, chemotherapy can be added to the protocol in a neoadjuvant approach to maximize the possibility of eradication. Other modalities explored to find new, synergistic combinations led to the investigation of local and whole body hyperthermia; however, the high costs of this treatment as well as the lengthiness of the procedure and the need for highly-skilled operators confined this therapy to a small number of research institutions [1]. Another critical point when evaluating local control modalities in cancer patients is the biological cost paid to accomplish such a goal. Many patients have to undergo disfiguring or mutilating surgeries and often the side effects of radiation therapy can leave sequelae that may lead to a poor quality of life. The most commonly reported side effects of radiation therapy are: 1) gradual side-effects, usually dose-dependent (local fibrosis, necrosis, nerve damage etc.) and 2) the so called ―statistically demonstrable side effects‖, also known as ―Radiation induced tumors‖ [2,3] A new treatment modality being further explored that can achieve high rates of remission without the associated problems of high financial and biological cost of previous procedures is electrochemotherapy (ECT). In vitro studies showed that the application of high voltage, exponentially-decaying electric pulses to cells in suspension could induce pores in the cell membrane thus resulting in cross-membrane flow of material (electroporation, electroinjection) or even in cell fusion if the cells were adjacent [4-7]. These methods were initially used to transfect bacterial cells with plasmids and subsequently exploited to produce monoclonal antibodies [8] through fusion of eukaryotic cells. Later, researchers realized that electroporation could enhance the transport of drugs and genes through the cytoplasmic membrane by exposing animal cells in culture and plant protoplasts to non-cytotoxic electric pulses [9-12] Subsequently, electroporation has been proven to be very effective at enhancing the in vitro cytotoxicity of anticancer molecules [10,13]. The first and most actively studied electroporation agent has been bleomycin. This drug can penetrate the cell membrane only through protein receptors due to its lipophobic nature, thus resulting in slow and quantitatively limited uptake under normal conditions [14]. The complex formed by bleomycin and its carrier is transported in the cytosol by means of endocytotic vesicles, but the mechanism of its release is still unknown. Over the past years, our group focused on the development of novel ECT protocols in pets affected by advanced cancer as a model for down-staged human patients. After preliminary studies involving also the development of custom-tailored electrodes, we studied the impact of ECT on several cohorts of canine and feline patients affected by spontaneously occurring tumors [15-19]. Our results on companion animals suggest that ECT is promising at controlling oral mucosal melanomas either as a single modality therapy or in conjunction with surgical cytoreduction [17]. Moreover, ECT can be employed not only to directly attack tumours but can also be used in an adjuvant fashion to treat residual disease, thus sterilizing the surgical field in case of incomplete excision as per radiation therapy [17-19]. Despite the consistent number of preclinical and clinical publications on this topic, there are few data on the

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Histopathological Analysis of Canine and Feline Cancer…

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histopathological modifications induced by this therapy [20]. Mir and coll. described massive necrosis induced by ECT in cats harboring post-vaccinal sarcomas, characterized by diffuse infiltration of the tumor perimeter by macrophages, lymphocytes and eosinophils [21]. The lack of extensive investigation in this field prompted us to run a thorough revision of our histological samples to gather a broader picture of patterns of tumor response and eventually to identify possible prognostic factors. Goal of this chapter is to summarize the data produced by our group on this topic in the last few years [22] and to update the cohort analyzed.

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MATERIAL AND METHODS Pets that entered in our studies received two sessions of ECT one week apart (two weeks for patients with cardiomyopathy) under sedation with medetodimine and ketamise as per manufacturer‘s instruction. Briefly the tumor‘s bed and the margins for ½ cm in all directions were infiltrated with bleomycin at the concentration of 1.5 mg/ml. Five minutes after the infiltration, trains of 8 biphasic electric pulses lasting 50 + 50 s each, with 1 ms interpulse intervals, were delivered by means of modified caliper electrodes [17]. Biopsies were collected before the beginning of ECT, after one session (one week) and at the completion of the protocol (two weeks). Histopathology specimens embedded in paraffin have been cut into 8 μm sections and stained following standard protocols, using Hematoxylin/Eosin, Hematoxylin/Van Gieson, and toluidine blue staining (to identify poorly differentiated mast cell tumors) [23]. The TUNEL reaction was performed using the peroxidase-based Apoptag kit (Oncor, Gaithersburg, MD), as previously described [24]. In order to confirm the diagnoses, histological examination of the biopsies were independently performed by two pathologists (FB and AB) that were not informed of the clinical outcome of the veterinary patients that were staged, treated and followed up by another investigator (EPS).

RESULTS The clinical outcome of pet cancer patients, object of this study, treated with ECT is briefly summarized in table 1. A predictable pattern of response was identified: among other factors such as tumor size and presence or absence of previous treatment(s), cancer destruction was dependent on the amount of extra-cellular connective substance. In a scale of sensitivity to ECT, melanoma seems to be the most responsive tumor, followed by hemangiopericytoma, mast cell tumor (MCT), carcinoma and sarcoma(s). Of interest, fibrosarcoma and MCT are very responsive when ECT is given as an adjuvant after surgical debulking, in order to treat residual disease. Patterns of response in the early phases of the treatment (after 1 session of ECT) involved an acute inflammatory response made up of neutrophils, lymphocytes and plasmacells, followed by extensive necrosis.

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Table 1. Tumor types treated with ECT in 215 companion animals with spontaneously occurring neoplasms Tumor type

Species Number of patients

Oral melanoma

Dog

10

Soft tissue sarcoma

Cat

90

Soft tissue sarcoma

Dog

40

Mast cell tumor

Dog

63

Squamous cell carcinoma** Cat

12

At the completion of the treatment (after two weeks), the tumor samples showed a dramatic decrease in cell number, with the majority of the remaining cells in apoptosis with no inflammatory response, while most of the residual tumor mass was made up of scar tissue. In three cases of feline sarcomas that experienced local failure, the tumor recurred as a less aggressive histotype: a neurofibroma-like lesion rather than an high grade sarcoma. Interestingly, we always detected lack of sufferance in the normal tissues surrounding the neoplasm. Table 2 summarizes the histopathological features of the ECT-treated cancers, that have been encountered in this study. In figure 1 several examples of the histopathological appearance of different tumors, are depicted (see the figure legend for the details).

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Table 2. Main histopathological features of the ECT-treated cancers Phase of treatment*

Histopathological pattern

Early phase (First session of ECT)

acute inflammatory response followed by necrosis

End of treatment (second session of ECT)

no acute inflammatory response, presence of mixed T and B lymphocytes, scar tissue and apoptosis of the residual tumor cells

* See the text for the detailed description of the treatment Modified from references n° 22.

DISCUSSION ECT has several advantages on other anti-tumor techniques: ease of administration, low cost, minimal toxicity (usually limited to mild focal inflammation at the treated site), and high response rate.

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Figure 1. A) A high grade feline fibrosarcoma situated on the dorsum of a cat before the ECT treatment (Haematoxylin and Eosin, original magnification X 10). B) The same lesion after completion of the ECT treatment (two weeks) is shown: note the complete disappearance of the neoplastic tissue, substituted by scar tissue (Haematoxylin/Van Gieson, original magnification X 20). C) A canine oral melanoma before ECT treatment (Haematoxylin and Eosin, original magnification X 10).D) The same tumor after completion of the ECT treatment (two weeks): most of the neoplastic cells are destroyed and substituted by scar tissue (Haematoxylin/Van Gieson, original magnification X 20).E) A canine cutaneous MCT before the ECT treatment (Haematoxylin and Eosin, original magnification X 40).F) The same lesion at the end of the adjuvant ECT treatment for incomplete surgical excision is shown: most of the residual neoplastic cells are apoptotic (TUNEL reaction, original magnification X 40)

Our morphological study shows on a broad selection of high grade tumors of companion animals a progressive and highly selective destruction that frequently allowed conservative surgeries in the case of extensive tumors. The histopathological analysis of the treated tumor revealed that cancer cells did not undergo the typical response to bleomycin consisting with enlargement and polynucleation [25], but evidenced marked necrosis circumscribed to the

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tumour tissue, and an associated acute inflammatory response mediated by lymphocytes and plasma cells. In the second phase of the treatment, neoplastic apoptotic cells are detected in most of the tumour mass, with no signs of acute inflammation. This apoptotic pattern could be a consequence not only of the ECT but also a phenomenon mediated by cellular immunity (mixed T and B lymphocyte population). This pattern of tumor lysis seems to play a key role in the prevention of local recurrence and distant dissemination for melanomas treated with this technique. In fact we recently described a vitiligo-like lesion in canine malignant melanomas of the oral cavity where the absence of any pigment at the treatment site in the long term survivors might imply the aiming of the immune system to melanin and other deep melanoma antigens [17]. Another patter of response, at the moment observed only in feline high grade sarcomas, suggests that ECT seems to promote a selection of tumor cells, leading to a local recurrence in the form of a less aggressive histotype: a neurofibroma-like lesion rather than an high grade sarcoma. The morphological analysis further confirm the efficacy and selectivity of this novel anticancer treatment as evidenced by the lack of sufferance elicited in the normal tissues surrounding the neoplasms. Studies are currently ongoing at our laboratory to further define the nature of the immune response elicited by the ECT treatment, in order to refine and ameliorate the efficacy of our protocols.

REFERENCES

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[1]

Strohbehn JW. Hyperthermia equipment evaluation. International Journal of hyperthermia.1994; 10: 429-432. [2] Banfi A, Lombardi F. Danni da radioterapia. Pag. 1331-1341 in: Bonadonna G, Robustelli della Cuna G. Medicina oncologica. Fifth edition.1994 Masson. [3] Hellman S. Principles of cancer management: radiation therapy. Pag 307-322 in: DeVita VT. ―Cancer. Principles and Practice of Oncology‖. 5th edition. 1997 Lippincott. [4] Kinoshita K Jr, Tsong Ty. Hemolysis of human erythrocytes by a transient electric field. Pro. Natl. Acad. Sci. USA 1977; 74: 1923-1927. [5] Senda M, Takeda J, Abe S, Nakamura T. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol. 1979; 20: 1441-1443. [6] Zimmermann U, Scheurich P. High frequency fusion of plant protoplasts by electric fields. Planta 1981; 151: 26-32. [7] Sugar IP, Neumann E. Stochastic model for electric field-induced membrane pores. Biophysical Chemistry 1984; 19: 211-225. [8] Lo MMS, Tsong TY, Conrad MK, Strittmatter SM, Hester LD, Snyder SH. Monoclonal antibody production by receptor-mediated electrically induced cell fusion. Nature 1984; 31: 792-794. [9] Mir LM, Banoun H, Paoletti C. Introduction of definite amounts of nonpermanent molecules into living cells after electropermeabilization: direct access to cytosol. Exp. Cell Res. 1988; 175: 15-25. [10] Orlowski S, Belehradec J, Paoletti C, Mir LM. Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs. Biochem. Pharmacol. 1988; 37: 4727-4733.

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[11] Neuman E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric field. EMBO J. 1982; 1: 841-845. [12] Fromm M, Taylor LP, WalbotV. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Nat. Acad. Sci. USA 1985; 82: 5824-5828. [13] Poddevin B, Orlowski S, Belehradek J Jr, Mir LM. Very high cytotoxicity of bleomycin introduced into the cytosol of cells in culture. Biochem. Pharmacol. 1991; 42(S): 67-75. [14] Pron G, Belehradec J Jr, Mir LM. Identification of a plasma membrane protein that specifically binds bleomycin. Biochem. Biophys. Res. Commun. 1993; 194: 333-7. [15] Spugnini E.P., Porrello A. (2003). Potentiation of chemotherapy in companion animals with spontaneous large neoplasms by application of biphasic electric pulses. J. Exp. Clin. Cancer Res. 22: 571-580. [16] Spugnini E.P., Citro G., Porrello A. (2005). Rational design of new electrodes for electrochemotherapy. J. Exp. Clin. Cancer Res. 24: 245-254. [17] Spugnini E.P., Dragonetti E., Vincenzi B., Onori N., Citro G., Baldi A. (2006). Pulse mediated chemotherapy enhances local control and survival in a spontaneous canine model of primary mucosal melanoma. Melanoma Res. 16: 23-27. [18] Spugnini EP, Baldi A, Vincenzi B, Bongiorni F, Bellelli C, Citro G, Porrello A. (2006) Intraoperative versus postoperative electrochemotherapy in high grade soft tissue sarcomas: a preliminary study in a spontaneous feline model. Cancer Chemother. Pharmacol. 59: 375-381. [19] Spugnini EP, Vincenzi B, Baldi F, Citro G, Baldi A. (2006c) Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors. Anticancer Res. 26: 4585-4589. [20] Salomskaite-Davalgiene S., Venslauskas M.S., Pauziene N. (2002). Histological analysis of electrochemotherapy influence in Lewis lung carcinoma. Medicina (Kaunas) 38: 540-544. [21] Mir L.M., Devauchelle P., Quintin-Colonna F., Delisle F., Doliger S., Fredelizi D., Belehradek J. Jr., Orlowski S. (1997). First clinical trial of cat soft-tissue sarcomas treatment by electrochemotherapy. Br. J. Cancer 76: 1617-1622. [22] Spugnini EP, Baldi F, Mellone P, Feroce F, D‘Avino A, Bonetto F, Vincenzi B, Citro G, Baldi A (2007). Patterns of tumor response in canine and feline cancer patients treated with electrochemotherapy: preclinical data for the standardization of this treatment in pets and humans. Journal of Translational Medicine 5:48. [23] Strefezzi R.DeF., Xavier J.G., Catão-Dias J.L. (2003) Morphometry of canine cutaneous mast cell tumors. Vet. Pathol. 40: 268-275. [24] Baldi A., Abbate A., Bussani R., Patti G., Melfi R., Angelini A., Dobrina A., Rossiello R., Silvestri F., Baldi F., Di Sciascio G. (2002). Apoptosis and post-infarction left venricular remodeling. Journal of Molecular and Cellular Cardiology 34: 165-174. [25] Tounekti O., Pron G., Belehradek J., Mir L.M. (1990). Bleomycin, an apoptosismimetic drug that induces two types of cell death depending on the number of molecules internalized. Cancer Res. 53: 5462-5469.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 15

CLINICAL APPLICATION OF ELECTROCHEMOTHERAPY – AN ADJUNCT TO SURGERY Declan M. Soden1, * Mira Sadadcharam1,*Patrick Forde1, and Gerald C. O’Sullivan1 1

Cork Cancer Research Centre, Leslie Quick Jnr. Laboratory, BioSciences Institute, University College Cork, Ireland

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1. INTRODUCTION Many cancers are resistant to multimodal treatments and there is a need for therapeutic innovations and discovery. The ideal treatment of cancer should effectively control local and systemically recurrent disease, be applicable to a diversity of tumour types and anatomical locations, facilitate multimodal and systemic therapies, be minimally intrusive and improve patient wellbeing and life expectancy by tumour control and cure. The first use of electrochemotherapy with bleomycin in humans was published in 1991 on head and neck tumour nodules [1]. Since that time, the therapy has undergone significant advances in terms of the systems used and the cancers demonstrated to be suitable for treatment [2-5]. A number of companies, including IGEA (IGEA, Carpi, Italy) and Inovio (Inovio Biomedical Corporation, CA, USA) have developed pulse generators with approval for use in humans. In the decade since their development, electrochemotherapy has become established as a safe and effective therapy in the treatment of a wide variety of cutaneous and subcutaneous lesions. Electrochemotherapy may be given with conventional anticancer treatments and experiences to date suggest it may also be a significant adjunct in combination with surgery. While all cancer types appear responsive to electrochemotherapy, experience to date has been confined to cutaneous malignancies, often recurrent and metastatic after multimodal therapies [1-3, 4-6]. The treatment is minimally disturbing for patients, is easy to perform in *

Both authors have contributed equally in the preparation of this manuscript

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an outpatient setting and is inexpensive. These encouraging clinical experiences promise the development of systems for treatment of intractable deep-seated malignancies. In this chapter, we describe electrochemotherapy and review the clinical experience and potential for further development.

2. SYSTEMS AND ELECTRODES EMPLOYED FOR CLINICAL APPLICATION Two companies have led the way in the development of clinically approved electroporation equipment and its associated electrodes. These are IGEA with its CliniporatorTM and Inovio with the MEDPULSER (see Figure 1). The electric field generated by these machines is largely determined by the geometry of the electrodes used.

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Figure 1. (L – R) Cliniporator (IGEA); MEDPULSER (Inovio).

Two main electrode designs are the plate and needle electrodes (Figure 2). Plate electrodes are suited for use in skin or superficial lesions. The depth of penetration of the effective electric field is rather small and depends on the distance between the electrodes: the greater the distance, the deeper the penetration of the electric field into the tissue. A caveat in the case of plate electrodes is that the electric field is substantially impeded by the resistance presented by the skin. In turn, needle electrodes are either arranged in two parallel rows or in a circular (hexagonal) array. Unlike plate electrodes, these are impaled transcutaneously into the target tissue [21].

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Irrespective of electrode design, the electric field is highest at the electrode surface, and rapidly decreases outside the electrode array. This phenomenon is more apparent in needle than plate electrodes (Figure 2). As such, if a tumour is larger than the distance between the electrodes, it necessitates the movement and placement of the electrodes adjacently for each consecutive pulse application in order to ensure sufficient coverage of the tumour site.

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3. APPROPRIATE DRUGS FOR ELECTROCHEMOTHERAPY The phospholipid bilayer of the cell membrane is a selectively permeable structure found in all cells, wherein channels and transport proteins regulate the flow of metabolites into and out of the cell interior. An impermeant or poorly permeant drug is one which cannot cross the cell membrane due to its hydrophilic nature and lack of specific channels or transport proteins [9]. Although most chemotherapeutic agents are permeable molecules for which the cell membrane does not pose a barrier, there are a few drugs which do not easily pass the cell membrane [29, 30]. Several chemotherapeutic agents have previously been tested on cells for potential application in combination with electroporation. These include doxorubicin, daunorubicin, etoposide, paclitaxel, actinomycin D, Adriamycin, Mitomycin C, 5-fluorouracil, Vincristine, Vinblastine, Gemcitabine, Cyclophosphamide, Carboplatin, Cisplatin and Bleomycin. Of these, bleomycin and cisplatin have emerged as the two commonest drugs used in clinical electrochemotherapy [19]. The reason for this is that they have the benefit of localizing toxicity to electroporated tissue, reducing systemic side effects and increasing logarithmically the effectiveness of the drug when used in combination with electroporation. In vitro studies using bleomycin have shown an enhancement of cytotoxicity of up to 700-fold [29, 31], while studies with cisplatin have shown an 80 fold increase in cytotoxicity [32, 33]. This lower but improved performance of cisplatin has been attributed to cisplatins‘ lipophilic nature which allows passage through the plasma membrane [34].

3.1. Bleomycin Bleomycins are a group of related basic glycopeptides which differ in the terminal amine substituent of the common structural unit, Bleomycin acid [35]. Bleomycin (Figure 3) was first discovered in 1962 by Hamao Umezawa who recognised its anti-cancer properties while screening culture filtrates of Streptomyces verticillus [35].

3.1.1. Indications The clinical utility of bleomycin has been well established on a wide variety of tumours including squamous cell carcinomas of the head and neck (tongue, buccal mucosa, larynx), penis, cervix, and vulva, Hodgkin‘s Disease, non-Hodgkin‘s lymphoma, as well as testicular carcinomas (embryonal cell, choriocarcinoma, and teratocarcinoma [6, 36, 37, 38].

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Figure 3. Chemical structure of Bleomycin.

3.1.2. Mechanism of Action Bleomycin binds to guanosine-cytosine-rich portions of DNA via association of the "S" tri-peptide and by partial intercalation of the bithiazole rings. A group of five nitrogen atoms arranged in a square-pyramidal conformation binds divalent metals including iron, the active ligand, and copper, an inactive ligand [39, 40]. This complex with iron reduces molecular oxygen to superoxide and hydroxyl radicals resulting in single- and double-stranded breaks in DNA [41] [39]. If only a few thousand bleomycin molecules are present in the cell, the cell is arrested in the G2-M phase, becomes enlarged and polynuclei and micronuclei are observed [39, 42]. The cell then dies in a slow process lasting about three doubling times [42]. If, however, the cell contains several million bleomycin molecules, it is killed within a few minutes by pseudoapoptosis, characterized by cell shrinkage, membrane blebbing, and chromatin condensation [39, 42, 43]. Preclinical results have shown an enhancement in the cytotoxic effect of bleomycin of several 1000 times when used in conjunction with electroporation [29, 31]. Variable resistance to bleomycin can be correlated with the presence of the bleomycin hydrolase enzyme, which is in the cysteine proteinase family. The enzyme replaces a terminal amine with a hydroxyl, thereby inhibiting iron binding and cytotoxic activity. The low concentration of enzyme in the skin and lung may explain the unique sensitivity of these tissues to bleomycin toxicity [41]. 3.1.3. Clinical Drug Dosage Present day clinical trials utilise doses of intravenous bleomycin that are similar to those used in conventional chemotherapeutic regimens (15 000IU/m2) [44, 45]. However, as electrochemotherapy is often a once-only treatment, the cumulative dose of bleomycin is much lower as compared to conventional chemotherapy, where multiple treatments are often necessary. Intralesional bleomycin doses are scaled based on tumour volume (500IU/cm3)

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[44, 45]. As such, unless the tumour is very large, or there are a large number of tumours requiring treatment, the overall dose tends to be significantly smaller than that required systemically.

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3.1.4. Route of Administration and Timing Bleomycin can either be administered systemically or intratumourally [23]. A number of variables dictate the choice between the two. As an example, in the case of a tumour with high interstitial pressures, or in a patient with multiple tumour nodules e.g. chest wall metastases from a breast adenocarcinoma, bleomycin should be administered systemically. Conversely, a number of factors such as poor circulation or a poorly vascularised tumour can adversely affect treatment outcome by restricting the amount of drug present at the tumour site. In an attempt to overcome such factors, bleomycin may be administered intratumourally. In addition, intratumoural administration of bleomycin would minimise the already greatly attenuated side effect and toxicity profile associated with chemotherapeutic doses used for electrochemotherapy. With regard to timing of drug administration, when the drug is delivered systemically, electric pulses need to be delivered to the tumour site during the pharmacokinetic peak, which has been reported to be between 8 to 28 minutes in humans [46]. For intratumoural applications, pulses need to be delivered from 1 to 10 minutes following drug administration [2, 47]. 3.1.5. Toxicity and Side Effects In patients with normal renal function, 60-70% of an administered dose is recovered from the urine as active bleomycin [30, 39, 48, 49]. Should the patient have a creatinine clearance of > 35 mL per minute, the serum or plasma half-life of Bleomycin is approximately 115 minutes. In patients with a creatinine clearance of < 35 mL per minute, the plasma or serum half-life increases exponentially as the creatinine clearance decreases [30, 39, 49]. These results suggest that severe renal impairment could lead to accumulation of the drug in blood, making renal impairment a relative contraindication for the administration of bleomycin. Pulmonary toxicities occur in 10% of treated patients. In approximately 1% of cases, this nonspecific bleomycin-induced pneumonitis progresses to pulmonary fibrosis, and death [39, 49]. Although this is age and dose related, the toxicity is unpredictable [41, 50].

3.2. Cisplatin In the 1960s, Rosenberg and van Camp et al discovered that electrolysis of a platinum electrode produced cisplatin, which inhibited binary fission in Escherichia coli (E. coli) bacteria. A series of experiments was then conducted to study the effects of various platinum coordination complexes on sarcomas artificially implanted in rats. This study found that cisdiamminedichloridoplatinum(II) (Figure 4) was the most effective out of this group, which started the medicinal career of cisplatin. Following its discovery over 3 decades ago, cisplatin represents one of the most successful drugs in chemotherapy [41].

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Figure 4. Chemical structure of Cisplatin.

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3.2.1 .Indications As a single agent, cisplatin is recommended in the treatment of urological cancers including transitional cell carcinomas of the bladder not amenable to surgery and/or radiation therapy as well as locally advanced or metastatic transitional cell carcinoma involving the renal pelvis, ureter, bladder and/or urethra. As a combination therapy, it is used in the palliative treatment of recurrent or metastatic squamous cell carcinomas of the head and neck, in the treatment of lung cancer, principally as a component of various chemotherapeutic regimens in the treatment on non-small cell lung cancer; as well as in the palliative treatment of recurrent or advanced squamous cell carcinoma of the cervix and metastatic testicular carcinomas. Cisplatin has also been used in the treatment of osteogenic sarcomas, neuroblastomas, and advanced oesophageal and prostatic carcinomas [41]. 3.2.2. Mechanism of Action Cisplatin exerts its anti-neoplastic activity when in the cis-configuration and its actions appears to be cycle-phase nonspecific, causing cell death in all cells. It is in those cells which turn over rapidly (tumour cells, skin cells, gastrointestinal cells, bone marrow cells) that cell death will occur at a faster rate than other cells with a slower turnover rate (e.g. muscle cells). Cisplatin complexes moves through cell membranes in an unionised form and this is achieved in the relatively high chloride concentration in the plasma. Intracellularly, the concentration of chloride ions is lower than in the plasma and the chloride ligands on the cisplatin complex are displaced by water. The result is the formation of positively charged platinum complexes that are toxic to cells. The cisplatin molecule binds to the DNA molecule at the guanine bases and thus inhibiting DNA synthesis, protein and RNA synthesis (the latter two are inhibited to a lesser degree).The drug forms intra- and inter-strand cross links in the DNA molecule. The tumour cells amass an overburden of mutations which lead eventually to the cell's death [51]. 3.2.3. Drug Dosage As with Bleomycin, the dosage for intratumoural Cisplatin therapy (1mg/cm3) is tumour volume dependent [44, 45]. Once again, unless the tumour is very large or there are a large number of tumours requiring treatment, the overall dose tends to be smaller than that required systemically.

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3.2.4. Route of Administration and Timing Cisplatin is only available in its injectable form and has been administered intratumourally in all clinical trials involving electrochemotherapy. One possible reason for this is that although intravenous, intra-arterial and intraperitoneal routes of administration have all been used in cisplatin therapy, poisoning is most likely occur by these three routes. As such, adequate pre-treatment and post treatment hydration (with intravenous fluids and diuresis) and maintenance of adequate urinary output must be ensured throughout cisplatin therapy to help minimise renal complications. The hypothesis behind this regimen is that it dilutes the cisplatin metabolite concentrations in the kidney and lessens the incidence of toxicity to the cells. Crucially, cisplatin and any platinum-containing products are rapidly and extensively bound to tissue and plasma proteins, including albumin, gamma-globulins and transferrin. Binding to tissue and plasma proteins appears to be essentially irreversible with the bound platinum remaining in plasma during the lifespan of the albumin molecule. Protein binding increases with time and less than 2 to 10% of platinum in blood remains unbound several hours after intravenous administration of cisplatin. The extent of protein binding is about 90% and this occurs essentially within the first two hours after a dose. The major toxicity caused during cisplatin treatment is dose related and cumulative. For example, renal tubular function impairment can occur during the second week of therapy and if higher doses or repeated courses of cisplatin are given then irreversible renal damage can occur [41, 51]. With regard to timing of drug administration, when the drug is delivered intratumourally, pulses need to be delivered from 1 to 10 minutes following drug administration [2, 47]. 3.2.5. Toxicity and Side Effects Numerous adverse effects during cisplatin therapy have been reported in the literature. Nephrotoxicity is dose related and can be severe. Cisplatin should be administered with adequate intravenous hydration and diuresis. Hypomagnesaemia and other electrolyte disturbances can be seen during cisplatin therapy which is secondary to cisplatin-induced renal dysfunction. Gastrointestinal side effects include marked nausea and vomiting. Diarrhoea is also a side effect of cisplatin therapy and occurs less frequently than nausea and vomiting. Ototoxicity has occurred in patients receiving cisplatin therapy and is manifested as tinnitus, with or without loss of hearing and occasional deafness. Ototoxicity tends to be more severe in children than in adults. Cisplatin has also caused vestibular ototoxicity, i.e. vertigo or vestibular dysfunction but it is a rare occurrence. Those patients with pre-existing vestibular dysfunction are most at risk. Peripheral neuropathies have been noted during cisplatin therapy and manifest themselves as paraesthesia of the upper and lower extremities and other sensory functions. Peripheral neuropathies generally occur only after prolonged (4 to 7 months) treatment but their incidence rises if other neurotoxic agents are used. Myelosuppression occurs in about 25 to 30% of patients and manifests itself as leucopenia, thrombocytopenia and anaemia. Myelosuppression can be cumulative and severe especially the patient has been previously treated with other anti-neoplastic agents. Cardiovascular effects are rare but can be debilitating. Symptoms include bradycardia, left bundle branch block and congestive heart failure. The vascular toxicities include

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myocardial infarction, coronary artery disease, renovascular lesions, cerebrovascular accidents and Raynaud's phenomenon [41, 50].

4. TREATMENT PROTOCOL 4.1. Eligibility Criteria The inclusion and exclusion criteria related to electrochemotherapy are as follows:

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Table 1. Inclusion/Exclusion Criteria for Patients Inclusion Criteria Histologically verified cancer of any type Progressive and/or metastatic disease Patients must have been offered standard treatments Electrochemotherapy can be considered either in cases of progression following standard treatments or in cases where patients do not wish to receive such standard treatment Measurable cutaneous or subcutaneous tumour nodules suitable for application of electric pulses Age at least 18 years Performance status (Karnofsky >70% OR WHO grade 2 AND allergic reactions to bleomycin or cumulative dose of 250mg BLM/m2 previously exceeded Chronic renal dysfunction (creatinine > 150µmol/L) Patients with a clinically manifested arrhythmia or with a pacemaker Patients with epilepsy Pregnancy or lactation

4.2. Anaesthesia The decision to perform electrochemotherapy under local or general anaesthesia is dependent upon a number of factors. These include the site and number of lesions requiring treatment, as well as the general medical condition of the patient. In the case of head and neck tumours for example, treatment is often carried out under general anaesthesia. When smaller,

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discrete tumours are involved, treatment may be carried out under local anaesthesia, often in combination with adrenaline. The adrenaline acts synergistically with the vascular effects of electroporation, causing local vasoconstriction and delaying systemic dissemination of the drug by local vasculature.

4.3. Patient Follow-Up In general, most patients treated with electrochemotherapy are discharged home the same day. Relative indications for overnight admission include patients who have previously had adverse reactions to anaesthesia, those who are a high anaesthetic risk and patients who have mitigating social circumstances. Patients are recalled approximately 6-8 weeks post-treatment at which time they are examined by their clinician. The number of treatments required will be dependent upon the size, number and responsiveness of the lesion(s) involved.

4.4. Response Criteria Tumour response is determined by tumour volume. Tumour volume (V) is calculated from measured dimensions using the formula: V = π x A x B2 / 6

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where (A) is the longest diameter of the lesion (in millimetres) and (B) is the longest diameter (in millimetres) perpendicular to A. Based on this tumour volume, response is determined in line with either WHO (Table 2) or RECIST (Table 3) criteria. Table 2. WHO Response Criteria [61] Response Classification Complete response (CR) Partial response (PR) No change (NC) Progressive disease (PD)

Tumour Volume Elimination of nodule >50% reduction in tumour volume 50% reduction in tumour volume >25% increase in tumour volume

Table 3. RECIST Response Criteria [61] Response Classification Complete response Partial response Progressive disease Stable disease

Tumour volume Complete disappearance of all targeted lesions > 30% reduction in the sum of the longest diameter of targeted lesions < 20% increase in the sum of the longest diameter of targeted lesions There is neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease

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5. EARLY CLINICAL EXPERIENCE

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5.1. Clinical Trials Involving Bleomycin Due to the encouraging results obtained from preclinical studies, information was rapidly translated into clinical electrochemotherapy trials [1,46, 53-59]. The earliest clinical study was published in 1991 by Mir et al involving patients with recurrent or progressive cutaneous metastases of head and neck squamous cell carcinomas located in the anterior cervical region or in the upper part of the thorax [1, 53]. The study protocol for this trial closely resembled optimised parameters from preclinical studies. Bleomycin was the chemotherapeutic of choice, and a 10mg/m2 bolus dose was administered to patients. Starting from 3.5 min following injection of the chemotherapeutic agent, pulses were administered to the first tumour. Subsequent tumours were treated in series with a one minute interval between each electrical pulse. The electrodes used consisted of two parallel stainless steel strips measuring 10mm in length x 6mm in width, with electrodes spaced 6mm apart. Either 4 or 8 pulses were administered with an electric field strength of 1300V/cm pulse duration of 100μs and a duty cycle of 1s. While most of the treated tumours were 2x2 cm in size or less, any nodules that proved to be larger than this were treated in sections. Among the many observations drawn from this trial, it was noted that maximal antitumor effects were obtained when pulses were administered between 8 and 28 minutes following injection of a bolus dose of bleomycin. This finding seemed to indicate the window during which bleomycin concentrations were highest in the interstitium of the tumour. The findings from this original trial prompted a number of other groups to launch their own clinical studies. These included a Phase I-II trial by Belehradek et al involving eight patients with 40 squamous cell carcinoma nodules of the head and neck. The pulse parameteres utilised in this trial were comparable to those in the study by Mir et al in 1991. The observations of this particular trial were that in addition to a 57% complete response rate, there was no evidence of local or systemic toxicity as well as good patient tolerance of electrochemotherapy. Nearly identical patient tolerance profiles were noted by Heller et al in a Phase I-II study involving three melanomas, two basal cell carcinomas and one metastatic breast adenocarcinoma [57, 60]. Once again, bleomycin was the chemotherapeutic agent of choice, and a dose of 10units/m2 was administered intravenously at a rate of 0.6-0.8mg/min. Five to fifteen minutes following drug administration, eight 99μs rectangular direct current pulses were delivered to tumour tissue. The results of this study were that in two of these patients, five out of six melanoma metastases responded, while the third patient had no response to treatment. The patient with the metastatic breast adenocarcinoma also had a complete clinical response. Aside from these studies, numerous other groups conducted their own trials into the clinical efficacy of electrochemotherapy including [46, 53-55, 58, 59]. Table 4 [53] summarizes some of the clinical trials involved in the development of clinical electrochemotherapy. Table 5 [53] summarizes the results of clinical studies on electrochemotherapy with bleomycin, given intravenously or intratumorally.

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Table 4. Summary of Preliminary ECT Clinical Trials (Taken from [62]) Trial location

No. of patients

Types of cancer

Villejuif, France Tampa, Florida

8

HNSCC

6

Tampa, Florida Tampa, Florida Ljubljana, Slovenia

8

Melanoma, BCC, adenocarcinoma Melanoma, BCC, KS Melanoma, BCC, SCC Melanoma

11 2

Ref

No. of tumors treated 40

Bleomycin administration

Electrode type

Intravenous bolus Intravenous perfusion

Parallel plate Parallel plate

25

Intratumor

24

Intravenous bolus Inatrevenous bolus

Parallel plate Needle array Parallel plate

18

24

Clinical tumour responses (%) NE CR PR OR 28 15 57 72 27.8

38.9

33.3

72.2

0

4

96

100

2.1

6.1

91.8

97.9

8.4

0

91.6

91.6

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Table 5. Results of clinical studies on electrochemotherapy with bleomycin, given intravenously or intratumorally (Taken from [62]) Tumour No. Pts No. Tumours Intravenous BLM dose: 10-15mg/m2 or 18-27 U/m2 Head and neck 17 77 squamous cell carcinoma Malignant melanoma 14 94 Basal cell carcinoma 2 6 Adenocarcinoma 4 31 (breast, salivary gland, hypernephroma) Total 37 208 Intratumoral BLM dose:0.2-0.55 mg/cm3 or 0.25-1.0 U/cm3 Head and neck 14 14 tumours (squamous, adeno and adenid cystic carcinoma) Malignant melanoma 11 106 Intratumoral BLM dose:0.2-0.55 mg/cm3 or 0.25-1.0 U/cm3 Squamous cell 1 1 carcinoma Kaposi sarcoma 1 4 Breast cancer 2 14 Bladder; trans. cell ca 1 17 Total 25 116

OR (%)

CR (%)

62

43

89 100 100

34 17 97

62-100

17-97

86

50

95

60

100

0

100 100 100 80-100

100 58 82 0-100

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5.2. Clinical Trials Involving Cisplatin In 1998, Sersa et al published the first clinical data on electrochemotherapy with intratumorally injected cisplatin [61]. This study was aimed at assessing the clinical efficiency of electrochemotherapy with cisplatin on cutaneous nodules of varying histologies, including malignant melanoma, squamous cell carcinoma and basal cell carcinoma. Four patients with 30 evaluable tumour nodules were enrolled in this study. Of these, 5 were left untreated, 19 were treated with electrochemotherapy, 1 with electric pulses alone, and 5 were treated with intratumoral injection of cisplatin alone. Following a period of 4 weeks, complete responses were obtained for all 19 tumour nodules treated with electrochemotherapy. The findings of this study were verified by [3, 5, 62, 63]. This study was followed up with studies assessing the clinical efficiency of electrochemotherapy using intravenously injected cisplatin. Specifically, a study by Sersa et al in 2000 was designed to verify the ability of electroporation in enhancing the antitumor efficiency of conventional cisplatin therapy in individuals with progressive malignant melanomas [3]. The study found that although good antitumor effect was noted, this did not translate through to a high objective response rate. One of the reasons postulated for this was the inclusion of larger tumour nodules where electrochemotherapy would be less effective. These findings were verified in studies conducted by [2, 55, 64]. Comparative studies have shown that electrochemotherapy with intratumoral administration of cisplatin is more effective than electrochemotherapy with intravenously injected cisplatin (Table 6). It has also been shown that intravenous cisplatin was used during electrochemotherapy, it was found to be less effective as compared to electrochemotherapy with bleomycin administered either intravenously or intratumorally [2, 55, 64].

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Table 6. Results of clinical studies on electrochemotherapy with cisplatin, given intravenously or intratumorally (Taken from [62]) Tumor No. Pts No. Tumours Intravenous cisplatin based chemotherapy protocol Malignant 9 27 melanoma Intratumoral cisplatin 1mg/cm3 Head and neck 1 squamous ca. Malignant 10 melanoma Basal cell 1 carcinoma Adenocarcinoma 2 (breast, ovary) Total 14

OR (%)

CR (%)

48

11

2

100

100

82

78

68

4

100

100

6

100

78

94

78-100

68-100

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6. ESOPE TRIAL (TAKEN FROM [20]) 6.1. Background Until relatively recently, the studies involving electrochemotherapy utilised varying treatment protocols for electrode design, pulse parameters and generators, as well as differing chemotherapeutic doses. This created the need for a study to look at these varying parameters and attempt to formulate a standard operating procedure to be utilised by all centres involved in electrochemotherapy. As a result of this, the ESOPE study (European Standard Operating Procedures of Electrochemotherapy) was launched. This prospective, non-randomised multicentre project was funded under the European Commission‘s 5th Framework Programme. The study ran from 31st March 2003 to 20th April 2005 and assessed treatment outcomes following electrochemotherapy based on tumour histology, chemotherapeutic agent, route of administration and electrode type.

6.2. Patient Population

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Patients were recruited from four centres, namely: Institute Gustave-Roussy (IGR), Villejuif, France, Institute of Oncology Ljubljana (OI), Ljubljana, Slovenia, University of Copenhagen at Herlev Hospital (HH), Herlev, Denmark, and the Cork Cancer Research Centre Biosciences Institute and Mercy University Hospital, National University of Ireland (CCRC), Cork, Ireland. Of the 62 patients who were eligible according to the inclusion criteria, only 42 patients were evaluable for response (Figure 5). These 42 patients who between them had 171 tumour nodules of varying histology formed the basis for the primary statistical analysis plan of the ESOPE study.

Figure 5. Breakdown of patient population involved in the ESOPE trial.

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6.3. Tumour Histologies Patients with cutaneous and subcutaneous primary and metastatic nodules including malignant melanoma, basal cell carcinomas, squamous cell carcinomas, cutaneous metastases from breast and colorectal adenocarcinomas were recruited into the trial. For the purposes of the trial, treated nodules were divided into ―melanoma‖ (n=98) and ―non-melanoma‖ (n=73) groups on the basis of tumour diagnosis. Overall, 57.3% of recruited tumour nodules were melanomas, while non-melanotic tumours comprised 42.7% of the patient population (Figure 6).

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Figure 6. Distribution of Tumour Histology.

6.4. Overall Response Electrochemotherapy with bleomycin revealed an objective response rate of 85% (73.3% complete response rate) (Figure 7). Electrochemotherapy with cisplatin resulted in 77% long lasting complete responses of tumour nodules. This held true over a variety of tumour histologies, drugs and routes of administration. There was also a trend towards higher antitumor activity in non-melanoma nodules (OR 90.4% versus 80.6%). These findings mimicked the clinical responses from previous clinical studies (Table 7). Examples of tumour responses to electrochemotherapy with either intravenous or intratumoral bleomycin can be seen in Figures 8 and 9. Table 7. Clinical response to electrochemotherapy in the ESOPE and previous studies Electrochemotherapy

Patients

Nodules

Before ESOPE study ESOPE study

247 41

1009 171

Positive (CR+PR) % 83 85

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Figure 7. Objective rate associated with electrochemotherapy with bleomycin as found in the ESOPE study.

Figure 8. Images of cutaneous chest wall metastases from a breast adenocarcinoma before (A) and after (B) electrochemotherapy demonstrating successful tumour ablation.

Figure 9 Images from an inoperable parotid squamous cell carcinoma before (A) and after (B) electrochemotherapy demonstrating good antitumor effect.

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6.5. Influence of Drug Delivery One objective of the ESOPE study was to investigate the influence of the route of drug administration (local or systemic) on the overall objective response rate (ORR). Even though the results of the Chi square test were only marginally significant, it was found that the systemic route of drug administration was associated with a higher objective response rate (ORR) and associated with a proportionally lower rate of non-responders (NR) (Figure 10)

Figure 10. Systemic drug delivery is associated with a higher objective response rate.

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6.6. Influence of Tumour Locale The study found that tumour nodules located on the thorax, abdomen and back (TAB) were associated with a higher objective response rate (ORR) as compared to tumours located on the head and neck and lower limb regions (Figure 11).

Figure 11. Objective response rates vary depending on tumour locale.

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6.7. Influence of Tumour Volume Regardless of the drug and route of drug administration used, treated tumour nodules were divided into three categories according to their size: (small < 0.1cm3, medium >0.1cm3 and 0.5cm3). No statistically significant difference was found between responses to electrochemotherapy according to tumour size (Figure 12), although when considering route of drug administration, systemic injection of bleomycin (32 nodules, 93.8% OR) resulted in a greater anti-tumour effect as compared to local injection of bleomycin or cisplatin (24 nodules, 75% OR).

Figure 12. Objective Response Rate is Independent of Tumour Size.

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6.8. Influence of Electrode Type Electrochemotherapy was performed using three different types of electrodes: Type I plate electrodes for superficial tumour nodules and Type II and III electrodes for deeperseated tumours. Analysis of tumour response showed no statistically significant difference in terms of treatment outcome between the three electrodes types (Figure 13).

Figure 13. No statistical significant difference in the objective response rate was noted between the three electrode types. Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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7. LIMITATIONS OF ELECTROCHEMOTHERAPY 7.1. Side Effects Related to Therapy The commonest side-effect associated with electrochemotherapy is the involuntary muscle contraction that occurs at the instant the electric pulses are applied [25, 55, 57, 64-66]. These contractions cease at the end of the pulse. While the contractions themselves are generally painless, patients often describe the sensation as uncomfortable. It has been noted that manually elevating the area of skin to be electroporated somewhat relieves this sensation [27]. There have also been reports of burning of the skin associated with the use of plate electrodes. This may be ameliorated by using an interface medium e.g. aqueous gel. Similar reports of burning are absent when using needle electrodes [64].

7.2. Patient Referral Criteria In its present application, electrochemotherapy is a treatment modality which is often utilised in a palliative setting once all other conventional therapies have failed. As such, a large number of the patients referred for treatment present with advanced disease, whereby the tumour burden, both local and distant, overwhelms any potential therapeutic effect. Active measures need to be taken in an attempt to educate patients and physicians alike as to the therapeutic potential of electrochemotherapy in an effort to recruit patients with earlier-stage disease to achieve better treatment outcome.

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7.3. Limitation of Clinical Utility To date, the clinical license of electrochemotherapy has been limited to the treatment of cutaneous and subcutaneous tumour nodules. This has mainly been due to limitations in electrode design, whereby electrodes have been designed as attachments to hand-held probes. As a result of this, more deep-seated and intraluminal tumours that might otherwise have been amenable to treatment, have been rendered inaccessible. In an attempt to address this issue, IGEA (IGEA, Carpi, Italy) have recently introduced a hand-held finger electrode (Figure 14) to treat oral head ad neck tumours e.g. lingual squamous cell carcinomas and para-stomal recurrences from laryngeal squamous cell carcinomas.

7.4. Need for Comparison Studies with other Therapeutic Modalities At present, there is a lack of published data which directly compares the clinical results of electrochemotherapy with various other therapeutic modalities targeted at local disease control e.g. isolated limb perfusion and radiotherapy.

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Figure 14. Finger electrode by IGEA (IGEA, Carpi, Italy).

8. DEVELOPMENTAL PERSPECTIVES

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8.1. Extension of Clinical Utility At present, the main limitation of electrochemotherapy is its restriction to the management of cutaneous and subcutaneous tumour nodules. Modification of electrode designs to allow for attachment to conventional endoscopic and laparoscopic equipment would allow for the clinical license of this therapy to be greatly extended [68]. If it were possible to deliver permeabilising electric pulses to luminal tumours e.g. intra-abdominal, intra-thoracic and genitourinary malignancies, tumours that would previously have been unresponsive or inaccessible to conventional therapies would be amenable to electrochemotherapy. As well as this, the vast majority of patients presenting with metastatic disease tend to be elderly and at high anaesthetic risk. A large number of these patients requiring palliative treatment have few clinically efficacious options available to them. These patients would stand to benefit from a treatment like electrochemotherapy that is both quick, minimally invasive and that has proven clinical efficiency. At present, the Cork Cancer Research Centre Biosciences Institute and Mercy University Hospital, National University of Ireland (CCRC), Cork, Ireland is working on the development and validation of a suction electrode device designed to extend the clinical utility of electrochemotherapy to the management of luminal tumours. If this system proves successful, many otherwise intractable of inaccessible tumours would be rendered amenable to electrochemotherapy.

8.2. Enhancement of Existing Therapeutic Regimens As cisplatin is a drug which is incorporated in many conventional chemotherapeutics regimens, electrochemotherapy could be used to increase the efficiency profile of many existing chemotherapeutic protocols. Electrochemotherapy can also be used as an effective

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means of pre-sensitising tumours to the effects of radiation therapy, allowing for a more targeted tumoricidal therapy with minimal collateral injury.

8.3. Gene Delivery Vector The fact that electroporation maintains cell viability makes it well suited for gene therapies where therapeutic gene expression requires a duration of normal cell function. Electrical delivery is currently the most efficient way of delivering non-viral gene constructs into tumours and results in tissue transfection efficiency in up to 15% of cells. Electrical delivery of immunogenes into the primary tumour offers the possibility of a neoadjuvant therapy which would induce systemic responses effective against minimal residual disease.

8.4. Microneedles

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Electroporation is also amenable to biomedical microstructures such as microneedle arrays. Microneedles consist of an array of electrically conductive needles measuring hundreds of microns in length. These are electrically isolated from each other thus enabling the generation of strong field strengths across short electrode-electrode spacings. The microneedle array can provide minimally invasive, painless insertion into skin and can act as an electrode after proper metallization process. Unlike conventional macro-needles, these have a shallow insertion depth, minimising pain during the application of the electric field. In addition, the voltage required for electroporation can be reduced due to the small scale, avoiding the use of a high voltage source, which is not suitable for portable applications.

8.5. Nanoparticles Nanoparticles have been shown to be able to selectively deliver high concentrations of antitumor drugs to tumor cells. As such, they hold a great deal of promise for the future of cancer therapy. The high concentrations of antitumor drugs that are achieved using nanoparticles as a delivery medium appear to persist for long periods within tumor cells and have more potent antitumour effects and less toxicity than similar agents administered systemically. Nanoparticles can also be used to package genes that can then be ―mailed‖ the tumor sites influencing various factors e.g. neo-angiogenesis. Electroporation can be used as a physical delivery system to further enhance the properties afforded by nanoparticles technology.

CONCLUSIONS Electrochemotherapy is an extremely effective physical drug delivery system which can be utilised in the treatment of a wide variety of recurrent, progressive or inoperable cutaneous and subcutaneous malignancies. It acts by exponentially enhancing the local cytotoxic effects

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of bleomycin. Electrochemotherapy is a quick, safe, inexpensive treatment with easily reproducible results that can be used to improve patient quality of life and minimise intrusion on their valuable remaining time. As such, there exists a need to prioritise physician education as there are many who stand to benefit from such a minimally invasive treatment. At present, the therapeutic remit of electrochemotherapy is limited to the management of palliative cutaneous and subcutaneous tumours. Future efforts should include looking to extend this clinical license to the management of primary tumours and earlier stage disease. The Cork Cancer Research Centre, Ireland has already taken steps towards the development of novel electrodes to permit the application of this technology to internal cancers. As well as this, there also exists the need for continued impetus towards greater therapeutic developments. The potential for using electrochemotherapy in combination with conventional therapies e.g. in a neoadjuvant setting prior to surgical resection, as well as the potential for combining electrochemotherapy with immunotherapies and gene therapies offers exciting prospects for the future advancement of this therapy.

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Mir, L.M., et al., [Electrochemotherapy, a new antitumor treatment: first clinical trial]. C R Acad Sci III, 1991. 313(13): p. 613-8. [2] Mir, L.M., et al., Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br.J. Cancer, 1998. 77(12): p. 2336-42. [3] Heller, R., et al., Effective treatment of B16 melanoma by direct delivery of bleomycin using electrochemotherapy. Melanoma Res., 1997. 7(1): p. 10-8. [4] Sersa, G., et al., Electrochemotherapy with cisplatin: clinical experience in malignant melanoma patients. Clin. Cancer Res., 2000. 6(3): p. 863-7. [5] Bloom, D.C. and P.M. Goldfarb, The role of intratumour therapy with electroporation and bleomycin in the management of advanced squamous cell carcinoma of the head and neck. Eur. J. Surg. Oncol., 2005. 31(9): p. 1029-35. [6] Sersa, G., et al., Electrochemotherapy with cisplatin: the systemic antitumour effectiveness of cisplatin can be potentiated locally by the application of electric pulses in the treatment of malignant melanoma skin metastases. Melanoma Res., 2000. 10(4): p. 381-5. [7] Larkin, J.O., et al., Electrochemotherapy: aspects of preclinical development and early clinical experience. Ann Surg., 2007. 245(3): p. 469-79. [8] Chang, D.C. and T.S. Reese, Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. Biophys. J., 1990. 58(1): p. 1-12. [9] Lopez, A., M.P. Rols, and J. Teissie, 31P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells. Biochemistry, 1988. 27(4): p. 1222-8. [10] Mir, L.M., H. Banoun, and C. Paoletti, Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp. Cell Res., 1988. 175(1): p. 15-25.

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[67] Rols, M.P., et al., Electrochemotherapy of cutaneous metastases in malignant melanoma. Melanoma Res., 2000. 10(5): p. 468-74. [68] Soden, D.M., et al., Successful application of targeted electrochemotherapy using novel flexible electrodes and low dose bleomycin to solid tumours. Cancer Lett, 2006. 232(2): p. 300-10.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 16

ELECTROPORATION IN CHRONIC LYMPHOCYTIC LEUKEMIA Femke Van Bockstaele†, Valerie Pede, Bruno Verhasselt and Jan Philippé* Department of Clinical Chemistry, Microbiology and Immunology Ghent University, Ghent, Belgium

ABSTRACT

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Chronic lymphocytic leukemia (CLL) is a quintessential example of a malignancy caused by defects in apoptosis rather than increased cell proliferation. As a consequence, most circulating leukemia cells are in the G0/G1 phase of the cell cycle, which seriously hampers gene transfer by commonly used viral vectors. Nevertheless, efficient gene transfer methods could contribute substantially to fundamental and clinical research in CLL, so many scientists researched methods for introducing different biomolecules into CLL cells. This chapter briefly reviews the results obtained with viral vectors in CLL and mainly focuses on the numerous applications of electroporation (and the related nucleofector technology) in CLL research. Survival and efficiency strongly depend on the nature of the transfected biomolecules and on the applied electrical parameters. Overexpression of genes can be obtained by electroporating plasmid DNA vectors, mRNA or proteins into CLL cells, whereas transfer of small interfering RNA (siRNA) or antisense molecules results in gene silencing. Both approaches play a crucial role in the elucidation of the function of genes or proteins in CLL pathobiology. Furthermore, electroporation of CLL cells with plasmid vectors encoding CD40 ligand (CD40L) results in cellular vaccines, which are currently evaluated in clinical gene therapy trials. In conclusion, electroporation-based approaches provide a valuable alternative to the expensive and labor intensive use of viral vectors. Especially in resting cells like CLL cells, electroporation has gained importance as a tool for gene transfer, with applications in fundamental as well as clinical research.

*

Corresponding author: Jan Philippé, Address: UZ Gent, 2P8, De Pintelaan 185, B-9000 Gent, Belgium, e-mail: [email protected], †Present address: Ablynx NV, Technologiepark 4, 9052 Zwijnaarde, Belgium.

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INTRODUCTION Chronic Lymphocytic Leukemia

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With an incidence rate of 4.0 per 100,000 people in the USA [1], CLL is the most common leukemia in adults in the Western world, accounting for nearly 30% of all leukemias.[2] The risk of developing this leukemia increases progressively with age. Median age at diagnosis is 65 and more than 90% of patients are older than 50.[2, 3] The disease is characterized by the clonal accumulation of mature B lymphocytes in blood, bone marrow and lymphoid organs, resulting in symptoms like lymphadenopathy, splenomegaly, fatigue, weight loss, recurrent infections, bleedings and anemia.[2-4] Infections like pneumonia and sepsis are the main cause of morbidity and mortality in CLL.[2] Furthermore, auto-immune complications can have a fatal outcome, and patients are more prone to develop secondary tumors like melanoma, colon cancer or lung carcinoma.[2, 4] The clinical course of CLL is extremely variable, with life expectancies ranging from a few years to decades. Although the great majority of patients presents with low grade or indolent CLL, about half of them will eventually develop a more progressive disease, which is reflected by aggravating clinical symptoms.[5] The identification of these early stage patients with a realistic risk for disease progression has long been a challenge in CLL research. As these patients might benefit from early treatment, it is crucial to assess patients‘ prognosis at diagnosis, allowing individual risk-adapted therapy. During the last decades many efforts have been made to identify prognostic markers in CLL and nowadays tens of different parameters linked to prognosis have been determined [6], including clinical staging systems [7, 8], morphological criteria [9], serum parameters [10-12], cytogenetic aberrations [13] and several molecular markers.[14-16]

Opportunities for Gene Transfer in CLL Research Although prognostic markers in CLL have mainly been studied from a clinical and practical point of view, the value of fundamental functional studies of these markers should not be underestimated. Research about the cellular and molecular mechanisms underlying the prognostic differences can provide useful insights into the biology and pathogenesis of CLL and may ultimately lead to the identification and characterization of new therapeutic targets. In order to perform such functional studies, gene transfer could be an extremely useful tool, since it allows selective overexpression of one or more specific genes of interest. Instead of comparing cells from two different patient subsets, gene transfer enables the comparison of two cell populations simply and solely differing in the expression of one specific gene, so the observed biological differences can be attributed unambiguously to the gene or protein under study. Of course this argumentation holds true for any gene or protein in any cell type, but in CLL gene transfer could be of special interest, given the existence of prognostic subgroups, identified by different prognostic markers. Furthermore, gene transfer can also contribute to a better understanding of the function of genes and proteins involved in important cellular processes like apoptosis, proliferation, migration or signal transduction.

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Next to functional studies, effective gene therapy also depends on efficient gene transfer methods. Gene therapy strategies show great promise in CLL [17], which is still considered incurable with currently available treatment options like chemotherapy and monoclonal antibodies. In general, gene therapy approaches in CLL aim at generating a cytotoxic T cell response against the leukemic cells after inducing the expression of immunostimulatory genes like CD40L on CLL cells by gene transfer. In contrast to solid tumors, CLL cells are easily accessible, so large numbers of cells can be obtained by blood collection or leukapheresis. This allows for ex vivo gene transfer before re-introducing the manipulated cells by intravenous infusion, so they can serve as an anti-leukemia vaccine. Although many factors are thus in favor of gene therapy in CLL, the development of these strategies is hampered by one major drawback: the difficulty in obtaining efficient, stable and non-cytotoxic gene transfer in CLL cells. In conclusion, both fundamental and clinical CLL research could strongly benefit from the availability of a reliable method for gene transfer in CLL. Current gene transfer methods can be divided into two categories: viral (or biological) and non-viral (or non-biological) methods. In the following section, a general overview of these methods is provided.

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Viral Versus Non-Viral Gene Transfer Methods Viral gene transfer is based on the use of modified viruses to deliver a gene of interest (GOI) into host cells, while non-viral vectors rely on chemical or physical techniques such as lipofection or electroporation. In general, the application of viral vectors is associated with a superior gene transfer efficiency and transgene expression stability compared to non-viral vectors. However, the latter are easier and less expensive to produce, cause less immunogenic responses and hold no risk for insertional mutagenesis. Since the strengths and weaknesses of both approaches are complementary (Table 1), the ‗ideal‘ technique for gene transfer does not exist. Viruses are naturally evolved vehicles carrying DNA or RNA. For their survival and replication they are completely dependent on efficient transfer of their genetic material into host cells. These viral mechanisms can be exploited and different cell types can be infected with modified viruses to introduce foreign genetic material. This process is also referred to as ‗transduction‘. Obviously, viruses need to be genetically engineered in order to avoid unwanted viral replication or disease. Generally, certain viral genes are replaced by one or more GOI‘s, so that the produced viruses can infect cells and introduce the GOI‘s, but cannot replicate and spread to other cells. Different viral vectors have been developed, each with their own advantages and disadvantages. The characteristics of the most commonly used viral vectors are summarized in Table 2.[18-23] Non-viral gene delivery methods have been developed in an attempt to circumvent different problems encountered with viral vectors [24-27]. The process of introducing foreign genetic material into target cells is called ‗transfection‘. Two main categories of transfection methods have been developed: (1) chemical methods, based on artificial vectors of DNA or RNA complexed with cationic lipids or polymers, referred to as ‗lipofection‘ and ‗polyfection‘, respectively. (2) Physical methods like particle bombardment, electroporation and nucleofection, where genetic material is literally forced into the target cells.

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Table 1. Main advantages and disadvantages of viral and non-viral gene transfer methods Viral gene transfer Advantages

Non-viral gene transfer

in general high efficiency less safety issues stable transgene expression is possible no risk for insertional mutagenesis limited effect on viability less immunogenic easy vector production relatively cheap vector production no size limits for transgene cell division is not required

Disadvantages safety issues risk for insertional mutagenesis immunogenic difficult vector production expensive vector production size limits for transgene cell division is often required

in general low efficiency only transient transgene expression possible adverse effects on viability

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Table 2. Properties of the most commonly used viral vectors

Oncoretrovirus

Lentivirus

Adenovirus

Adeno-associated virus

Herpes simplex virus

Genome

ssRNA

ssRNA

dsDNA

ssDNA

dsDNA

Genome size

~7-10 kb

~7-10 kb

~36 kb

~4.7 kb

~152 kb

Insert size

< 8 kb

< 8 kb

< 30 kb

< 4 kb

< 100 kb

Site

genome

genome

episome

genome/episome

episome

Expression

stable

stable

transient

stable/transient

transient

Mitosis

required

not required

not required

not required

not required

Main advantages

integration

integration

high titer virus preparations

integration

large insert capacity

stable expression stable expression

large insert capacity

stable expression

infects only dividing cells

immunogenic

small insert capacity cytopathic

Main problems

insertional mutagenesis

insertional mutagenesis

difficult to produce

immunogenic difficult to produce

ss = single stranded, ds = double stranded, kb = kilobases.

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Most of these methods have been applied in CLL research, with varying success. In the following sections, both viral and non-viral gene transfer in CLL will be discussed, with emphasis on electroporation-based gene delivery.

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VIRAL GENE TRANSFER IN CLL CELLS So far, no efficient retroviral transduction of CLL cells has been reported, due to the intrinsic quiescent nature of CLL cells [28, 29] combined with the restriction of retroviruses to infect only actively dividing cells.[30] Human immunodeficiency virus (HIV) derived pseudotyped lentiviral vectors were shown to be an efficient transfer systems for human lymphocytes, including B cell lineages [31, 32] and activated primary B cells.[33] However, cell cycle activation, at least from G0 to G1 appears necessary for lentiviral transduction [34] and quiescent primary B cells could not be transduced with lentiviral vectors.[33] Up to now, no reports with regard to efficient lentiviral transduction of CLL cells have been published. The first successful viral gene delivery in CLL cells was achieved with adenoviral vectors.[35, 36] Although these vectors should be able to infect quiescent or post-mitotic cells, CLL cells required pre-activation with an anti-CD40 antibody [35, 36] or CD40L combined with interleukin-4 (IL-4) [36] in order to permit efficient transduction. Nevertheless, transduction efficiencies up to 70% could be obtained with these approaches. Integrin expression needed for vector internalization increased after stimulation with antiCD40 [35] or after co-culture with a human embryonic fibroblast cell line.[37] The latter method allowed efficient adenoviral gene transfer of enhanced green fluorescent protein (EGFP), IL-2 and human CD40L.[37] Further research building on the results of Cantwell et al. demonstrated efficient gene transfer of murine CD40L into CLL cells by replication defective adenoviruses [38], which eventually resulted in the first phase I clinical trial using adenoviral vectors in CLL patients.[17] Not only immunotherapy related research, but also more fundamental functional studies benefited from the development of adenoviral vectors. The group of Kipps used these vectors to introduce ZAP70, ZAP70 mutants and several genes related to apoptosis into CLL cells in order to elucidate the function of these proteins in the biology of CLL.[39-43] Since adenoviral vectors could elicit toxic [44] or immunogenic [45] cellular responses, some researchers turned to adeno-associated viruses for gene transfer in CLL. Efficient transgene expression after transduction with recombinant adeno-associated virus (rAAV) was first demonstrated in 2002 by Wendtner et al..[46] However, transfer of the transduced CLL cells on irradiated CD40L expressing feeder cells appeared to be a prerequisite for efficient transduction.[46, 47] Efficiency could be increased from ~25% to ~60% by adding stimulatory CpG-oligodeoxynucleotides to the co-culture system [48] and later it was demonstrated that the time-consuming and laborious use of the feeder cell line could be replaced by crosslinking of the B cell receptor (BCR).[49] CLL cells were found to be highly sensitive for infection with an herpes simplex virus-1 (HSV-1) derived viral vector,[50] indicating the potential use of HSV vectors for gene transfer in CLL, which was later confirmed by another study.[51] Finally, the use of amplicon vectors also allowed efficient transgene expression in CLL cells.[52, 53]

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The natural tropism of Epstein Barr virus (EBV) for B cells was exploited in one study, based on the use of helper virus-free recombinant EBV vectors to deliver transgenes in CLL cells. Two days after infection, transgene expression was observed in up to 85% of cells, without the need for pre-activation.[54]

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ELECTROPORATION OF PLASMID DNA IN CLL CELLS Although electroporation of mammalian cells was reported for the first time in 1982 [55], the first paper describing successful electroporation of primary B-CLL cells appeared only 8 years later [56]. After attempts with calcium phosphate precipitation, DEAE-dextran transfection, polybrene-DMSO-glycerol transfection, lipofection and liposome fusion, these authors turned to electroporation for the introduction of plasmid DNA into primary tonsillar B cells and CLL cells. After optimization, efficient gene transfer could be demonstrated in both cell types, using a CAT reporter assay. Viability was strongly affected by the procedure and was between 20% and 30% at 24 hours after electroporation. Remarkably, in contrast to the tonsillar B cells, CLL cells had to be pre-activated with TPA (12-O-tetradecanoylphorbol 13acetate) for 3 days before electroporation, in order to obtain a transfection efficiency of 510%. The relatively low transfection efficiency and survival still remain the main problems encountered with electroporation of plasmid DNA in CLL cells. The decreased viability post electroporation is probably due to DNA related cytotoxicity [57], and not to the cointroduction of bacterial components [58], since the use of endotoxin free plasmid preparations does not seem to improve cell survival post electroporation.[59, 60] On the other hand, the lack of an active (post-)-transcriptional cellular machinery in CLL cells might be responsible for the low transgene expression levels. Nevertheless, since viral methods also suffer from specific drawbacks in CLL cells, the possibilities of electroporation-based methods have been explored further in CLL research. In 2005, MaxCyte Inc. presented the first results obtained with a new electroporationbased gene delivery system (MaxCyte GT), that can be scaled up to a wide range of volumes, is configured as a sterile and closed system and is specifically designed for clinical gene therapy.[61-63] The applicability of this method was proven with EGFP and CD40L encoding plasmids. EGFP marker gene expression could be obtained in 52% of the processed CLL cells, whereas CD40L expression could be detected in 56% of the CLL cells. A decrease in average viability from 83% to 68% was observed when cells were electroporated with plasmid DNA, but not when FITC (fluorescein isothiocyanate)-dextran, mRNA or siRNA were introduced in the cells, illustrating once more the cytotoxic effects of plasmid DNA. In further preclinical experiments, it was demonstrated that CLL cells electroporated with CD40L could induce an allogeneic immune response. Furthermore, co-culture of CD40L expressing CLL cells and normal CLL bystander cells resulted in the upregulation of immunostimulatory genes in the bystander CLL cells.[61, 62] Since these results indicated that the manipulated CLL cells could serve as an effective anti-leukemia vaccine, a phase I/II clinical trial was initiated. Final results and conclusions of this study are not yet available, but interim results point out that the vaccine was well tolerated by the patients and that white

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blood cell counts remained stable.[62, 64] Up to present, this is the only clinical gene therapy trial in CLL patients based on electroporation, and as far as we know results are promising. Although electroporation of CLL cells with plasmid DNA was proven to be feasible, it was rarely used in fundamental research. This changed when a novel electroporation based technique called nucleofection was introduced in 2004.[65] Amaxa‘s Nucleofector technology is a non-viral gene transfer method, based on a cell type specific combination of electrical parameters and solutions, designed to deliver different biomolecules directly into the cell‘s nucleus. Both the electroporation device and the buffers are provided by Amaxa. Nucleofection programs are indicated by a unique letter-number combination, so no data with regard to the exact electrical parameters like voltage or capacitance can be retrieved. Despite these limitations, nucleofection has proven to be a valuable tool for gene transfer in primary cells and hard-to-transfect cell lines. In a pilot study, nucleofection of plasmid vectors resulted in transgene expression in 5-43% of CLL cells, with an average viability of 50%.[65] During the last few years, nucleofection has become the most frequently used method for gene transfer experiments in CLL. Reported efficiency rates after nucleofection with DNA vectors range from 20% to 70%, with a maximum viability of 60% (Table 3). Viability can be improved by transferring the CLL cells to stromal feeder cells after nucleofection with plasmid DNA, but is consistently lower than in mock nucleofected cells (without DNA).[59] The relatively low survival rates after nucleofection of plasmid DNA were only recently examined in more detail.[59] It was demonstrated that cell viability correlated with the amount as well as the size of the plasmid used for nucleofection. Furthermore, the structure of the plasmid also affected viability, since cells nucleofected with linearized plasmid vectors showed a survival similar to that of control nucleofected samples. However, transgene expression levels decreased when linearized plasmids were used instead of circular plasmids.[59]

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Table 3. Electroporation and nucleofection of CLL cells Method

Biomolecule

Settings

Survival (%)

Efficiency (%)

Time point

Ref.

EP

pDNA

625 V/cm,960 µF

20-30%

5-10%

24h

[56]

NF

pDNA

U15

50%

5-43%

5h

[65]

NF

pDNA

U16

50%

20%

18h

[77]

EP

pDNA

ND

25%

60-70%

24h

[61] [62]

NF

pDNA

U15

ND

ND

48h

[74]

NF

pDNA

U15

20-60%

50-70%

24h

[59]

NF

pDNA

U13

> 50%

60%

16-20h

[78]

NF

pDNA

U13

ND

40-60%

5h

[79]

NF

pDNA

U15

20-55%

30-55%

24h

[80]

NF

pDNA

U15

30-45%

30%

18h

[81]

NF

pDNA

U13

26%

36%

24h

[60]

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Table 3. (Continued)

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Method

Biomolecule

Settings

Survival (%)

Efficiency (%)

Time point

Ref.

NF

pDNA

U13

ND

40-60%

5h

[66]

NF

pDNA

U15

ND

20-30%

30h

[82]

EP

pDNA

ND

88%

56%

ND

[64]

NF

pDNA

ND

ND

ND

12h

[83]

EP

mRNA

ND

90%

90%

3h

[62]

NF

mRNA

U15

45-80%

80-90%

24h

[59]

EP

mRNA

1250 V/cm, 150 µF

70%

90%

24h

[60]

NF

mRNA

U13

35%

ND

48h

[66]

EP

antisense

750-1250 V/cm, 900 µF ND

ND

24h

[68]

EP

antisense

750-1250 V/cm, 900 µF 70%

100%

24h

[67]

EP

antisense

ND

ND

ND

ND

[69]

EP

dsODN

1100 V/cm, 10 ms

60-80%

75-100%

24h

[72]

NF

siRNA

ND

ND

ND

24h

[73]

NF

siRNA

U15

ND

89%

24h

[74]

NF

siRNA

ND

ND

80%

ND

[70]

NF

siRNA + pDNA U13

ND

ND

48h

[75]

NF

siRNA

U15

~28%

ND

48h

[81]

NF

siRNA

U13

52%

ND

24h

[66]

NF

siRNA

X5

> 90%

37.7%

3h

[71]

EP

protein

750-1250 V/cm, 900 µF 68%

58%

ND

[68]

EP

protein

625V/cm, 900 µF

58%

0.5h

[76]

89%

EP = electroporation, NF = nucleofection pDNA = plasmid DNA, ND = not determined, dsODN = double stranded oligonucleotide

ELECTROPORATION OF MRNA IN CLL CELLS Given the adverse effects associated with the introduction of DNA in (CLL) cells, other ways to establish overexpression of target genes were explored. One option is the use of mRNA instead of DNA. mRNA of virtually any gene can be generated easily by in vitro transcription from a DNA template containing the coding region of the GOI. Consequently,

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nuclear import of DNA is avoided, so it suffices to deliver the mRNA directly in the cytoplasm. Only the translation of mRNA to protein needs then to be carried out by the cellular mechanisms. In CLL research, only a few research groups have yet adopted this approach, using both electroporation [60, 62] and nucleofection [59, 66] (Table 3). Survival as well as efficiency rates of mRNA transfection are clearly superior to the results obtained with plasmid DNA vectors. Viability is not or only marginally affected by the procedure, with values similar to non-electroporated or mock electroporated samples. Up to 90% of mRNA electroporated CLL cells show clearly detectable levels of transgene expression. Up to now, EGFP, CD40L, CD79b, ZAP70, ZAP70-EGFP fusion, Akt and MEK2 have all been successfully overexpressed in CLL cells after electroporation of the corresponding mRNA.[59, 60, 62, 66] It is evident that stable transfection cannot be obtained by mRNA electroporation, and in dividing cells the introduced mRNA and the resulting protein are distributed among the daughter cells with each cell division, leading to a dilution effect. However, given the fact that the majority of circulating CLL cells are arrested in the G0/G1 phase of the cell cycle, the latter phenomenon does not seem to be of much importance in CLL. If future research demonstrates that mRNA electroporation does not induce any unwanted side-effects, this method is not only of great value in in vitro experiments, buts also holds promise for gene therapy applications.

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ELECTROPORATION OF SIRNA AND ANTISENSE OLIGO’S IN CLL CELLS Not only increasing or introducing the expression of a certain GOI, but also the inhibition or silencing of gene expression can provide important clues to the molecular and cellular function of a GOI. Therefore, electroporation and nucleofection have been applied to introduce antisense or siRNA molecules into CLL cells (Table 3). In general, transfer of these small RNA molecules is highly efficient and has little or no effect on cell survival. Different genes related to apoptosis (e.g. Mcl-1 [66-68], FLIP [69], BAFF [70], Bcl2A1, Bcl-X [71], Bcl-XL and XIAP [66]), signaling (e.g. STAT1 [72] and IL-10 [73]) or other cellular pathways (e.g. Mda-7 [74] and c-abl [75]) were efficiently silenced after electroporation or nucleofection of antisense or siRNA.

ELECTROPORATION OF PROTEINS IN CLL CELLS Next to introducing DNA or mRNA into CLL cells, a third way to study the overexpression of a GOI is to introduce the protein itself instead of its genetic code. This way, functioning cellular mechanisms for DNA transcription, mRNA translation and/or protein modification are no longer a prerequisite for efficient transgene expression. In CLL research, the use of electroporation to introduce foreign proteins into CLL cells is limited (Table 3), although positive results were obtained. After optimization of the electroporation buffer, voltage and capacitance, efficiency of protein transfer to CLL cells was nearly 60%, with survival rates ranging from 70 to 90%.[68, 76] The molecular weight of the introduced

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proteins had no influence on electroporation efficiency nor cell survival; proteins of up to ~150 kDa could be delivered in CLL cells by electroporation.[76] This approach was used to study possible defects in apoptotic pathways, responsible for chemotherapy resistance, by electroporating different pro- and anti-apoptotic proteins in CLL cells [68].

CONCLUSION Electroporation has proven to be a valuable tool in CLL research. As these cells are hard to infect by commonly used viral vectors, electroporation and nucleofection are the most useful alternatives for gene transfer in CLL cells. Electroporation is a versatile method, which may be used for the introduction of different types of nucleic acids, but also of larger biomolecules like proteins. For overexpression studies, the use of mRNA is preferred to that of DNA, since it is associated with significantly less pronounced cytotoxic effects. The application of electroporation in overexpression and silencing studies with similar experimental setups should be able to generate unequivocal results regarding the cellular and molecular function and importance of the protein(s) under study. This research will not only improve the basic understanding of CLL‘s pathobiology, but may ultimately lead to the identification and characterization of new therapeutic targets in CLL. Furthermore, it is clear that electroporation-based methods are suitable for gene therapy strategies in CLL, which need to be evaluated further in clinical trials. Eventually, not only researchers but even patients may benefit from electroporation in CLL.

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In: Electroporation in Laboratory and Clinical Investigations ISBN 978-1-61668-327-6 Editors: Enrico P. Spugnini and Alfonso Baldi © 2012 Nova Science Publishers, Inc.

Chapter 17

FUTURE DEVELOPMENTS IN ELECTROPORATION: RECOMBINANT CLOSTRIDIA AS VIABLE AND TARGETED TUMOUR THERAPEUTICS Tam H Nguyen1, Siyu Cao2, Shu-Feng Zhou3, and Ming Q Wei 2* 1

Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute, The University of Queensland, St Lucia, QLD 4072, Australia 2 Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical Research, School of Medical Science, Griffith University, Gold Coast campus, Southport, Qld 4222, Australia 3 School of Health Sciences, RMIT University, Victoria 3083, Australia

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ABSTRACT The importance of bacteria in everyday life is underscored by their wide usage in numerous processes and applications, such as alcohol and food fermentation, waste disposal, and production of therapeutic molecules and vaccines. However, the recognition of the potential usefulness of bacteria as bona fide therapeutics has been less enthusiastic. In the quest to develop novel tumour therapeutics that can be specifically targeted and have greater efficacy and fewer side effects, recombinant clostridia are regaining attention as viable therapeutic candidates. The importance of the Clostridia in terms of their innate potential role in tumour targeted therapy has prompted many studies. These include their mechanisms of tumour targeting, the way they cause oncolysis, and methods in genetic manipulation of these organisms. This chapter summarizes the recent advances in the use of bacteria as viable therapeutics, and discusses how these advances could be potentially improved by the use of genetic engineering tools, such as electroporation (more detailed methods, see chapters 5 and 8). We predict that recombinant Clostridia could potentially provide an excellent platform for the development of a range of bacterial-targeted therapeutics for a variety of solid tumours. *

Corresponding author: Professor Ming Q Wei, Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical Research, School of Medical Science, Griffith University, Gold coast campus, Southport, Qld 4222, Australia Fax: +61 7 55528908, E-mail address: [email protected] (M.Q. Wei).

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INTRODUCTION

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Bacterial Targeted Tumour Therapy - Past and Present Spontaneous tumour regression is not a myth. Although there might be other reasons for such an event to occur, the correlation of bacterial infection and spontaneous tumour regression dates back almost two hundred years. As early as 1813, Vautier observed tumour regression and cure in patients with gas gangrene manifestation, a bacterial infection that is now known to be caused by Clostridial bacterial species. This observation, for the first time, suggested that bacteria such as Clostridia could target tumours and induce antitumour effects. The first deliberate use of bacteria to treat cancer patients was performed by the German physician W. Busch in 1868 (Hall 1998). He induced an ―erysipelas‖ (Streptococcus pyrogenes) infection in a female patient with inoperable sarcoma and reported that the primary tumour had shrunk to half its size within a week of initial infection. However, this treatment was administered without the knowledge of the aetiology of erysipelas and the patient died 9 days post-infection. In 1883, another German surgeon Friedrich Fehleisen, repeated the treatment and furthermore, discovered that the cause of erysipelas was Streptpcoccus pyrogenes (Fehleisen 1882). Several years later, a young American surgeon named William B Coley was treating a patient with malignant round-celled sarcoma of the neck who was accidentally infected with erysipelas. Like the previous two physicians, he also observed a striking regression or the tumour in this patient. These encouraging observations of tumour regression inspired Coley to begin deliberately infecting (treating) cancer patients with erysipelas, and so convinced was he about the effectiveness of this treatment that he devoted much of his life to studying the use of bacteria in cancer treatment. Coley initially used live S. pyrogenes in his treatments but later resorted to using a combination of heatkilled S. pyrogenes and S. marcesens, now commonly referred to as ―Coley‘s toxins‖, which gave him more effective and reproducible results (Coley 1893, 1894, 1991). The efforts of Coley and his predecessors gave promising results for the prospect of treating cancer tumours and laid the foundation for later work by others in developing the field now known as tumour immunotherapy. Other attempts to use bacteria for tumour therapy have included the use of Clostridia histolyticus spores to induce oncolysis and tumour regression in transplantable mouse sarcoma (Parker et al. 1947). More recently, developments in the field have included the use of other anaerobic bacteria such as Bifidobacterium (Kimura et al. 1980; Yazawa et al. 2000), and facultative anaerobes such as E. coli (Yu et al. 2004), Salmonella (Clairmont et al. 2000; Soghomonyan et al. 2005). The focus of most of the research relating to the use of these bacteria for cancer therapy is on improving intratumoral targeting and controlled delivery of therapeutics. Once these issues are satisfactorily addressed, it is envisaged that bacteria will play a prominent role in the treatment of cancer.

The Unique Microenvironment of Solid Tumours Almost 90% of all human cancers are solid tumours. Over the last century, our understanding of the pathogenesis and patho-physiological features of solid tumours has advanced dramatically. This includes important scientific breakthroughs which have

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contributed greatly to the difficult and challenging task of creating a successful treatment for cancer. Several key findings relating to tumour biology have been invaluable to the research effort to find a cure. (I) Tumours must undergo angiogenesis to grow beyond microscopic size (Folkman 1971); (II) The proliferation and survival of cancer cells is mediated by gainof-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes (Weinberg 2007); (III) Cancer cells have an elevated rate of glycolysis and a decreased rate of oxidative metabolism as compared to normal cells (Warburg 1930); (IV) Most solid tumours contain regions with very low oxygen concentration, and that such intratumoral hypoxic conditions play an important role in tumour development and further progression (Harris 2002).

Mechanisms of Bacteria Targeting- Solid Tumour Microenvironment

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Since most anticancer drugs are designed to target highly proliferating cells, hypoxiainduced inhibition of tumour cell proliferation is one of the major reasons why chemotherapy has not been as effective as expected. During the last decade, a number of studies have shown that tumour hypoxia is directly involved in tumour progression and correlates with a more aggressive phenotype (Subarsky and Hill 2003; Chan and Giaccia 2007; Sullivan and Graham 2007). Both clinical studies and studies in animal models have indicated that solid tumour hypoxia plays an important role in distant metastasis (Hockel et al. 1996; Cairns et al. 2001; Fyles et al. 2002; Cairns and Hill 2004). In addition, findings from Graeber and colleagues (Graeber et al. 1996) suggested that tumour hypoxia might predispose tumours to a more malignant phenotype by selecting for mutant p53.

Figure 1. Primary tumour hypoxia. There are two types of hypoxia in solid tumour: diffusion limited hypoxia and perfusion limited hypoxia. While diffusion limited hypoxia is a chronic process, perfusion limited hypoxia is transient and fluctuating. (Adapted from Lunt et al., 2009)

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It has been demonstrated that hypoxia mainly exerts its impact on cells via a transcription factor called hypoxia-inducible factor (HIF)-1 (Wang et al. 1995). HIF-1 belongs to the basic helix-loop-helix superfamily of eukaryotic transcription factors. It is a heterodimer composed of an α and β subunit. There are three human HIF α genes, HIF-1α, HIF-2α, and HIF-3α. While the expression of its β subunit (also known as aryl hydrocarbon receptor nuclear translocator, ARNT) is constitutive and not influenced by oxygen levels, all three α subunits are sensitive to and controlled by oxygen levels (Gruber and Simon 2006). Under normoxia conditions, HIF-1α is unstable and almost undetectable. Its conserved prolyl residues within the oxygen-dependant degradation domain are initially hydroxylated by prolyl hydroxylases and subsequently targeted for degradation through the ubiquitin-proteasome system by binding to von Hippel-Lindau (VHL) protein (Zhou et al. 2006). However, under hypoxic conditions, prolyl hydroxylase is rendered inactive leading to HIF-1α accumulation followed by dimerization with HIF-1β. Studies have shown HIF-1 plays an extremely important role in tumour progression, angiogenesis, and metastasis (Hockel et al. 1996; Maxwell et al. 1997; Manalo et al. 2005). High levels of HIF-1α have been directly linked to poor prognosis in patients with various malignant tumours such as non-small cell lung carcinoma, breast, stomach, ovarian, and cervical tumours (Birner et al. 2000; Birner et al. 2001; Schindl et al. 2002; Takahashi et al. 2003; Liu et al. 2006). Additionally, the induction of HIF-1 is also partially responsible for the resistance of hypoxic tumour cells to chemotherapy and radiation therapy (Zhou et al. 2006). Tumour hypoxia is a therapeutic concern since it can reduce the effectiveness of drugs and radiotherapy, where well oxygenated cells requiring one third of the dose of hypoxic cells to achieve a given level of cell killing. Cancer stem cells might be the cause of tumour recurrence, sometimes many years after the appearance of the successful treatment of a primary tumour. Thus, the primary objective of such a treatment system will be to provide sufficient selective toxicity to both kill cancer stem cells and cells of hypoxic fractions of the tumour.

Low Extracellular pH in Solid Tumour Low extracellular pH is another key feature of the microenvironment of tumours. While normal cells produce their energy through mitochondrial respiration, tumour cells obtain their energy mainly via glycolysis due to abnormal vasculature and the resultant hypoxia (Fantin et al. 2006). However, it has been demonstrated that tumour cells are able to maintain high rates of anaerobic-like glycolysis even in the presence of oxygen (Warburg 1956). Additionally, a recent study has indicated that not all highly glycolytic tumour regions are necessarily hypoxic (Rajendran et al. 2004). A possible explanation for this phenomenon might be that stabilization of HIF-1α via inactivation of VHL (Kaelin 2005) and thus leading to lack of hypoxia. HIF-1 has been found to control the expression of many genes that regulate pH such as CA-IX, the glucose transporters (Glut-1 and 3), and important enzymes in the glycolytic pathway such as aldolase, enolase and lactate dehydrogenase (Bartrons and Caro 2007; Semenza 2007). Glycolysis-induced lactic acid accumulation results in the increased acidity of the tumour microenvironment. Several studies have demonstrated a correlation between a low extracellular pH and increased tumour invasion or metastases (Schlappack et al. 1991; Rofstad et al. 2006). More importantly, the acidic tumour microenvironment could select for

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cells with mutant p53 leading to clonal expansion of aberrant cell populations (Gatenby and Gillies 2004).

Elevated Interstitial Fluid Pressure (IFP) of Solid Tumour Another important patho-physiological feature of solid tumours is elevated IFP. While microvascular pressure is the principal driving force (Boucher and Jain 1992), several factors are involved in the elevated IFP in solid tumours . Firstly, the walls of newly formed tumour vessels triggered by angiogenesis often have big pores resulting in increased permeability. This high vessel wall permeability promotes the movement of fluid from inside the vessels to the extra-cellular matrix (Bermudes et al., 2001). Secondly, due to lack of functional lymphatic vessels in tumours (Padera et al. 2002), fluid accumulates in tumour interstitial contributing to elevated IFP. Thirdly, the stroma surrounding the tumour cells differs from that of normal tissues and is characterized by an abnormal extracellular matrix composition, increased microvessel density, and an infiltration of macrophages and other inflammatory cells, leading to what is frequently referred to as ‗reactive stroma‘. A consequence of the infiltration of the inflammatory cells of the immune system is that various cytokines are released in the tumour tissue, such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-)and vascular endothelial cell growth factor (VEGF) as well as other angiogenic factors. These factors can subsequently act on different cell types in the tumour and affect the IFP (Heldin et al. 2004).

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Table 1. Overview of the most profound physiological differences of solid tumours from healthy tissues Structure/characteristics

Emerging abnormalities

Vasculature

Irregular, arterio-venous shunts, blind ends, incomplete endothelial linings, vessel leakage

Centre of the tumour

Necrosis

Hypoxia fraction

Hypoxia is a pathophysiologic consequence of the structurally and functionally disturbed vasculature and the deterioration of diffusion

Cancer Stem Cell

Re-emerge of cancer

Lymph cytes

Infiltrating, immunosuppression,

Interstitial fluid pressure (IFP)

Elevated up to 100 mm Hg

Oxygen tension gradient

Normal tissue pO2 ranges between 10 and 80 mm Hg vs. regions of pO2 < 5 mm Hg in cancer tissues

pH

Acidic, low extracellular pH (pHe)

Increased tumour IFP can act as a barrier to various anti-cancer drug agents leading to insufficient drug delivery and reduced efficacy. Indeed, several studies have demonstrated Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

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improved uptake of chemotherapeutic drugs following reduction of tumour IFP (Pietras et al. 2002; Vlahovic et al. 2007). A direct relationship between IFP and disease progression, however, has not been conclusively demonstrated.

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Anaerobic Bacteria as Anti-Cancer Agents Clearly, hypoxia is one of the prominant characteristic pathological events in solid tumours (Brown and Giaccia 1998). Although tumour hypoxia induces problems such as resistance to chemotherapy and radiation therapy and increased angiogenesis and/or metastases, it also provides a perfect condition for the growth of obligate anaerobic bacteria. To date, three classes of anaerobic bacteria have been tested for their anti-cancer capacity: (1) the lactic acid-producing, Gram-positive obligate, anaerobic, non-sporulating bacteria represented by Bifidobacteria; (2) the intracellular, Gram-negative facultative anaerobic bacteria represented by Salmonella and (3) the saccharolytic/proteolytic, Gram-positive, strictly anaerobic, spore forming bacteria represented by Clostridium. Among the members of Bifidobacterium, three species were the focus of studies including B. longum, B. infantis and B. adolescentis. They are all common flora of human intestine and regarded as ―safe bacteria‖. Tests using Bifidobacteria in cancer therapy by intravenous injection of B. longum into mice implanted with Ehrlich ascites tumours have shown that the bacteria are highly selective and localised mainly within the tumour. Moreover, virtually no bacteria were detected in other organs 96 hours after injection. Unfortunately, no obvious oncolytic effect was observed in this study (Kimura et al. 1980). Meanwhile, another study also showed that B. adolescentis triggered tumour apoptosis and inhibited the occurrence and development of colorectal carcinoma in vivo (Wang et al. 1999). Furthermore, B. longum was also engineered by Fujimori‘s group to deliver the suicide gene cytosine deaminase/5-C prodrug combination as well as the endostatin gene to tumour models through intravenous injection (Yazawa et al. 2000). The drawback of using bifidobacteria is that they do not form spores and as a result, are more susceptible to hostile conditions and are inconvenient to handle and store. Besides bifidobacteria, the intracellular, Gram-negative facultative anaerobic Salmonella typhimurium has also been studied as a possible anticancer agent. Since most Salmonella strains are pathogenic due to substantial immuno-stimulation produced by Salmonella lipopolysaccharide and other virulence factors, Salmonella strains used for anticancer therapy were all genetically attenuated. Furthermore, additional genetic modifications have been applied to attenuated Salmonella enabling them to express a variety of genes coding for therapeutic proteins such as cytosine deaminase (King et al. 2002). To date, two clinical trials have been carried out since the development of attenuated Salmonella as a new anticancer agent (Toso et al. 2002; Nemunaitis et al. 2003). However, both trials failed to show sufficient clinical efficacy, possibly due to insufficient tumour colonization by the bacteria. In addition to Bifidobacteria and Salmonella, Clostridium have also been investigated for the purpose of anti-cancer treatment. Clostridium are Gram-positive, strictly anaerobic, spore forming bacteria. Importantly, members of the Clostridium family are mostly motile and peritrichously flagellated, properties which are advantagous for better tumour colonization. In 1813, the observed recovery of cancer patients who were also concurrently infected with gas gangrene, an infection caused by clostridium, represented one of the first lines of evidence to suggest an anti-cancer effect for this bacterium. However it wasn‘t until 1947 that

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Clostridium was first investigated for its anti-cancer potential. Experiments using C. histolyticum spores directly injected into mouse sarcoma induced tumour oncolysis and shrinkage (Parker et al. 1947). Later experiments using C. tetani further confirmed that clostridial spores only germinate and colonize in solid tumours (Malmgren and Flanigan 1955). This study also demonstrated that direct injection of clostridial spores into tumours was not necessary, and that intravenous administration of spores was enough to induce similar anti-tumour effects. In 1959, a non-pathogenic strain of C. butyricum M-55 was isolated by Moese and colleages (Moese and Moese 1959). This clostridial strain is now known as the C. sporogenes strain (ATCC13732). The intravenous administration of the non-pathogenic strain spores also induced tumour oncolysis (Minton 2003). Furthermore, C. novyi ATCC19402 and C. sordellii ATCC9714 were also tested as anti-cancer agents due to their extensive colonization ability within tumour necrotic regions (Dang et al. 2001). One possible mechanism of the clostridial anti-tumour effect had been atributed to their fast speed of dividing. This quick rate of multiplification is much faster than that of cancer cell division, enabling rapid build-up of bacterial mass. This will effectively compete for limited nutrients, starving cancer cells and attenuating growth. Another mechanism might be due to the action of hydrolytic enzymes including proteases and lipases, which kill the hypoxic tumour cells. An additional possible mechanism is that Clostridial spores might induce the host‘s immune response to eliminate tumour cells (Van Mellaert et al. 2006). However, although administration of clostridial spores induced tumour lysis in most cases, complete tumour control was not reported. An outer rim of solid tumour consistently persisted resulting in tumour regrowth (Minton 2003). Moreover, Clostridium induced oncolysis frequently caused experimental animal death, possibly due to systemic toxicity triggered by the release of necrotic tumour debris and bacterial inflammation (Minton 2003).

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Designer Bacteria - Genetic Manipulation to Improve Anticancer Potency Bacteria have enormous capacity to adopt and evolve. Recent advances in recombinant DNA technology have made directional evolvement easier, reigniting interest in genetic manipulation and the use of genetically modified anaerobic bacteria as anticancer agents. Amongst the three major classes of bacteria, Bifidobacterium has been primarily used as a targeted gene delivery vehicle after genetic modification. B. longum was first engineered by Professor Fujimori‘s group in Japan to deliver the bacterial cytosine deaminase (CD)/5-FC suicide gene combination as well as the endostatin gene to tumour models using intravenous injection (Fujimori 2006; Yazawa K et al. 2000; 2001). Professor Xu‘s group in China has used to great effect the oral route for administration of these bacterial vehicles (Fu et al. 2005). Similarly, B adolescentis and B. infantis have also been successfully engineered by two separate Chinese groups (Wang et al. 1999). All Salmonella strains tested so far are classified as designer bacteria as only attenuated strains have been used in human (Forbes et al. 2003; Spreng et al. 2006). S. typhimurium was the first to be attenuated and a S. enterica serovar Typhi strain was registered as a live oral vaccine against typhoid fever more than two decades ago. This vaccine strain was found to induce long-term hinderence of tumour growth in a broad range of human and mouse tumours implanted in mice, even up to several weeks after untreated mice had died from the tumours. Further genetic modification has focused on the delivery of therapeutic genes, such as those

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encoding herpes simplex thymidine kinase (TK) (Forbes et al. 2003), E coli CD (Li et al. 2001), tumor necrosis factor α (TNFα) (Lin et al. 1999), and colicin E3 (Clairmont et al. 2000). A phase I study of VNP20009, a lipid-A attenuated strain in humans with melanoma was recently reported. The bacteria were well tolerated. However, there were no antitumor responses observed, and bacteria were only cultured from tumour tissue in 12.5% of patients (Toso et al. 2002). Modification of the kinetics of infusion also failed to improve response or colonization in a small number of subsequently treated patients (Toso et al. 2002). There are two metabolically different types of Clostridia that have been used for genetic modification: The first type was the proteolytic Clostridia, represented by C. sporogenes (Liu et al. 2002; Nuyts et al. 2002). Obviously, a prerequisite for such manipulations is a means of introducing recombinant DNA into individual viable bacterial cells. The techniques needed to genetically modify C. sporogenes were considered rather demanding initially. The presence of DNases produced by many species of Clostridium has contributed to the low or inconsistent transformation efficiences. Therefore, the real success was only reported when Prof. Brown‘s group introduced E. coli CD into C. sporogenes NCIMB10696 by strain specific electroporation (Liu et al. 2002). Intravenous injection of the spores showed super capacity of tumour colonisation and at least 108 CFU/g of tumour tissue was obtained, accompanied by tumour inhibition, which was enhanced by the use of 5-FU. Unfortunately, for reasons unknown, this inhibitory effect did not last. C. sporogenes was considered ―pathogenic‖ because of its extraordinary capacities in tumour colonisation and liquefaction, thus, the use of less aggressive Clostridia was considered to be more advantageous. Prof. Jozef Anne‘s group was the driving force behind the use of saccharolytic Clostridia, including C. beijerinckii, acetobutylicum and butyricum. These strains have industry value and are ―truly non-pathogenic‖ and easy to manipulate. Therapeutic genes, encoding cytokine, TNFα, CD, and nitroreductase have been introduced and a considerable amount of heterologous proteins were efficiently expressed and secreted at the tumour site. However, no significant tumour inhibition was observed in vivo in solid tumour models (Barbe et al. 2004; Van Mellaert et al. 2006). Several factors may explain the lack of antitumour effects, such as insufficient recombinant gene expression and secretion at tumour sites, or, possibly the presence of low numbers of colonising bacteria. At this stage, very little work has been published on the use of these groups of non pathogenic bacteria as successful anticancer agents.

Electroporation for Genetic Manipulation For bacteria to be effective tools for cancer therapy they should ideally be amenable to genetic modification. Genetic modification is a prerequisite for the optimal design of bacteria with effective therapeutic properties, and considerable research has been undertaken in the past decade to achieve such aims. In the case of Clostridia, efforts to genetically manipulate this genus have focused on the use of electroporation for tumour-targeted delivery of therapeutic genes and toxins. Clostridia, however, have proven to be difficult to manipulate. Schlechte and Elbe reported the first attempt at modification of Clostridium (C. sporogenes) whereby an E. coli gene encoding the bacteriocin Colicin E3 was introduced (Schlechte and Elbe 1988). Their results indicated, however, that the recombinant strains were sub-optimal and it was later discovered that the plasmids and electrotransformation conditions used at the

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time were not suitable for electroporation of C. sporogenes. Subsequent to this, Liu and colleagues described a modified protocol for electrotransformation of C. sporogenes NCIMB 10696 using polyethylene glycol-based transfection buffer containing DNase inhibitors (Liu et al. 2002). However, despite the use of DNase inhibitors, which was aimed at overcoming problems associated with the negative effects of exogenous and secreted endonucleases on DNA transfer, the frequency of transfer was still very low. Other studies demonstrating successful transformation of Clostridia using electroporation have been reported, however, these cases have involved strains that were either not suitable for tumour therapy such as C. paraputrificum M-21 (Sakka et al. 2003), or did not confer high levels of hypoxia-associated tumour colonization such as C. perfringens (Jiraskova et al. 2005). More recently, attempts to circumvent these limitations in Clostridia transformation have proved to be more successful. Theys and colleagues were able to obtain C. sporogenes transformants bearing vectors expressing NTR (nitroreductase) at high frequencies using a conjugative transfer method (Theys et al. 2006). The same method was used, for the first time, to successfully transform C. novyi-NT with a plasmid expressing single chain antibodies specific for human HIF-1α (Groot et al. 2007). The conjugative transfer method used in both studies was modified from the one used previously to transform C. defficile (Purdy et al. 2002) and involves the coculturing of plasmid-bearing E. coli donor cells with Clostridia cells, followed by counter selection of the E. coli donor cells to obtain recombinant Clostridia transformants. Importantly, these two studies have demonstrated high levels of expression and functionality of the respective gene products. Thus the conjugative transfer method represents a promising technique for transforming high colonizing strains of Clostridia with therapeutic DNA plasmids, however, continued development of electroporation techniques for the same purpose needs to be continued to complement the initial and future success of the conjugative transfer method.

CONCLUSION After initial excitment and then many decades of neglect, anaerobic bacteria have now remerged as promising potential candidates for solid tumour therapy. Cancer is difficult to treat with conventional approaches due to regions of poor vasculature present transport limitations for oxygen, nutrients, and therapeutic drugs. Bacteria-based therapies have the potential to overcome such limitations due to their inherent ability to actively target intratumoural hypoxic microenvironments. The obligate anaerobic genera Clostridia, has been widely and convincingly demonstrated to selectively target to, colonise, and germinate in hypoxic regions of solid tumours, resulting in initiation of oncolysis of the tumour. Furthermore, facultative anaerobes such as Salmonella can also selectively target to tumours via mechanisms of chemotactic attraction towards chemicals secreted by distinct microenvironments of tumours and preferential growth. The complementary nature of the distinct targeting mechanisms of obligate and facultative anaerobes confers a substantial advantage to anti-tumour therapy. Obligate anaerobes strictly target hypoxic regions that are only present in tumours and not normal tissue, while also having the ability to target metastases and regions outside necrotic regions. Indeed, the true potential of these bacteria can only be realised with adequate advances in methods to genetically manipulate and modify

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the bacteria to be ideal and potent cancer therapies. In this regard, there are several issues that should be addressed, (i) specificity of targeting to tumour tissues, (ii) minimization of toxicity associated bacteria administration and activity, and (iii) the ability to deliver therapeutic genes and drugs. It has been highlighted that the Clostridial strains have the greatest ability to colonize and germinate in hypoxic tumours, but not every strain could easily be manipulated. C. sporogenes is inherently difficult to electroporate. However, it is reassuring that advances in genetic manipulation techniques such as electroporation are continuing with great pace, to address some of the aforementioned issues. It is envisaged that once these issues are reconciled, anaerobic bacteria will be positioned as one of the principal tools for combating cancers.

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Pietras K, Rubin K, Sjoblom T, Buchdunger E, Sjoquist M et al. (2002) Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res 62(19): 5476-5484. Purdy D, O'Keeffe TA, Elmore M, Herbert M, McLeod A et al. (2002) Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Molecular Microbiol 46(2): 439-452. Rajendran JG, Mankoff DA, O'Sullivan F, Peterson LM, Schwartz DL et al. (2004) Hypoxia and glucose metabolism in malignant tumours: evaluation by [18F]fluoromisonidazole and [18F]fluorodeoxyglucose positron emission tomography imaging. Clin Cancer Res 10(7): 2245-2252. Rofstad EK, Mathiesen B, Kindem K, Galappathi K (2006) Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res 66(13): 6699-6707. Sakka K, Kawase M, Baba D, Morimoto K, Karita S et al. (2003) Electro-transformation of Clostridium paraputrificum M-21 with some plasmids. Journal of Bioscience and Bioengineering 96(3): 304-306. Schindl M, Schoppmann SF, Samonigg H, Hausmaninger H, Kwasny W et al. (2002) Overexpression of hypoxia-inducible factor 1alpha is associated with an unfavourable prognosis in lymph node-positive breast cancer. Clin Cancer Res 8(6): 1831-1837. Schlappack OK, Zimmermann A, Hill RP (1991) Glucose starvation and acidosis: effect on experimental metastatic potential, DNA content and MTX resistance of murine tumour cells. Br J Cancer 64(4): 663-670. Schlechte H, Elbe B (1988) Recombinant plasmid DNA variation of Clostridium oncolyticum--model experiments of cancerostatic gene transfer. Zentralblatt fur Bakteriologie, Mikrobiologie, und Hygiene 268(3): 347-356. Semenza GL (2007) HIF-1 mediates the Warburg effect in clear cell renal carcinoma. J Bioenerg Biomembr 39(3): 231-234. Soghomonyan SA, Doubrovin M, Pike J, Luo X, Ittensohn M et al. (2005) Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK. Cancer Gene Ther 12(1): 101-108. Spreng S, Dietrich G and G. Weidinger. (2006) Rational design of Salmonella-based vaccination strategies, Methods 38(2): 133–143. Subarsky P, Hill RP (2003) The hypoxic tumour microenvironment and metastatic progression. Clin Exp Metastasis 20(3): 237-250. Sullivan R, Graham CH (2007) Hypoxia-driven selection of the metastatic phenotype. Cancer Metastasis Rev 26(2): 319-331. Takahashi R, Tanaka S, Hiyama T, Ito M, Kitadai Y et al. (2003) Hypoxia-inducible factor1alpha expression and angiogenesis in gastrointestinal stromal tumour of the stomach. Oncol Rep 10(4): 797-802. Theys J, Pennington O, Dubois L, Anlezark G, Vaughan T et al. (2006) Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. British J Cancer 95(9): 1212-1219. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP et al. (2002) Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20(1): 142-152.

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Van Mellaert L, Barbe S, Anne J (2006) Clostridium spores as anti-tumour agents. Trends Microbiol 14(4): 190-196. Vlahovic G, Ponce AM, Rabbani Z, Salahuddin FK, Zgonjanin L et al. (2007) Treatment with imatinib improves drug delivery and efficacy in NSCLC xenografts. British J Cancer 97(6): 735-740. Wang GL, Jiang BH, Rue EA, Semenza GL (1995) Hypoxia-inducible factor 1 is a basichelix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 92(12): 5510-5514. Wang L, Pan L, Shi L, Sun Y, Zhang Y et al. (1999) Roles of bifidobacterium on prevention of experimental colorectal carcinoma and induction of apoptosis. Zhonghua Yu Fang Yi Xue Za Zhi 33(6): 337-339. Warburg O (1930) The metabolism of Tumours. London: Arnold Constable. Warburg O (1956) On respiratory impairment in cancer cells. Science 124(3215): 269-270. Weinberg RA (2007) The biology of cancer. New York: Garland Science. Yazawa K, Fujimori M, Amano J, Kano Y, Taniguchi S (2000) Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors. Cancer Gene Ther 7(2): 269-274. Yazawa K, Fujimori M, and N. Nakamura et al., (2001) Bifodobacterium longum as a delivery system for cancer gene therapy of chemically induced rat mammary tumours. Breast Cancer Res Treat 66: 165–170 Yu YA, Shabahang S, Timiryasova TM, Zhang Q, Beltz R et al. (2004) Visualization of tumours and metastases in live animals with bacteria and vaccinia virus encoding lightemitting proteins. Nature Biotech 22(3): 313-320. Zhou J, Schmid T, Schnitzer S, Brune B (2006) Tumour hypoxia and cancer progression. Cancer Lett 237(1): 10-21.

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INDEX # 20th century, 219

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A acid, 94, 99, 103, 109, 137, 147, 150, 202, 275 acidity, 318 acidosis, 327 acquired immunity, 184 action potential, 26, 55 activation energy, 76 activity level, 226, 229 adenine, 226, 237 adenocarcinoma, 174, 175, 178, 179, 186, 188, 191, 192, 195, 196, 202, 277, 282, 283, 287 adenoma, 252 adenosine, 25 adenovirus, 229, 310 ADP, 32, 161 adrenal gland, 179 adrenaline, 281 adsorption, 32, 102, 138, 164, 193 advancement, 26, 219, 235, 293 adverse effects, 13, 27, 56, 279, 302, 306 adverse event, 2, 16, 26 aetiology, 316 AFM, 107, 113 agar, 146 agonist, 44 Agrobacterium, 59, 155, 156, 165, 166 AIDS, 233, 240 alanine, 136 albumin, 279 aldolase, 318 allergic reaction, 280 alopecia, 252, 261

alternating current (AC), 16, 212 alternating current sine-waves (ACSWs), 16 alters, 197 aluminium, 102, 108, 109, 111, 145 amine, 275, 276 amino, 136, 163 amino acids, 136, 163 amplitude, 20, 47, 48, 50, 51, 52, 53, 54, 55, 57, 69, 88, 90, 91, 92, 106, 143, 223, 242 anaerobic bacteria, 316, 320, 321, 323, 326 anemia, 215, 220, 222, 226, 229, 300 aneurysm, 56, 66 angiogenesis, 221, 228, 230, 241, 292, 317, 318, 319, 320, 325, 326, 327 animal welfare, 230 anthrax, 224, 231, 241 antibiotic, 139 antibody, 213, 214, 226, 240, 270, 303 anti-cancer, 168, 232, 275, 319, 320 anticancer drug, 28, 116, 129, 199, 270, 295, 317 antigen, 23, 40, 41, 184, 192, 193, 195, 202, 203, 222, 223, 224, 225, 231, 234, 242, 311 antigen-presenting cell, 23, 41, 224, 242 antisense, 28, 299, 306, 307 antisense RNA, 28 antitumor, 28, 40, 41, 50, 57, 59, 68, 79, 116, 171, 184, 189, 193, 194, 195, 196, 197, 198, 199, 202, 203, 282, 284, 286, 287, 292, 293, 296, 322, 324, 327 antitumor agent, 324 APC, 190 apex, 47 apoptosis, 7, 24, 41, 96, 128, 171, 202, 268, 271, 295, 299, 300, 303, 307, 310, 311, 312, 313, 320, 328 apoptotic pathways, 308 aqueous humor, 229 aqueous pathways, 22, 39, 45, 68 aqueous solutions, 86

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Index

330 Arabidopsis thaliana, 162 arrest, 174, 175, 186, 197 arrhythmia, 56, 280 artery(ies), 21, 43, 56, 66, 240 arthritis, 227, 237, 238 aryl hydrocarbon receptor, 318 ascites, 16, 173, 320 assessment, 17, 20, 39, 40, 53, 73, 75, 79, 151 asymmetry, 18, 47, 48, 141 atomic force, 108, 112 atoms, 10, 12, 276 ATP, 25, 32, 90, 161, 197 attachment, 150, 291 avian influenza, 225, 231, 240

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B bacillus, 325 Bacillus subtilis, 150, 151, 153 bacteria, 6, 28, 67, 83, 95, 109, 112, 133, 134, 135, 136, 137, 139, 140, 142, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 218, 277, 315, 316, 320, 321, 322, 323, 328 bacterial infection, 316 bacterial strains, 138 bacterium, 82, 145, 146, 147, 148, 320 barriers, 6, 11, 39, 310 basal cell carcinoma, 116, 174, 178, 196, 282, 284, 286, 296 base, 38, 59, 230, 239, 299, 303, 304, 308 basic research, 13, 162 beneficial effect, 66, 145, 172, 228, 229 benefits, 24, 65, 217, 220 benign, 65, 247, 266 biochemical processes, 99, 100, 106, 108, 109 biochemistry, 161 biological processes, 168 biological samples, 121 biological systems, 10 biomarkers, 229, 239 biomedical applications, 27, 59 biomolecules, 33, 97, 299, 305, 308 biosynthesis, 150 biotechnological applications, 160 biotechnology, 133, 135, 136, 146, 155, 156, 157, 161, 162 birefringence, 31 bladder cancer, 173, 198 blood, 14, 20, 21, 22, 23, 26, 38, 39, 41, 49, 56, 59, 63, 64, 65, 66, 67, 72, 76, 79, 82, 112, 126, 129, 191, 213, 228, 238, 277, 279, 294, 300, 301, 310, 325 blood circulation, 65

blood flow, 14, 21, 22, 23, 41, 49, 64, 65, 72, 294 blood stream, 67 blood vessels, 20, 23, 56, 59, 63, 65, 66, 67, 76, 79, 82 body weight, 177, 229 Boltzmann constant, 9 Boltzmann distribution, 10 bone, 20, 168, 179, 278, 300 bone marrow, 168, 278, 300 boundary value problem, 9 brachytherapy, 266 bradycardia, 279 brain, 28, 130, 182, 201 brain tumor, 130, 182 breakdown, 4, 19, 26, 30, 31, 32, 48, 81, 95, 97, 108, 111, 112, 141 breast cancer, 266, 296, 325, 327 breast carcinoma, 168, 175, 195 breeding, 155, 160 BSR, 148 buccal mucosa, 275 bundle branch block, 279

C Ca2+, 68, 101, 103, 140 cachexia, 229 calcitonin, 238 calcium, 96, 101, 304 cancer cells, 49, 65, 81, 167, 179, 185, 263, 269, 317, 321, 328 cancer progression, 328 candidates, 173, 315, 323 capsule, 172, 186, 188 carcinoma, 83, 129, 130, 168, 173, 175, 178, 181, 182, 183, 191, 193, 194, 198, 200, 223, 242, 251, 252, 253, 255, 267, 268, 271, 278, 283, 284, 300, 318, 320, 324, 327, 328 cardiomyopathy, 267 cartilage, 222 castor oil, 194 catheter, 240 cattle, 219, 220, 222, 224, 230, 231, 241 CD8+, 192, 203, 231 cDNA, 227, 236, 239 cell assembly, 48 cell biology, 28, 133, 134, 135, 149 cell culture, 157, 163, 214 cell cycle, 299, 303, 307 cell death, 7, 8, 15, 19, 32, 45, 63, 64, 66, 68, 76, 81, 82, 85, 87, 88, 89, 108, 134, 139, 184, 185, 190, 202, 271, 278, 295 cell division, 156, 302, 307, 321

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Index Cell electropermeabilization, 1, 2 cell fusion, 52, 57, 81, 116, 130, 149, 155, 266, 270 cell killing, 86, 318 cell line(s), 15, 36, 41, 51, 66, 85, 174, 175, 178, 179, 181, 194, 196, 303, 305, 312, 326 cell membranes, 3, 4, 18, 22, 30, 31, 38, 42, 56, 61, 63, 64, 66, 81, 85, 95, 115, 116, 124, 125, 126, 128, 129, 130, 141, 152, 169, 182, 217, 278 cell movement, 165 cell size, 6, 18, 19, 38, 46, 51, 64, 158 cell surface, 24, 142, 168, 169 cellular immunity, 184, 231, 270 cellulose, 97 cervical cancer, 324 cervix, 275, 278, 325 chemical, 10, 14, 24, 25, 65, 68, 99, 100, 102, 103, 109, 122, 146, 147, 156, 168, 169, 301 chemical properties, 25, 122 chemical reactions, 100, 102, 103, 109 chemicals, 135, 137, 161, 323 chemokine receptor, 326 chemokines, 313 chemotherapeutic agent, 2, 49, 116, 167, 172, 173, 175, 176, 177, 178, 182, 193, 255, 275, 282, 285 chlorine, 102, 103, 108, 109, 172 chloroplast, 155, 159, 160, 163, 164 cholesterol, 117 choriocarcinoma, 275 chromium, 105 chromosome, 150 chronic lymphocytic leukemia, 308, 309, 310, 311, 312, 313 chronic renal failure, 229, 239 circulation, 192, 206, 277 clinical adverse events, 2 clinical application, 14, 22, 73, 116, 184, 205, 206, 214, 231 clinical oncology, 263 clinical symptoms, 300 clinical trials, 26, 27, 49, 116, 172, 174, 217, 219, 231, 232, 233, 234, 248, 276, 279, 282, 308, 320 clone(ing), 146, 147, 149, 175, 176, 177, 185 closure, 89 clustering, 125 clusters, 18, 122, 123 coding, 222, 224, 306, 320 codon, 218, 224 collagen, 38, 227, 237, 238, 249 colon, 168, 172, 175, 181, 182, 183, 190, 191, 192, 196, 199, 200, 201, 202, 300 colon cancer, 172, 181, 183, 196, 199, 300 colonization, 320, 322, 323 colorectal adenocarcinoma, 286

331

colorectal cancer, 173, 181, 195 combination therapy, 192, 278 combined effect, 13, 23, 199 complexity, 13, 18, 19, 22, 27, 124 complications, 56, 228, 279, 300 composition, 13, 14, 48, 99, 100, 102, 137, 140, 158, 185, 186, 319 compounds, 24, 85, 86, 93, 109, 116, 124, 126, 218 comprehension, 155 conductance, 4, 6, 19, 31, 37, 38, 46, 294 conduction, 20, 25, 97, 124 conductivity, 4, 5, 6, 18, 20, 21, 23, 39, 46, 56, 66, 69, 72, 73, 76, 78, 100, 130, 141, 143, 169 configuration, 2, 7, 18, 20, 54, 64, 69, 72, 74, 112, 142, 278 congestive heart failure, 279 conjugation, 149 connective tissue, 45, 65, 186, 188, 249 consensus, 1, 223, 224, 225, 226, 231, 234, 240, 241, 242, 243 contamination, 37, 110, 312 control group, 210, 261 cooling, 21, 39, 144 copper, 100, 105, 176, 276 cornea, 96 coronary artery disease, 280 correlation, 69, 81, 316, 318 corrosion, 110 cost, vii, 25, 55, 219, 220, 234, 263, 266, 268 cost effectiveness, 219 creatine, 26 creatine phosphokinase, 26 creatinine, 212, 277, 280 critical analysis, 156 critical value, 12, 141 crop, 150, 156, 157, 160 crops, 156, 157, 159, 163 CSF, 191, 197 culture, 112, 129, 143, 146, 147, 148, 157, 197, 199, 266, 270, 271, 275, 295, 303, 304 culture media, 143 cure, 155, 167, 171, 176, 177, 178, 179, 180, 181, 182, 183, 185, 188, 189, 190, 191, 192, 195, 199, 200, 217, 251, 252, 273, 316, 317 cycles, 55, 262, 327 cyclophosphamide, 173, 174 cysteine, 136, 276 cytokines, 23, 193, 319 cytomegalovirus, 28 cytoplasm, 5, 46, 119, 120, 121, 140, 141, 158, 307 cytosine, 159, 276, 320, 321, 326 cytoskeleton, 19, 38, 126, 131 cytostatic drugs, 191

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Index

332 cytotoxic agents, 168 cytotoxic drugs, 45, 168, 185 cytotoxicity, 28, 43, 116, 129, 196, 199, 266, 270, 271, 275, 295, 304, 311

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D damages, 7, 56, 144 danger, 41, 67, 184 death rate, 181, 189 debulking surgery, 194 decay, 36, 143, 144, 145 decibel, 18 decomposition, 171 decontamination, 67 defecation, 248 defects, 11, 47, 51, 115, 116, 124, 125, 126, 127, 169, 299, 308 defibrillation, 53, 55, 56, 57, 59, 67 defibrillator, 56 deformation, 31, 126, 130 degradation, 117, 140, 162, 218, 310, 318 denaturation, 21, 65, 144 dendritic cell, 18, 40, 191, 192, 193, 195, 312 deoxyribonucleic acid, 49 depolarization, 19, 55 depth, vii, 3, 274, 292 dermis, 119 detectable, 120, 122, 123, 183, 193, 307 detection, 56, 162, 181 diabetes, 228, 238 diabetic nephropathy, 228, 238 diabetic neuropathy, 228 dielectric constant, 11, 25 dielectric strength, 104, 141 differential equations, 24, 39 diffusion, 7, 8, 9, 19, 22, 24, 32, 34, 49, 52, 74, 89, 97, 116, 118, 124, 127, 128, 129, 141, 144, 151, 152, 214, 317, 319 diffusion process, 22 dimerization, 318 discoidal erythrocytes, 17 disease progression, 174, 223, 229, 242, 300, 320 diseases, 49, 205, 214, 217, 220, 228, 230, 232, 235, 308, 309, 324 disinfection, 94 distribution function, 92, 94 divergence, 142 diversity, 2, 20, 273 DNA strand breaks, 232 DNA transcription, 307 DNAs, 151 DNase, 161, 323

dogs, 66, 116, 172, 173, 202, 218, 219, 220, 222, 225, 226, 229, 239, 243, 246, 248, 251, 252, 254, 255, 257, 258, 259, 260, 261, 262, 263 dosage, 196, 278 Drosophila, 164 drug delivery, 20, 29, 39, 48, 49, 57, 100, 103, 111, 115, 116, 117, 127, 129, 130, 134, 150, 151, 180, 195, 249, 288, 292, 319, 325, 326, 328 drug resistance, 249 drugs, 2, 8, 13, 25, 43, 45, 58, 64, 67, 116, 117, 123, 128, 129, 167, 168, 171, 173, 176, 177, 181, 182, 183, 184, 185, 195, 196, 216, 218, 228, 263, 266, 275, 277, 286, 292, 295, 318, 320, 323 dyes, 2, 13 dysphagia, 26

E EAE, 229 ECM, 55 edema, 23 electric charge, 20, 25, 108 electric circuits, 199 electric conductivity, 71 electric current, 51, 99, 100, 101, 112, 156, 171, 216 electrical breakdown, 32, 95, 117, 294 electrical conductivity, 21, 70, 72, 73, 76, 78 electrical fields, 42, 50, 67, 96 electrical properties, 20, 30, 195 electrical resistance, 20 electricity, 82, 175 electrocardiogram, 56, 59 electrochemical deposition, 117 electrochemical treatment (EChT), 103, 108, 112, 171, 172, 202, 203 electrode surface, 108, 113, 172, 275 electrolysis, 14, 99, 100, 171, 172, 196, 200, 277 electrolyte, 103, 279 electromagnetic, 43, 83, 119, 129, 174 electromagnetic fields, 43, 83 electromagnetic waves, 119, 174 electron, 30, 57, 115, 118, 119, 128, 149, 293 electron microscopy, 30, 57, 115, 118, 119, 125, 126, 128, 149, 174, 293 electropermabilization protocols, 25 electrophoresis, 23, 24, 32, 42, 49, 51, 205, 206, 209, 211, 214, 216 electroporating zygotic wheat embryos, 134 elucidation, 299 encephalomyelitis, 229 encoding, 40, 41, 197, 207, 221, 222, 223, 224, 225, 226, 227, 228, 231, 233, 237, 238, 241, 242, 299, 304, 322, 328

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Index endocardium, 56 endocrine, 215 endothelial cells, 19, 229 endothelium, 23, 239 enzyme(s), 89, 90, 111, 136, 206, 276, 318, 321, 327 eosinophils, 267 epidermis, 20, 178 epilepsy, 280 epithelial cells, 178 epithelium, 17 EPS, 267 Epstein Barr, 304, 311 erysipelas, 316, 324, 325 erythrocyte membranes, 31 erythrocytes, 17, 83, 91, 95, 96, 97, 111, 115, 122, 125, 126, 127, 129, 270, 294 erythropoietin, 218, 220, 235, 236, 237 eukaryotic, 1, 28, 156, 266, 318 eukaryotic cells, 1, 266 evagination, 47 experimental autoimmune encephalomyelitis, 239 exponentially enhanced pulse (EEP), 15 extracellular matrix, 18, 63, 65, 67, 79, 192, 319 extravasation, 249, 255

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F fat, 20, 76 fatty acids, 117 fermentation, 148, 315 fiber(s), 170, 186, 188, 210 fibrillation, 56 fibroblasts, 18, 19, 36, 38, 42 fibrosarcoma, 83, 173, 183, 191, 196, 251, 267, 269 fibrosis, 266, 277 finite element method, 54, 95 fluctuations, 7, 47, 80, 88, 124 fluid, 16, 19, 24, 30, 51, 89, 171, 193, 319, 325 fluorescence, 36, 117, 118, 141, 207, 212 fluorine, 181 fluorophores, 111 food, 50, 67, 83, 85, 86, 95, 100, 155, 163, 230, 315 food industry, 50, 67 food production, 230 food products, 100 formation, 2, 11, 12, 22, 27, 30, 32, 33, 56, 66, 67, 85, 86, 87, 88, 93, 95, 96, 100, 103, 104, 116, 118, 119, 123, 124, 126, 127, 129, 141, 169, 171, 206, 278 formula, 5, 6, 7, 8, 9, 17, 25, 26, 46, 68, 281 fragments, 222, 242 free energy, 6 freeze-fracturing, 115, 118, 125

333

freezing, 30, 57, 128, 146, 149, 293 frequency distribution, 86 fungi, 2 fusion, 48, 131, 163, 164, 226, 234, 237, 238, 241, 266, 270, 304, 307

G ganglioneuroblastoma, 251 gangrene, 320 gas gangrene, 316, 320 gastrocnemius, 207 gastrointestinal tract, 44 gene expression, 24, 28, 36, 40, 49, 155, 156, 157, 158, 160, 161, 162, 163, 166, 205, 206, 207, 209, 210, 211, 212, 213, 215, 221, 224, 229, 236, 241, 292, 304, 307, 309, 311, 313, 322, 324, 326 gene pool, 155 gene silencing, 299 gene therapy, 14, 23, 29, 35, 44, 48, 50, 59, 100, 129, 134, 206, 213, 214, 216, 217, 218, 219, 220, 221, 225, 227, 228, 229, 230, 235, 237, 238, 240, 241, 243, 263, 299, 301, 304, 305, 307, 308, 309, 310, 311, 312, 325, 326, 328 gene transfer, 2, 16, 25, 32, 35, 37, 40, 43, 61, 96, 116, 134, 155, 156, 157, 158, 163, 164, 165, 166, 205, 206, 207, 209, 211, 212, 213, 214, 215, 216, 218, 226, 228, 229, 230, 236, 237, 238, 239, 240, 299, 300, 301, 302, 303, 304, 305, 308, 309, 310, 311, 312, 327 general anaesthesia, 280 genetic code, 307 genetic disorders, 160 genetic information, 156, 159 genome, 133, 147, 148, 157, 159, 160, 302 genus, 322 geometry, 6, 14, 46, 54, 55, 69, 76, 142, 274 gland, 255 glioblastoma, 174 glioma, 90, 173, 174 glucagon, 228 glucose, 112, 147, 228, 238, 318, 324, 327 glucose tolerance, 228 glutamate, 226, 237 glycerol, 44, 139, 140, 143, 146, 147, 148, 304 glycine, 135, 136, 147, 149, 150 glycol, 156, 158, 164, 323 glycolysis, 317, 318, 325 glycopeptides, 275 glycoproteins, 124, 184 green alga, 156 growth factor, 221, 228, 229, 230, 238, 240, 319 growth hormone, 44, 222, 226, 229, 238, 239, 241

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Index

334 growth rate, 150, 174 growth temperature, 138 guanine, 278 guidelines, 69, 76, 214, 309

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H hair, 119, 177 hair follicle, 119 half-life, 277 harvesting, 137 HBV, 225, 231, 241 HBV infection, 231 head and neck cancer, 29, 43, 178, 197, 199 heat capacity, 21, 72, 73, 78 heat generation, 2, 21, 26, 72 heat shock protein, 310 heat transfer, 21, 80 heating rate, 72 hematocrit, 220, 226 hematology, 308 hemoglobin, 47 hemophilia, 205, 213, 214, 221, 230, 240 hepatic necrosis, 200, 202 hepatitis, 224, 231, 232, 241 hepatocellular carcinoma, 83, 193, 203, 206, 215 hepatocyte growth factor (HGF), 228, 238 hepatocytes, 206 hepatoma, 92, 173, 197 herpes simplex, 303, 311, 322 heterogeneity, 3, 19, 20, 76 histological examination, 267 histology, 34, 66, 285, 309 history, 249 HIV, 224, 226, 231, 232, 233, 240, 242, 303, 310 HIV-1, 224, 226, 240, 242, 310 homeostasis, 64, 228 homogeneity, 17, 48, 246 hormone, 44, 222, 226, 229, 238, 239 horses, 44, 173, 200, 219, 220, 222, 227, 238, 247, 254 host, 41, 150, 153, 156, 157, 184, 190, 193, 199, 206, 218, 301, 321 human brain, 198 human condition, 229 human immunodeficiency virus, 166, 224, 242 hybrid, 160, 162, 310 hybridization, 155 hybridoma, 218 hydrogen, 11, 101, 103, 104, 172 hydrogen gas, 103, 104 hydrophilic electropores, 47 hydrophobicity, 169

hydroxyl, 102, 109, 276 hyperglycemia, 228 hypernephroma, 283 hypersensitivity, 191 hypertension, 222, 241, 324 hyperthermia, 175, 193, 266, 270 hypothesis, 18, 24, 25, 65, 118, 124, 126, 127, 209, 279 hypoxia, 221, 240, 317, 318, 320, 323, 324, 325, 326, 327, 328 hypoxia-inducible factor, 318, 324, 326, 327 hypoxic cells, 318

I IFN, 192, 225, 229, 239 IFN-β, 229 immune defense, 168 immune reaction, 23, 180, 184, 189, 190 immune response, 23, 44, 63, 67, 79, 168, 179, 180, 183, 184, 185, 186, 189, 190, 191, 192, 193, 197, 199, 200, 202, 218, 223, 224, 226, 230, 231, 235, 240, 241, 242, 270, 304, 310, 321 immune system, 23, 79, 184, 189, 193, 200, 248, 249, 270, 319 immunity, 40, 54, 180, 184, 185, 188, 189, 190, 191, 192, 194, 195, 196, 197, 199, 200, 202, 203, 222, 223, 226, 241, 242, 311 immunization, 193, 199, 206, 222, 223, 226, 240, 242 immunodeficiency, 303 immunogenicity, 41, 188, 189, 206, 219, 224, 225, 231, 233, 236, 240, 242, 243 immunoglobulin, 309 immunomodulatory, 192 immunostimulatory, 185, 301, 304 immunosuppression, 189, 319 immunosuppressive agent, 189 immunotherapy, 41, 191, 192, 193, 194, 195, 196, 303, 311, 316 impulses, 80, 157, 158 in vitro exposure, 125 incubation time, 85, 89 incubator, 146 indolent, 300 induction, 2, 4, 5, 11, 12, 34, 51, 116, 141, 142, 164, 170, 180, 184, 195, 200, 213, 229, 231, 312, 318, 328 infarction, 271 infection, 155, 303, 304, 310, 311, 316, 320, 326 inflammation, 23, 26, 40, 192, 218, 227, 229, 260, 261, 268, 270, 321 inflammatory cells, 188, 190, 319

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Index influenza, 223, 224, 225, 231, 234, 242, 243 influenza a, 225 influenza virus, 223, 225, 231, 242 inhibition, 41, 137, 174, 177, 195, 223, 227, 229, 242, 307, 310, 317, 322 inhibitor, 137, 227, 237 inhomogeneity, 108 injury mechanisms, 81 inoculation, 168, 177, 178, 182, 185, 186, 189, 224, 242 inositol, 110 insecurity, 163 insertion, 25, 292 insulin, 112, 227, 228, 229, 238 integration, 10, 41, 150, 157, 159, 160, 302 integrity, 19, 65, 161, 218 interface, 8, 11, 21, 54, 70, 74, 99, 101, 124, 290 interference, 8, 22, 162 interferon, 229, 239, 259, 263 internalization, 49, 51, 57, 171, 182, 303 internalizing, 184 intervention, 56 intramuscular injection, 227, 228, 237 intravenous fluids, 279 intravenously, 49, 180, 181, 282, 283, 284 ion transport, 33, 118, 129 ionization, 199 ionizing radiation, 192, 196 ions, 1, 2, 13, 19, 24, 25, 45, 52, 68, 90, 91, 92, 97, 100, 101, 102, 103, 104, 105, 108, 109, 110, 111, 143, 144, 171, 278 IP-10, 239 iron, 105, 112, 276 irradiation, 198, 201 isolation, 149 issues, 2, 3, 8, 9, 13, 18, 22, 24, 26, 63, 112, 181, 218, 220, 302, 316, 324

J joint destruction, 227

K K+, 19, 38, 40, 90, 91, 101, 103 Kaposi sarcoma, 283 keratin, 117 keratinocyte, 228 keratinocytes, 119 kidney, 179, 228, 229, 238, 279 kinase activity, 310

335

kinetics, 3, 6, 11, 23, 32, 85, 86, 89, 94, 95, 118, 141, 142, 149, 164, 178, 195, 212, 213, 322

L lactate dehydrogenase, 318 lactation, 280 lactic acid, 136, 147, 148, 318, 320 lactose, 148 larynx, 173, 199, 275 lead, 4, 12, 23, 50, 51, 56, 88, 99, 100, 108, 124, 192, 209, 219, 227, 266, 277, 278, 300, 308 leakage, 68, 85, 86, 90, 93, 144, 171, 319 leptin, 228 lesions, 66, 168, 195, 198, 202, 247, 257, 260, 261, 273, 274, 280, 281 leukemia, 15, 36, 299, 300, 301, 304, 308, 310, 311, 313 life expectancy, 177, 273 lifetime, 4, 32, 50, 51, 95, 111, 131, 170 ligand, 40, 237, 276, 299, 309, 310, 311 light, 4, 31, 82, 115, 192, 328 light scattering, 4, 31 lipases, 321 lipid peroxidation, 129 lipids, 4, 47, 115, 121, 124, 126, 127, 301 liposomes, 31, 90, 119, 134, 168, 196 liquid phase, 4 Listeria monocytogenes, 94, 95 lithium, 135, 151 liver, 35, 41, 58, 65, 66, 73, 79, 172, 173, 175, 181, 182, 183, 191, 192, 195, 196, 198, 200, 201, 202, 203, 213, 215, 228, 236, 238, 294, 325 liver cancer, 198, 325 liver cells, 215, 236 liver metastases, 172, 201 localization, 117, 326, 328 loci, 191 locus, 239 longevity, 6, 239 low temperatures, 80, 140 low-intensity electric fields (LIEF), 171 luciferase, 207, 209, 210, 211, 212, 221 lung cancer, 172, 278, 326 lung metastases, 178, 184, 186, 191 lymph, 66, 179, 253, 255, 300, 309, 324, 327 lymph node, 66, 179, 253, 255, 309, 324, 327 lymphadenopathy, 300 lymphatic system, 178 lymphocytes, 23, 122, 185, 186, 188, 190, 191, 192, 267, 268, 270, 300, 303, 310, 311 lymphoid, 168, 202, 300, 313 lymphoid organs, 168, 300

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Index

336 lymphoma, 60, 223, 242, 257, 258, 259, 260, 261, 263, 308, 313 lysis, 7, 26, 136, 141, 270, 321 lysozyme, 135, 136, 150

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M macromolecules, 14, 15, 24, 32, 42, 47, 49, 52, 59, 68, 106, 109, 110, 116, 119, 124, 127, 130, 133, 135, 142, 156, 157, 158, 168, 169, 175, 193, 200, 206, 208, 217 macrophages, 18, 185, 189, 191, 198, 267, 319 magnesium, 101 magnetic resonance imaging, 172 major histocompatibility complex, 184 majority, 3, 17, 87, 90, 268, 291, 300, 307 malaria, 224, 243 malignancy, 178, 186, 225, 243, 299 malignant cells, 58, 68, 172, 192 malignant melanoma, 29, 43, 174, 201, 248, 270, 284, 286, 293, 294, 296, 297 malignant tumors, 65, 172, 174, 175, 203 mammalian cells, 2, 31, 32, 39, 42, 43, 59, 61, 65, 113, 130, 151, 195, 198, 200, 202, 218, 304 manganese, 105 mannitol, 90, 91, 139, 140 mapping, 55 marker genes, 165 mass, 78, 183, 184, 193, 227, 228, 238, 268, 270, 321 materials, 10, 54, 100, 105, 201 maternal inheritance, 159 matrix, 56, 121, 122, 125, 222, 263, 265, 319 MCP-1, 240 median, 178, 179, 248, 249, 251, 259 medicine, 20, 42, 50, 52, 68, 134, 167, 173, 241, 242 medium composition, 146 melanin, 270 melanoma, 4, 30, 34, 43, 82, 115, 116, 117, 119, 120, 121, 122, 123, 125, 126, 127, 128, 130, 168, 172, 173, 174, 175, 176, 177, 185, 190, 193, 194, 195, 197, 199, 203, 206, 215, 219, 232, 236, 248, 249, 254, 266, 267, 268, 269, 270, 271, 282, 283, 284, 286, 293, 295, 296, 300, 322, 327 mellitus, 228 membrane permeability, 85, 86, 93, 108, 121, 169, 208, 209 membrane pores, 1, 19, 25, 47, 152, 270 membranes, 7, 11, 16, 18, 30, 32, 33, 34, 42, 52, 56, 58, 59, 67, 95, 97, 112, 115, 116, 120, 121, 122, 123, 124, 126, 127, 128, 141, 163, 218 mesophyll, 165, 166 messengers, 121

metabolism, 21, 72, 317, 324, 327, 328 metabolites, 181, 275 metal ions, 54, 102, 104, 105, 106 metastasis, 66, 176, 254, 317, 318, 324, 326, 327 metastatic disease, 168, 180, 181, 253, 259, 280, 291, 326 methodology, 133, 134 Mg2+, 101, 103, 140, 209, 210 MHC, 41, 184 microenvironments, 138, 323 microinjection, 134, 164, 169, 201 microorganism(s), 81, 85, 86, 88, 89, 93, 94, 95, 112, 136, 138, 140, 146, 151, 152 microscope, 207 microscopy, 30, 57, 108, 112, 115, 117, 118, 119, 128, 149, 293 microstructures, 292 migration, 11, 18, 24, 38, 203, 235, 300 mitochondria, 119, 159, 160, 161, 163, 165 mitochondrial DNA, 161 model system, 175, 193 modelling, 33 molecular biology, vii, 36, 133, 136, 146, 148, 151, 155, 161 molecular dynamics, 10, 33, 34 molecular mass, 26 molecular medicine, 32, 39, 80 molecular oxygen, 276 molecular uptake, 2, 6, 13, 15, 16, 24, 25, 29, 37, 52 molecular weight, 24, 142, 169, 307 molecules, vii, 1, 2, 7, 11, 12, 14, 15, 19, 20, 22, 23, 24, 25, 28, 31, 40, 45, 47, 52, 56, 88, 89, 90, 92, 97, 101, 102, 106, 108, 112, 116, 124, 127, 129, 134, 138, 139, 140, 142, 145, 156, 158, 159, 161, 164, 168, 169, 170, 171, 175, 192, 200, 206, 218, 232, 249, 266, 270, 271, 275, 276, 293, 295, 299, 307, 315 monoclonal antibody, 198, 201 morbidity, 15, 172, 228, 300 morphology, 119, 126, 309 mortality, 158, 172, 180, 181, 219, 230, 300 mortality rate, 181 mosaic, 162, 165 motif, 159, 164 mRNA(s), 40, 41, 159, 160, 162, 163, 186, 299, 304, 306, 307, 308, 312 multiple factors, 19 multiple sclerosis, 229 muscles, 53, 57, 207, 221, 230, 237, 239, 240 muscular dystrophy, 230, 239 mutagenesis, 160, 161, 206, 301, 302 mutant, 160, 317, 319 mutation(s), 162, 230, 278, 309, 317

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Index mycobacteria, 137 mycosis fungoides, 259 myelin, 63, 67 myoblasts, 221, 240 myocardial infarction, 280 myocardium, 56 myocyte, 207

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N Na+, 19, 40, 90, 101, 103 NaCl, 102, 104, 108, 147 nanometer, 206 nanometers, 7 nanoparticles, 292 NATO, 37 natural killer cell, 40 necrosis, 56, 66, 171, 172, 183, 185, 188, 192, 212, 232, 237, 266, 267, 268, 269, 326 negative effects, 323 negative outcomes, 26 neoplasm, 251, 254, 257, 261, 268 neoplastic tissue, 269 nerve, 20, 26, 53, 80, 97, 266 neurofibroma, 268, 270 neurons, 112 neuropathy, 280 neutral, 24, 169, 209 neutrophils, 267 nickel, 105 nitrogen, 276 NK cells, 174 NMR, 58, 293 nodules, 54, 58, 174, 203, 257, 259, 273, 277, 280, 282, 284, 285, 286, 288, 289, 290, 291, 296 non-Hodgkin‘s lymphoma, 275 nuclear genome, 159 nuclear membrane, 25 nucleation, 4 nuclei, 119, 120, 121, 155, 218 nucleic acid, 44, 169, 208, 221, 230, 240, 308, 312 nucleoprotein, 225 nucleotides, 182 nucleus, 25, 134, 305, 309 nude mouse, 115 numerical analysis, 23 nutrients, 321, 323

O occlusion, 66 oncogenes, 317

337

oocyte, 201 operon, 160 optimization, 2, 3, 6, 7, 13, 15, 22, 23, 50, 99, 117, 127, 162, 217, 218, 231, 232, 235, 304, 307 oral cavity, 13, 270 organ, 13, 171, 172, 193, 222, 241 organelles, 96, 119, 120, 121, 155, 156, 159, 161, 163, 165 organic compounds, 108 organism, 22, 147, 156, 172 organs, 13, 20, 66, 211, 320 ornithine, 200 osmolality, 142 osmosis, 24, 42 osmotic pressure, 48, 49, 112, 141 osteoarthritis, 227, 238 osteogenic sarcoma, 278 osteomyelitis, 26 ototoxicity, 279 oxidation, 101, 102, 108, 111 oxidative stress, 158 oxygen, 102, 192, 317, 318, 323, 326

P p53, 311, 317, 319 paclitaxel, 26, 173, 275 pain, 26, 54, 211, 227, 232, 234, 292 palliate, 248 pancreas, 238 parallel, 24, 33, 54, 72, 117, 119, 142, 143, 170, 186, 274, 282 parotid, 287 partial differential equations, 24 particle bombardment, 160, 301 pasteurization, 95, 100, 111, 112 pathogenesis, 300, 316 pathology, vii, 229 pathophysiology, 38 pathways, 8, 19, 22, 39, 45, 68, 123, 129, 169, 307, 313 PCR, 161, 186 PCT, 312 pelvis, 278 penis, 275 peptide, 18, 228, 238, 276 perforation, 165 perfusion, 21, 23, 72, 80, 283, 290, 317 peripheral blood, 125, 309 peritoneum, 176 permeability, 1, 6, 7, 49, 55, 64, 66, 100, 119, 127, 128, 129, 134, 135, 141, 169, 170, 171, 182, 319 permeation, 2, 36, 123, 124, 173, 202

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338

Index

permit, 29, 293, 303 permittivity, 58 PET, 327 pH, 14, 15, 35, 99, 100, 102, 103, 104, 108, 109, 110, 112, 140, 318, 319, 327 phage, 150 pharmacokinetics, 194, 296 pharmacology, 296 phenotype, 317, 327 phenotypic variations, 156 phosphate(s), 104, 162, 234, 304 phosphatidylserine, 23, 33, 34 phospholipids, 31, 124, 127, 130 physical mechanisms, 9 physical properties, 25, 73, 76 physicians, 26, 290, 316 physicochemical properties, 15, 100 physics, 2, 51, 60 physiological mechanisms, 2 physiology, iv, 35, 147, 155, 161, 324, 325 pigmentation, 248 pigs, 129, 191, 195, 217, 218, 219, 220, 221, 226, 231, 240 pilot study, 44, 201, 222, 238, 305 plants, 111, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 plasma cells, 270 plasma membrane, 1, 3, 22, 28, 30, 42, 49, 52, 64, 119, 121, 122, 123, 125, 126, 134, 170, 171, 200, 205, 206, 216, 271, 275, 295 plasma membranes, 1, 3, 123, 126 plasma proteins, 279 plasminogen, 223, 242 plasmolysis, 158 plastid, 160, 166 platform, 171, 315 platinum, 54, 55, 100, 102, 105, 112, 172, 177, 191, 195, 277, 278, 279, 296 pneumonia, 300 pneumonitis, 277 polar, 11, 12, 20, 46, 124, 126, 134 polarity, 47, 53, 209, 210, 211, 213, 246 pollen, 112, 155, 158, 159, 160, 162 pollination, 159 polycarbonate, 145 polymer(s), 25, 96, 124, 135, 152, 237, 301 polypeptide, 124 polyploid, 159 polypropylene, 148 population, 8, 17, 87, 88, 89, 91, 93, 144, 157, 182, 185, 186, 190, 192, 213, 219, 226, 270, 285, 286 pore formation, 2, 11, 12, 27, 85, 86, 87, 88, 93, 95, 96

portability, 12 position effect, 159 positive correlation, 15 positron emission tomography, 327 potassium, 92, 96, 97, 101 potato, 161, 163 precipitation, 106, 109, 110, 304 predictability, 81, 202 preparation, iv, 113, 133, 134, 136, 138, 144, 147, 160, 227, 273 prepuce, 262 preservation, 16, 66, 85, 86, 193, 251 prevention, 217, 219, 270, 328 primary cells, 305, 312 primary tumor, 116, 168, 175, 176, 177, 179, 180, 182, 183, 184, 185, 186, 188, 189, 190, 191, 192 primate, 219, 225, 236, 241 principles, 32, 34, 42, 48, 79, 82, 151, 213, 239, 309 probability, 10, 15, 22, 52, 56, 89, 191 probability distribution, 10 probe, 65, 162, 171 prognosis, 300, 318, 324, 327 pro-inflammatory, 22, 228 prokaryotic cell, 28 proliferation, 66, 172, 174, 184, 197, 202, 299, 300, 317 promoter, 28, 164 prophylactic, 217 prostate cancer, 36, 168, 179, 180, 196, 200, 202, 222, 232, 242 prostate carcinoma, 175 prostatitis, 66 protection, 139, 161, 191, 192, 193, 222, 225, 231, 234, 240, 242 protein folding, 150 proteinase, 276

Q QRS complex, 56 quadriceps, 207, 209, 210, 211, 212 quality of life, 220, 226, 227, 229, 230, 266, 293 quantification, 38, 149 quantum-chemical calculations, 10

R radiation, 37, 83, 174, 192, 201, 248, 257, 266, 270, 278, 292, 294, 296, 318, 320 radiation therapy, 192, 248, 257, 266, 270, 278, 292, 318, 320 radicals, 276

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Index radio, 52, 156, 164 radiotherapy, 184, 290, 318 radius, 5, 8, 9, 16, 25, 46, 68, 88, 89, 91, 92, 118, 127, 171 random numbers, 10 reactions, 41, 99, 100, 101, 102, 103, 104, 108, 109, 110, 150, 280, 281, 294 reactive oxygen, 158, 192, 228, 238 reactivity, 13, 22, 184, 191, 193 real time, 117, 118 receptors, 168, 266 recognition, 159, 160, 184, 193, 310, 315 recombinant DNA, 113, 151, 158, 200, 321, 322 recombination, 160 recommendations, iv, 54 recovery, 22, 39, 139, 160, 228, 320 rectum, 13, 66 recurrence, 66, 79, 183, 249, 250, 251, 259, 270, 318 red blood cells, 4, 96, 97, 111, 115, 119, 125, 126, 127, 131 redistribution, 80, 122, 123, 125, 127 regenerate, 136, 156 regeneration, 65, 66, 156, 157, 160, 164, 240 regression, 64, 66, 138, 173, 174, 191, 195, 197, 316 regression analysis, 138 regrowth, 252, 321 rehabilitation, 186 relevance, 1, 18, 24, 175, 309 remission, 174, 251, 252, 253, 257, 260, 261, 266 renal dysfunction, 279, 280 renal failure, 215, 226, 237 replication, 152, 160, 162, 164, 198, 301, 303, 311 research institutions, 266 researchers, 1, 16, 27, 69, 156, 157, 160, 266, 303, 308 resection, 65, 192, 196, 202 residual disease, 177, 190, 251, 266, 267, 292 residues, 159, 318 resistance, 6, 9, 19, 20, 22, 23, 68, 117, 118, 140, 143, 144, 145, 160, 168, 185, 189, 190, 201, 202, 218, 274, 276, 308, 311, 312, 318, 320, 327 resolution, 10, 12, 63, 66, 67, 79, 118, 130 respiration, 318 responsiveness, 201, 281 restenosis, 66 resting potential, 19, 88 reticulum, 121 retina, 17, 112 retrovirus(es),303, 310 reversed polarity, 47 rheumatoid arthritis, 227 right ventricle, 56

339

risk, 27, 56, 66, 108, 159, 257, 259, 279, 281, 291, 300, 301, 302, 309 RNA(s), 2, 23, 106, 109, 159, 160, 161, 162, 163, 164, 165, 181, 278, 299, 301, 307, 310 RNA processing, 161 RNA splicing, 163 rodents, 2, 13, 195, 220 room temperature, 106, 118 roughness, 108, 112, 113 routes, 279, 286 rubidium, 97

S safety, 56, 202, 205, 206, 213, 214, 236, 239, 302 salivary gland, 283 salts, 101, 143 SARS, 223, 242 SARS-CoV, 223 saturated fat, 117 saturated fatty acids, 117 saturation, 138 scaling law, 69 scanning electron microscopy, 125, 126, 174 scar tissue, 83, 262, 268, 269 science, vii, 33, 245 scope, 3, 8, 20, 27, 230 search terms, 221 secretion, 128, 206, 220, 229, 322 seed, 160 selectivity, 19, 38, 97, 128, 270 sensitivity, 43, 61, 83, 263, 267, 276, 312 sensors, 170 sepsis, 300 sequencing, 161 serum, 25, 42, 43, 226, 228, 277, 300, 309 serum albumin, 42 shape, 13, 15, 17, 18, 25, 35, 45, 46, 47, 48, 53, 54, 58, 64, 68, 69, 71, 73, 91, 122, 123, 126, 127, 129, 130, 158, 173, 246 shock, 19, 38, 39, 56, 57, 59, 67, 134, 158, 190, 192, 206 shoots, 157 showing, 22, 31, 92, 112, 122, 230, 251 shrimp, 222, 241 side effects, 16, 26, 56, 119, 260, 266, 275, 279, 315 signal transduction, 300 signaling pathway, 313 sine wave, 211 single chain, 323 siRNA, 299, 304, 306, 307

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340

Index

skeletal muscle, 16, 17, 21, 26, 35, 39, 40, 41, 43, 206, 212, 214, 215, 216, 219, 220, 221, 227, 230, 236, 237, 238, 239, 240 skeleton, 124 skin, 3, 15, 20, 22, 29, 39, 40, 42, 43, 54, 58, 111, 112, 117, 119, 128, 129, 130, 134, 151, 178, 194, 198, 200, 206, 213, 216, 217, 221, 226, 234, 241, 274, 276, 278, 280, 290, 292, 293, 294, 296 skin cancer, 178 smooth muscle, 56, 66 smooth muscle cells, 56, 66 SMS, 59 sodium, 101, 104 soft tissue sarcomas, 29, 60, 249, 250, 254, 263, 271 software, 10, 54 solid tumors, 36, 60, 173, 175, 197, 201, 245, 301, 324 solution, 6, 8, 35, 54, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 133, 142, 143, 148, 159, 170, 176, 227, 230, 234 somatic cell, 156 species, 13, 51, 65, 86, 95, 101, 108, 113, 124, 133, 134, 135, 136, 137, 138, 140, 145, 148, 152, 155, 156, 158, 160, 192, 206, 217, 219, 220, 226, 228, 230, 232, 238, 316, 320, 322 specific heat, 72 sperm, 96 spleen, 185, 186, 191 splenomegaly, 186, 189, 190, 300 spore, 320, 326 squamous cell carcinoma, 60, 116, 168, 174, 175, 194, 196, 199, 203, 251, 254, 275, 278, 282, 283, 284, 286, 287, 290, 293 stability, 4, 19, 30, 136, 159, 160, 162, 301, 324 stabilization, 2, 5, 6, 12, 169, 318 stabilizers, 139 standard deviation, 104 standardization, 254, 271 state, 5, 6, 17, 18, 32, 45, 46, 49, 80, 87, 95, 102, 124, 125, 141, 157, 184, 201, 228, 245 states, 4, 6, 16, 20, 25, 47, 124, 206, 211 steel, 54, 99, 100, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 170, 176, 178, 180, 282 stem cells, 318 stenosis, 56 sterile, 145, 146, 304 stimulus, 3, 6, 176, 181, 206 stomach, 318, 327 stratum corneum, 20, 22, 39, 40, 115, 117, 118, 119, 128, 129, 130, 131 streptococci, 151 stress, 48, 52, 124, 136, 158, 169, 200, 202, 222, 230 stroma, 192, 319, 327

structural changes, 63, 64, 116, 117, 123, 126 structural modifications, 141 structure, 30, 38, 47, 50, 57, 118, 119, 120, 122, 123, 125, 128, 137, 149, 192, 275, 276, 278, 293, 305, 310, 312 subcutaneous tissue, 54, 58 subgroups, 300 succession, 53 sucrose, 89, 90, 91, 136, 139, 143, 147, 148 sugarcane, 158, 162 surface area, 46, 71 surface tension, 4, 8 surfactant, 39 surgical resection, 27, 172, 253, 293 surgical technique, 63 surveillance, 200 survival rate, 100, 108, 135, 140, 144, 145, 157, 158, 182, 183, 305, 307 survivors, 178, 185, 186, 270 susceptibility, 4, 136, 311 suspensions, 19, 38, 60, 137, 144, 148, 158, 326 swelling, 24, 121, 142, 227 symmetry, 17, 37, 263 symptoms, 228, 300 synchronization, 59 synergistic effect, 192 synthesis, 136, 137, 156, 181, 278

T T cell, 41, 184, 189, 193, 194, 195, 197, 203, 226, 228, 231, 240, 301, 313 T lymphocytes, 228 target, 12, 20, 38, 65, 76, 95, 156, 222, 223, 226, 230, 232, 241, 242, 251, 274, 301, 306, 316, 317, 323 tau, 167 technical assistance, 128 techniques, 13, 20, 26, 49, 50, 55, 65, 66, 67, 68, 79, 115, 155, 156, 157, 158, 160, 164, 218, 268, 301, 322, 323, 324 technology(ies), 50, 54, 85, 86, 94, 110, 113, 133, 134, 143, 151, 155, 160, 161, 200, 217, 219, 231, 232, 292, 293, 299, 305, 321 TEM, 118, 119, 120, 121, 125 temperature, 4, 6, 8, 9, 14, 20, 21, 39, 40, 64, 65, 69, 70, 72, 73, 74, 75, 76, 79, 80, 88, 93, 97, 99, 100, 112, 118, 128, 138, 144, 145, 149, 158, 170 temperature dependence, 118 tendon, 20 tension, 4, 124, 319, 328 testing, 36 testis, 215

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Index TGF, 228, 319 thalassemia, 236 theoretical approaches, 1 theory of electropermeabilization, 1 therapeutic effects, 128, 193, 227 therapeutic targets, 300, 308 therapeutics, 32, 82, 235, 315, 316 thermal energy, 9 thermal properties, 64, 69 thin sectioning, 115 thorax, 282, 288 threonine, 135 thrombocytopenia, 279 thrombus, 56, 66 thyroid, 253 tibialis anterior, 230 time lags, 219 tinnitus, 279 TLR, 192 TLR4, 191 TLR9, 40, 44 TNF, 191, 201, 227, 229, 237 TNF-alpha, 191, 201, 227 TNF-α, 229 tobacco, 158, 162, 164, 165, 166 topology, 151, 158 total energy, 25, 69 toxic products, 324 toxicity, 26, 136, 157, 194, 200, 205, 206, 211, 214, 232, 235, 268, 275, 276, 277, 279, 282, 292, 318, 321, 324 toxin, 193, 326 TPA, 304 traits, 155, 156 transcription, 161, 163, 306, 307, 313, 318 transcription factors, 318 transduction, 14, 23, 26, 40, 165, 215, 235, 301, 303, 310, 311 transfection, 14, 24, 36, 41, 48, 49, 50, 51, 54, 60, 108, 112, 134, 142, 152, 196, 205, 206, 207, 212, 215, 218, 219, 223, 238, 240, 242, 292, 301, 304, 307, 323 transfer RNA, 165 transferrin, 279 transforming growth factor, 228, 319 transgene, 156, 157, 158, 159, 160, 163, 206, 207, 218, 219, 226, 230, 234, 237, 301, 302, 303, 304, 305, 307, 310, 311 transgenic adenocarcinoma of mouse prostate (TRAMP), 168, 175, 179, 180, 188, 189, 196 transition temperature, 22 transitional cell carcinoma, 278 translation, 159, 165, 245, 257, 307

341

translocation, 25, 34 transmembrane region, 222 transmission, 159, 160, 224, 243 transplant, 195 transplantation, 230, 312 transport, 2, 7, 15, 19, 24, 31, 34, 38, 39, 42, 47, 49, 51, 65, 95, 112, 116, 117, 118, 119, 124, 129, 130, 144, 149, 157, 181, 202, 207, 216, 266, 275, 323 transverse section, 212 treatment methods, 189 trial, 28, 43, 79, 135, 174, 194, 197, 198, 200, 219, 220, 222, 223, 227, 232, 242, 245, 254, 271, 282, 285, 286, 293, 296, 303, 304, 326 tropism, 304 tumor cells, 67, 96, 116, 121, 122, 125, 168, 171, 173, 174, 175, 179, 180, 182, 184, 185, 189, 190, 191, 192, 193, 268, 270, 292, 312 tumor growth, 49, 121, 129, 168, 173, 174, 175, 181, 185, 190, 196, 199, 201 tumor metastasis, 174, 198 tumor necrosis factor, 227, 237, 322 tumor progression, 179, 181 tumor resistance, 186, 189 tumour growth, 191, 321, 325, 326 tumour suppressor genes, 317 tungsten, 105 turgor, 139 type 2 diabetes, 228 typhoid, 321 typhoid fever, 321 tyrosine, 313

U ubiquitin-proteasome system, 318 ultrasound, 63, 65, 66, 79, 80, 81, 82, 192, 203 ultrastructure, 119 underlying mechanisms, 200 unipolar pulse, 15, 47, 52, 53, 54 universal gas constant, 76 universality, 12 unmasking, 265 updating, 309 ureter, 278 urethra, 66, 278 urine, 277 uveitis, 229, 239

Electroporation in Laboratory and Clinical Investigations, Nova Science Publishers, Incorporated, 2010. ProQuest Ebook Central,

Index

342

V

W waste, 315 waste disposal, 315 water, 4, 6, 8, 11, 12, 25, 34, 45, 47, 67, 80, 83, 88, 101, 102, 103, 104, 108, 109, 124, 143, 147, 158, 278 weight gain, 229, 230 weight loss, 300 white blood cell count, 305 wires, 54, 176 workers, 6, 8, 24, 134, 136, 137, 139, 140, 142, 145 World Health Organization (WHO), 280, 281, 308 worldwide, 234 wound dehiscence, 261 wound healing, 221, 228, 238, 241

X xenografts, 30, 117, 130, 328

Y yeast, 28, 32, 97, 98, 134, 138, 147, 164, 218 yield, 19, 66, 71, 85, 86, 140, 145, 147, 219, 220

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vaccine, 41, 44, 184, 218, 219, 220, 221, 222, 223, 224, 225, 226, 230, 231, 232, 233, 235, 236, 240, 241, 242, 243, 247, 301, 304, 321 vagina, 223, 242 validation, 17, 18, 291 variables, 1, 3, 5, 7, 12, 13, 25, 27, 144, 218, 249, 277 variations, 16, 17, 20, 25, 139 Vascular endothelial growth factor (VEGF), 221, 228, 230, 240, 319 vasculature, 66, 281, 318, 319, 323 vasoconstriction, 281 vector, 17, 68, 149, 161, 235, 302, 303, 310, 326 velocity, 9, 134 vertigo, 279 vesicle, 118, 119, 121, 126, 127 vessels, 23, 56, 67, 319 video microscopy, 47 viral gene, 165, 215, 218, 235, 292, 301, 302, 303, 305 viral vectors, 206, 299, 301, 302, 308, 309 virus infection, 231 viruses, 225, 240, 301, 303 viscosity, 4, 209 visualization, 71, 118, 121, 122, 165 vitiligo, 270 vomiting, 279

vulva, 275

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