Electroporation-Based Technologies and Treatments Editors: Peter Kramar Damijan Miklavčič Book of the Electroporation-Based Technologies and Treatments Edited by: Peter Kramar Damijan Miklavčič 1st edition www.ebtt.org _____________________________________________________ Kataložni zapis o publikaciji (CIP) pripravili v Narodni in univerzitetni knjižnici v Ljubljani COBISS.SI-ID=20751107 ISBN 978-961-243-402-1 (pdf) _____________________________________________________ URL: http://www.ebtt.org/book Copyright © 2020 is retained by the authors. Anyone may freely copy and distribute this material for educational purposes, but may not sell the material for profit. For questions about this book, contact prof. Damijan Miklavčič, University of Ljubljana, Faculty of Electrical Engineering, damijan.miklavcic@fe.uni-lj.si. Publisher: Založba FE, Ljubljana Fakuleta za elektrotehniko, Ljubljana Editor: prof. dr. Sašo Tomažič 1st electronic edition Preface This book was created to fill the need for a concise introduction to electroporation and electroporation based technologies and treatments for those who are starting out, including students. The field of electroporation is a truly interdisciplinary field as it requires basic and advanced knowledge in physics, chemistry, biology, engineering and medicine and is thus very difficult to start in without this broad interdisciplinary insight. Most of the material is based on Electroporation-Based Technologies and Treatments Postgraduate Course and International Workshop held in Ljubljana bi-annually since 2003, and since 2011 every year (see history at www.ebtt.org). Other material comes from the expertise of the contributing authors and their experience in teaching and training students in postdocs in this interdisciplinary field. We thank all of the authors who have contributed material to this book. Damijan Miklavčič Chapter 1 ................................................................................................ 7 Cell in Electric Field – Induced Transmembrane Voltage .............. Tadej Kotnik ..................................................................................... Chapter 2 .............................................................................................. 21 Electric Properties of Tissues and their Changes during Electroporation ........................................................................................ Damijan Miklavčič, Bor Kos ............................................................ Chapter 3 .............................................................................................. 39 In vitro Cell Electropermeabilization ................................................ Justin Teissié ..................................................................................... Chapter 4 .............................................................................................. 55 Nucleic acids electrotransfer in vitro ................................................. Marie-Pierre Rols .............................................................................. Chapter 5 .............................................................................................. 73 Molecular Dynamics Simulations of Lipid Membranes Electroporation ........................................................................................ Mounir Tarek .................................................................................... Chapter 6 ............................................................................................ 105 Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization ...................................................................................... P. Thomas Vernier ............................................................................ Chapter 7 ............................................................................................ 120 Gene electrotransfer in vivo ................................................................ Maja Čemažar ................................................................................... Chapter 8 ............................................................................................ 134 Electrotransfer of DNA vaccine ......................................................... Véronique Préat and Gaëlle Vandermeulen ...................................... Chapter 9 ............................................................................................ 139 Electrochemotherapy from bench to bedside: principles, mechanisms and applications ................................................................. Gregor Serša...................................................................................... Chapter 10 .......................................................................................... 154 Electrochemotherapy in clinical practice; Lessons from development and implementation - and future perspectives ............... Julie Gehl .......................................................................................... Chapter 11 .......................................................................................... 160 Development of devices and electrodes ............................................. Damijan Miklavčič, Matej Reberšek ................................................ Chapter 12 .......................................................................................... 178 Electroporation and electropermeabilisation - pieces of puzzle put together .............................................................................................. Lluis M Mir ....................................................................................... Chapter 1 Cell in Electric Field – Induced Transmembrane Voltage Tadej Kotnik University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Abstract: Under physiological conditions, a resting voltage in the range of tens of millivolts is continually present on the cell plasma membrane. An exposure of the cell to an external electric field induces an additional component of transmembrane voltage, proportional to the strength of the external field and superimposing onto the resting component for the duration of the exposure. Unlike the resting voltage, the induced voltage varies with position, and also depends on the shape of the cell and its orientation with respect to the electric field. In cell suspensions, it also depends on the volume fraction occupied by the cells. There is a delay between the external field and the voltage induced by it, typically somewhat below a microsecond, but larger when cells are suspended in a low-conductivity medium. As a consequence of this delay, for exposures to electric fields with frequencies above 1 MHz, or to electric pulses with durations below 1 µs, the amplitude of the induced voltage starts to decrease with further increase of the field frequency or further decrease of the pulse duration. With field frequencies approaching the gigahertz range, or with pulse durations in the nanosecond range, this attenuation becomes so pronounced that the voltages induced on organelle membranes in the cell interior become comparable, and can even exceed the voltage induced on the plasma membrane. The cell and its plasma membrane A biological cell can be considered from various aspects. We will skip the most usual description, that of a biologist, and focus on two more technical ones, electrical and geometrical. 8 Tadej Kotnik From the electrical point of view, a cell can roughly be described as an electrolyte (the cytoplasm) surrounded by an electrically insulating shell (the plasma membrane). Physiologically, the exterior of the cell also resemble an electrolyte. If a cell is exposed to an external electric field under such conditions, in its very vicinity the field concentrates within the membrane. This results in an electric potential difference across the membrane, termed the induced transmembrane voltage, which superimposes onto the resting transmembrane voltage typically present under physiological conditions. Transmembrane voltage can affect the functioning of voltage-gated membrane channels, initiate the action potentials, stimulate cardiac cells, and when sufficiently large, it also leads to cell membrane electroporation, with the porated membrane regions closely correlated with the regions of the highest induced transmembrane voltage [1]. With rapidly time-varying electric fields, such as waves with frequencies in the megahertz range or higher, or electric pulses with durations in the submicrosecond range, both the membrane and its surroundings have to be treated as materials with both a non-zero electric conductivity and a non-zero dielectric permittivity. From the geometrical point of view, the cell can be characterized as a geometric body (the cytoplasm) surrounded by a shell of uniform thickness (the membrane). For suspended cells, the simplest model of the cell is a sphere surrounded by a spherical shell. For augmented generality, the sphere can be replaced by a spheroid (or an ellipsoid), but in this case, the requirement of uniform thickness complicates the description of the shell substantially. If its inner surface is a spheroid or an ellipsoid, its outer surface lacks a simple geometrical characterization, and vice versa. 1 Fortunately, this complication does not affect the steady-state voltage induced on the plasma membrane of such cells, which can still be determined analytically. Spheres, spheroids, and ellipsoids may be reasonable models for suspended cells, but not for cells in tissues. No simple geometrical body can model a typical cell in a tissue, and furthermore every cell generally differs in its shape from the rest. With irregular geometries and/or with cells close to each other, the induced voltage cannot be determined analytically, and thus cannot be formulated as an explicit function. This 1 This can be visualized in two dimensions by drawing an ellipse, and then trying to draw a closed curve everywhere equidistant to the ellipse. This curve is not an ellipse, and if one is content with an approximation, the task is actually easier to accomplish by hand than with basic drawing programs on a computer. Cell in Electric Field – Induced Transmembrane Voltage 9 deprives us of some of the insight available from explicit expressions, but using modern computers and numerical methods, the voltage induced on each particular irregular cell can still be determined quite accurately. Resting transmembrane voltage Under physiological conditions, a voltage in the range of –90 mV up to –40 mV is always present on the cell membrane [2,3]. This voltage is caused by a minute deficit of positive ions in the cytoplasm relative to the negative ones, which is a consequence of the transport of specific ions across the membrane. The most important actors in this transport are: (i) the Na-K pumps, which export Na+ ions out of the cell and simultaneously import K+ ions into the cell; and (ii) the K leak channels, through which K+ ions can flow across the membrane in both directions. The resting transmembrane voltage reflects the electrochemical equilibrium of the action of these two mechanisms, and perhaps the easiest way to explain the occurrence of this voltage is to describe how the equilibrium is reached. The Na-K pump works in cycles. In a single cycle, it exports three Na+ ions out of the cell and imports two K+ ions into it. This generates a small deficit of positive ions in the cytoplasm and a gradient of electric potential, which draws positive ions into the cell, and negative ions out of the cell. But at the same time, the pump also generates concentration gradients of Na+ and K+, which draw the Na+ ions into the cell, and the K+ ions out of the cell. The K+ ions are the only ones that possess a significant mechanism of passive transport through the membrane, namely the K leak channels, and through these the K+ ions are driven towards the equilibration of the electrical and the concentration gradient. When this equilibrium is reached, the electrical gradient across the membrane determines the resting transmembrane voltage, which is continually present on the membrane. The unbalanced ions responsible for the resting transmembrane voltage represent a very small fraction of all the ions in the cytoplasm, so that the osmotic pressure difference generated by this imbalance is negligible. Also, the membrane acts as a charged capacitor, with the unbalanced ions accumulating close to its surface, so that the cytoplasm can in general be viewed as electrically neutral. 10 Tadej Kotnik Induced transmembrane voltage When a biological cell is placed into an electric field, this leads to a local distortion of the field in the cell and its vicinity. As outlined in the introductory section of this paper, due to the low membrane conductivity, in the vicinity of the cell the field is concentrated in the cell membrane, where it is several orders of magnitude larger than in the cytoplasm and outside the cell. This results in a so-called induced transmembrane voltage, which superimposes to the resting component. In the following subsections, we describe in more detail the transmembrane voltage induced on cells of various shapes and under various conditions. In each considered case, the principles of superposition allow to obtain the complete transmembrane voltage by adding the resting component to the induced one. Spherical cells For an exposure to a DC homogeneous electric field, the voltage induced on the cell membrane is determined by solving Laplace's equation. Although biological cells are not perfect spheres, in theoretical treatments they are usually considered as such. For the first approximation, the plasma membrane can also be treated as nonconductive. Under these assumptions, the induced transmembrane voltage ∆Φm is given by a formula often referred to as the (steady-state) Schwan’s equation [4], 3 ∆Φ = θ (1) m ER cos , 2 where E is the electric field in the region where the cell is situated, R is the cell radius, and θ is the angle measured from the center of the cell with respect to the direction of the field. voltage is proportional to the applied electric field and to the cell radius. Furthermore, it has extremal values at the points where the field is perpendicular to the membrane, i.e. at θ = 0° and θ = 180° (the “poles” of the cell), and in-between these poles it varies proportionally to the cosine of θ (see Fig. 1, dashed). The value of ∆Φm given by Eq. (1) is typically established several μs after the onset of the electric field. With exposures to a DC field lasting hundreds of microseconds or more, this formula can safely be applied to yield the maximal, steady-state value of the induced transmembrane Cell in Electric Field – Induced Transmembrane Voltage 11 voltage. To describe the transient behavior during the initial microseconds, one uses the first-order Schwan’s equation [5], 3 ∆Φ = θ − − τ m ER cos (1 exp( t / m)) , (2) 2 where τm is the time constant of membrane charging, R εm τ = m σ σ (3) i e 2 d + Rσm σ + σ i 2 e with σi, σm and σe the conductivities of the cytoplasm, cell membrane, and extracellular medium, respecti-vely, εm the dielectric permittivity of the membrane, d the membrane thickness, and R again the cell radius. In certain experiments in vitro, where artificial extracellular media with conductivities substantially lower than physiological are used, the factor 3/2 in Eqns. (1) and (2) decreases in value, as described in detail in [6]. But generally, Eqns. (2) and (3) are applicable to exposures to sine (AC) electric fields with frequencies below 1 MHz, and to rectangular electric pulses longer than 1 µs. To determine the voltage induced by even higher field frequencies or even shorter pulses, the dielectric permittivities of the electrolytes on both sides of the membrane also have to be accounted for. This leads to a further generalization of Eqns. (2) and (3) to a second-order model [7-9], and the results it yields will be outlined in the last section of this paper. Spheroidal and ellipsoidal cells Another direction of generalization is to assume a cell shape more general than that of a sphere. The most straightforward generalization is to a spheroid (a geometrical body obtained by rotating an ellipse around one of its radii, so that one of its orthogonal projections is a sphere, and the other two are the same ellipse) and further to an ellipsoid (a geometrical body in which each of its three orthogonal projections is a different ellipse). To obtain the analogues of Schwan’s equation for such cells, one solves Laplace’s equation in spheroidal and ellipsoidal coordinates, respectively [10-12]. Besides the fact that this solution is by itself somewhat more intricate than the one in spherical coordinates, the generalization of the shape invokes two additional complications outlined in the next two paragraphs. 12 Tadej Kotnik Figure 1: Normalized steady-state ∆Φm as a function of the polar angle θ for spheroidal cells with the axis of rotational symmetry aligned with the direction of the field. Solid: a prolate spheroidal cell with R2 = 0.2 × R 1. Dashed: a spherical cell, R2 = R1 = R. Dotted: an oblate spheroidal cell with R2 = 5×R 1. A description of a cell is geometrically realistic if the thickness of its membrane is uniform. This is the case if the membrane represents the space between two concentric spheres, but not with two confocal spheroids or ellipsoids. As a result, the thickness of the membrane modeled in spheroidal or ellipsoidal coordinates is necessarily nonuniform. By solving Laplace's equation in these coordinates, we thus obtain the spatial distribution of the electric potential in a nonrealistic setting. However, under the assumption that the membrane conductivity is zero, the induced transmembrane voltage obtained in this manner is still realistic. Namely, the shielding of the cytoplasm is then complete, and hence the electric potential everywhere inside the cytoplasm is constant. Therefore, the geometry of the inner surface of the membrane does not affect the potential distribution outside the cell, which is the same as if the cell would be a homogeneous non-conductive body of the same shape. 2 A more rigorous discussion of the validity of this approach 2 As a rough analogy, when a stone is placed into a water stream, the streamlines outside the stone are the same regardless of the stone’s interior composition. Due to the fact that stone is impermeable to water, only its outer shape matters in this respect. Similarly, when the membrane is nonconductive, or “impermeable to electric current”, Cell in Electric Field – Induced Transmembrane Voltage 13 can be found in [10]. Fig. 1 compares the transmembrane voltage induced on two spheroids with the axis of rotational symmetry aligned with the direction of the field, and that induced on a sphere. Figure 2: Normalized steady-state ∆Φm as a function of the normalized arc length p for spheroidal cells with the axis of rotational symmetry aligned with the direction of the field. Solid: a prolate spheroidal cell with R2 = 0.2 × R 1. Dashed: a spherical cell, R2 = R1 = R. Dotted: an oblate spheroidal cell with R2 = 5×R 1. For nonspherical cells, it is generally more revealing to express ∆Φm as a function of the arc length than as a function of the angle θ (for a sphere, the two quantities are directly proportional). For uniformity, the normalized version of the arc length is used, denoted by p and increasing from 0 to 1 equidistantly along the arc of the membrane. This is illustrated in Fig. 2 for the cells for which ∆Φm(θ) is shown in Fig. 1, and all the plots of ∆Φm on nonspherical cells will henceforth be presented in this manner. only the outer shape of the cell affects the current density and the potential distribution outside the cell. 14 Tadej Kotnik Figure 3: Normalized steady-state ∆Φm( p) for a prolate spheroidal cell with R2 = 0.2 × R 1. Solid: axis of rotational symmetry (ARS) aligned with the field. Dashed: ARS at 45° with respect to the field. Dotted: ARS perpendicular to the field. Figure 4: Normalized steady-state ∆Φm( p) for an oblate spheroidal cell with R2 = 5 × R 1. Solid: axis of rotational symmetry (ARS) aligned with the field. Dashed: ARS at 45° with respect to the field. Dotted: ARS perpendicular to the field. Cell in Electric Field – Induced Transmembrane Voltage 15 The second complication of generalizing the cell shape from a sphere to a spheroid or an ellipsoid is that the induced voltage now also becomes dependent on the orientation of the cell with respect to the electric field. To deal with this, one decomposes the field vector into the components parallel to the axes of the spheroid or the ellipsoid, and writes the induced voltage as a corresponding linear combination of the voltages induced for each of the three coaxial orientations [11,12]. Figs. 3 and 4 show the effect of rotation of two different spheroids with respect to the direction of the field. Irregularly shaped cells For a cell having an irregular shape, the induced transmembrane voltage cannot be determined exactly, as for such a geometry Laplace's equation is not solvable analytically. Using modern computers and finite-elements tools such as COMSOL Multiphysics, the voltage induced on a given irregular cell can still be determined numerically, as described in detail in [13,14]. While the results obtained in this manner are quite accurate, they are only applicable to the particular cell shape for which they were computed. Fig. 5 shows examples of two cells growing in a Petri dish and the voltages induced on their membranes. Figure 5: Normalized steady-state ∆Φm( p) for two irregularly shaped cells growing on the flat surface of a Petri dish. 16 Tadej Kotnik Cells in dense suspensions In dilute cell suspensions, the distance between the cells is much larger than the cells themselves, and the local field outside each cell is practically unaffected by the presence of other cells. Thus, for cells representing less than 1 % of the suspension volume (for a spherical cell with a radius of 10 µm, this means up to 2 million cells/ml), the deviation of the actual induced transmembrane voltage from one predicted by Schwan's equation is negligible. However, as the volume fraction occupied by the cells gets larger, the distortion of the local field around each cell by the presence of other cells in the vicinity becomes more pronounced, and the prediction yielded by Schwan's equation less realistic (Fig. 6). For volume fractions over ten percent, as well as for clusters and lattices of cells, one has to use appropriate numerical or approximate analytical solutions for a reliable analysis of the induced transmembrane voltage [15,16]. Regardless of the volume fraction they occupy, as long as the cells are suspended, they are floating freely, and their arrangement is rather uniform. Asymptotically, this would correspond to a face-centered cubic lattice, and this lattice is also the most appropriate for the analysis of the transmembrane voltage induced on cells in suspension. For even larger volume fractions of the cells, the electrical properties of the suspension start to resemble that of a tissue, but only to a certain extent. The arrangement of cells in tissues does not necessarily resemble a face-centered lattice, since cells can form specific structures (e.g. layers). In addition, cells in tissues can be directly electrically coupled (e.g. through gap junctions). These and other specific features of the interactions between cells in tissues and electric fields will be considered in more detail in the paper that follows this one. High field frequencies and very short pulses The time constant of membrane charging (τm) given by Eq. (3) implies that there is a delay between the time courses of the external field and the voltage induced by this field. As mentioned above, τm (and thus the delay) is somewhat below a microsecond under physiological conditions, but can be larger when cells are suspended in a low-conductivity medium. For alternating (AC) fields with the oscillation period much longer than τm, as well as for rectangular pulses much longer than τm, the amplitude of the induced voltage remains unaffected. However, for AC Cell in Electric Field – Induced Transmembrane Voltage 17 fields with the period comparable or shorter than τm, as well as for pulses shorter than τm, the amplitude of the induced voltage starts to decrease. To illustrate how the amplitude of the induced transmembrane voltage gets attenuated as the frequency of the AC field increases, we plot the normalized amplitude of the induced voltage as a function of the field frequency. For a spherical cell, the plot obtained is shown in Fig. 6. The low-frequency plateau and the downward slope that follows are both described by the first-order Schwan's equation, but the high-frequency plateau is only described by the second-order model [7-9], in which all electric conductivities and dielectric permittivities are accounted for. Figure 6: Normalized steady-state ∆Φm(θ) for spherical cells in suspensions of various densities (intercellular distances). Solid: The analytical result for a single cell as given by Eq. (1). Dashed: numerical results for cells arranged in a face-centered cubic lattice and occupying (with decreasing dash size) 10%, 30%, and 50% of the total suspension volume. 18 Tadej Kotnik Figure 7: The amplitude of normalized steady-state ∆Φm as a function of the frequency of the AC field. The dashed curve shows the first-order, and the solid curve the second-order Schwan's equation. Note that both axes are logarithmic. With field frequencies approaching the GHz range, or with pulse durations in the nanosecond range, the attenuation of the voltage induced on the cell plasma membrane becomes so pronounced that this voltage becomes comparable to the voltage induced on organelle membranes in the cell interior. In certain circumstances, particularly if the organelle interior is electrically more conductive than the cytosol, or if the organelle membrane has a lower dielectric permittivity than the cell membrane, the voltage induced on the membrane of this organelle can temporarily even exceed the voltage induced on the plasma membrane [17]. In principle, this could provide a theoretical explanation for a number of recent reports that very short and intense electric pulses (tens of ns, millions or tens of millions of V/m) can also induce electroporation of organelle membranes [18-20]. References [1] T. Kotnik, G. Pucihar, D. Miklavčič. Induced transmembrane voltage and its correlation with electroporation-mediated molecular transport. J. Membrane Biol. 236: 3-13, 2010. [2] K.S. Cole. Membranes, Ions and Impulses. University of California Press, Berkeley, USA, 1972. Cell in Electric Field – Induced Transmembrane Voltage 19 [3] H.L. Atwood, W.A. Mackay. Essentials of Neurophysiology. BC Decker, Toronto, Canada, 1989. [4] H.P. Schwan. Electrical properties of tissue and cell suspensions. Adv. Biol. Med. Phys. 5: 147-209, 1957. [5] H. Pauly, H.P. Schwan. Über die Impedanz einer Suspension von kugelförmigen Teilchen mit einer Schale. Z. Naturforsch. 14B: 125-131, 1959. [6] T. Kotnik, F. Bobanović, D. Miklavčič. Sensitivity of transmembrane voltage induced by applied electric fields — a theoretical analysis. Bioelectrochem. Bioenerg. 43: 285-291, 1997. [7] C. Grosse, H.P. Schwan. Cellular membrane potentials induced by alternating fields. Biophys. J. 63: 1632-1642, 1992. [8] T. Kotnik, D. Miklavčič, T. Slivnik. Time course of transmembrane voltage induced by time-varying electric fields — a method for theoretical analysis and its application. Bioelectrochem. Bioenerg. 45: 3-16, 1998. [9] T. Kotnik, D. Miklavčič. Second-order model of membrane electric field induced by alternating external electric fields. IEEE Trans. Biomed. Eng. 47: 1074-1081, 2000. [10] T. Kotnik, D. Miklavčič. Analytical description of transmembrane voltage induced by electric fields on spheroidal cells. Biophys. J. 79: 670-679, 2000. [11] J. Gimsa, D. Wachner. Analytical description of the transmembrane voltage induced on arbitrarily oriented ellipsoidal and cylindrical cells. Biophys. J. 81: 1888-1896, 2001. [12] B. Valič, M. Golzio, M. Pavlin, A. Schatz, C. Faurie, B. Gabriel, J. Teissié, M.P. Rols, D. Miklavčič. Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment. Eur. Biophys. J. 32: 519-528, 2003. [13] G. Pucihar, T. Kotnik, B. Valič, D. Miklavčič. Numerical determination of the transmembrane voltage induced on irregularly shaped cells. Annals Biomed. Eng. 34: 642-652, 2006. [14] G. Pucihar, D. Miklavčič, T. Kotnik. A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells. IEEE T. Biomed. Eng. 56: 1491-1501, 2009. [15] R. Susil, D. Šemrov, D. Miklavčič. Electric field induced transmembrane potential depends on cell density and organization. Electro. Magnetobiol. 17: 391-399, 1998. [16] M. Pavlin, N. Pavšelj, D. Miklavčič. Dependence of induced transmembrane potential on cell density, arrangement, and cell position inside a cell system. IEEE Trans. Biomed. Eng. 49: 605-612, 2002. [17] T. Kotnik, D. Miklavčič. Theoretical evaluation of voltage inducement on internal membranes of biological cells exposed to electric fields. Biophys. J. 90: 480-491, 2006. [18] K.H. Schoenbach, S.J. Beebe, E.S. Buescher. Intracellular effect of ultrashort electrical pulses. Bioelectromagnetics 22: 440-448, 2001. [19] S.J. Beebe, P.M. Fox, L.J. Rec, E.L. Willis, K.H. Schoenbach. Nanosecond, high-intensity pulsed electric fields induce apoptosis in human cells. FASEB J. 17: 1493-1495, 2003. [20] E. Tekle, H. Oubrahim, S.M. Dzekunov, J.F. Kolb, K.H. Schoenbach, P. B. Chock. Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses. Biophys. J. 89: 274-284, 2005. 20 Tadej Kotnik Acknowledgement This work was supported by the Slovenian Research Agency. Tadej Kotnik was born in Ljubljana, Slovenia, in 1972. He received a Ph.D. in Biophysics from University Paris XI and a Ph.D. in Electrical Engineering from the University of Ljubljana, both in 2000. He is currently a Full Professor and former Vice-dean for Research at the Faculty of Electrical Engineering of the University of Ljubljana. His research interests include cell membrane electrodynamics, as well as theoretical and experimental study of related biophysical phenomena, particularly membrane electroporation and gene electrotransfer. Tadej Kotnik is the first author of 23 articles in SCI-ranked jour¬nals cited over 1200 times excluding self-citations, and a co-author of additional 28 such articles cited over 800 times excluding self-citations. His h-index is 26. In 2001 he received the Galvani Prize of the Bioelectrochemical Society. Chapter 2 Electric Properties of Tissues and their Changes during Electroporation Damijan Miklavčič, Bor Kos University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Abstract: Passive electric properties of biological tissues such as permittivity and conductivity are important in applied problems of electroporation. The current densities and pathways resulting from an applied electrical pulse are dictated to a large extent by the relative permittivity and conductivity of biological tissues. We briefly present some theoretical basis for the current conduction in biologic materials and factors affecting the measurement of tissue dielectric properties that need to be taken into account when designing the measurement procedure. Large discrepancies between the data reported by different researchers are found in the literature. These are due to factors such as different measuring techniques used, the fact that macroscopic tissue properties show inhomogeneity, dispersions, anisotropy, nonlinearity, as well as temperature dependence and changes over time. Furthermore, when biological tissue is exposed to a high electric field, changes in their electric properties occur. Introduction The electrical properties of biological tissues and cell suspensions have been of interest for over a century. They determine the pathways of current flow through the body and are thus very important in the analysis of a wide range of biomedical applications. On a more fundamental level, knowledge of these electrical properties can lead to the understanding of the underlying, basic biological processes. To analyze the response of a tissue to electric stimulus, data on the conductivity and relative permittivity of the tissues or 22 Damijan Miklavčič, Bor Kos organs are needed. A microscopic description of the response is complicated by the variety of cell shapes and their distribution inside the tissue as well as the different properties of the extracellular media. At low frequency, the electric conductivity is determined by mainly by extracellular ion concentration and their mobility. Therefore, a macroscopic approach is most often used to characterize field distributions in biological systems. However, even on a macroscopic level the electrical properties are complicated. They can depend on the tissue orientation relative to the applied field (directional anisotropy), the frequency of the applied field (the tissue is neither a perfect dielectric nor a perfect conductor) or they can be time and space dependent (e.g., changes in tissue conductivity during electroporation) [1]–[3]. Biological materials in the electric field The electrical properties of any material, including biological tissue can be broadly divided into two categories: conducting and insulating. In a conductor the electric charges move freely in response to the application of an electric field whereas in an insulator (dielectric) the charges are fixed and are thus not free to move – the current does not flow. If a conductor is placed in an external electric field, charges will redistribute within the conductor until the resulting internal field is zero. In the case of an insulator, there are no free charges so net migration of charge does not occur. In polar materials, the positive and negative charge centers in the molecules (e.g. water) do not coincide. An applied field, E0, tends to orient the dipoles and produces a field inside the dielectric, Ep, which opposes the applied field. This process is called polarization. Most materials contain a combination of dipoles and free charges. Thus the electric field is reduced in any material relative to its free-space value. The resulting internal field inside the material, E, is then E = E − E 0 p The resulting internal field is lowered by a significant amount relative to the applied field if the material is an insulator and is essentially zero for a good conductor. This reduction is characterized by a factor εr, which is called the relative permittivity or dielectric constant, according to E0 E = εr Electric Properties of Tissues and their Changes during Electroporation 23 In practice, most materials, including biological tissue, actually display some characteristics of both, insulators and conductors, because they contain dipoles as well as charges which can move, but in a restricted manner [4], [5]. On a macroscopic level we describe the material as having a permittivity, ε, and a conductivity, σ. The permittivity characterizes the material’s ability to trap or store charge or to rotate molecular dipoles whereas the conductivity describes its ability to transport charge. The permittivity also helps to determine the speed of light in a material so that free space has a permittivity ε0 =8.85 x 10-12 F/m. For other media: ε = ε ε r 0 The energy stored per unit volume in a material, u, and the power dissipated per unit volume, p, are: 2 E ε 2 E σ u = p = 2 2 Consider a sample of material which has a thickness, d, and cross-sectional area, A (Figure 1). A d Figure 1: A considered theoretical small part of a material. If the material is an insulator, then we treat the sample as a capacitor with capacitance (C); if it is a conductor, then we treat it as a conductor with conductance (G): A C = ε ⋅ A d G = σ⋅ d A simple model for a real material, such as tissue, would be a parallel combination of the capacitor and conductor. If a constant (DC) voltage V is applied across this parallel combination, then a conduction current IC = GV will flow and an amount of charge Q=CV will be stored. However, if an alternating (AC) voltage was applied to the combination: 24 Damijan Miklavčič, Bor Kos V(t)=V cos(ωt) 0 The charge on the capacitor plates is now changing with frequency f. We characterize this flow as a displacement current: dQ I = = -ωCV sin(ωt) d 0 dt The total current flowing through the material is the sum of the conduction and displacement currents, which are 90° apart in phase, with the displacement currents being “ahead” of the applied voltage. The total current is I = Ic + Id, hence dV A I=GV+C⋅ = (σ + iωε)V ⋅ dt d The actual material, then, can be characterized as having an admittance, Y*, given by: * Y = G + iωC = (A d)(σ + iωε) where * indicates a complex-valued quantity. In terms of material properties we define a corresponding, complex-valued conductivity * σ = (σ + iωε) Describing a material in terms of its admittance emphasizes its ability to transport current. Alternatively, we could emphasize its ability to restrict the flow of current by considering its impedance Z*=1/Y*, or for a pure conductance, its resistance, R=1/G. We can also denote total current as: dV iσ A I = (ε − )iωε = C r 0 ωε d 0 dt We can define a complex-valued, relative permittivity: iσ ' ' * ε = ε − = ε − iε r r r ωε0 with εr'=εr and εr''=σ/(ωε0). The complex conductivity and complex permittivity are related by: * * σ = iωε = iωε ε * 0 r We can consider the conductivity of a material as a measure of the ability of its charge to be transported throughout its volume in a response to the applied electric field. Similarly, its permittivity is a measure of the ability Electric Properties of Tissues and their Changes during Electroporation 25 of its dipoles to rotate or its charge to be stored in response to the applied field. Note that if the permittivity and conductivity of the material are constant, the displacement current will increase with frequency whereas the conduction current does not change. At low frequencies the material will behave like a conductor, but capacitive effects will become more important at higher frequencies. For most materials, however, σ* and ε* are frequency-dependent. Such a variation is called dispersion and is due to the dielectric relaxation – the delay in molecular polarization following changing electric field in a material. Biological tissues exhibit several different dispersions over a wide range of frequencies [4]. Dispersions can be understood in terms of the orientation of the dipoles and the motion of the charge carriers. At relatively low frequencies it is relatively easy for the dipoles to orient in response to the change in applied field whereas the charge carriers travel larger distances over which there is a greater opportunity for trapping at a defect or interface like cell membrane [6]. As the frequency increases, the dipoles are less able to follow the changes in the applied field and the corresponding polarization disappears. In contrast, the charge carriers travel shorter distances during each half-cycle and are less likely to be trapped. As frequency increases, the permittivity decreases and, because trapping becomes less important, the conductivity increases. In a heterogeneous material, such as biological tissue, several dispersions are observed as illustrated in Figure 2. In short, alpha dispersion in the kilohertz range is due to cell membrane effects such as gated channels and ionic diffusion and is the first of the dispersions to disappear with tissue death. Beta dispersion can be observed around the megahertz range due to the capacitive charging of cell membranes. Above beta dispersion the impedance of cell membranes drops drastically, allowing the electric current to pass through not only extracellular, but also intracellular space. This dispersion is particularly interesting as it is also apparent in the conductivity of the material. The last, gamma dispersion (above the gigahertz range) is due to dipolar mechanisms of water molecules in the material. 26 Damijan Miklavčič, Bor Kos Figure 2: Typical frequency dependence of the complex permittivity and complex conductivity of a heterogeneous material such as biological tissue. Measurements of dielectric properties of tissues There is a large discrepancy between various data on electrical properties of biological materials found in the literature. The measurement of tissue dielectric properties can be complicated due to several factors, such as tissue inhomogeneity, anisotropy, the physiological state of the tissue, seasonal, age and disease-linked changes and electrode polarization [1]. Inhomogeneity of tissues Tissue is a highly inhomogeneous material. The cell itself is comprised of an insulating membrane enclosing a conductive cytosol. A suspension of cells can be regarded at low frequencies simply as nonconducting inclusions in a conducting fluid [6]. The insulation is provided by the cell membrane. At frequencies in the MHz range capacitive coupling across this membrane becomes more important, allowing the electric current to pass not only around the cell, but also through it. In tissue, the cells are surrounded by an extracellular matrix, which can be extensive, as in the case of bone, or minimal, as in the case of epithelial tissue. Tissue does not contain cells of a single size and function. The tissue is perfused with blood and linked to the central nervous system by neurons. It is thus difficult (if not impossible) to extrapolate from the dielectric properties of a cell suspension to those of an intact tissue. Electric Properties of Tissues and their Changes during Electroporation 27 Anisotropy of tissues Some biological materials, such as bone and skeletal muscle, are anisotropic. Therefore, when referring to measured conductivity and permittivity values, one needs to include data on the orientation of the electrodes relative to the major axis of the tissue; e.g., longitudinal, transversal or a combination of both. For example, muscles are composed of fibers, very large individual cells aligned in the direction of muscle contraction. Electrical conduction along the length of the fiber is significantly easier than conduction in the direction perpendicular to the fibers. Therefore, muscle tissue manifests typical anisotropic electric properties. The longitudinal conductivity is significantly higher than the transverse conductivity (can be up to 8 times higher) [7]. Moreover, tissue anisotropy is frequency dependent. Namely, if the frequency of the current is high enough, the anisotropic properties disappear. Specifically for muscle tissue, that happens in the MHz frequency range, i.e. at beta dispersion. Physiological factors and changes of tissue Any changes in tissue physiology should produce changes in the tissue electrical properties. This principle has been used to identify and/or monitor the presence of various illnesses or conditions [8], [9]. Tumors generally have higher water content than normal cells because of cellular necrosis but also irregular and fenestrated vascularization. Higher conductivity of tumors in the MHz frequency range has been identified as a potential target for imaging applications [10]. In addition, there may be differences in the membrane structure. Also, fat is a poorer conductor of electricity than water. Changes in the percentage of body fat or water are reflected in tissue impedance changes [8]. It has also been reported that pathological changes in the liver, such as fatty liver disease or liver cirrhosis can influence the measured properties of tissue at 100 MHz frequencies [9]. Further, tissue death or excision results in significant changes in electrical properties. Tissue metabolism decreases after the tissue has been excised and often the temperature falls. If the tissue is supported by temperature maintenance and perfusion systems, the tissue may be stabilized for a limited period of time in a living state in vitro (ex vivo). If the tissue is not supported, however, irreversible changes will occur, followed by cell and tissue death. For these reasons considerable caution 28 Damijan Miklavčič, Bor Kos must be taken in the interpretation of electrical measurements which were performed on excised tissues. The electrical properties of tissue also depend on its temperature. The mobility of the ions which transport the current increases with the temperature as the viscosity of the extracellular fluid decreases. The rapid increase of conductivity with temperature was suggested to be used e.g. to monitor the progress of hyperthermia treatment. Also, possible other changes, such as cell swelling and edema, or blood flow occlusion, all affect tissue properties. Electrode polarization Electrode polarization is a manifestation of molecular charge organization which occurs at the tissue/sample-electrode interface in the presence of water molecules and hydrated ions. The effect increases with increasing sample conductivity [11]. In a cell suspension a counterion layer can form at each electrode. The potential drop in this layer reduces the electric field available to drive charge transport in the bulk suspension, resulting in apparently lower suspension conductivity. As the frequency increases, the counterion layer is less able to follow the changes in the applied signal, the potential drop at the sample-electrode interface decreases, and the apparent conductivity of the suspension increases. Thus electrode polarization is more pronounced at lower frequencies, and at lower amplitudes of the measurement voltage signal. The process is more complicated in tissue. Insertion of invasive electrodes can first cause the release of electrolytes due to trauma from the surrounding tissue and later the development of a poorly-conductive wound region may occur. This region can shield part of the electrode from the ionic current and thus reduce the polarization effects compared to an ionic solution equivalent in conductivity to the intracellular fluid. The material of the electrode plays an important role in determining its polarization impedance, the relative importance of which decreases with increasing frequency. It is considered a good practice to measure tissue impedance in-vivo after waiting a sufficient time for the electrode polarization processes to stabilize. A typical time might be on the order of thirty minutes. Two different basic electrode set-ups are used to measure the electric properties of biological materials; the two-electrode and the four-electrode method. Electric Properties of Tissues and their Changes during Electroporation 29 Two-electrode method: Suitable for alternating current (AC) measurements. Cannot be used as such for direct current (DC) measurements because of the electrode polarization, which consequently gives incorrect results for the conductivity of the sample between the electrodes. For AC measurements the frequency range over which electrode polarization is important depends to some extent on the system being measured and the electrode material. For cell suspensions it is important up to nearly 100 kHz whereas for tissue measured in vivo it is significant only up to about 1 kHz. By varying the separation of the electrodes, the contribution of the electrode polarization can be determined and eliminated, however this is a method best applicable to liquid samples, since it requires parallel plate electrodes. Four-electrode method: Can be used for both DC and AC measurements. Two pairs of electrodes are used: the outer, current electrodes and the inner, voltage electrodes. The current from the source passes through the sample. Voltage electrodes of known separation are placed in the sample between the current electrodes. By measuring the current as the voltage drop across a resistor in series with the sample and the voltage drop across the inner electrodes, one can determine the conductivity of the sample between the inner electrodes. The advantage of this method is that the polarization on the current electrodes has no influence on the voltage difference between the voltage electrodes. Polarization at the voltage electrodes is negligible for both DC and AC due to the high input impedance of the measurement system. The drawback is that measurement results are interpreted based on the assumption of tissue being homogeneous in the entire region where measurement is performed. Electrical response of tissue to electric field Changes in tissue conductivity have been observed in vivo if the tissue is subjected to a high enough electric field. Having said that, we can use the dielectric properties of liver and try to calculate the theoretical electrical response to a short rectangular voltage pulse having the duration of 100 μs and the rise time of 1 μs (typical pulse parameters used for electrochemotherapy). We used the parallel RC circuit to model the electrical response of the tissue (see Figure 3). 30 Damijan Miklavčič, Bor Kos Figure 3: Parallel RC circuit: a theoretical representation of tissue response to electic pulses. The complications arise from the facts that i) the pulse parameters (the pulse duration, the rise and the fall time) determine the content of its frequency spectrum and ii) the tissue conductivity and permittivity are frequency dependent. The obtained response for the first pulse is presented in Figure 4. At the onset of voltage pulse, capacitive transient displacement current is observed. As membranes charge, voltage across them rises and the measured current decreases. Soon steady state is reached and current stabilizes through the conductance of extracellular fluid. Since the model describing dielectric dispersions is linear, change of the applied voltage proportionally scales the amplitude of the current. We can compare this calculated response with the measured response on rat liver in vivo for the same pulse as above and different pulse amplitudes spanning up to electroporative field strengths (Figure 5) [12]. For the lowest applied voltage we can see a good agreement with calculated response. As the field intensity is increased, the electrical response of tissue is no longer linear, meaning that the current is increasing faster than the voltage. The current is also increasing during the pulse itself, as can be seen from the current rise at the higher voltages in Figure 5. Measuring the passive electrical properties of electroporated tissues could provide real time feedback on the outcome of the treatment [12], [13]. However, care must be taken in the interpretation of the current rise during the application of electroporation treatments, due to the natural variability of the tissue dielectric properties, and because conductivity rise can also be a product of tissue heating [14], [15]. Electric Properties of Tissues and their Changes during Electroporation 31 Figure 4: Calculated tissue response during delivery of rectangular voltage pulse with the duration of 100 μs having the rise time of 1 μs and the amplitude of 120 V. Plate electrodes with 4.4 mm interelectrode distance were assumed. Figure 5: Measured tissue response during delivery of 100 μs rectangular pulses of different amplitudes to rat liver in vivo. Adapted from Cukjati et al. [10]. Pulses were generated using Jouan GHT1287B; plate electrodes with 4.4 mm interelectrode distance were used. The measured response is consistent with the hypothesis that the bulk tissue conductivity should also increase measurably since on a cellular level electroporation causes the increase of membrane conductance [16]. In 32 Damijan Miklavčič, Bor Kos measuring ex vivo tissue and phantom tissue made of gel like material [17] and ex-vivo liver tissue [18] using MREIT we were able to demonstrate that electric conductivity changes due to membrane electroporation are amplitude dependent and occur in tissue only but not in phantom tissue. It is not clear, however, to which value tissue conductivity increases as a consequence of plasma membrane electroporation. It has been stipulated that this could be close to the value in beta dispersion range [19]. Figure 6: Conductivity of tissues as a function of the local electric field strength. Further, in applications where electric pulses to skin or tissues underneath (such as subcutaneous tumor) are applied externally, through skin, one might expect very high voltage amplitudes would be required to breach the highly resistive skin tissue and permeabilize tissues underneath. Namely, tissues between the electrodes can be seen as serially connected resistors. Applying voltage on such a circuit (voltage divider) causes the voltage to be distributed between the resistors proportionally to their resistivities [20]. Upon applying electric pulses, almost the entire applied voltage thus rests across the most resistive (least conductive) tissue, in our case the outermost layers of the skin. That would results in a very high electric field strength in skin tissue, while the electric field in other tissues would remain insufficient for successful cell electroporation. If our goal is the electrochemotherapy of the underlying tumor, one might wonder how a successful electrochemotherapy of subcutaneous tumors is possible when external plate electrodes are used. The answer lies in the increase in bulk conductivities of tissues during electroporation, a phenomenon that was also observed in vivo. This conductivity increase leads to a change in electric field distribution, which exposes the tumor to an electric field high Electric Properties of Tissues and their Changes during Electroporation 33 enough for successful cell membrane permeabilization [21]. To further support this hypothesis, we described this process with a numerical model, taking into account the changes of tissue bulk electrical properties during electroporation. In Figure 6 six steps of the electroporation process in the subcutaneous tumor model for the voltage of 1000 V between the electrodes are shown. The electric field distribution shown in V/cm. Step 0 denotes the electric field distribution as it was just before the electroporation process started, thus when all the tissues still had their initial conductivities. When the voltage is applied to the electrodes, the electric field is distributed in the tissue according to conductivity ratios of the tissues in the model. The field strength is the highest in the tissues with the lowest conductivity, where the voltage drop is the largest. In our case, almost the entire voltage drop occurs in the skin layer which has a conductivity of about 10-100 times lower than the tissues lying underneath. If we look at the last step of the sequential analysis, step 5, at 1000 V (Figure 7) the tumor is entirely permeabilized, in some areas the electric field is also above the irreversible threshold. Figure 7: Six steps of the sequential analysis of the electroporation process in the subcutaneous tumor model at 1000 V between two plate electrodes with distance of 8 mm [21]. Time intervals between steps are in general not uniform. Different steps follow a chronological order but do not have an exact time value associated with them. The electric field distribution is shown in V/cm. A similar situation can be encountered when applying electric pulses on a skin fold with external plate electrodes as a method to enhance in vivo 34 Damijan Miklavčič, Bor Kos gene transfection in skin [22]. Skin consists of three main layers: epidermis, dermis and subcutaneous tissue (Figure 8). The epidermis is made up of different layers, but the one that defines its electrical properties the most is the outermost layer, the stratum corneum. Although very thin (typically around 20 μm), it contributes a great deal to the electrical properties of skin. Its conductivity is three to four orders of magnitude lower than the conductivities of deeper skin layers. Again, when electric pulses are applied on skin fold through external plate electrodes, almost the entire applied voltage rests across the stratum corneum, which causes a very high electric field in that layer, while the electric field in deeper layers of skin – the layers targeted for gene transfection – stays too low. Similarly as in the case of subcutaneous tumors, the increase in bulk conductivities of skin layers during electroporation exposes the skin layers below stratum corneum to an electric field high enough for a successful permeabilization [23]. Figure 8: Schematics of a skinfold as described in a numerical model. Only one quarter of the skinfold is presented here. Electric Properties of Tissues and their Changes during Electroporation 35 Tissue properties during high frequency electroporation High frequency electroporation is recently gaining considerable attention after it was pointed out in the literature, that electroporation with pulse durations between 1 μs and 100 μs is relatively unexplored [24]. The shorter pulses, combined with some pause between pulses have the potential of reducing nerve and muscle stimulation present in “classical” electroporation pulses [25]. Since the pulse protocols of high frequency electroporation typically employ larger number of bipolar pulses of short duration, the frequency content of these pulses is can be markedly different from the typical 100 μs electroporation puslses [26]. One possible advantage of this is that there could be a lower conductivity contrast between e.g. tumor and surrounding healthy tissue, however the tissue main frequencies of these pulse trains are still low enough, that electroporation causes a significant rise in the conductivity due to membrane electroporation . Conclusions Theoretical explanation of the process of electroporation offers useful insight into the understanding of the underlying biological processes and allows for predicting the outcome of the treatment [27]. Therefore, due effort needs to be invested into measurements of tissue electrical properties and their changes during electroporation [28]. Further, one of the concerns associated with electroporation could be the amount of resistive heating in the tissue. Excessive heating is unwanted not only to avoid skin burns and assure patient safety, but also to avoid damage to viable cells. Potential excess of the resistive heating during electroporation has been demonstrated [29], therefore thermal aspect in treatment planning and theoretical analysis of specific applications of electroporation-based treatments should be considered [26]. In order to stay within the safety limit while achieving successful treatment, heating needs to estimated, by means of theoretical models, as a part of treatment planning [30]. 36 Damijan Miklavčič, Bor Kos References [1] D. Miklavčič, N. Pavšelj, and F. X. Hart, “Electric Properties of Tissues,” in Wiley Encyclopedia of Biomedical Engineering, M. Akay, Ed. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2006, p. ebs0403. [2] K. R. Foster and H. P. Schwan, “Dielectric properties of tissues and biological materials: a critical review,” Crit Rev Biomed Eng, vol. 17, no. 1, pp. 25–104, 1989. [3] C. Gabriel, A. Peyman, and E. H. Grant, “Electrical conductivity of tissue at frequencies below 1 MHz,” Phys. Med. 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Martin et al. , “Irreversible electroporation in locally advanced pancreatic cancer: A call for standardization of energy delivery: IRE Technique in Pancreatic Cancer,” Journal of Surgical Oncology, vol. 114, no. 7, pp. 865–871, Dec. 2016. [15] A. H. Ruarus, L. G. P. H. Vroomen, R. S. Puijk, H. J. Scheffer, T. J. C. Faes, and M. R. Meijerink, “Conductivity Rise During Irreversible Electroporation: True Permeabilization or Heat?,” CardioVascular and Interventional Radiology, vol. 41, no. 8, pp. 1257–1266, Aug. 2018. [16] M. Pavlin and D. Miklavcic, “Effective conductivity of a suspension of permeabilized cells: A theoretical analysis,” Biophys. J. , vol. 85, no. 2, pp. 719–729, 2003. [17] M. Kranjc, F. Bajd, I. Serša, and D. Miklavčič, “Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation,” Physiol Meas, vol. 35, no. 6, pp. 985–996, Jun. 2014. Electric Properties of Tissues and their Changes during Electroporation 37 [18] M. Kranjc, F. Bajd, I. Sersa, E. J. Woo, and D. Miklavcic, “Ex vivo and in silico feasibility study of monitoring electric field distribution in tissue during electroporation based treatments,” PLoS ONE, vol. 7, no. 9, p. e45737, 2012. [19] R. E. Neal 2nd, P. A. Garcia, J. L. Robertson, and R. V. Davalos, “Experimental characterization and numerical modeling of tissue electrical conductivity during pulsed electric fields for irreversible electroporation treatment planning,” IEEE Trans Biomed Eng, vol. 59, no. 4, pp. 1076–1085, Apr. 2012. [20] N. Pavšelj and D. Miklavčič, “Numerical modeling in electroporation-based biomedical applications,” Radiol. Oncol. , vol. 42, no. 3, pp. 159–168, Sep. 2008. [21] N. Pavselj, Z. Bregar, D. Cukjati, D. Batiuskaite, L. M. Mir, and D. Miklavcic, “The Course of Tissue Permeabilization Studied on a Mathematical Model of a Subcutaneous Tumor in Small Animals,” IEEE Trans. Biomed. Eng. , vol. 52, no. 8, pp. 1373–1381, Aug. 2005. [22] N. Pavselj and V. Preat, “DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse,” J. Controlled Release, vol. 106, no. 3, pp. 407–415, Sep. 2005. [23] N. Pavšelj, V. Préat, and D. Miklavčič, “A Numerical Model of Skin Electropermeabilization Based on In Vivo Experiments,” Ann Biomed Eng, vol. 35, no. 12, pp. 2138–2144, Sep. 2007. [24] J. C. Weaver, K. C. Smith, A. T. Esser, R. S. Son, and T. R. Gowrishankar, “A brief overview of electroporation pulse strength-duration space: a region where additional intracellular effects are expected,” Bioelectrochemistry, vol. 87, pp. 236–243, Oct. 2012. [25] C. B. Arena et al. , “High-frequency irreversible electroporation (H-FIRE) for nonthermal ablation without muscle contraction,” Biomed Eng Online, vol. 10, p. 102, 2011. [26] S. Bhonsle, M. F. Lorenzo, A. Safaai-Jazi, and R. V. Davalos, “Characterization of Nonlinearity and Dispersion in Tissue Impedance During High-Frequency Electroporation,” IEEE Transactions on Biomedical Engineering, vol. 65, no. 10, pp. 2190–2201, Oct. 2018. [27] D. Miklavcic et al. , “Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy,” Biomed Eng Online, vol. 9, p. 10, 2010. [28] J. Langus, M. Kranjc, B. Kos, T. Šuštar, and D. Miklavčič, “Dynamic finite-element model for efficient modelling of electric currents in electroporated tissue,” Scientific Reports, vol. 6, p. 26409, May 2016. [29] I. Lackovic, R. Magjarevic, and D. Miklavcic, “Three-dimensional finite-element analysis of joule heating in electrochemotherapy and in vivo gene electrotransfer,” IEEE Trans. Dielect. Electr. Insul. , vol. 16, no. 5, pp. 1338–1347, Oct. 2009. [30] B. Kos, P. Voigt, D. Miklavcic, and M. Moche, “Careful treatment planning enables safe ablation of liver tumors adjacent to major blood vessels by percutaneous irreversible electroporation (IRE),” Radiol Oncol, vol. 49, no. 3, pp. 234–241, Sep. 2015. 38 Damijan Miklavčič, Bor Kos Acknowledgement This work was supported by the Slovenian Research Agency. Damijan Miklavčič was born in Ljubljana, Slovenia, in 1963. He received a Masters and a Doctoral degree in Electrical Engineering from University of Ljubljana in 1991 and 1993, respectively. He is currently Professor and the Head of the Laboratory of Biocybernetics at the Faculty of Electrical Engineering, University of Ljubljana. His research areas are biomedical engineering and study of the interaction of electromagnetic fields with biological systems. In the last years he has focused on the engineering aspects of electroporation as the basis of drug delivery into cells in tumor models in vitro and in vivo. His research includes biological experimentation, numerical modeling and hardware development for electrochemotherapy, irreversible electroporation and gene electrotransfer. Bor Kos was born in Ljubljana, Slovenia, in 1983. He received a Doctoral degree in Electrical Engineering from University of Ljubljana in 2013. He is currently Assistant Professor at the Faculty of Electrical Engineering, University of Ljubljana. Research field of assist. prof. Bor Kos is bioelectromagnetics and biomedical engineering with main focus being on numerical modelling of electroporation. Since the first treatment of deep-seated tumours with electrochemotherapy has been performed at the Institute of Oncology of Ljubljana, Bor Kos has been constantly engaged in the development, validation and advancement of numerical treatment planning for electroporation-based treatments. For his contributions to the field, he received the 2018 Alessandro Chiabrera award, presented by EBEA. He is involved in international research collaborations and publications with colleagues from Denmark, Hungary, France, Germany, Italy, Israel, Poland, Spain, United Kingdom and the USA. Chapter 3 In vitro Cell Electropermeabilization Justin Teissié IPBS UMR 5089 CNRS and Université de Toulouse, Toulouse, France Abstract: Electropulsation (delivery of short lived electric pulses) is one of the most successful non-viral methods to introduce foreign molecules in living cells in vitro. This lecture describes the factors controlling electropermeabilization to small molecules (< 4 kDa). Pulse durations are selected from submicroseconds to a few milliseconds. The description of in vitro events brings the attention of the reader on the processes occurring before, during and after electropulsation of cells. The role of the different electrical parameters (Field strength, pulse duration, delay between pulses) is delineated. The kinetic of the processes affecting the cell surface is described outlining that most of the exchange across the membrane takes place after the pulse during the so called resealing. Cell contribution to this critical step is tentatively explained. The membrane events appear to be controlled by the cellular metabolism. Introduction The application of electric field pulses to cells leads to the transient permeabilization of the plasma membrane (electropermeabilization). This phenomenon brings new properties to the cell membrane: it becomes permeabilized, fusogenic and exogenous membrane proteins can be inserted. It has been used to introduce a large variety of molecules into many different cells in vitro [1, 2]. The present lecture is reporting what is called “classical electropermeabilization”. This meant that it is relevant of the effect of field pulses lasting from µs to several ms, with a rising time of a few hundreds of ns. In this time domain, dielectric spectroscopy of a cell shows that the 40 Justin Teissié membrane can be considered as a non conductive insulator (indeed some active leaks may be present). The physics of the process was part of Prof. Kotnik lecture. One of the limiting problems remains that very few is known on the physicochemical mechanisms supporting the reorganisation of the cell membrane. Electropermeabilization is not simply punching holes in a one lipid bilayer. The physiology of the cell is controlling many parameters. The associated destabilisation of the membrane unpermeability is a stress for the cells and may affect the cell viability. This lecture explains the factors controlling electropermeabilization to small molecules (< 4 kDa). The events occurring before, during and after electropulsation of cells are described. Preambule: what is a biological membrane? The main target of cell electropermeabilization is the cell membrane, more precisely the plasma membrane. Organelles may be affected when they are shielded by the plasma membrane or by a back effect of the transport linked to the plasma membrane permeabilization (uptake of ions, leakage of secondary metabolites. In many approaches such as molecular dynamics simulations, the description of a biological membrane is limited to a lipid bilayer. This is far from the biological complexity and should be used only for soft matter investigations. When the process is applied to a cell (and to a tissue), a more sophisticated description of the biological membrane organization is needed. It is a complex assembly between proteins and a mixture of lipids. It results from a network of weak forces resulting in a complex pattern of lateral pressure across the membrane. A lot of lateral and rotational movements of the membrane components on the submicrosecond timescale is present. Spontaneous transverse movements are energy driven or result from membrane traffic related events (endocytosis, exocytosis). The distribution of lipids is not homogeneous as assumed in the fluid matrix model but localized specific accumulations are detected (rafts). This is due to the fact that a biological membrane is an active entity where a flow of components is continuously occurring (so called membrane traffic). Endocytosis and exocytosis are processes involved in the membrane organization. They are affected by stresses applied on the cell. The mechanical signals are transduced by the membrane. This costs a lot of energy provided by the cell metabolism. Another consequence is the ionic gradient across the membrane resulting from the balance between active pumping and spontaneous leaks. A final aspect is that damages to the In vitro Cell Electropermeabilization 41 membrane are repaired not only by an intra-membraneous process (as for a viscoelastic material) but by a patching process mediated by cytosolic vesicles. It is therefore very difficult to provide an accurate physical description of a biological membrane at the molecular level. Either oversimplifying approximations are used (using lipid vesicles, a soft matter approach) or a phenomenological description is provided with fitting to physical chemical equations (a life science approach). Both are valid as long as you keep aware of the limits in accuracy. The present lecture will be within the life science approach to give the suitable informations for Clinical and well as biotechnological applications. A- A biophysical description and a biological validation A-1 The external field induces membrane potential difference modulation An external electric field modulates the membrane potential difference as a cell can be considered as a spherical capacitor [3]. The transmembrane potential difference (TMP) induced by the electric field after a (capacitive) charging time, ∆Ψi is a complex function g(λ) of the specific conductivities of the membrane (λm), the pulsing buffer (λo) and the cytoplasm (λi), the membrane thickness, the cell size (r) and packing. Thus, ∆Ψ = f ⋅ g (λ)⋅ r ⋅ E ⋅cosθ (1) i in which θ designates the angle between the direction of the normal to the membrane at the considered point on the cell surface and the field direction, E the field intensity, r the radius of the cell and f, a shape factor (a cell being a spheroid). Therefore, ∆Ψi is not uniform on the cell surface. It is maximum at the positions of the cell facing the electrodes. These physical predictions were checked experimentally by videomicroscopy by using potential difference sensitive fluorescent probes [4-6]. More locally on the cell surface, it is affected by the local curvature and the associated defects in packing.This description is valid with dilute cell suspensions. In dense systems, self shielding in the cell population affects the local field distribution and reduces the local (effective) field distribution [7]. Stronger field intensities are needed to get the same induced potential. Another factor affecting the induced potential differences is the shape of the cells and their 42 Justin Teissié relative orientation to the field lines. When the resulting transmembrane potential difference ∆Ψ (i.e. the sum between the resting value of cell membrane ∆Ψo and the electroinduced value ∆Ψi) reaches locally 250 mV, that part of the membrane becomes highly permeable for small charged molecules and transport is detected [3, 8]. One more parameter is that as the plasma membrane must be considered as a capacitor, there is a membrane charging time that may affect the magnitude of the TMP when the pulse duration is short (submicrosecond) or in poorly conducting pulsing buffers. A-2 Parameters affecting electropermeabilization A-2-1 Electric field parameters Permeabilization is controlled by the field strength. Field intensity larger than a critical value (Ep,r) must be applied to the cell suspension. From Eq. (1), permeabilization is first obtained for θ close to 0 or π. Ep,r is such that: ∆Ψ = f ⋅ g λ ⋅ r ⋅ E (2) i, perm ( ) p, r Permeabilization is therefore a local process on the cell surface. The extend of the permeabilized surface of a spherical cell, Aperm, is given by: Ep, 1 r − E A = A (3) perm tot 2 where Atot is the cell surface and E is the applied field intensity. Increasing the field strength will increase the part of the cell surface, which is brought to the electropermeabilized state. These theoretical predictions are experimentally directly supported on cell suspension by measuring the leakage of metabolites (ATP) [9] in a population or at the single cell level by digitised fluorescence microscopy [10, 11]. The permeabilized part of the cell surface is a linear function of the reciprocal of the field intensity. Permeabilization, due to structural alterations of the membrane, remained detected on a restricted cap at the cell surface. In other words, the cell obeys the physical predictions! The area affected by the electric field depends also on the shape (spheroid) and on the orientation of the cell with the electric field lines [12]. Changing the field orientation between the different pulses increases the fraction of the cell surface which is permeabilized. Experimental results obtained either by monitoring conductance changes on cell suspension [13] or by fluorescence observation at the single In vitro Cell Electropermeabilization 43 cell level microscopy [10, 11] shows that the density of the local alterations is strongly controlled by the pulse duration. An increase of the number of pulses first leads to an increase of local permeabilization level. The field strength controls the geometry of the part of the cell which is permeabilized. This is straightforward for spherical cells (and validitated by fluorescence microscopy) but more complicated with other cell shapes. Within this cap, the density of defects is uniform and under the control of the pulse(s) duration. A-2-2 Cell size The induced potential is dependent on the size of the cell (Eq (1)). The percentage of electropermeabilized cells in a population, where size heterogeneity is present, increases with an increase in the field strength. The relative part of the cell surface which is permeabilized is larger on a larger cell at a given field strength [13]. Large cells are sensitive to lower field strengths than small one. Plated cells are permeabilized with Ep value lower than when in suspension. Furthermore, large cells in a population appear to be more fragile. An irreversible permeabilization of a subpopulation is observed when low field pulses (but larger than Ep) are applied. Another characteristic is that the ‘loading’ time is under the control of the cell size [14]. A-3 Forces acting on the membrane The external electrical field pulse generates a net transient mechanical force which tends to stretch the spherical membrane [15]. This force appears due to Maxwell stresses existing in the spherical dielectric shell which cause deformation. The total radial force acts on the membrane during the transient process and tends to stretch the microorganism. It can even lead to rupture of the membrane resulting in the death of the microorganism [16]. But as the cellular elasticity is based upon the actin cytoskeleton, this stretching would affect the internal cell organization by signal transduction. 44 Justin Teissié B- Structural Investigations B-1 P31 NMR investigations of the polar head region of phospholids NMR of the phosphorus atom in the phosphatidylcholine headgroup was strongly affected when lipid multilayers were submitted to electric field pulses. It is concluded that the conformation of the headgroup was greatly affected while no influence on the structure and dynamics of the hydrocarbon chains could be detected [17]. On electropermeabilized CHO cells, a new anisotropic peak with respect to control cells was observed on 31 P NMR spectroscopic analysis of the phospholipid components [18]. A reorganization of the polar head group region leading to a weakening of the hydration layer may account for these observations. This was also thought to explain the electric field induced long lived fusogenicity of these cells. B-2 Structural approaches with advanced technologies Atomic Force Microscopy (AFM) has been extensively used to image live biological samples at the nanoscale cells in absence of any staining or cell preparation. [19]. AFM, in the imaging modes, can probe cells morphological modifications induced by EP. In the force spectroscopy mode, it is possible to follow the nanomechanical properties of a cell and to probe the mechanical modifications induced by EP. transient rippling of membrane surface were observed as consequences of electropermeabilization and a decrease in membrane elasticity by 40% was measured on living CHO cells [20]. An inner effect affected the entire cell surface that may be related to cytoskeleton destabilization. Due to the nonlinear and coherent nature of second harmonic generation (SHG) microscopy, 3D radiation patterns from stained neuronal membranes were sensitive to the spatial distribution of scatterers in the illuminated patch, and in particular to membrane defect formation. Higher scatterers (membrane alterations) densities, lasting < 5 milliseconds, were observed at membrane patches perpendicular to the field whereas lower density was observed at partly tangent locations [21, 22]. Higher pore densities were detected at the anodic pole compared to cathodic pole. Coherent Anti-stokes Raman Scattering (CARS) is a label free spectroscopy. It was recently used to confirm that proteins were affected along electropermeabilization [23, 24]. In vitro Cell Electropermeabilization 45 CARS results are indicative of an alteration of the interfacial water molecules, a direct consequence of the fusogenicity of electropermeabilized membranes [25]. C-Practical aspects of electropermeabilization C-1 Sieving of electropermeabilization Electropermeabilization allows a post-pulse free-like diffusion of small molecules (up to 4 kDa) whatever their chemical nature. Polar compounds cross easily the membrane. But the most important feature is that this reversible membrane organisation is nevertheless long-lived in cells. Diffusion is observed during the seconds and minutes following the ms pulse. Most of the exchange took place after the pulse [10, 11]. Resealing of the membrane defects and of the induced permeabilization is a first order multistep process, which appears to be controlled by protein and organelles reorganisation. But as for other macroscopic damage to a plasma membrane, electropermeabilization has been shown to cause internal vesicles (lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis. Membrane resealing is thus a cellular process. C-2 Associated transmembrane exchange Molecular transfer of small molecules (< 4 kDa) across the permeabilized area is mostly driven by the concentration gradient across the membrane. Electrophoretic forces during the pulse may contribute [10]. Concentration driven diffusion of low weight polar molecules after the pulse can be described by using the Fick equation on its electropermeabilized part [9]. This gives the following expression for a given molecule S and a cell with a radius r: 2 φ( S, t) = 2π r ⋅ P ⋅ S ∆ ⋅ X ( N, T ) − − k ⋅ N T ⋅ t (4) S ( Ep, 1 r E ) exp( ( , ) ) where Φ(S, t) is the flow at time t after the N pulses of duration T (the delay between the pulses being short compared to t), Ps is the permeability coefficient of S across the permeabilized membrane and ∆S is the concentration difference of S across the membrane. X is reporting the density of conducting defects in the field affected cap on the cell surface. Ep depends on r (size). The delay between pulses is clearly playing a role 46 Justin Teissié in the definition of X but this remains to be investigated in details. Characterization of electropermeabilization is clearly dependent on the transport of S through Ps and the sensibility of its detection. For a given cell, the resealing time (reciprocal of k) is a function of the pulse duration but not of the field intensity as checked by digitised videomicroscopy [9]. A strong control by the temperature is observed. The cytoskeletal integrity should be preserved [27]. Resealing of cell membranes is a complex process which is controlled by the ATP level. Starved cells are fragile. An open question is to know if it is a self-resealing or other components of the cell are involved. Organelle fusion may be involved as in the case of other membrane repair occurring with after laser induced damage. Resealing is complex as permeabilization cancellation is obtained when bipolar pulses with a short interpulse delay are delivered [29,30]. C-3 Cellular responses When cells are submitted to short lived electric field pulses, a leakage of metabolites from the cytoplasm is observed which may bring a loss in viability. This can occur just after the pulse (short term death) or on a much longer period when cells have resealed (long term death) [27]. Reactive oxygen species (ROS) are generated at the permeabilized loci, depending on the electric field parameters [28]. These ROS can affect the viability. This is a major drawback for the transfer of sensitive species (nucleic acids). Adding antioxydants is a safe approach [31]. When a cell is permeabilized, an osmotic swelling may result, leading to an entrance of water into the cell. This increase of cell volume is under the control of the pulse duration and of course of the osmotic stress [32]. There is a loss of the bilayer membrane asymmetry of the phospholipids on erythrocytes [33] due to the induced osmotic swelling bringing hemolysis. A mechanical stress is present during the pulse delivery when high fields are present as shown by the occurrence of shock waves [34]. and result in a biological response [35]. Conclusion All experimental observations on cell electropermeabilization are in conflict with a naive model where it is proposed to result from holes punched in a lipid bilayer (see [36] as a recent review). Biochemical modifications such as lipid oxidation may be present as suggested by In vitro Cell Electropermeabilization 47 membrane blebbings formed just after the pulse delivery [37, 38]. Structural changes in the membrane organization supporting permeabilization remains poorly characterized. New informations appear provided by coarse grained computer-based simulations. Nevertheless it is possible by a careful cell dependent selection of the pulsing parameters to introduce any kind of polar molecules in a mammalian cell while preserving its viability. The processes supporting the transfer are very different for different molecules. Transfer is electrophoretically mediated during the pulse and is mostly present after the pulse driven by diffusion for small charged molecules (drugs) [39, 9]. SiRNA are only transferred by the electrophoretic drag present during the pulse [40]. DNA plasmids are accumulated in spots on the electropermeabilized cell surface during the pulse and slowly translocated in the cytoplasm along the microtubules by a metabolic process [41, 42]. Cell membrane electropermeabilization is a complex process. To improve our knowledge, one has to very careful in the description of the experimental protocol [43]. Acknowledgement Supports from the CNRS and the region Midi Pyrénées must be acknowledged. This state of the art in Electropermeabilization is mostly due to the collective work of scientists and students in my former group of “Cell Biophysics” in Toulouse. Discussions with many colleagues were appreciated. Research conducted in the scope of the EBAM European Associated Laboratory (LEA) and in the framework of COST Action TD1104. References [1] Potter, H., Application of electroporation in recombinant DNA technology, in Methods in Enzymology, vol. 217, I. Academic Press, Editor. 1993. [2] Orlowski, S. and L.M. Mir, Cell electropermeabilization: a new tool for biochemical and pharmacological studies. Biochim Biophys Acta, 1993. 1154(1): 51-63. [3] Teissié, J. and M.P. Rols, An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization. Biophys J, 1993. 65(1): 409-13. [4] Gross, D., L.M. Loew, and W.W. Webb, Optical imaging of cell membrane potential changes induced by applied electric fields. Biophys J, 1986. 50: 339-48. [5] Lojewska, Z., et al., Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields. Biophys J, 1989. 56(1): 121-8. [6] Hibino, M., et al., Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential. Biophys J, 1991. 59(1): 209-20. 48 Justin Teissié [7] Pucihar, G., et al., Electropermeabilization of dense cell suspensions. Biophys J. 2007 36(3): 173-185 [8] Teissié, J. and T.Y. Tsong, Electric field induced transient pores in phospholipid bilayer vesicles. Biochemistry, 1981. 20(6): 1548-54. [9] Rols, M.P. and J. Teissie, Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. Biophys J, 1990. 58(5): 1089-98. [10] Gabriel, B. and J. Teissie, Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane. Biophys J, 1997. 73(5): 2630-7. [11] Gabriel, B. and J. Teissie, Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse. Biophys J, 1999. 76(4): 2158-65. [12] Valič B, Golzio M, Pavlin M, Schatz A, Faurie C, Gabriel B, Teissié J, Rols MP, Miklavčič D. Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment. Eur. Biophys. J. 32: 519-528, 2003 [13] Kinosita, K., Jr. and T.Y. Tsong, Voltage-induced conductance in human erythrocyte membranes. Biochim Biophys Acta, 1979. 554(2): 479-97. [14] Sixou, S. and J. Teissie, Specific electropermeabilization of leucocytes in a blood sample and application to large volumes of cells. Biochim Biophys Acta, 1990. 1028(2): 154-60. [15] Winterhalter M and Helfrich W Deformation of spherical vesicles by electric fields J. Colloid Interface Sci. 1988. 122 583–6 [16] Harbich W. and Helfrich W Alignment and opening of giant lecithin vesicles by electric fields Z Naturforsch 1991 34a, , 133-1335. [17] Stulen G. Electric field effects on lipid membrane structure. Biochim Biophys Acta. 1981 ; 640(3):621-7 [18] Lopez A, Rols MP, Teissie J.31P NMR analysis of membrane phospholipid organization in viable, reversibly electropermeabilized Chinese hamster ovary cells. Biochemistry. 1988 ;27(4):1222-8 [19] Pillet F, Chopinet L, Formosa C, Dague E Atomic Force Microscopy and pharmacology: From microbiology to cancerology Biochimica et Biophysica Acta 1840 (2014) 1028–1050 [20] Chopinet L, Roduit C, Rols MP, Dague E Destabilization induced by electropermeabilization analyzed by atomic force microscopy Biochimica et Biophysica Acta 2013 1828 2223–2229 [21] Zalvidea D, Claverol-Tinturé E Second Harmonic Generation for time-resolved monitoring of membrane pore dynamics subserving electroporation of neurons Biomedical Optics Express 2011 / Vol. 2, No. 2 / 305-314 [22] Moen, EK. Ibey, BL. Beier HT Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging Biophysical Journal 2014 106 L37–L40 [23] Azan A, Untereiner V, Gobinet C, Sockalingum GD, Breton M, Piot O, Mir LM. Demonstration of the Protein Involvement in Cell Electropermeabilization using Confocal Raman Microspectroscopy. Sci Rep. 2017 ;7:40448. doi: 10.1038/srep40448. [24] Azan A, Untereiner V, Descamps L, Merla C, Gobinet C, Breton M, Piot O, Mir LM. Comprehensive Characterization of the Interaction between Pulsed Electric Fields and In vitro Cell Electropermeabilization 49 Live Cells by Confocal Raman Microspectroscopy. Anal Chem. 2017 ;89(20):10790-10797. doi: 10.1021/acs.analchem.7b02079. [25] Azan A, Scherman M, Silve A, Breton M, Leray I, Dorval N, Attal-Trétout B, Mir LM Interfacial water probing by CARS spectroscopy on biological samples exposed to intense pulsed electric fields 2015 URSI AT-RASC Conference, ISBN: 9789090086286 [26] Gabriel, B. and J. Teissie, Control by electrical parameters of short- and long-term cell death resulting from electropermeabilization of Chinese hamster ovary cells. Biochim Biophys Acta, 1995. 1266(2): 171-8. [27] Teissié, J. and M.P. Rols, Manipulation of cell cytoskeleton affects the lifetime of cell membrane electropermeabilization. Ann N Y Acad Sci, 1994. 720: 98-110. [28] Gabriel, B. and J. Teissie, Generation of reactive-oxygen species induced by electropermeabilization of Chinese hamster ovary cells and their consequence on cell viability. Eur J Biochem, 1994. 223(1): 25-33. [29] Ibey BL, Ullery JC, Pakhomova ON, Roth CC, Semenov I, Beier HT, Tarango M, Xiao S, Schoenbach KH, Pakhomov AG, Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses, Biochem. Biophys. Res. Commun. 2014 443 568–573.. [30] Schoenbach KH, Pakhomov AG, Semenov I, Xiao S, Pakhomova ON, Ibey BL, Ion transport into cells exposed to monopolar and bipolar nanosecond pulses, Bioelectrochemistry 2015 103 44–51. [31] Markelc B, Tevz G, Cemazar M, Kranjc S, Lavrencak J, Zegura B, Teissie J, Sersa G. Muscle gene electrotransfer is increased by the antioxidant tempol in mice. Gene Ther. 2011. doi: 10.1038 [32] Golzio, M., et al., Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J, 1998. 74(6): 3015-22. [33] Haest, C.W., D. Kamp, and B. Deuticke, Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells. Biochim Biophys Acta, 1997. 1325(1): 17-33. [34] Barnes RA, Roth CC, Beier HT, Noojin G, Valdez C, Bixler J, Moen E, Shadaram M, Ibey BL.Probe beam deflection optical imaging of thermal and mechanical phenomena resulting from nanosecond electric pulse (nsEP) exposure in-vitro. Opt Express. 2017;25(6):6621-6643. doi: 10.1364/OE.25.006621. [35] Roth CC, Glickman RD, Martens SL, Echchgadda I, Beier HT, Barnes RA Jr, Ibey BL.Adult human dermal fibroblasts exposed to nanosecond electrical pulses exhibit genetic biomarkers of mechanical stress. Biochem Biophys 2017;9:302-309. doi: 10.1016/j.bbrep.2017.01.007 [36] Teissie J, Golzio M, Rols MP Mechanisms of cell membrane electropermeabilization: a minireview of our present (lack of ?) knowledge.Biochim Biophys Acta, 2005 .1724(3): 270-80 [37] Gass GV, Chernomordik LV Reversible large-scale deformations in the membranes of electrically-treated cells: electroinduced bleb formation. Biochim Biophys Acta. 1990 1023(1):1-11 [38] Escande-Géraud ML, Rols MP, Dupont MA, Gas N, Teissié J. Reversible plasma membrane ultrastructural changes correlated with electropermeabilization in Chinese hamster ovary cells. Biochim Biophys Acta. 1988 939(2):247-59 [39] Pucihar G, Kotnik T, Miklavcic D, Teissié J. Kinetics of transmembrane transport of small molecules into electropermeabilized cells Biophys J. 2008; 95(6) :2837-48 50 Justin Teissié [40] Paganin-Gioanni A, Bellard E, Escoffre JM, Rols MP, Teissié J, Golzio M. Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells. Proc Natl Acad Sci U S A. 2011;108(26): 10443-7. [41] Wolf H, Rols MP, Boldt E, Neumann E, Teissié J.Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. Biophys J. 1994; 66(2):524-31. [42] Golzio M, Teissie J, Rols MP. Direct visualization at the single-cell level of electrically mediated gene delivery.Proc Natl Acad Sci U S A. 2002; 99(3): 1292-7. [43] Cemazar M, Sersa G, Frey W, Miklavcic D, Teissié J Recommendations and requirements for reporting on applications of electric pulse delivery for electroporation of biological samples. Bioelectrochemistry. 2018; 122:69-76. doi: 10.1016/j.bioelechem.2018.03.005. APPENDIX - Transmembrane transport Introduction A Membrane transport complies with basic thermodynamic principles. A general principle of thermodynamics that governs the transfer of substances through membranes and other surfaces is that the exchange of free energy, Δ G, for the transport of a substance of concentration C1 to another compartment where it is present at C2 is: 𝐶𝐶 ∆𝐺𝐺 = 𝑅𝑅𝑅𝑅 log 2 𝐶𝐶1 ( T, temperature; R, gas constant i.e. 8.3145 J/mol·K.) When C2 is less than C1, Δ G is negative, and the process is thermodynamically favorable. As the energy is transferred from one compartment to another, an equilibrium will be reached where C2=C1 (Δ G=0). However, there are circumstances, relevant for the in vivo functioning of biological membranes, under which this equilibrium will not be reached. A membrane electrical potential can exist which can influence ion distribution. For example, for the transport of ions from the exterior to the interior of a cell, 𝐶𝐶 ∆𝐺𝐺 = 𝑅𝑅𝑅𝑅 log 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 + 𝑍𝑍𝑍𝑍∆𝑃𝑃 (1) 𝐶𝐶𝑜𝑜𝑜𝑜𝑜𝑜𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 Where F is Faraday's constant, Z the charge of the ion and Δ P the transmembrane potential. If Δ P is negative and Z is positive, the contribution of the term ZFΔ P to Δ G will be negative, that is, it will favor the transport of cations out of the cell. So, if the potential difference is maintained, the equilibrium state Δ G=0 will not correspond to an equimolar concentration of ions on both sides of the membrane. In vitro Cell Electropermeabilization 51 Passive diffusion Simple diffusion and osmosis are in some ways similar. Simple diffusion is the passive movement of solute from a high concentration to a lower concentration until the concentration of the solute is uniform throughout and reaches equilibrium. Osmosis is much like simple diffusion but it specifically describes the movement of water (not the solute) across a permeable membrane until there is an equal concentration of solute on both sides of the membrane. Simple diffusion and osmosis are both forms of passive transport 𝑑𝑑𝐶𝐶 𝐽𝐽 = −𝐷𝐷 (2) 𝑑𝑑𝑑𝑑 Electrophoretic drift When an external electric field E is present, it has an action on molecules in the buffer. If the molecule is charged (nucleic acids, ions, dyes), it will migrate in an electric field to the electrode of opposite charge. Consider the simple case of a charged particle (+ Q) moving in an electric field ( E) in a poorly conducting medium, such as water. If the particle is moving at a constant velocity toward the cathode (- electrode), the net force Ftot on the particle is 0 (since F=ma, and the acceleration ( a) of the particle is 0 at constant velocity). Two forces are exerted on the particle, the force exerted on the charged particle by the field Fe, which is in the direction of the motion (toward the cathode), and the frictional force on the charged particle, Ff, which retards its motion toward the cathode. Therefore: 𝑍𝑍𝑡𝑡𝑡𝑡𝑡𝑡 = 𝑍𝑍𝑒𝑒 + 𝑍𝑍𝑓𝑓 = 0, (3) where Fe = QE (the electric force) and Ff = -fv (the frictional force), where v is the velocity of the particle, and f is a constant called the frictional coefficient. The last equation shows that the force Ff hindering motion toward the cathode is proportional to the velocity of the particle (Note: in the case of negatively charged particles such as nucleic acids, the directions of the forces are inverted and the direction of motion as well). The frictional coefficient depends on the size and shape of the molecule. The larger the molecule, the larger the frictional coefficient (i.e. more resistance to motion of the molecule). The frictional coefficient for a spherical particle is given by 𝑓𝑓 = 6𝜋𝜋𝜋𝜋𝑅𝑅𝑠𝑠 (4) 52 Justin Teissié where η is the viscosity, and Rs (Stokes radius) is the radius of the hydrated sphere. From (1), (2), and (3), Fe = Ff, or 𝑄𝑄𝑄𝑄 = 𝑓𝑓𝑓𝑓 (5) Hence v/E = Q/f = U = electrophoretic mobility, or 𝑣𝑣 𝑄𝑄 𝑈𝑈 = = (6) 𝐸𝐸 2𝜋𝜋𝜋𝜋𝑅𝑅𝑆𝑆 Counter ions in the solution (from salts) form a cloud around the charged macromolecule, and partially shield the charged particle from the electric field E. When the field is delivered across a “porous” membrane, friction is critical. “Porous” means that transient structural defects are present. The smaller molecules can pass through the membrane defects more readily than larger molecules, so there is an additional sieving mechanism that contributes to the effective transport Note: Electrode nomenclature might be confusing to some of you. As mentioned above, cations move towards the cathode (where reduction occurs), so the cathode must be negative. Likewise, anion move towards the anode (where oxidation occurs), so the anode must be positive Facilitated diffusion Facilitated diffusion, also called carrier-mediated osmosis, is the movement of molecules across the cell membrane via special transport proteins that are embedded within the cellular membrane. Large, insoluble molecules, such as glucose, vesicles and proteins require a carrier molecule to move through the plasma membrane. Facilitated diffusion is a passive process: the solutes move down their concentration gradient and do not require the expenditure of cellular energy Active and co-transport In active transport a solute is moved against a concentration or electrochemical gradient by transport proteins that consume metabolic energy, usually ATP. In primary active transport the hydrolysis of the energy provider (e.g. ATP) takes place directly in order to transport the solute in question (ATPase enzymes). In secondary active transport, the In vitro Cell Electropermeabilization 53 energy provider acts indirectly; the energy is stored in an electrochemical gradient to transport a target compound against its gradient. Primary active transport is mediated by the formation of a substrate-transporter complex; Therefore, each transport protein has an affinity constant for a solute. This is equivalent to the case of an enzyme to the Michaelis-Menten constant. 𝐽𝐽 𝐽𝐽 = 𝑚𝑚𝑚𝑚𝑚𝑚𝑆𝑆 (7) 𝐾𝐾𝑚𝑚+𝑆𝑆 Some important features of active transport in addition to its ability to intervene even against a gradient, its kinetics and the use of ATP, are its high selectivity. Pumps A pump is a protein that hydrolyses ATP to transport a particular solute through a membrane, and in doing so, generating an electrochemical gradient membrane potential. This gradient is of interest as an indicator of the state of the cell through parameters such as the Nernst potential 𝑅𝑅𝑅𝑅 [𝑖𝑖𝑡𝑡𝑖𝑖 𝑡𝑡𝑜𝑜𝑡𝑡𝑠𝑠𝑖𝑖𝑑𝑑𝑒𝑒 𝑐𝑐𝑒𝑒𝑐𝑐𝑐𝑐] 𝑄𝑄 = ln (8) 𝑧𝑧𝑧𝑧 [𝑖𝑖𝑡𝑡𝑖𝑖 𝑖𝑖𝑖𝑖𝑠𝑠𝑖𝑖𝑑𝑑𝑒𝑒 𝑐𝑐𝑒𝑒𝑐𝑐𝑐𝑐] With active pumping, the Goldman equation gives the resting potential ∑𝑁𝑁 𝑃𝑃 +� 𝑀𝑀 − �𝐴𝐴−� 𝑅𝑅𝑅𝑅 𝑖𝑖 +∑ 𝑃𝑃 𝑀𝑀+�𝑀𝑀𝑖𝑖 𝑜𝑜𝑜𝑜𝑜𝑜 𝑗𝑗 𝐴𝐴 𝑗𝑗 𝑄𝑄 𝑖𝑖 𝑗𝑗 𝑖𝑖𝑖𝑖 𝑚𝑚 = ln � � (9) 𝑧𝑧 ∑𝑁𝑁 𝑃𝑃 +� 𝑀𝑀 − − 𝑖𝑖 +∑ 𝑃𝑃 �𝐴𝐴 � 𝑀𝑀+�𝑀𝑀𝑖𝑖 𝑗𝑗 𝐴𝐴 𝑗𝑗 𝑖𝑖 𝑖𝑖𝑖𝑖 𝑗𝑗 𝑜𝑜𝑜𝑜𝑜𝑜 Em is the transmembrane potential Pion is the permeability for that ion, [ion]out is the extracellular concentration of that ion, [ion]in is the intracellular concentration of that ion. Transport by vesicles Specialized vesicles mediate the transport by complex interactions with the membrane. Their intra-vesicular cargo is delivered to the other side of the membrane. This is called transcytosis (endo and exocytosis). The process is active meaning it requires energy and the action of the cell machinery. 54 Justin Teissié Suggested reading and watching • https://www.khanacademy.org/test-prep/mcat/cells/transport-across-a-cell-membrane/a/passive-transport-and-active-transport-across-a-cell-membrane-article • http://www.sumanasinc.com/webcontent/animations/content/vesiclebudding.html • http://www.like2do.com/learn?s=Membrane_vesicle_trafficking • Popescu I. Aurel, Biophysics. Current Status and Future Trends, Publishing House of the Romanian Academy, 2016 Teissié Justin was born 24 March 1947 in Poitiers, France. Got a degree in Physics at the Ecole superieure de Physique et de Chimie Industrielles de Paris (ESPCI) in 1970. Got a PhD in Macromolecular Chemistry on a project on fluorescence detection of action potential under the supervision of Prof. Monnerie (ESPCI) and Changeux (Institut Pasteur) in 1973. Got a DSC in Biophysics on a project on fluorescence characterisation of Langmuir Blodgett films in Toulouse in 1979. Was a Post Doc at the Medical School of the John Hopkins University in Baltimore in 1979-81. Present position: Directeur de recherches au CNRS emeritus. Author of more than 250 papers. Chapter 4 Nucleic acids electrotransfer in vitro Marie-Pierre Rols Institut de Pharmacologie et de Biologie Structurale, Toulouse, France Abstract: Cell membranes can be transiently permeabilized by application of electric pulses. This process, called electropermeabilization or electroporation, allows hydrophilic molecules, such as anticancer drugs and nucleic acids, to enter into targeted cells and tissues. The knowledge of the processes involved in membrane permeabilization and in gene transfer is mandatory for this promising method to be efficiently and safely used. The behavior of the membranes and the cells both while the electric field is on and after its application has therefore to be addressed. The description of the full mechanisms takes benefit from studies performed on different biological models (lipid vesicles, cells in 2D and 3D culture) and from different microscopy tools that allow to visualize the processes. Single cell imaging experiments revealed that the uptake of molecules (antitumor drugs, nucleic acids) takes place in well-defined membrane regions and depends on their chemical and physical properties (size, charge). Small molecules can freely cross the electropermeabilised membrane and have a free access to the cytoplasm. Heavier molecules, such as plasmid DNA, face physical barriers (plasma membrane, cytoplasm crowding, nuclear envelope) which engender a complex mechanism of transfer. Gene electrotransfer indeed involves different steps, occurring over relatively large time scales. As will be presented, these steps include the initial interaction with the electropermeabilised membrane, the crossing of the membrane, the transport within the cell towards the nuclei and finally gene expression. Introduction Gene therapy is a treatment option for a number of diseases as inherited disorders and cancer. Despite the fact that a lot of methods of vectorization 56 Marie-Pierre Rols have been developed during the last decades, the technique has still to be improved to be both efficient and safe (1). Among the different approaches, electroporation appears as the most promising one. This physical method can be efficiently used for the targeted deliver of molecules in a wide range of cells and tissues (2). Electroporation is nowadays a well-known technique of cell transfection used in the laboratories. Vaccination and oncology gene therapy are major fields of application of DNA electrotransfer in clinics (3, 4). Translation of preclinical studies into clinical trials started 10 years ago. The first clinical trial of plasmid electroporation carried out in patients with metastatic melanoma has shown hopeful results (5). The method has also been successfully used for the treatment of companion animals. However, despite the fact that the pioneering work on plasmid DNA electrotransfer in cells was initiated more than 30 years ago (6), many of the mechanisms underlying membrane electropermeabilization and DNA electrotransfer remain to be elucidated. Even if in vitro electrotransfer is efficient in almost all cell lines, in vivo gene delivery and expression in tumors can be not as efficient as in viral vectorization. It is therefore mandatory, for increasing gene transfer and expression while preserving safety, to increase knowledge about the mechanisms. This chapter aims to describe the basics aspects of membrane electropermeabilization and gene delivery in cells and by doing so to give some tips to perform experiments and optimize protocols. Membrane electropermeabization The basics Cells have a resting transmembrane potential which is uniform all along their plasma membrane. Exposure of living cells to short and intense electric pulses induces position-dependent changes of this transmembrane potential. Being dependent on the angle between the electric field direction and the normal to the membrane, the electric field effects are not uniform along the membrane. Maximum effects are present at the poles of the cells facing the electrodes when the resulting transmembrane potential reaches a threshold value. Above this threshold, permeabilization of the cell membrane occurs. Electropermeabilization of the plasma membrane is a prerequisite for gene electrotransfer since nucleic acids are highly charged and large molecules that cannot enter cells. Nucleic acids electrotransfer in vitro 57 The way to conduct an experiment Electropermeabilization can be performed in different manners depending on the way cells are grown. For cells grown on Petri dish, culture medium can be removed and replaced by a low ionic, iso-osmotic buffer. This pulsation buffer allows to limit the Joule effect and therefore help to preserve the cell viability. The composition of this medium is generally a 10 mM phosphate buffer, 250 mM sucrose and 1 mM MgCl2. On a practical point of view, the bottom of the Petri dish can serve as an electropulsation chamber. For cells in suspension, cells resuspended in the pulsing buffer are placed in purchased cuvettes or in “home-made” chambers that can be easily obtained by placing the electrodes on the bottom of the Petri dish (see Figure 1). The electric pulses are delivered through a set of electrodes connected to the pulse generator. In most experiments, square-wave electric pulses generators are used. Contrary to exponential decay generators, they allow the independent control of the amplitude of the electric field pulses E and their duration T. This is important for mammalian cells which have no cell wall and therefore are more affected by electric pulses than bacteria and yeast. The electric pulse parameters have to be selected considering the characteristics of the cells in particular their size. One key step to further ensure DNA electrotransfer and expression is to determine the best electric conditions allowing both the permeabilization of the plasma membrane and the preservation of the cell viability. Fig. 1 Tips for your experiments. Cells are pulsed on Petri dish or on cuvettes. Permeabilized and viable cells are plotted to define the optimum conditions ((1) E < Ep or just above, poor permeabilization; (2) E>> Ep, viability loss; (3) best values). The use of video microscopy allows visualization of the permeabilization phenomenon at the single cell level. Fluorescent indicators of membrane 58 Marie-Pierre Rols permeabilization, such as Propidium Iodide (PI), are very convenient to detect the electrotransfer of molecules into the cytoplasm. They can simply be added to the cells before application of the electric pulses. The uptake of the fluorescent dye into the cells is the signature of membrane electropermeabilization. Whatever the value of the pulses duration T, permeabilization only appears above a threshold value of pulse intensity E, called Ep. Therefore, the first experiment to perform consists to submit the cells to increasing values of E and determine the permeabilization efficiency (i.e. the percentage of cells that have been electropermeabilized, cells which nuclei become fluorescent). For E Ep, the formation of transient permeable structures facing the electrodes allows the exchange of molecules; Propidium iodide is observed to rapidly access the cell interior in the region of the cells facing the electrodes, mainly at the anode facing site; (iii) after electropulsation, membrane can stay permeable before resealing occurs (7). Life-time of permeabilization can be assayed by adding the fluorescent dyes at various times following the pulses. If the cell membrane is still permeable, then the cell will be fluorescent. Resealing Nucleic acids electrotransfer in vitro 59 varies from a few seconds (when cells are put at 37°C just after pulsation) to several hours (when cells are maintained on ice) according to the experimental conditions (temperature and pulse parameters). However, one has to take into account that viability can be affected since ATP release will occur. It is therefore better to avoid to maintain the cells at low temperature after pulse delivery. Whatever the molecules used to detect permeabilization (if they are small enough and charged), a direct transfer into the cell cytoplasm is observed. When added after electropulsation, molecules can still penetrate into the cells but less efficiently because electric field acts on both the permeabilization of the membrane and on the electrophoretic drag of the charged molecules from the bulk into the cytoplasm. The electrotransfer mechanism involved is indeed specific for the physico-chemical properties of the molecule (8). Progress in the knowledge of the involved mechanisms, in particular in the elucidation of membranes structures that are responsible for molecules transfer, is still a biophysical challenge. Hydrophilic pores have been proposed to be created and their formation confirmed by molecular dynamics modelling. But their existence in permeabilized cells has still to be proven. Phospholipid scrambling and changes on lateral mobility of proteins have been observed suggesting that part of the membrane surface is occupied by defects or pores and that these structures propagate rapidly over the cell surface (9). One can also took advantage of atomic force microscopy to directly visualize the consequences of electropermeabilization and to locally measure the membrane elasticity. Results obtained both on fixed and living CHO cells give evidence of an inner effect affecting the entire cell surface that may be related to cytoskeleton destabilization. Thus, AFM appears as a useful tool to investigate basic process of electroporation on living cells in absence of any staining (10, 11). 60 Marie-Pierre Rols PI uptake DNA/Membra ne complexes Figure 2: Molecule electrotransfer mechanisms. Left: During electric pulses application: Plasma membrane is electropermeabilized facing the 2 electrodes (PI uptake). DNA aggregates are formed. This interaction takes place only on the membrane facing the cathode. Right: About 2 h after electric pulses application, DNA molecules are present around the nucleus. Finally, eGFP expression is detected for hours. The arrow indicates the direction of the electric field. The fact that the entire cell surface is affected is not so obvious since permeabilization is only induced in specific regions of the cells. So, even if the entire mechanisms of membrane electropermeabilization (or electroporation) is not fully understood, and the existence of the exact structures responsible for molecules uptake still a debate, this physical method of vectorization has become one of the most efficient for gene delivery. Mechanisms of electrotransfer of dna molecules into cells What is known about the process The first electroporation-mediated gene transfer experiment was published more than 30 years ago (6). Translation to the clinic benefited from increased knowledge of the mechanisms involved in the electrotransfer of nucleic acids during the last 3 decades. As for electropermeabilization, single-cell studies aided in describing the process of DNA electrotransfer. In addition to membrane permeabilization, DNA electrotransfer is dependent on DNA electrophoresis. The oligonucleotide must indeed be present during the pulse to be later on transferred in the cytoplasm. The electrophoretic mobility of pDNA is not dependent on its number of base Nucleic acids electrotransfer in vitro 61 pairs. Short pulses with high field strength can be used but are less effective than long pulses with lower field strength. Therefore, pulses parameters have to be determined to lead the membrane to be permeable (E > Ep) while preserving as much as possible cell viability (above 30-50 %). Reporter genes are useful to optimize the protocol. As for electropermeabilization, single-cell microscopy and fluorescent plasmids can be used to visualize and determine the different steps of electrotransfection. Plasmids can be labeled with fluorescent dyes to allow visualization of its electrotransfer. DNA molecules, which are negatively charged, migrate electrophoretically when submitted to the electric field. Under electric fields which are too small to permeabilize the membrane (E Ep), the DNA interacts with the plasma membrane. DNA/membrane interaction Interaction only occurs at the pole of the cell opposite the cathode and this demonstrates the importance of electrophoretic forces in the initial phase of the DNA/membrane interaction. When the DNA-membrane interaction occurs, the formation of “microdomains” whose dimensions lie between 0.1 and 0.5 µm is observed (Figure 2). Also seen are clusters or aggregates of DNA which grow during the application of the field. However once the field is cut the growth of these clusters stops. DNA electrotransfer can be described as a multi-step process: the negatively charged DNA migrates electrophoretically towards the plasma membrane on the cathode side where it accumulates. This interaction, which is observed for several minutes, lasts much longer than the duration of the electric field pulse. Translocation of the plasmid from the plasma membrane to the cytoplasm and its subsequent passage towards the nuclear envelope take place with a kinetics ranging from minutes to hours. Dynamic of the process DNA/membrane interaction and as a direct consequence gene expression depend on electric pulse polarity, repetition frequency and duration. Both are affected by reversing the polarity and by increasing the repetition frequency or the duration of pulses. These observations revealed the existence of 2 classes of DNA/membrane interaction: (i) a metastable DNA/membrane complex from which DNA can leave and return to external medium and (ii) a stable DNA/membrane complex, where DNA cannot be 62 Marie-Pierre Rols removed, even by applying electric pulses of reversed polarity. Only DNA belonging to the second class leads to effective gene expression (12). Dynamics of membrane/complexes formation has been poorly understood because direct observations have been limited to time scales that exceed several seconds. However, experimental measurement of the transport of plasmid DNA and propidium iodide with a temporal resolution of 2 ms has been performed thanks to high speed and sensitive camera and allowed the visualization of the DNA/membrane interaction process during pulse application (13). Plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane during the first pulse. Increasing the number of pulses do not lead to the creation of new sites, but to the increase in the amount of DNA. The formation of plasmid complexes at fixed sites suggested that membrane domains may be responsible for DNA uptake and their lack of mobility (as directly observed under the microscope or quantify by Fluorescence Return After Photobleaching (FRAP) measurements) could be due to their interaction with the actin cytoskeleton. As will be described later in this chapter, several publications reported evidences for the involvement of cytoskeleton (14, 15). The dynamics of the entire process is reported in Table 1. If pulse delivery occurs in a relative short time scale (µs to ms), the subsequent traffic of plasmid DNA occurs during the minutes and hours following pulse delivery. Table 1. Kinetics of the different steps involved in gene delivery. Time Scale Steps involved in DNA electro-mediated delivery Reference µs Plasma membrane facing the electrodes is (7) permeabilized ms Electrophoretic migration of DNA towards the (7, 13) membrane s DNA/membrane complex formation (12) min Conversion of the metastable form of the (13) DNA/membrane complex to a stable one hour DNA translocation/diffusion across the membrane (14, 15) day DNA transport towards the nucleus along the (16) cytoskeleton DNA transfer through the cytoplasm The process of plasmid transfer through the cellular cytoplasm to the nuclear envelope is a complex process (17). In principle micro sized Nucleic acids electrotransfer in vitro 63 aggregates of DNA or vesicles filled with DNA could be too large to pass through the pores formed by electroporation. However individual DNA molecules, while they can pass through electropores, have a limited mobility within the cell and may well be totally degraded before reaching the nucleus. It is possible and worth investigating the possibility that the actin cytoskeleton reacts to the presence of DNA aggregates and plays an important role in the subsequent intracellular transport. It seems reasonable that only aggregates beyond a certain size (a few hundred nanometers) can induce a biological cellular response and can be transported by the cell. In addition, the fact that the DNA is in aggregate form means that the DNA in the center of the aggregate is relatively protected from degradation. Therefore, for gene therapy purposes, it is optimal for DNA to enter the cell as single molecules, but the subsequent transport toward the nucleus is, for biological (possibly by inducing a response of the actin cytoskeleton) and physical (diminishing enzymatic degradation) reasons, optimized if the DNA is in a micro-sized aggregate form. Even if the first stage of gene electrotransfection, i.e. migration of plasmid DNA towards the electropermeabilised plasma membrane and its interaction with it, becomes understood, guidelines to improve gene electrotransfer can not only result from the way pulses parameters have been selected. Expression of the pDNA is controlled by the viability of the pulsed population and successful expression of the plasmid depends on its subsequent migration into the cell. Therefore, the intracellular diffusional properties of plasmid DNA, as well as its metabolic instability and nuclear translocation, represent cell limiting factors that must be taken into account. The cytoplasm is composed of a network of microfilament and microtubule systems, along with a variety of subcellular organelles present in the cytosol. The mesh-like structure of the cytoskeleton, the presence of organelles and the high protein concentration means that there is substantial molecular crowding in the cytoplasm which hinders the diffusion of plasmid DNA. These apparently contradictory results might be reconciled by the possibility of a disassembly of the cytoskeleton network that may occur during electropermeabilisation, and is compatible with the idea that the cytoplasm constitutes an important diffusional barrier to gene transfer. In the conditions induced during electropermeabilisation, the time a plasmid DNA takes to reach the nuclei is significantly longer than the time needed for a small molecule (hours compared to minutes). Therefore, plasmid DNA present in the cytosol after being electrotransferred can be lost before reaching the nucleus, for example because of cell division. Finally, after the cytoskeleton, the nuclear envelope will represent the last, but by no means the least important, obstacle for the expression of the plasmid DNA. 64 Marie-Pierre Rols Passage through the nuclear envelope and gene expression Figure 3: Schematic representation of the mechanism of DNA electrotransfer. During the electric pulses, (1) the plasma membrane is permeabilized, (2) DNA is electrophoretically pushed onto the cell membrane, which results in (3) DNA-membrane interactions. After resealing of the membrane, (4) DNA is internalized by endocytosis and other means where actin may take shape of bursts of polymerization. (5) While being actively transported in the cytoplasm by actin and tubulin networks, DNA aggregates pass through the endosomal compartments. Free DNA interacts with adapter protein in order to be transported by motor proteins. For gene expression to occur, (6) DNA has to escape from endosomal compartments. Once in the perinuclear region, (7) DNA crosses the nuclear envelope to be expressed and (8) yield proteins released. A high transport does not always result in a high level in expression. The relatively large size of plasmid DNA makes it unlikely that the nuclear entry occurs by passive diffusion. Single particle tracking experiments of Nucleic acids electrotransfer in vitro 65 DNA aggregates in living cells showed how electrotransferred DNA is transported in the cytoplasm towards the nucleus. The modes of DNA aggregates motion in CHO cells have been analyzed. Fast active transport of the DNA aggregates occurs over long distances. Tracking experiments in cells treated with different drugs affecting both the actin and the tubulin network clearly demonstrate that transport is related to the cellular microtubule network (Figure 3, (16)). Active transport of DNA aggregates Several studies point towards the contribution of endocytosis in the electrotransfer of DNA, but more investigations have to be performed in order to understand what type(s) of endocytosis would be involved. It is necessary to understand as well how electric fields could stimulate such processes. Also notably, any endocytosis model would only explain the internalization of large molecules as it does not support the free membrane crossing of small molecules. It has therefore to be considered to occur in parallel to another model valid for small molecule transmembrane exchange. One model that could reconcile all the DNA internalization models would be that DNA accumulates where pores are formed and that its electrophoretically driven insertion in the membrane pulls the pore and the plasma membrane around. This would generate membrane curvature that could be recognized as an emerging endocytic vesicle and induce a similar response from the cell as for an endocytic process, with the recruitment of actin, clathrin, caveolin, dynamin and other endocytic regulators (18, 19). Electrotransferred DNA trajectories possess portions of active transport interrupted by phases of nearly immobility (15). During the phases of active transport, DNA aggregates featured a motion on average having a velocity of 250 nm/s, persisting for 6 s and leading to a displacement of 1.3 µm. However, the distributions were rather broad with velocities from 50 nm/s to 3400 nm/s, displacements from 0.1 µm to 12 µm and active transport durations from 2 s to 30 s. These ranges are in agreements with other types of intracellular particle dynamics as observed for viruses, polyplexes, lipoplexes, receptors, endosomes and mitochondria. Lower velocities were shown to correspond to actin-associated transport. Indeed, after disruption of the microtubules using the nocodazole drug, active transport of the DNA still occurred and the measured velocities were in the range expected for myosin motors operating on actin – between 50 nm/s and 300 nm/s for myosin VI and between 250 nm/s and 500 nm/s for myosin V. In addition to motor driven transport, actin-related movement could be also due to bursts of actin polymerization which was reported to drive viruses, bacteria 66 Marie-Pierre Rols or endosomes from the plasma membrane to the cytosol with mean velocities ranging from 50 to 600 nm/s. New challenges to increase gene expression As mentioned above, the dense latticework of the cytoskeleton impedes free diffusion of DNA in the intracellular medium. Electrotransferred plasmid DNA, containing specific sequences could then use the microtubule network and its associated motor proteins to move through the cytoplasm to nucleus (20). Clear limits of efficient gene expression using electric pulses are therefore due to, in addition to the passage of DNA molecules through the plasma membrane, to the cytoplasmic crowding and transfer through the nuclear envelope. One of the key challenge for electromediated gene therapy is to pinpoint the rate limiting steps in this complex process and to find strategies to overcome these obstacles. One of the possible strategies to enhance DNA uptake into cells is to use short (10-300 ns) but high pulse (up to 300 kV/cm) induce effects that primarily affect intracellular structures and functions. As the pulse duration is decreased, below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. An idea, to improve transfection success, is thus to perform classical membrane permeabilization allowing plasmid DNA electrotransfer to the cell cytoplasm, and then after, when DNA has reached the nuclear envelope, to specifically permeabilize the nuclei using these short strong nanopulses. Thus, when used in conjunction with classical electropermeabilisation, nanopulses gave hope to increase gene expression (21). However this work was not yet replicated. Another idea is to combine electric pulses and ultrasound assisted with gas microbubbles. Although electroporation induced the formation of DNA aggregates into the cell membrane, sonoporation induced its direct propulsion into the cytoplasm. Twenty-four hours later, cells that received electrosonoporation demonstrated a four-fold increase in transfection level and a six-fold increase in transfection efficiency compared with cells having undergone electroporation alone (22). Sonoporation can therefore improve the transfer of electro-induced DNA aggregates by allowing its free and rapid entrance into the cells (23). Lipid vesicles and spheroids as other models to study gene electrotransfer Coming back to a mechanistic point of view and due to the complexity of the composition of the plasma membrane, other experimental tools can be useful to characterize the membranes domains observed during gene Nucleic acids electrotransfer in vitro 67 electrotransfer. For that purpose, giant unilamellar vesicles (GUV) represent a convenient way to study membrane properties such as lipid bilayer composition and membrane tension (24). They offer the possibility to study and visualize membrane processes due to their cell like size in absence of any constraint due to cell cytoskeleton. They can be obtained by simple methods such as electroformation and their composition can be very simple (one type of phospholipids) or more complex (several lipids including cholesterol). Experiments showed a decrease in vesicle radius which was observed as being due to lipid loss during the permeabilization process. Three mechanisms responsible for lipid loss were directly observed: pore formation, vesicle formation and tubule formation, which may be involved in molecules uptake. However, no interaction between plasmid DNA and the GUV membrane could be observed; a direct transfer of DNA into the GUVs took place during application of the electric pulses (25). That gives clear evidence that “lipid bubble” is not always relevant as a cell and a tissue is not a simple assembly of single cells. Therefore, it is necessary to develop and use different models, from simple lipid vesicles to tumor multicellular tumor spheroids more closed to the in vivo situation, for the understanding of the membrane permeabilization and DNA electrotransfer process in tissues. Each of this model has advantage and limits. Together combined they can help in the study of the full processes (table 2). Table 2. What models can address about electropermeabilization and gene delivery processes. Model Membrane permeabilization DNA electrotransfer GUV Direct visualization of membrane Failed to address permeabilization and its consequences DNA/membrane (deformation, lipid loss) interaction (DNA is directly transferred inside the vesicle) 2D Cell culture Kinetics of permeabilization and its Visualization of consequences (lateral and transverse DNA/membrane mobility of lipids and proteins) complex formation and access to DNA traffic into the cells 3D Cell culture Molecules diffusion and transfer that Allow to address DNA mimic in vivo complex situation delivery in 3D and (contacts between cells, junctions, mimic what happens in extracellular matrix) vivo (decrease in gene expression from the periphery to the core) 68 Marie-Pierre Rols Even if the high majority of studies underlying molecule transfer by electric fields have been performed on 2D cell culture in Petri dish or in cells cultured in suspension, 3D multicellular spheroids represent a nice, relevant, cheap, easy-to-handle in vitro model. Upon growth, spheroids display a gradient of proliferating cells. These proliferating cells are located in the outer cell-layers and the quiescent cells are located more centrally. This cell heterogeneity is similar to that found in avascular micro regions of tumors (26). Confocal microscopy allowed to visualize the repartition of permeabilized cells in spheroids submitted to electric pulses. Results revealed that cells were efficiently permeabilized, whatever their localization in the spheroid, even those in the core, mimicking previously observed in vivo situations. Propidium iodide uptake was observed to be present but spatially heterogeneous within the 3D multicellular spheroid after electroporation, with a progressive decrease from peripheral to interior cells. In the case of large molecules as plasmid DNA, spheroids allowed showing that electrophoresis, and not tissue deformation or electroosmosis, is the driving force for interstitial transport. In addition, and at the opposite of cells in 2D cultures, only cells on one side of the outer leaflet expressed the reporter gene (27). This low expression is in fair agreement with in vivo experiments on tumors. Close contacts between cells and extracellular matrix may act as physical barrier that limit/prevent (uniform) DNA distribution and explain the absence of gene expression in the inner region of spheroid. The limited access of plasmid-DNA to central region of spheroid remains a significant barrier to efficient gene delivery in tissues. Taken together, these results, in agreement with the ones obtained by the group of R. Heller (28), indicate that the spheroid model is more relevant to an in vivo situation than cells cultured as monolayers and therefore can be useful to address the mechanisms of DNA electrotransfer. In order to assess the effects of the extracellular matrix composition and organization, as well as intercellular junctions and communication, other 3D reconstructed human connective tissue model can be used. Cell sheets, reconstructed in vitro by a tissue engineering approach, presents multiple layers of primary dermal fibroblasts embedded in a native, collagen-rich Extra Cellular Matrix (ECM) and can be a useful tool to study skin DNA electrotransfer mechanisms. Cells within this standardized 3D tissue can be efficiently electropermeabilized by milliseconds electric pulses (29, 30). Moreover such a tissue-engineered dermal model recapitulates the mechanical properties of human native dermal tissue unlike the classically used monolayer and spheroid models (31). A better comprehension of gene electrotransfer in such a model tissue would help improve electrogene therapy approaches such as the systemic delivery of therapeutic proteins and DNA vaccination. Nucleic acids electrotransfer in vitro 69 Conclusions “Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Every day in research institutes and clinical centres around the world, scientists use kits and protocols based on viral vectors, lipid transfection agents, and electroporation, among other options. The complex mechanisms of established methods and their often unpredictable impact on cell behaviour have dramatically limited the scope of biological experiments and reduced efficacy of potentially promising cell therapy concepts. The biomedical research community would benefit greatly from a more mechanistic and transparent understanding of intracellular delivery, both to further the development of more robust techniques and to realize key medical and industrial applications” (32). In this context, the so- called electroporation technology is probably the most promising one. Classical theories of electropermeabilization present some limits to give a full description of the transport of molecules through membranes. Certain effects of the electric field parameters on membrane permeabilization, and the associated transport of molecules, are well established but a great deal of what happens at the molecular level remains speculative. Molecular Models of Lipid Bilayers and Electropore Formation are giving interesting new insight into the process. Electroinduced destabilization of the membrane includes both lateral and transverse redistribution of lipids and proteins, leading to mechanical and electrical modifications which are not yet fully understood. One may suggest that such modifications, that may vary according to the micro environment, can be involved in the subsequent transport of molecules interacting with them such as the DNA molecules. Experimental verification of the basic mechanisms leading to the electropermeabilization and other changes in the membrane, cells and tissues remain a priority given the importance of these phenomena for processes in cell biology and in medical applications. In vivo gene electrotransfer will face other challenges such as the necessity to control electric field distribution and gene expression both in space (targeted DNA delivery to the cells) and in time. Guidelines for successful DNA delivery are still required but we can be optimistic that further working to improve gene electrotransfer mechanisms will yield effective treatments. 70 Marie-Pierre Rols References [1] Verma, I. M., and M. D. Weitzman. 2005. Gene therapy: twenty-first century medicine. Annu Rev Biochem 74:711-738. [2] Yarmush, M. L., A. Golberg, G. Sersa, T. Kotnik, and D. Miklavcic. 2014. Electroporation-based technologies for medicine: principles, applications, and challenges. Annual review of biomedical engineering 16:295-320. [3] Lambricht, L., A. Lopes, S. Kos, G. Sersa, V. Preat, and G. Vandermeulen. 2016. Clinical potential of electroporation for gene therapy and DNA vaccine delivery. Expert Opin Drug Deliv 13:295-310. [4] Sersa, G., J. Teissie, M. Cemazar, E. Signori, U. Kamensek, G. Marshall, and D. Miklavcic. 2015. Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer. Cancer immunology, immunotherapy : CII. [5] Daud, A. I., R. C. DeConti, S. Andrews, P. Urbas, A. I. Riker, V. K. Sondak, P. N. Munster, D. M. Sullivan, K. E. Ugen, J. L. Messina, and R. Heller. 2008. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 26:5896-5903. [6] Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. Embo J 1:841-845. [7] Golzio, M., J. Teissie, and M. P. Rols. 2002. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci U S A 99:1292-1297. [8] Paganin-Gioanni, A., E. Bellard, J. M. Escoffre, M. P. Rols, J. Teissie, and M. Golzio. 2011. Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells. Proc Natl Acad Sci U S A 108:10443-10447. [9] Escoffre, J. M., E. Bellard, C. Faurie, S. C. Sebai, M. Golzio, J. Teissie, and M. P. Rols. 2014. Membrane disorder and phospholipid scrambling in electropermeabilized and viable cells. Biochim Biophys Acta 1838:1701-1709. [10] Chopinet, L., C. Roduit, M. P. Rols, and E. Dague. 2013. Destabilization induced by electropermeabilization analyzed by atomic force microscopy. Biochim Biophys Acta 1828:2223-2229. [11] Chopinet, L., C. Formosa, M. P. Rols, R. E. Duval, and E. Dague. 2013. Imaging living cells surface and quantifying its properties at high resolution using AFM in QI (TM) mode. Micron 48:26-33. [12] Faurie, C., M. Rebersek, M. Golzio, M. Kanduser, J. M. Escoffre, D. Pavlin, J. Teissie, D. Miklavcic, and M. P. Rols. 2010. Electrically mediated gene transfer and expression are controlled by the life-time of DNA/Membrane complex formation. Journal of Gene Medicine 12:117-125. [13] Escoffre, J. M., T. Portet, C. Favard, J. Teissie, D. S. Dean, and M. P. Rols. 2011. Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery. Biochim Biophys Acta 1808:1538-1543. [14] Rosazza, C., J. M. Escoffre, A. Zumbusch, and M. P. Rols. 2011. The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells. Mol Ther 19:913-921. Nucleic acids electrotransfer in vitro 71 [15] Rosazza, C., A. Buntz, T. Riess, D. Woll, A. Zumbusch, and M. P. Rols. 2013. Intracellular tracking of single plasmid DNA-particles after delivery by electroporation. Mol Ther. [16] Rosazza, C., H. Deschout, A. Buntz, K. Braeckmans, M. P. Rols, and A. Zumbusch. 2016. Endocytosis and Endosomal Trafficking of DNA After Gene Electrotransfer In Vitro. Molecular therapy. Nucleic acids 5:e286. [17] Lechardeur, D., and G. L. Lukacs. 2006. Nucleocytoplasmic Transport of Plasmid DNA: A Perilous Journey from the Cytoplasm to the Nucleus. Hum Gene Ther 17:882-889. [18] Rosazza, C., S. H. Meglic, A. Zumbusch, M. P. Rols, and D. Miklavcic. 2016. Gene Electrotransfer: A Mechanistic Perspective. Curr Gene Ther 16:98-129. [19] Rems, L., and A. Miklavcic. 2016. Titorial: electroporation of cells in complex materials and tissue. J Appl Phys 119:201101. [20] Vaughan, E. E., and D. A. Dean. 2006. Intracellular trafficking of plasmids during transfection is mediated by microtubules. Mol Ther 13:422-428. [21] Beebe, S. J., J. White, P. F. Blackmore, Y. Deng, K. Somers, and K. H. Schoenbach. 2003. Diverse effects of nanosecond pulsed electric fields on cells and tissues. DNA Cell Biol 22:785-796. [22] Escoffre, J. M., K. Kaddur, M. P. Rols, and A. Bouakaz. 2010. In vitro gene transfer by electrosonoporation. Ultrasound Med Biol 36:1746-1755. [23] Delalande, A., S. Kotopoulis, M. Postema, P. Midoux, and C. Pichon. 2013. Sonoporation: mechanistic insights and ongoing challenges for gene transfer. Gene 525:191-199. [24] Riske, K. A., and R. Dimova. 2005. Electro-deformation and poration of giant vesicles viewed with high temporal resolution. Biophys J 88:1143-1155. [25] Portet, T., C. Favard, J. Teissie, D. Dean, and M. P. Rols. 2011. Insights into the mechanisms of electromediated gene delivery and application to the loading of giant vesicles with negatively charged macromolecules. Soft Matter 7:3872-3881. [26] Sutherland, R. M. 1988. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240:177-184. [27] Gibot, L., and M. P. Rols. 2013. Progress And Prospects: The Use Of 3D Spheroid Model As A Relevant Way To Study And Optimize Dna Electrotransfer. Curr Gene Ther. [28] Marrero, B., and R. Heller. 2012. The use of an in vitro 3D melanoma model to predict in vivo plasmid transfection using electroporation. Biomaterials. [29] Madi, M., M. P. Rols, and L. Gibot. 2015. Efficient In Vitro Electropermeabilization of Reconstructed Human Dermal Tissue. J Membr Biol. [30] Madi, M., M. P. Rols, and L. Gibot. 2016. Gene Electrtransfer in 3D Reconstructed Human Dermal Tissue. Curr Gene Ther 16:75-82. [31] Pillet, F., L. Gibot, M. Madi, M. P. Rols, and E. Dague. 2017. Importance of endogenous extracellular matrix in biomechanical properties of human skin model. Biofabrication 9:025017. [32] Stewart, M. 2016. In vitro and ex vivo strategies for intracellular delivery. Nature 538:183-192. 72 Marie-Pierre Rols Acknowledgement This research was performed in the scope of the EBAM European Associated Laboratory (LEA) and is a result of networking efforts within COST TD1104. Experiments are due to the works of the PhD students and post-docs I have/had the pleasure to supervise and/or work with: Muriel Golzio, Cécile Faurie, Emilie Phez, Jean-Michel Escoffre, Thomas Portet, Chloé Mauroy, Louise Chopinet, Elisabeth Bellard, Christelle Rosazza, Amar Tamra, Moinecha Madi, Luc Wasungu, Flavien Pillet, Laure Gibot and Nathalie Joncker. Marie-Pierre Rols was born in Decazeville, the “gueules noires” city of the Duc Decazes, France, in 1962. She received a Masters in Biochemistry, a Ph.D. in Cell Biophysics and the Habilitation à Diriger les Recherches from the Paul Sabatier University of Toulouse in 1984, 1989 and 1995, respectively. She is currently Director of Research at the IPBS-CNRS laboratory in Toulouse, “cellular biophysics” group leader and head of the “Structural Biology and Biophysics” Department. She is member of the board of the SFNano, ISEBTT, BES societies and of the LIA EBAM. Her research interests lie in the fields of membrane electropermeabilization in cells and tissues from the basics to the development of applications. Marie-Pierre Rols is the author of more the 150 articles in peer-reviewed journals. Chapter 5 Molecular Dynamics Simulations of Lipid Membranes Electroporation Mounir Tarek Theory, Simulations and Modeling, CNRS- Université de Lorraine France Abstract: Currently, computational approaches enable to follow, at the atomic scale, the local perturbation lipid membranes undergo when they are subject to external electric field. We describe here the molecular dynamics simulation methods devised to perform in silico experiments of membranes subject to nanosecond, megavolt-per-meter pulsed electric fields and of membranes subject to charge imbalance, mimicking therefore the application of low voltage – long duration pulses. At the molecular level, the results show the two types of pulses produce similar effects: provided the TM voltage these pulses create are higher than a certain threshold, hydrophilic pores stabilized by the membrane lipid head groups form within the nanosecond time scale across the lipid core. The simulations are further used to characterize the transport of charged species through these pores. The results obtained are believed to capture the essence of the several aspects of the electroporation phenomena in bilayers’ membranes, and could serve as an additional, complementary source of information to the current arsenal of experimental tools. Introduction Electroporation disturbs transiently or permanently the integrity of cell membranes [1–3]. These membranes consist of an assembly of lipids, proteins and carbohydrates that self–organize into a thin barrier that separates the interior of cell compartments from the outside environment [4]. The main lipid constituents of natural membranes are phospholipids that arrange themselves into a two-layered sheet (a bilayer). Experimental 74 Mounir Tarek evidence suggests that the effect of an applied external electric field to cells is to produce aqueous pores specifically in the lipid bilayer [5–9]. Information about the sequence of events describing the electroporation phenomenon can therefore be gathered from measurements of electrical currents through planar lipid bilayers along with characterization of molecular transport of molecules into (or out of) cells subjected to electric field pulses. It may be summarized as follows: Long and intense electrical pulses induce rearrangements of the membrane components (water and lipids) that ultimately lead to the formation of aqueous hydrophilic pores [5–10] whose presence increases substantially the ionic and molecular transport through the otherwise impermeable membranes [11]. In erythrocyte membranes, large pores could be observed using electron microscopy [12], but in general, the direct observation of the formation of nano-sized pores is not possible with conventional techniques. Furthermore, due to the complexity and heterogeneity of cell membranes, it is difficult to describe and characterize their electroporation in terms of atomically resolved processes. Atomistic simulations in general, and molecular dynamics (MD) simulations in particular, have proven to be effective for providing insights into both the structure and the dynamics of model lipid membrane systems in general [13–18]. Several MD simulations have recently been conducted in order to model the effect of electric field on membranes [19–23], providing perhaps the most complete molecular model of the electroporation process of lipid bilayers. MD simulations of lipid membranes Molecular dynamics (MD) refers to a family of computational methods aimed at simulating macroscopic behaviour through the numerical integration of the classical equations of motion of a microscopic many-body system. Macroscopic properties are expressed as functions of particle coordinates and/or momenta, which are computed along a phase space trajectory generated by classical dynamics [24,25]. When performed under conditions corresponding to laboratory scenarios, MD simulations can provide a detailed view of the structure and dynamics of a macromolecular system. They can also be used to perform “computer experiments” that cannot be carried out in the laboratory, either because they do not represent a physical behaviour, or because the necessary controls cannot be achieved. MD simulations require the choice of a potential energy function, i.e. terms by which the particles interact, usually referred to as a force field. Those most commonly used in chemistry and biophysics, e.g. GROMOS Molecular Dynamics Simulations of Lipid Membranes Electroporation 75 [26] CHARMM [27] and AMBER [28], are based on molecular mechanics and a classical treatment of particle-particle interactions that precludes bond dissociation and therefore the simulation of chemical reactions. Classical MD force fields consist of a summation of bonded forces associated with chemical bonds, bond angles, and bond dihedrals, and non-bonded forces associated with van der Waals forces and electrostatic interactions. The parameters associated with these terms are optimized to reproduce structural and conformational changes of macromolecular systems. Conventional force fields only include point charges and pair-additive Coulomb potentials, which prevent them from describing realistic collective electrostatic effects, such as charge transfer, electronic excitations or electronic polarization, which is often considered as a major limitation of the classical force fields. Note that constant efforts are undertaken on the development of potential functions that explicitly treat electronic polarizability in empirical force fields [29–31] but none of these “polarizable” force fields is widely used in large-scale simulations for now, the main reasons for that being the dramatic increase of the computational time of simulation and additional complications with their parameterization. In this perspective, classical force fields provide an adequate description of the properties of membrane systems and allow semi-quantitative investigations of membrane electrostatics. MD simulations use information (positions, velocities or momenta, and forces) at a given instant in time, t, to predict the positions and momenta at a later time, t + ∆t, where ∆t is the time step, of the order of a femtosecond, taken to be constant throughout the simulation. Numerical solutions to the equations of motion are thus obtained by iteration of this elementary step. Computer simulations are usually performed on a small number of molecules (few tens to few hundred thousand atoms), the system size being limited of course by the speed of execution of the programs, and the availability of computer power. In order to eliminate edge effects and to mimic a macroscopic system, simulations of condensed phase systems consider a small patch of molecules confined in a central simulation cell, and replicate the latter using periodic boundary conditions (PBCs) in the three directions of Cartesian space. For membranes for instance the simulated system would correspond to a small fragment of either a black film, a liposome or multilamellar oriented lipid stacks deposited on a substrate [32,33]. Traditionally, phospholipids have served as models for investigating in silico the structural and dynamical properties of membranes. From both a theoretical and an experimental perspective, zwitterionic phosphatidylcholine (PC) lipid bilayers constitute the best characterized systems [34–37]. More recent studies have considered a variety of 76 Mounir Tarek alternative lipids, featuring different, possibly charged, head groups [38][39–42], and more recently mixed bilayer compositions [43–49]. Despite their simplicity, bilayers built from PC lipids represent remarkable test systems to probe the computation methodology and to gain additional insight into the physical properties of membranes [14,17,50,51]. Modeling membranes electroporation The effects of an electric field on a cell may be described considering the latter as a dielectric layer (cell surface membrane) embedded in conductive media (internal: cytoplasm and external: extracellular media). When relatively low-field pulses of microsecond or millisecond duration are applied to this cell (by placing for instance the cell between two electrodes and applying a constant voltage pulse) the resulting current causes accumulation of electrical charges at both sides of the cell membrane. The time required to charge the surface membrane is dependent upon the electrical parameters of the medium in which it is suspended. For a spherical cell it is estimated using equivalent network RC circuits in the 100 ns time scale [19,52–55]. A charging time constant in the range of hundreds of nanoseconds was also obtained from derivations based on the Laplace equation (see e.g. [56] for the first-order analysis on a spherical vesicle; [57] for the second-order analysis; and [58] for the second-order analysis for two concentric spherical vesicles i.e. modeling an organelle). If on the other hand, the pulse duration is short enough relative to the charging time constant of the resistive-capacitive network formed by the conductive intracellular and extracellular fluids and the cell membrane dielectric, which is the case for nanosecond pulses, then the response of the system is mainly dielectric and is linked to the polarization of the interfacial water (see below). Simulations allow ones to perform in silico experiments under both conditions, i.e. submitting the system either to Nanosecond, megavolt-per-meter pulsed electric fields or to charge imbalance, mimicking therefore the application of low voltage – long duration pulses. In the following we will describe the results of such simulations. Molecular Dynamics Simulations of Lipid Membranes Electroporation 77 Figure. 1 Protocols for atomistic modelling of cell membranes or liposomes lipid bilayers (A) electroporation; (B) nsPEFs protocol: the system is modeled in absence of salt, and subject to an electric field Eapp perpendicular to the bilayer (z axis). Note that in some studies ions were also considered; (C) μs-msPEFs protocol introduced in the double bilayer setup: a charge imbalance ∆ Q is set across each bilayer and the scheme is implemented using classical PBCs. To prevent ions from migrating through the periodic boundary conditions, the simulation box (in blue) is extended in the direction perpendicular to the bilayer (z axis) to create a vacuum slab in the air/water interface protocol (D). Electroporation induced by dirrect effect of an electric field In simulations, it is possible to apply “directly” a constant electric field E perpendicular to the membrane (lipid bilayers) plane. In practice, this is done by adding a force F = q E to all the atoms bearing a charge [59– i i q 63]. MD simulations adopting such an approach have been used to study membrane electroporation [19–23], lipid externalization [64], to activate voltage-gated K+ channels [65] and to determine transport properties of ion channels [66–69]. 78 Mounir Tarek The consequence of such perturbation stems from the properties of the membrane and from the simulations set-up conditions: Pure lipid membranes exhibit a heterogeneous atomic distributions across the bilayer to which are associated charges and molecular dipoles distributions. Phospholipid head-groups adopt in general a preferential orientation. For hydrated PC bilayers at temperatures above the gel to liquid crystal transition, the phosphatidyl-choline dipoles point on average 30 degrees away from the membrane normal [70]. The organization of the phosphate (PO - + 4 ), choline (N(CH3)3 ) and the carbonyl (C=O) groups of the lipid head group give hence rise to a permanent dipole and the solvent (water) molecules bound to the lipid head group moieties tend to orient their dipoles to compensate the latter [71]. The electrostatic characteristics of the bilayer may be gathered from estimates of the electrostatic profile 𝜙𝜙(𝑧𝑧) that stems from the distribution of all the charges in the system. 𝜙𝜙(𝑧𝑧) is derived from MD simulations using Poisson’s equation and expressed as the double integral of 𝜌𝜌(𝑧𝑧), the molecular charge density distributions: 1 𝑧𝑧 Δ𝜙𝜙(𝑧𝑧) = 𝜙𝜙(𝑧𝑧) − 𝜙𝜙(0) = − ∬ 𝜌𝜌�𝑧𝑧"�𝑑𝑑𝑧𝑧"𝑑𝑑𝑧𝑧′ . 𝜖𝜖0 0 Figure. 2 Electrostatic potential profiles φ(z) along the membrane normal (z axis) of a POPC lipid bilayer. Bilayer (A) at rest, (B) subject to a transverse electric field (nsPEF protocol), and (C) bilayer set with a charge imbalance (μs-msPEF protocol). z=0 represents the center of the lipid bilayer. The contributions to the electrostatic profile from water (blue), lipid (yellow), ions (green) are reported next to the total one (black). The dashed arrows in panel C indicate the positions of the lipid/water interfaces and the solid arrows the position of the water/air interfaces. Note that the TM voltage Um (potential difference between the upper and lower water baths) in the nsPEF protocol is mainly due to water dipoles reorientation, while in the μs-msPEF protocol it is mainly due to the charge (ions) distribution. For lipid bilayers, most of which are modelled without consideration of a salt concentration, an applied electric field acts specifically and primarily on the interfacial water dipoles (small polarization of bulk water molecules). The reorientation of the lipid head groups appears not to be affected at very short time scales [21,72], and not exceeding few degrees Molecular Dynamics Simulations of Lipid Membranes Electroporation 79 toward the field direction at longer time scale [22]. Hence, within a very short time scale - typically few picoseconds [21] –a transverse field 𝑄𝑄�⃑ induces an overall TM potential ∆𝑽𝑽 ( cf. Fig 2). It is very important to note here that, because of the MD simulation setup (and the use of PBCs), E induces a voltage difference ∆𝑽𝑽 ≈ �𝑄𝑄�⃑�. 𝐿𝐿𝑍𝑍 over the whole system, where L z is the size of the simulation box in the field direction. In the example shown in Fig 2, L is ~ 10 nm. The electric field (0.1 V.nm-1) applied to the POPC z bilayer induces ∆𝑽𝑽 ~ 1V. MD simulations of pure lipid bilayers have shown that the application of electric fields of high enough magnitude leads to membrane electroporation, with a rather common poration sequence: The electric field favours quite rapidly (within a few hundred picoseconds) formation of water defects and water wires deep into the hydrophobic core [20]. Ultimately water fingers forming at both sides of the membrane join up to form water channels (often termed pre-pores or hydrophobic pores) that span the membrane. Within nanoseconds, few lipid head-groups start to migrate from the membrane-water interface to the interior of the bilayer, stabilizing hydrophilic pores (~1 to 3 nm diameter). All MD studies reported pore expansion as the electric field was maintained. In contrast, it was shown in one instance [21] that a hydrophilic pore could reseal within few nanoseconds when the applied field was switched off. Membrane complete recovery, i.e. migration of the lipid head group forming the hydrophilic pore toward the lipid/water interface, being a much longer process, was not observed. More recently systematic studies of pore creation and annihilation life time as a function of field strength have shed more light onto the complex dynamics of pores in simple lipid bilayers [22,73]. Quite interestingly, addition of salt has been shown to modulate these characteristic time scales [74]. Figure. 3 Pore evolution in a POPC bilayer: The POPC headgroups are shown as cyan and white beads, the lipids tails are not show for clarity. The pore creation, in MD simulations, takes places in the range of nanoseconds. For typical MD system sizes (128 lipids; 6 nm x 6 nm membrane cross section), most of the simulations reported a single pore formation at high 80 Mounir Tarek field strengths. For much larger systems, multiple pore formation with diameters ranging from few to 10 nm could be witnessed [20,21]. Such pores are in principle wide enough to transport ions and small molecules. One attempt has so far been made to investigate such a molecular transport under electroporation [21]. In this simulation, partial transport of a 12 base pairs DNA strand across the membrane could be followed. The strand considered diffused toward the interior of the bilayer when a pore was created beneath it and formed a stable complex DNA/lipid in which the lipid head groups encapsulate the strand. The process provided support to the gene delivery model proposed by Golzio et al. [75] in which, an ‘‘anchoring step’’ connecting the plasmid to permeabilized cells membranes that takes place during DNA transfer assisted by electric pulses, and agrees with the last findings from the same group [76]. More recently, (see sections below) it was shown that even a single 10 ns electric pulses of high enough magnitude can enhance small siRNA transport through lipid membranes [77]. The eletroporation process takes place much more rapidly under higher fields, without a major change in the pore formation characteristics. The lowest voltages reported to electroporate a PC lipid bilayer are ~ 2 V [22][72]. Ziegler and Vernier [23] reported minimum poration external field strengths for 4 different PC lipids with different chain lengths and composition (number of unsaturations). The authors find a direct correlation between the minimum porating fields (ranging from 0.26 V.nm-1 to 0.38 V.nm-1) and the membrane thickness (ranging from 2.92 nm to 3.92 nm). Note that estimates of electroporation thresholds from simulations should, in general be considered only as indicative since it is related to the time scale the pore formation may take. A field strength threshold is “assumed” to be reached when no membrane rupture is formed within the 100 ns time scale. Electroporation induced by ionic salt concentration gradients Regardless of how low intensity millisecond electrical pulses are applied, the ultimate step is the charging of the membrane due to ions flow. The resulting ionic charge imbalance between both sides of the lipid bilayer is locally the main effect that induces the TM potential. In a classical set up of membrane simulations, due to the use of 3d PBCs, the TM voltage cannot be controlled by imposing a charge imbalance 𝑄𝑄𝑠𝑠 across the bilayer, even when ions are present in the electrolytes. Several MD simulations protocols that can overcome this limitation have been recently devised (Fig. 1): Molecular Dynamics Simulations of Lipid Membranes Electroporation 81 The double bilayer setup: It was indeed shown that TM potential gradients can be generated by a charge imbalance across lipid bilayers by considering a MD unit cell consisting of three salt-water baths separated by two bilayers and 3d-PBCs [78] (cf. Fig. 1.C). Setting up a net charge imbalance between the two independent water baths at time t=0 induces a TM voltage ∆𝑽𝑽 by explicit ion dynamics. The single bilayer setup: Delemotte et al. [79] introduced a variant of this method where the double layer is not needed, avoiding therefore the over-cost of simulating a large system. The method consists in considering a unique bilayer surrounded by electrolyte baths, each of them terminated by an air/water interface [43]. The system is set-up as indicated in Fig. 1.D. First, a hydrated bilayer is equilibrated at a given salt concentration using 3d periodic boundary conditions. Air water interfaces are then created on both sides of the membrane, and further equilibration is undertaken at constant volume, maintaining therefore a separation between the upper and lower electrolytes. A charge imbalance 𝑄𝑄𝑠𝑠 between the two sides of the bilayer are generated by simply displacing at time t=0 an adequate number of ions from one side to the other. As far as the water slabs are thicker than 25-30 Å, the presence of air water interfaces has no incidence on the lipid bilayer properties and the membrane “feels” as if it is embedded in infinite baths whose characteristics are those of the modelled finite solutions. Fig. 2 reports the electrostatic potential profiles along the normal to the membrane generated from MD simulations a POPC bilayer in contact with 1M NaCl salt water baths at various charge imbalances 𝑄𝑄𝑠𝑠, using the single bilayer method. For all simulations, the profiles computed at the initial stage. show plateau values in the aqueous regions and, for increasing 𝑄𝑄𝑠𝑠, an increasing electrostatic potential difference between the two electrolytes indicative of a TM potential ∆𝑽𝑽. Quite interestingly, the profiles show clearly that, in contrast to the electric field case where the TM voltage is mainly due to the water dipole reorientation, most of the voltage drop in the charge imbalance method is due to the contribution from the ions. Indeed the sole collapse of the electrostatic potential due to the charge imbalance separation by the membrane lipid core accounts for the largest part of ∆𝑽𝑽. Using the charge imbalance set-up, it was possible for the first time to directly demonstrate in silico that the simulated lipid bilayer behaves as a capacitor [79,80]. Simulations at various charge imbalances 𝑄𝑄𝑠𝑠 show a 82 Mounir Tarek linear variation of ∆𝑽𝑽 from which the capacitance can be estimated as 𝐶𝐶 = 𝑄𝑄𝑠𝑠. ∆𝑽𝑽−1. The capacitance values extracted from simulations are expected to depend on the lipid composition (charged or not) and on the force field parameters used and as such constitute a supplementary way of checking the accuracy of lipid force field parameters used in the simulation. Here, in the case of POPC bilayers embedded in a 1M solution of NaCl, the later amounts to 0.85 µF.cm-2 which is in reasonable agreement with the value usually assumed in the literature e.g. 1.0 µF.cm-2 [78,81] and with recent measurements for planar POPC lipid bilayers in a 100 mM KCl solution (0.5 µF.cm-2). For large enough induced TM voltages, the three protocols lead to electroporation of the lipid bilayer. As in the case of the electric field method, for ∆𝑽𝑽 above 1.5-2.5 Volts, the electroporation process starts with the formation of water fingers that protrude inside the hydrophobic core of the membrane. Within nanoseconds, water wires bridging between the two sides of the membrane under voltage stress appear. If the simulations are further expended, lipid head-groups migrate along one wire and form a hydrophilic connected pathway (Fig.3). Because salt solutions are explicitly considered in these simulations, ion conduction through the hydrophilic pores occurred following the electroporation of the lipid bilayers. Details about the ionic transport through the pores formed within the bilayer core upon electroporation could be gathered. The MD simulations of the double bilayer system [82,83], and the results presented here for the single bilayer set-up show that both cations and anions exchange through the pores between the two baths, with an overall flux of charges directed toward a decrease of the charge imbalance. Ions translocation through the pores from one bulk region to the other lasts from few tens to few hundreds picoseconds, and leads to a decrease of the charge imbalance and hence to the collapse of ∆𝑽𝑽. Hence, for all systems, when the charge imbalance reached a level where the TM voltage was down to a couple of hundred mV, the hydrophilic pores “close” in the sense that no more ionic translocation occurs (Fig 4.F). The final topology of the pores toward the end of the simulations remain stable for time spans exceeding the 10 nanoseconds scale, showing as reported in previous simulations [21] that the complete recovery of the original bilayer structure requires a much longer time scale. Molecular Dynamics Simulations of Lipid Membranes Electroporation 83 Figure. 4 Left Sequence of events following the application of a TM voltage to a POPC lipid bilayer using the charge imbalance method (panels A to F). Note the migration of Na+ (yellow) and Cl- (cyan) ions through the formed hydrophilic pores that are lined with lipid phosphate (magenta) and nitrogen (blue) head group atoms. Panel F represents the state of a non conducting pore reached when the exchange of ions between the two baths lowered 𝑄𝑄𝑠𝑠 and therefore ∆𝑉𝑉 to values ≈ 200 mV. Right Topology of the nanometer wide hydrophilic pores formed under high transmembrane ∆𝑉𝑉 imposed by the charge imbalance method in the planar bilayer (A). The arrows highlight the subsequent ionic flow through the pores. Note that in order to maintain ∆𝑽𝑽 constant the modeler needs to maintain the initial charge imbalance by “injecting” charges (ions) in the electrolytes at a paste equivalent to the rate of ions translocation through the hydrophilic pore. This protocol is, in particular for the single bilayer setup, adequate for performing simulations under constant voltage (low voltage, ms duration) or constant current conditions, which is suitable for comparison to experiments undertaken under similar conditions [84]. Internal electric field distribution and origin of membranes electroporaiton In order to determine the detailed mechanism of the pore creation, it is helpful to probe the electric filed distribution across the bilayer, both at rest and under the effect of a TM voltage. Figure 5.A displays the electrostatic potential profiles for a lipid bilayer subject to increasing electric fields that generate TM potentials ranging from 0 V to ~ 3V. At 0 V, the lipid bilayer is at rest and the profiles reveal, in agreement with experiment [85], the existence of a positive potential difference between the membrane interior and the adjacent aqueous phases. 84 Mounir Tarek Figure. 5 (A) Electrostatic potential profiles across a lipid bilayer subject to electric fields of 0 V/nm (dotted line) 0.06 V/nm (thin line) and 0.30 V/nm (bold line), or to a charge imbalances ∆Q. (B) Corresponding electric field profiles. (C) 2d (out of plane) maps of the electric field distribution. The local electric field direction and strength are displayed as white arrows. Note that at 0mV, due to the bilayer dipole potential at rest, the larger electric fields are located at the lipid water interfaces and are oriented toward the solvent, and no electric field is present in the lipid core. When the bilayer is subject to a TM potential, a net electric field appears in the hydrocarbon region. The latter promotes dipolar orientation and penetration of water molecules (Right panel) inside the bilayer. At rest, the voltage change across the lipid water interfaces gives rise locally to large electric fields (in the present case up to 1.5 V.nm-1) oriented toward the bulk, while at the center of the bilayer, the local electric field is null (Fig. 5.B,C). When external electric fields of magnitudes respectively of 0.06 and 0.30 V.nm-1 are applied, reorientation of the water molecules gives rise to TM potentials of respectively ~ 0.75 and 3 V. Figs 5.B and C reveal the incidence of such reorganization on the local electric field both at the interfacial region and within the bilayer core. In particular one notes Molecular Dynamics Simulations of Lipid Membranes Electroporation 85 that the field in the membrane core has risen to a value ~ 1 V.nm-1 for the highest ∆𝑽𝑽 imposed. For the charge imbalance method, the overall picture is similar, where again, the TM voltages created give rise to large electric fields within the membrane core, oriented perpendicular to the bilayer. Qualitatively, in both methods, the cascade of events following the application of the TM voltage, and taking place at the membrane, is a direct consequence of such a field distribution. Indeed, water molecules initially restrained to the interfacial region, as they randomly percolate down within the membrane core, are subject to a high electric field, and are therefore inclined to orient their dipole along this local field. These molecules can then easily hydrogen bond among themselves, which results in the creation of single water files. Such fingers protrude through the hydrophobic core from both sides of the membrane. Finally, these fingers meet up to form water channels (often termed pre-pores or hydrophobic pores) that span the membrane. As the TM voltage is maintained, these water wires appear to be able to overcome the free energy barrier associated to the formation of a single file of water molecules spanning the bilayer (estimated to be ~ 108 kJ/mol in the absence of external electric field [86]. As the electrical stress is maintained, lipid head group migrate along the stable water wires and participate in the formation of larger “hydophilic pores”, able to conduct ions and larger molecules as they expend. Ziegler et al. [23] have shown clearly that the orientation of the lipid headgroups (dipoles) is not a determinant factor in the EP process. The general assumption that the lipid headgroups have a marginal role in the formation of the electropores, is consistent with studies on octane [20] as well as vacuum slabs [87] electroporation: These works have shown that, as in lipid bilayers, water columns can form in any water/low-dielectric/water system subject to high electric fields. Experimental evidence shows that pores do close when the PEF is turned off. The kinetics of this process determines how long leakage from or delivery to targeted cells can last. MD simulations indicate that this process initiates with a collapse of the pore (closure) due to a rapid leakage of water outwards to the bulk, followed by a much slower reorganization that leads to lipid headgroups re-partitioning toward the external hydrophilic leaflets. Resealing kinetics is independent of the magnitude of the pore initiation electric fields. In general, complete recovery of the original bilayer structure requires a much longer time scale [21,87,88], spanning from nanoseconds to hundreds of nanoseconds, and depends critically on the structure of the bilayer [89]. Note that addition of salt to systems undergoing the nsPEF protocol has been shown to modulate the characteristic time scales of the whole pore life cycle [88,90]. 86 Mounir Tarek COMPLEX BILYAER MODELS: EP THRESHOLDS AND PORE FEATURES Electroporation thresholds Since the pioneering simulations [21,91], which considered simple lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dimyristoyl-phosphatidylcholine (DMPC), a variety of lipid bilayers have been modeled in order to understand the key elements that might modulate their electroporation thresholds. The increase of the EP threshold upon addition of cholesterol [92–94] was studied using the E field [95] and charge imbalance protocols [93]. For the former, a steady increase of the EP threshold coincides with an increase in cholesterol concentration: a two folds higher electric field was necessary for the electroporation of bilayers with the addition of 50 mol% cholesterol. Under μs-msPEFs conditions, the EP threshold was showed to level-off above 30 mol % cholesterol. Generally, the increase of the EP threshold has been linked to the increase of the stiffness of the bilayer [92,94] . In a series of papers [96,97] Tarek’s group investigated the effect on the EP threshold of ester and ether linkages, of branched (phytanoyl) tails, and of bulky (glucosyl-myo and myo inositol) lipid head groups. The authors have found that the EP threshold of a lipid bilayer depends not only on the ‘‘electrical’’ properties of the membrane, i.e. its dipole potential or membrane capacitance, but also on the nature of lipids hydrophobic tails. The authors report that there is a correlation between the lateral pressure in the water/lipid interface and the EP threshold. They suggest that an increase of the lateral pressure (in the branched lipid membrane compared with the simple lipid bilayers) hinders the local diffusion of water molecules toward the interior of the hydrophobic core, which lowers the probability of pore formation, increasing therefore the electroporation threshold. Comparing specifically the Archeal lipids (glucosyl-myo and myo inositol head groups) to normal PC lipid, the higher electroporation thresholds for the former was attributed [96,97] to the strong hydrogen-bonding network stabilizing the head-group head group interactions. Likewise, Gurtovenko et al. [98] reported higher EP threshold for phosphatidylethanolamine (PE) lipid bilayers compared to phosphatidylcholine (PC) lipid bilayers. This effect was linked to inter-lipid hydrogen bonding taking place in the PE bilayer, which leads to a denser packed water/lipid interface and more ordered hydrocarbon lipid chains. Considering an asymmetric bilayer, composed by PC and PE lipid leaflets, the authors observed that the initial electroporation feature, i.e. the water Molecular Dynamics Simulations of Lipid Membranes Electroporation 87 column formation is also asymmetric, with initial steps taking place primarily at the PC leaflet. Studying more complex composition membranes, Piggot et al. [99] reported that the Gram-positive bacterial S. aureus cell membrane is less resistant to poration than the Gram-negative bacterial E.coli outer membrane (EcOM). The higher EP threshold of the EcOM was linked to the reduced mobility of the Lipopolysacharide molecules that are located in the outer leaflet. Additional factors, such as cholesterol, the presence of impurities, and other compounds, can modify the permeation properties of membrane models by acting on their stability. Pore features The MD results support the hypothesis that following the application of a high transmembrane voltage, the cell membrane is permeabilized by the formation of conducting hydrophilic pores stabilized by the lipid headgroups. The properties of the lipids play a determinant role in the electropores life-time and in its structural characteristics (e.g. size, shape, morphology) [87]. Other studies, considering various lipid bilayers, challenged the standard pore morphology. Tarek and coauthors pointed out that a peculiar EP process may be possible in which large long living ion-conducting water columns are not stabilized by lipid headgroups [93,97,100]. These “hydrophobic” conducting pores originate from constraints of a different nature in the lipid bilayer. The first report [100] focused on a palmitoyl-oleyl-phosphatidylserine (POPS) bilayer characterized by negatively charged headgroups. When this system was subject to a charge imbalance high enough to electroporate the bilayer, the migration of lipids along the water column turn out to be largely hindered (Fig. 5, second panel [100]). Similar conclusions were drawn for PC lipid bilayers containing more than 30 mol % cholesterol [93] or for Archaeal lipids [97] (Fig. 5). This peculiar morphology was ascribed to the repulsion of negatively charged headgroups in the first case [100], to the condensing effect of cholesterol in the second [93], and to the steric hindrance of the bulky headgroups coupled with the branched tails in the latter [97]. 88 Mounir Tarek Figure 6. Various morphologies of conducting pores revealed by MD simulations. Note that beside the POPC zwitterionic lipids, pores formed in POPS, a negatively charged lipid, with addition of cholesterol, or in the complex Archaea lipids (sugar like head groups), the electropores are not stabilized by the lipid head groups. Pores stabilization When dealing with the characteristics of electropores (e.g. size, conductance, transport of molecules) one would expect the pore to be in an energetically favorable state, i.e. one that corresponds to a stable configuration. In order to understand if the pore can be considered in a steady state for a given TM voltage and characterize its size and conductance, the two MD procedures, (introduced in previous sections) need to be improved. Indeed, the main drawback of these two protocols, as usually used, resides in the impossibility of maintaining a stable pore. In the electric field method, the pore tends to expand, leading to the breakdown of the bilayer, when it reaches the dimensions of the simulation cell box. The charge imbalance protocol, on the other hand, suffers from an important shortcoming: The imbalance is not re-set during the simulation. Thus, in the studies carried out both with the double and single bilayer schemes, the charge imbalance imposed at the beginning decreases significantly within several tens/hundreds ps (depending on the system size) of EP due to an exchange of ions through the pore. The decrease of the charge imbalance results in a TM voltage drop, which ultimately leads to pore collapse and resealing. When using the nsPEF protocol, the lowering of the electric field intensity after pore creation was shown to result in its stabilization [22]. Using the same strategy, Fernández et al. [95] could modulate the size of the pore and showed that it depends only on the strength of the stabilizing electric field. More recently our group [101] used a scheme to maintain a constant charge imbalance, refining thereby the μs-msPEFs approach to obtain size-controlled steady pores. The protocol used is identical to the procedure proposed by Kutzner et al. [84] to study the transport in ion channels using the double layer scheme. In this procedure, named “swapping”, the number of ions in the two solution baths is frequently Molecular Dynamics Simulations of Lipid Membranes Electroporation 89 estimated and, if the latter differs from the initial setup, a “swapping” event takes place: An ion of one solution is exchanged by a water molecule of the other solution bath (see the supplementary material for more information). Note that to overcome the limitation of simulating the bilayer in the NVT ensemble (constant volume), the swapping procedure can be coupled with the NPγT ensemble (constant surface tension) to maintain the bilayer surface tension constant (null) and mimic, therefore, experimental conditions [101]. Pore characterization A first attempt to link experimental evidence of pore conductance and radius estimation was carried out by Kramar et al. using a linear rising current technique combined with MD simulations performed under similar conditions [102]. Their findings suggest that the opening and closing of a single pore under conductance in the 100-nS scale would be possible for a pore diameter of ~5 nm. More systematic investigations, using the nsPEF [95,103] and μs-msPEF [101] modified protocols allowed to better characterize the conductance of electropores. For simulations carried out under the two protocols and when applying TM voltages below the EP threshold, the pore formed could be stabilized to different radii for tens of ns. Quite interestingly, the pore radii, and the pore conductance were found to vary almost linearly with the applied voltage. Moreover, the pores were found to be more selective to cations than to anions [101,103,104]. This selectivity arises from the nature of the lipid molecules constituting the pore: The negatively charged phosphate groups that form the walls of the pore attract sodium ions, which hinders their passage across the bilayer, but also makes the pore interior electrostatically unfavorable for other sodium ions [105]. This, already, suggests that the transport through electropores is sensitive to the type of solutes, showing a different affinity for different charged species. TRANSPORT OF MOLECULES Although numerous molecules are implicated in EP and/or concerned by its applications (e.g. drugs, genetic material, dyes, …), very few have been investigated with MD simulations. Apart from few studies in which electropore-mediated flip-flop of zwitterionic PC lipids [106–108] was reported, most simulations concerned charged species for which transport involved electrophoresis [21,77,109]. In the following, we discuss the results obtained using the two simulations protocols. 90 Mounir Tarek nsPEFs nsPEFs can induce externalization of phosphatidylserine (PS), a phospholipid usually confined to the inner leaflet of the plasma membrane that and can trigger several recognition, binding and signaling functions. MD studies of PS bilayers [19,110] showed how PS externalization is a pore-mediated event occurring exclusively with an electrophoretic drift. A decade ago, Tarek [21] reported the first MD simulation on the transport of a short DNA double strand using high intense electric fields. It was shown that the uptake occurred only in presence of the pore by electrophoretic drift. Since then, to our knowledge, only two MD studies have been reported on the transport of molecules under nsPEFs. In 2012 Breton et al. [77] showed that a single 10 ns high-voltage electric pulse can permeabilize giant unilamellar vesicles (GUVs) and allows the delivery of a double-stranded siRNA (-42e charge, 13.89 kDa) through the formed pore, by electrophoresis (Fig. 7 [77]). Comparing experimental evidence with MD simulations they could show in particular that: (i) following the application of an electric field, the siRNA is pushed toward the lipid headgroups forming an siRNA- phospholipids headgroups complex that remains stable even when the pulse is switched off; (ii) no transport is detected for electric fields applied below the EP threshold; (iii) when the Eapp is above the EP threshold (Eth) the siRNA is electrophoretically pulled through the electropore and translocated within a 10 ns time scale; (iv) if the Eth is turned off before the complete transition, the pore collapses around the molecule which is, hence, trapped. Recently, Salomone et al. [109] used a combination of nsPEFs and the chimeric peptides (CM18-Tat11) as efficient delivery vectors for plasmid DNA using endocytotic vesicles. To provide molecular details about the processes taking place, the authors modeled the peptide and its fragments. They reported from MD simulations that, when subject to high electric fields, Tat11, a small cationic peptide (residues 47-57 of HIV-1 Tat protein; +8e charge, 1.50 kDa) can translocate through an electroporated bilayer within few nanoseconds without interacting with the phospholipid headgroups. In contrast, the amphipathic peptide CM18, even when located near a preformed pore, remains anchored to the lipid headgroups and does not translocate during a 12 ns high electric field pulse. Molecular Dynamics Simulations of Lipid Membranes Electroporation 91 Figure 7: A single 10 ns high-voltage electric pulse can permeabilize lipid vesicles and allow the delivery of siRNA to the cytoplasm. Combining experiments and molecular dynamics simulations has allowed us to provide the detailed molecular mechanisms of such transport and to give practical guidance for the design of protocols aimed at using nanosecond-pulse siRNA electro-delivery in medical and biotechnological applications [77]. μs-msPEFs We present below the latest results from MD simulations of the uptake of molecules through lipids bilayers subject to μs-msPEFs. We focus our attention on Tat11 and the siRNA double strand to compare their mechanism of transport to the one reported using the nsPEFs [77,109]. These data have been reported in [111]. Transport of siRNA In 2011 Paganin-Gioanni et al. [76] investigated siRNA uptake by murine melanoma cells, when subject to electric pulses (1 Hz of repetition frequency) using time lapse fluorescence confocal microscopy. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. Noting that when added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cell, the authors concluded that the siRNA transport takes place during the electric pulse and is due to electrophoresis through electropores. The same group reported also that 0.17 kV/cm - 5 ms pulses, named EGT, are more effective in terms of silencing than the more intense less lasting HV pulses (1.3 kV/cm - 0.1 ms). They showed on the other hand that a double pulse procedure, consisting of one HV followed by a long below-EP-threshold 92 Mounir Tarek pulse does not increase the efficiency of the delivery. All together, their evidence suggests that, for msPEFs, the key factors for an efficient delivery are the voltage above the EP threshold and the duration of the pulse. In order to investigate the siRNA transfer into cells under conditions similar to the μs-msPEFs experiments, we have performed a set of simulations where the system was subject to several voltages (see Table 1). We first electroporate a bilayer patch by submitting it to a high charge imbalance. Once the pore was large enough (arbitrary value of ~2 nm radius) we lowered ΔQs to stabilize it to different radii as in [101]. These configurations were then used to start the simulations with siRNA placed near the pore mouth and were continued at the desired voltage. Table 1 Pore radius R and crossing time tc estimated at specific TM voltages (Um) for the two molecules considered. The pore radius (diameter) is estimated as the minimum lipid to lipid distance along the pore lumen System ts (ns) Um (V) R (nm) tc (ns) POPC_1024+siRNA 100 0.16 ± 0.16 2.0 ± 0.6 > 100 35 0.55 ± 0.19 3.3 ± 0.2 32.5 POPC_1024+Tat11 40 0.43 ± 0.16 1.6 ± 0.2 32.8 14 0.70 ± 0.24 2.0 ± 0.1 11.3 ts – simulation time; Um – transmembrane voltage create by the charge imbalance; R – minimum pore radius maintained by a given Um (see SM); tc – crossing time of the molecule through the electropore. For the lowest transmembrane voltages Um run, the siRNA approached the large pore (~4 nm diameter) mouth then started sliding through it while interacting with the lipid headgroups lining it. The complete translocation of the siRNA did not occur however within the first 100 ns of the run. In a completely independent run, we repeated the simulation by maintaining a higher voltage, namely 0.55 V. The siRNA approach, pore entry and sliding under these conditions (Fig. 7) were similar to the lower voltage run. However, at 0.55 V despite its anchoring to the lipid headgroups, a complete translocation from the upper to the lower water bath occurred in ~30 ns. Two factors contributed probably to this speed up. Compared to the previous conditions, not only the electrophoretic force pulling the siRNA is indeed higher, but the pore size increases too under this higher voltage. All together the simulations mimicking μs-msPEFs experiments, demonstrate that the translocation of siRNA through the pore driven by the application of TM voltages above 0.5 V takes place in the nanosecond time scale, as reported for the nsPEFs. Noticeably, in both simulations carried Molecular Dynamics Simulations of Lipid Membranes Electroporation 93 out under electric field or under the charge imbalance, the siRNA remains anchored to the lower leaflet of the membrane after translocation without diffusing in the bulk solution even if the voltage is maintained. Experiments performed on mouse melanoma cells applying ms-long pulses evidenced that tuning the duration of the pulse is essential for an efficient siRNA uptake. In fact the authors found more effective the EGT (0.17 kV/cm, 5 ms) class of pulses than the HV (1.3 kV/cm, 0.1 ms) one. No direct measurement of the TM voltage was carried out during these experiments and the authors assume that it is around 0.25 V, since it was observed that the EP threshold value is always about 0.20 mV for many different cell systems [112]. Corroborated by our findings, one can speculate that the transport of siRNA when subject to longer pulses could be facilitated by the formation of a pore population having larger diameters. This population of larger pores would allow siRNAs to flow through the pore and to access directly the cytoplasm increasing the transport efficiency. Transport of Tat11 The translocation for Tat11 differs from the highly charged siRNA because no specific interactions between this peptide and the lipid headgroups take place during the process, resulting in a faster uptake. Under a TM voltage Um ~0.70 V, the molecule, initially parallel to the membrane and located near the pore opening, first rotates to align its dipole along the local electric field (Fig. 10, t = 0 ns), then drifts though the center of the pore with a radius of 2 nm (Fig. 10, t = 8 ns), over the same time scale reported by the nsPEFs procedure [109]. The Tat11 reaches the lower bath where it freely diffuses (Fig. 8, t = 12 ns). At lower Um (~0.43 V) Tat11 translocates in 32.8 ns (see Table 1), presumably as a consequence of a higher hindrance of the pore (the pore radius decreases to 0.4 nm) and of a reduction of the electrophoretic drift. Considering a patch of 256 lipids, and applying an electric field that generates a 1.6 V across the bilayer, Salomone et al. [109] reported that Tat11 translocates through an electropore within 10 ns. This seems inconsistent with our results since one should expect that under our conditions, i.e. subject to a voltage Um of ~0.43 V, the time needed for Tat11 transport would be much longer. Indeed, if one considers only the ratio of electrophoresis, translocation of Tat11 should be three times slower at the lower voltage. In addition, a second inconsistency concerns the sizes of the pores created. Indeed in [109] the pore created has a radius of ~1.7 nm, much smaller than one expected from our results: we generated a pore of radius ~1.6 nm under Um ~0.43 V (Table 1). We have recently reported size effects in simulations of lipid bilayers electroporation, and shown 94 Mounir Tarek specifically that patches of 256 lipids are too small to study electroporation: Pores generated in MD simulations using such patches are much smaller than those generated using larger patches (1024 lipid). Figure. 8 The process of Tat11 transport in three frames corresponding to 0, 8, and 12 ns. In the right panel the top view clearly shows no interactions between the molecule and the pore walls. The POPC headgroups are shown as mauve and violet beads, the tails as purple lines; sodium and chloride ions are colored in yellow and gray; Tat11 is green (adapted from [111]). Despite these discrepancies, it is very interesting to note that both when applying both an electric field and charge imbalance, the translocation of a small charged molecule such as Tat11 occurs on the tens of nanosecond time scale. Discussion and perspectives A current goal in improving our understanding of EP is the development of a comprehensive microscopic description of the phenomenon, not an easy task due to the nanoscale dimensions of the lipid electropore and the short time scale (nanoseconds) of pore creation, which present challenges to direct experimental observations. For these reasons, molecular dynamics simulations have become extremely important to study EP in atomic detail. In the last decade, a large number of MD simulations have hence been conducted in order to model the effect of electric fields on membranes, providing perhaps the most complete molecular model of the EP process of lipid bilayers. Our investigation of the electrotransfer of small charged molecules, siRNA (-42e) and Tat11 (+8e) through a cell membrane model subject to microsecond pulse electric fields (μs-msPEFs) provided a novel insight. For transmembrane voltages of few hundred millivolts we report for siRNA a complete crossing translocation from one side of the bilayer to the other within several tens of nanoseconds despite its strong anchoring with the zwitterionic phospholipids headgroups. Tat11 on the other hand, is Molecular Dynamics Simulations of Lipid Membranes Electroporation 95 transported (within ~10 ns) without any interaction with the pore. Interestingly, for both molecules, we found that the transport process takes place at the same time scale (nanosecond) as much shorter pulses (nsPEFs) that we previously reported. Importantly, we recall that experiments are performed on cells, while our investigation concerns lipid bilayers. In cells, one should also consider the cytoskeleton and possible interactions with molecules e.g. siRNA in its way to the cytosol, slowing down the process of translocation. In summary, we have designed MD protocols suitable for the characterization of the transport of uncharged and charged species driven by μs-msPEFs that can help to shed light on the uptake mechanism of drugs by cell membranes. Systematic studies carried out with this protocol in presence of other relevant drugs (e.g. bleomycin) or dyes (e.g. propidium iodide, YO-PRO,…) are expected to drastically broaden our understanding of the uptake mechanism, thus providing further insights may lead to improvements in related experimental techniques and therapeutic effectiveness. It is worth mentioning another aspect that needs to be considered as well when studying electric field effect on cells. It has been suggested over a decade ago, that membranes can be oxidized upon electroporation. Experimental evidence reports, indeed, that pulsed electric fields can increase the extent at which lipid acyl chain peroxidation occurs. 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The authors would like to acknowledge very fruitful and insightful discussion with Damijan Miklavcic, Luis Mir and Thomas Vernier. Research conducted in the scope of the EBAM European Associated Laboratory (LEA). M.T acknowledges the support of the French Agence Nationale de la Recherche, under grant (ANR-10_, BLAN-916-03-INTCELL), and the support from the “Contrat Plan État-Region Lorraine 2015-2020” sub-project MatDS. Mounir Tarek born in Rabat-Morocco. He received a Ph.D. in Physics from the University of Paris in 1994. He is a senior research scientist (Directeur de Recherches) at the CNRS. For the last few years, he worked on large-scale state-of-the-art molecular simulations of lipid membranes and TM proteins probing their structure and dynamics. Chapter 6 Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization P. Thomas Vernier Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, USA Introduction To utilize the diverse effects of electric fields on biological systems we must understand the causes. In particular, we want to know the details of the interactions between electric fields and biomolecular structures. By looking at very short time scales (nanoseconds) and at single events (non-repetitive stimuli), we reduce the number of larger-scale disturbances and concentrate on reversible perturbations. The analysis is primarily in the time domain, but pulse spectral content may be important for some applications. Of course, some important effects of electropulsation may be a consequence of irreversible processes driven by longer electric field exposures (microseconds, milliseconds). Short-pulse studies can help to dissect these processes. 106 P. Thomas Vernier 1E+6 Control cells ) 8E+5 L ells/m 6E+5 Total pulsed cel s t (cnu 4E+5 ll Co 0 pulses, TB-negative Ce 2E+5 0 pulses, all cells 50 pulses, TB-negative TB-negative pulsed cells 50 pulses, all cells 0E+0 0 20 40 60 80 100 120 Minute s Af ter Exposure Figure 1. Nanoelectropulsed Jurkat T lymphoblasts recover over 2 hrs from initial Trypan blue permeabilization after exposure to 50, 20 ns, 4 MV/m pulses at 20 Hz. Although modeling is of necessity a significant component of bioelectrics investigations, experimental observations are fundamental, and to conduct experiments in nanosecond bioelectrics, one must be able to generate and accurately monitor the appropriate electrical stimuli, a non-trivial engineering challenge. We will discuss cause and effect here from both scientific and engineering perspectives, using data from experiments and simulations. It is commonplace in electrical engineering, and increasingly so in biology, to attack a problem with a combination of modeling and experimental tools. In nanosecond bioelectrics, observations (in vitro and in vivo) give rise to models (molecular and continuum), which drive experiments, which adjust and calibrate the models, which feed back again to empirical validation. This feedback loop focuses investigations of a very large parameter space on the critical ranges of values for the key variables. Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization 107 + Electric field propagation + cH2O = 225 mm/ns Dielectric + + 4 ps Shell Dielectric + H (1 mm cuvette) polarization + O − t + ∆ψ 3 t E t a cos 1 e τ θ + m ( ) = ( ) H − m Dipole 8 ps m 2 + (H O) 2 Orientation Electrolyte relaxation τ = 1 1 a C + D = E ε m m m σ 2 σ 5 ns e i D = D = D 1 2 3 – ε – ε ε τ = 1 3 e – E = E = E 2 1 3 σ Interface – ε ε charging 2 2 – – Mobile Charge 50 ns Dielectric – Migration – Stack – Figure 2. Timeline representing the sequence of events following electrical polarization of a biological tissue or aqueous suspension of cells. The dielectric properties of the system are important in the sub-nanosecond regime. For longer times the distribution of fields and potentials is dominated by the migration of charged species. Nanosecond bioelectrics From longstanding theory that models the cell as a dielectric shell [1–4] came the notion that sub-microsecond electric pulses could “bypass” the cell membrane, depositing most of their energy inside the cell instead of in the plasma membrane, the primary target of longer pulses. This idea was investigated experimentally beginning in the late 1990s, and apparently confirmed [5–6]. Even though one early report indicated that the electric field-driven conductive breakdown of membranes can occur in as little as 10 ns [7], and a theoretical analysis demonstrated that pulses with field amplitudes greater than about 1 MV/m will produce porating transmembrane potentials within about 2 ns [8], and a well-grounded model predicted “poration everywhere” in the nanosecond regime [9], procedures used to detect electroporation of the plasma membrane (and the loss of membrane integrity in general) produced negative results for pulses with durations less than the charging time constant of a small cell in typical media (< 100 ns). 108 P. Thomas Vernier ≥ 550 MV/m 450 MV/m ≤ 350 MV/m Figure 3. Electric field-driven intrusion of water into a simulated lipid bilayer. In addition to highlighting the limitations of traditional experimental methods for observing membrane permeabilization, this apparent discrepancy between model and observation points also to inadequacies in the dielectric shell model itself, at time scales below the membrane (cell) charging time. Higher-frequency effects associated with the dielectric properties of high-permittivity aqueous media and low-permittivity biological membranes [10–13] are negligible for the electropermeabilizing conditions that are most commonly studied (μs, kV/m pulses), but for nanosecond pulses they cannot be ignored. Several lines of experimental evidence indicate that nanosecond electric pulses cause changes in the integrity and organization of the cell membrane. Trypan blue permeabilization. While remaining propidium-negative, the cell volume of Jurkat T lymphoblasts exposed to a series of 50, 20 ns, 4 MV/m pulses increases, and they become permeable to Trypan blue (TB) (Figure 1). With increasing time after pulse exposure, these weakly TB-positive cells become again impermeable to TB. Similar observations have been reported for B16 murine melanoma cells exposed to sub-nanosecond (800 ps) pulses at very high fields [14]. Nanosecond porating transmembrane potentials. Fluorescence imaging with a membrane potential-sensitive dye indicates that porating transmembrane potentials are generated during nanoelectropulse exposure [15]. Nanoelectropulse-induced PS externalization. Loss of asymmetry in membrane phospholipid distribution resulting from phosphatidylserine (PS) externalization occurs immediately after nanoelectropulse exposure [16], Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization 109 consistent with membrane reorganization driven directly by nanosecond-duration electric fields and a mechanism in which nanometer-diameter pores provide a low-energy path for electrophoretically facilitated diffusion of PS from the cytoplasmic leaflet of the plasma membrane to the external face of the cell [8]. Simulations link PS externalization and nanoporation. In molecular dynamics (MD) simulations of electroporation, hydrophilic pores appear within a few nanoseconds [17], and PS migrates electrophoretically along the pore walls to the anode-facing side of the membrane [18–19], an in silico replication of experimental observations in living cells [20]. Nanoelectropermeabilization. The first direct evidence for nanoelectropermeabilization was obtained by monitoring influx of YOPRO-1 (YP1) [21], a more sensitive indicator of membrane permeabilization than propidium (PPD) [22]. Additional direct evidence comes from patch clamp experiments, which reveal long-lasting increases in membrane conductance following exposure to 60 ns pulses [23–25]. Nanosecond activation of electrically excitable cells. Electrically excitable cells provide a highly responsive environment for nanoelectropulse biology. Adrenal chromaffin cells [26] and cardiomyocytes [27] react strongly to a single 4 ns pulse, and muscle fiber has been shown to respond to a 1 ns stimulus [28]. Nanosecond bioelectrics and the dielectric stack model. Figure 2 depicts a time line of events in an aqueous suspension of living cells and electrolytes between two electrodes after an electric pulse is applied. Water dipoles re-orient within about 8 ps. The field also alters the electro-diffusive equilibrium among charged species and their hydrating water, with a time constant that ranges from 0.5 to 7 ns, depending on the properties of the media. Pulses shorter than the electrolyte relaxation time do not generate (unless the field is very high) enough interfacial charge to produce porating transmembrane potentials. The dielectric shell model in this regime can be replaced with a simpler, dielectric stack model, in which the local electric field depends only on the external (applied) electric field and the dielectric permittivity of each component of the system. Nanoelectropermeabilization and continuum models. MD simulations at present provide the only available molecular-scale windows on electropore formation in lipid bilayers. Current models perform reasonably well, but simulations of electroporation still contain many assumptions and simplifications. To validate these models, we look for intersections between all-atom molecular assemblies, continuum representations of cell suspensions and tissues, and experimental observations of cells and whole organisms. For example, a leading continuum model assumes an 110 P. Thomas Vernier exponential relation between the transmembrane potential and several indices of electropore formation [29]. The MD results in Figure 3, showing water intrusion into the membrane interior as a function of applied electric field, qualitatively demonstrate this same non-linear relation between field and poration. The challenge is to achieve a quantitative congruency of the coefficients. Nanosecond experiments, models Experiments and molecular models of membrane permeabilization. Figure 4 shows a simple and direct response of cells to pulse exposure — swelling [25,30,31]. Electropermeabilization of the cell membrane results in an osmotic imbalance that is countered by water influx into the cell and an increase in cell volume. This phenomenon, initiated by electrophysical interactions with basic cell constituents — ions, water, and phospholipids — on a much shorter time scale (a few nanoseconds) than usually considered by electrophysiologists and cell biologists, provides a simple, direct, and well-defined connection between simulations and experimental systems. By correlating observed kinetics of permeabilization and swelling with rates of pore formation and ion and water transport obtained from molecular simulations and continuum representations, we are improving the accuracy and applicability of the models Figure 4. Differential interference contrast (DIC) images of Jurkat T lymphoblasts before (A) and 30 s after (B) exposure to 5 ns, 10 MV/m electric pulses (30 pulses, 1 kHz). Note swelling, blebbing, and intracellular granulation and vesicle expansion, results of the osmotic imbalance caused by electropermeabilization of the cell membrane. Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization 111 Figure 5. Electropore creation sequence. (A) Molecular dynamics representation of a POPC lipid bilayer. Small red and white spheres at the top and bottom of the panel are water oxygen and hydrogen atoms. Gold and blue spheres are head group phosphorus and nitrogen, respectively, and grey spheres are phospholipid acyl oxygens. For clarity, atoms of the hydrocarbon chains in the interior of the bilayer are not shown. In the presence of a porating electric field, a water intrusion appears (B) and extends across the bilayer (C). Head groups follow the water to form a hydrophilic pore (D). The pore formation sequence, from the initiation of the water bridge to the formation of the head-group-lined pore takes less than 5 ns. Molecular dynamics and macroscale (continuum) models. Figure 5 shows the main steps in the electric field-driven formation of a nanopore in a typical MD simulation of a porating phospholipid bilayer, part of a larger scheme for the step-by-step development (and dissolution) of the electrically conductive defects that contribute at least in part to what we call a permeabilized membrane [32]. These molecular simulations permit us to conduct virtual experiments across a wide parameter space currently 112 P. Thomas Vernier inaccessible in practice to direct observation. Although we cannot yet align the detailed energetics and kinetics that can be extracted from MD simulations with laboratory results, it is possible to compare MD data with the predictions of the macroscale models used to describe electroporation. Figure 6 shows how pore initiation time (time between application of porating electric field and the appearance of a membrane-spanning water column (Fig. 5C)) varies with the magnitude of the electric field in MD simulations [32]. The value of the electric field in the membrane interior, extracted from simulations by integrating the charge density across the system, is used as a normalizing quantity. Figure 6. Electropore initiation time is a nonlinear function of the magnitude of the porating electric field. Pore initiation time (time required to form the water bridge shown in Fig. 1C) is exponentially dependent on the applied electric field, expressed here as the electric field observed in the lipid bilayer interior in molecular dynamics simulations. Error bars are standard error of the mean from at least three independent simulations. Data are from Tables 4 and 5 of [32]. Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization 113 Figure 7. Sodium and chloride ions migrating through a lipid nanopore in the presence of an external electric field. This membrane internal field results from the interaction of the applied external field with the interface water and head group dipoles, which also create the large dipole potential found in the membrane interior even in the absence of an applied field [33]. The nonlinear decrease in pore initiation time with increased electric field may be interpreted as a lowering of the activation energy for the formation of the pore-initiating structures described above. We can use simulation results like those in Fig. 6 to reconcile molecular dynamics representations with continuum models, and ultimately both of these to experiment. For example, the relation between electric field and pore creation rate is described in the Krassowska-Weaver stochastic pore model in the following expression, − E( r V , m ) k T B K = Ae , (1) pore where Kpore is the pore creation rate, A is a rate constant, E(r,Vm) is the energy of a pore with radius r at transmembrane potential Vm, and kB, and T are the Boltzmann constant and the absolute temperature [29,34–36]. One of our objectives is to reconcile the pore creation rate in (1) with our simulated pore initiation times, reconciling the two models. We are in the process also of validating the stochastic pore model expression for pore density, dN β ( 2 ψ m ) N = ∆ α e 1− , (2) dt Neq where N and Neq are pores per unit area, instantaneous and equilibrium values, α and β are empirical electroporation model parameters, and ∆ψ m is the transmembrane potential. Computing power is needed not only to enable simulations of larger systems. The large variability in pore initiation time indicated by the error 114 P. Thomas Vernier bars in Fig. 6 means that independent simulations of each condition must be repeated many times to ensure valid results. (A surprising number of conclusions in the existing literature have been published on the basis of single simulations.) Because of the complexity of all of the structures, systems, and processes which comprise the permeabilized membrane of a living cell (the electropermeome), a comprehensive analytical understanding of permeabilization (pore?) lifetime remains a major challenge for both models and experimental approaches. Better models can contribute also to our understanding of practical problems in bioelectrics. For example, despite years of study, controversy remains regarding the effects, or lack of effects, of exposures to low levels of radio-frequency (RF) electromagnetic fields [37,38]. Part of the reason for failure to establish certainty on this issue arises from the difficulty of conducting experiments with a sufficient number of variables and a sufficient number of samples to generate reliable data sets. With accurate simulation tools, honed by reconciliation with experiment, we can explore the large variable and statistical space in which suspected biophysical effects might occur, narrowing the range of experimental targets and focusing on systems in which effects are most likely and in which mechanisms will be clear. Experiments and molecular models of ion conductance. The earliest identified and most direct indicators of electric field-driven membrane permeabilization are changes in electrical properties, including an increase in ion conductance [39,40]. Data from careful experimental work can be interpreted as measured values corresponding to the conductance of a single pore [41–44]. By combining continuum models of electroporation with this experimental data and with established values for ion electrophoretic mobilities and affinities between ions and phospholipids, we can draw conclusions about pore geometry and areal density. But the inaccessibility (so far) of membrane electropores to direct observation and manipulation of their physical structure prevents us from definitively bridging the gap between model and experiment. A recently developed method for stabilizing electropores in molecular dynamics simulations of phospholipid bilayers [45] allows extraction of ion conductance from these model systems and thus provides a new and independent connection between models and experiments, in this case from the atomically detailed models of lipid electropores constructed with molecular dynamics. Figure 7 shows one of these stabilized pores with electric field-driven ions passing through it. Although the magnitude of the conductance measured in these simulations is highly dependent on the accuracy of the ion and water models Nanoscale and Multiscale Membrane Electrical Stress and Permeabilization 115 and their interactions with the phospholipid bilayer interface (and there is much room for improvement in this area), initial results are consistent with expectations from both continuum models and experimental observations. Nanosecond excitation Nanoelectrostimulation of neurosecretory and neuromuscular cells. Applications of pulsed electric fields in the clinic, particularly in electrochemotherapy and gene electrotransfer, are well known and described in detail in other parts of this course. We note here a potential biomedical application specifically of nanosecond electric pulses, the activation and modulation of the activity of neurosecretory and neuromuscular processes, an area which remains relatively unexplored. The sensitivity of electrically excitable cells to nanoelectropulses raises the possibility that very low energy (nanosecond, megavolt-per-meter pulses are high power, but low total energy because of their brief duration) devices for cardiac regulation (implanted pacemakers and defibrillators), remote muscle activation (spinal nerve damage), and neurosecretory modulation (pain management) can be constructed with nanoelectropulse technology. Figure 8 demonstrates functional activation of an adrenal chromaffin cell after a single 5 ns, 5 MV/m pulse [46,47]. Figure 8. Immunocytochemical labeling of dopamine-β-hydroxylase (DβH) using an anti-DβH antibody coupled with a fluorescently-tagged 2o antibody. DβH is externalized by exocytotic fusion of vesicles with plasma membrane. Left panel, control. Center panel, 2 min after treatment with the pharmacological stimulant DMPP. Right panel, 2 min after a single, 5 ns, 5 MV/m pulse. 116 P. Thomas Vernier Acknowledgment Collaborative insights from Francesca Apollonio, Delia Arnaud-Cormos, Maura Casciola, Gale Craviso, Rumiana Dimova, M. Laura Fernández, Wolfgang Frey, Julie Gehl, Martin Gundersen, Loree Heller, Richard Heller, Volker Knecht, Malgorzata Kotulska, Philippe Leveque, Zachary Levine, Micaela Liberti, Carmela Marino, Caterina Merla, Damijan Miklavčič, Lluis Mir, Andrei Pakhomov, Olga Pakhomova, Uwe Pliquett, Ramon Reigada, Marcelo Risk, Marie-Pierre Rols, Stefania Romeo, Maria Rosaria Scarfi, Aude Silve, Esin Sözer, Mounir Tarek, Justin Teissié, Peter Tieleman, Mayya Tokman, Jim Weaver, and Olga Zeni (and very important to me but too many to name members of their research groups), and modeling and experimental expertise from Maura Casciola, Federica Castellani, Ming-Chak Ho, Zachary Levine, Paolo Marracino, Stefania Romeo, Esin Sözer, and Yu-Hsuan Wu contributed to this work. Funding is provided by the Frank Reidy Research Center for Bioelectrics at Old Dominion University and the Air Force Office of Scientific Research (FA9550-15-1-0517, FA9550-14-1-0123). Computing resources were provided by the USC Center for High-Performance Computing and Communications (http://www.usc.edu/hpcc/) and Old Dominion University High-Performance Computing (http://www.odu.edu/hpc/). References [1] Sher, L. D., E. Kresch, and H. P. Schwan. 1970. On the possibility of nonthermal biological effects of pulsed electromagnetic radiation. Biophys. J. 10:970-979. [2] Drago, G. P., M. Marchesi, and S. Ridella. 1984. The frequency dependence of an analytical model of an electrically stimulated biological structure. Bioelectromagnetics 5:47-62. [3] Plonsey, R., and K. W. Altman. 1988. Electrical stimulation of excitable cells - a model approach. Proceedings of the IEEE 76:1122-1129. [4] Schoenbach, K. H., R. P. Joshi, J. F. Kolb, N. Y. Chen, M. Stacey, P. F. Blackmore, E. S. 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Yoon, I. Chatterjee, and P. T. Vernier, "Nanosecond electric pulse-induced increase in intracellular calcium in adrenal chromaffin cells triggers calcium-dependent catecholamine release," Ieee Transactions on Dielectrics and Electrical Insulation, vol. 16, pp. 1294-1301, Oct 2009. [47] G. L. Craviso, S. Choe, P. Chatterjee, I. Chatterjee, and P. T. Vernier, "Nanosecond electric pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated Ca2+ channels," Cell Mol Neurobiol, vol. 30, pp. 1259-65, Nov 2010. P. Thomas Vernier is Research Professor at the Frank Reidy Research Center for Bioelectrics at Old Dominion University and Adjunct Research Professor in the Ming Hsieh Department of Electrical Engineering at the University of Southern California. His research and industrial experience includes ultraviolet microscopy analysis of S-adenosylmethionine metabolism in the yeast Rhodotorula glutinis, molecular biology of the temperature-sensitive host restriction of bacterial viruses in Pseudomonas aeruginosa, low-level environmental gas monitoring, wide-band instrumentation data recording, and semiconductor device modeling and physical and electrical characterization. He currently concentrates on the effects of nanosecond, megavolt-per-meter electric fields on biological systems, combining experimental observations with molecular dynamics simulations, and on the integration of cellular and biomolecular sensors, carbon nanotubes, and quantum dots with commercial integrated electronic circuit fabrication processes. Vernier received his Ph.D. in Electrical Engineering from the University of Southern California in 2004, and is a member of the American Chemical Society, American Society for Microbiology, Bioelectromagnetics Society, Biophysical Society, European BioElectromagnetic Association, and Institute of Electrical and Electronics Engineers. Chapter 7 Gene electrotransfer in vivo Maja Čemažar Institute of Oncology Ljubljana, Slovenia Abstract: Gene electrotransfer consists of administration of nucleic acids (DNA, RNA oligonucleotides…) followed by application of electric pulses to the specific tissue in order to enable delivery of nucleic acids into cells and consequently the therapeutic action of delivered genetic material. Due to the size of nucleic acids, the electrical parameters of gene electrotransfer vary greatly depending on the tissue to be transfected and also on the desired level and duration of expression as well as accompanied tissue damage. Besides optimization of electrical parameters for specific application, design of therapeutic plasmid DNA or RNA molecules can also influence the therapeutic outcome. Initial studies on gene electrotransfer were mainly focused on the evaluation of electrical parameters for efficient gene delivery to different tissues, such as skin, muscle, liver and tumors using various reporter genes encoding fluorescent proteins, luciferase and β- galactosidase. Therapeutic field of gene electrotransfer is mainly divided into two fields: DNA vaccination and cancer gene therapy. DNA vaccination against infectious diseases and cancer on one-hand and antiangiogenic and immunomodulating gene therapies against cancer on the other hand are the prevalent areas of research. Furthermore, increasing number of clinical trials, especially in USA, are registered using electroporation for delivery of therapeutic plasmid DNA. The perspectives of therapeutic gene electrotransfer for cancer therapy lie mainly in different combination with standard local therapies, such as radiation therapy or electrochemotherapy, with the aim to turn local treatments into systemic ones. In addition, a lot of preclinical work is dedicated to optimization of therapeutic plasmid DNAs, development of new electrodes and evaluation of electrical parameters, which will lead to better planning and design of clinical trials. Gene electrotransfer in vivo 121 Introduction The in vitro application of electroporation for the introduction of DNA into the cells was evaluated and tested in 1982 by Neumann et al [1], 6 years before the use of electroporation for delivery of antitumor chemotherapeutic drugs (electrochemotherapy) into the tumor cells [2]. However, in vivo studies only slowly followed and the first in vivo study was performed in 1991 by Titomirov et al [3], evaluating the usefulness of exponentially decaying pulses for delivery of genes to the mouse skin. Later on, the transfection of brain, liver, tumor and muscle using different reporter genes were successfully demonstrated using different types of electric pulses [3– 7]. Due to the physicochemical properties and the size of nucleic acids compared to small chemotherapeutic drugs, the mechanism of entry of nucleic acids is different than that of small molecules. In tissues, other, tissue and cell related parameters also influence the transfection efficiency, such as cell size, shape and organization in the tissues, presence of the extracellular matrix and tissue heterogeneity (presence of different types of cells in the particular tissue). In addition, the construction of plasmid and its administration can also influence the level of transfection as well as its duration. Therefore, a vast amount of studies in the field of in vivo gene electrotransfer were dedicated to evaluation of different parameters of electric pulses for different tissue type as well as for different application (Figure 1). Currently, therapeutic use of gene electrotransfer is focused in mainly two fields: DNA vaccination and cancer gene therapy [8,9]. Preclinical gene electrotransfer of reporter genes Reporter genes used in preclinical studies on gene electrotransfer were mainly encoding either different fluorescent proteins or luciferase. Both enable to visualize the transfection of tissues (gene expression in cells in tissues) in vivo using different types of in vivo imaging, either whole body imaging or at the cellular level [10,11]. Most of the studies were performed in muscle and skin, as these tissues are easily accessible and therefore represent an obvious target tissue for DNA vaccination. Besides easy accessibility for gene electrotransfer, muscle cells are long lived and they can produce relatively high quantities of therapeutic proteins that are also released into the blood stream, thus acting systemically. On the other hand, skin also represent a great target tissue, not only due to the easy accessibility, but mainly because of the numerous immune cells present in 122 Maja Čemažar the skin that can elicit effective immune response of the organisms needed for DNA vaccination [12,13](Figure 2). Figure 1: Different parameters can influence the transfection efficiency and therapeutic outcome of gene electrotransfer. As mentioned in the introduction, numerous different parameters of electric pulses were used, either short (~100µs) high voltage (in the range of ~1000 V) electric pulses or long (up to 100 ms) low voltage (up to few 100 V) pulses were used. Moreover, even a combination of high voltage and low voltage pulses were tested and showed improved transfection in skin and muscle compared to single type of pulses used for transfection [14– 16]. In tumors, the combination of pulses did not result in improved transfection [18]. In addition, the influence of orientation and polarity of the applied electric pulses were also evaluated in tumors, demonstrating that increased transfection efficiency is obtained only by changing the electrode orientation, but not pulse polarity[19]. The main type of electrodes used in the studies was either plate or needle and more recently also non-invasive multielectrode arrays [15,19,20]. Other types of electrodes that were tested for gene electrotransfer were spatula electrodes for gene delivery to muscle [22] and other types of noninvasive electrodes, such as needle free, meander and contact electrodes for skin delivery [21–24]. Selection of electrode is very important for appropriate electric field distribution in the tissue which is a prerequisite for effective gene electrotransfer[24, 25]. Gene electrotransfer in vivo 123 Figure 2: Gene electrotransfer to skin. A injection of plasmid DNA subcutaneously. A bubble on the skin will be formed. B If using plate electrodes, they are positioned in a way that the bubble is encompassed between the two plates. C Intravital confocal microscopy of cells in mouse skin expressing DsRED fluorescent protein at the depth of 30 µm. Besides electrical parameters, the type of the nucleic acid used for electrotransfer can also affect the transfection efficiency. Namely, it was shown that smaller siRNA can more easily crossed the plasma membrane compared to larger plasmid DNA molecules, however the duration of the expression (or effect) is shorter [26–28]. Therefore, the plasmid DNA are still the most often used in gene electrotransfer studies. To improve the safety and targeting of the plasmid DNA delivery as well as to minimize the undesired tissue damage, the plasmids with tissue specific promoters, devoid of antibiotic resistance gene and with minimal or no bacterial backbone were constructed and evaluated in combination with electroporation [29–32]. Due to the size of plasmid DNA and the presence of nucleases in the blood and also tissues, the most suitable was of plasmid DNA administration is local injection. The distribution of the plasmid DNA in different tissues has different time frame, therefore it is also very important the timing between the injection of plasmid DNA and application of electric pulses. For muscle is was shown that it should be as soon as possible, while for the tumors, depending on the histological type, it can be up to 30 min after the injection of the plasmid [33–35]. Improved distribution and consequently better transfection efficiency can be achieved also by pretreatment of muscles and tumors with extracellular matrix degrading enzymes, such as hyaluronidase and collagenase [36,37]. In vitro, it was shown that size, orientation and shape of the cells influence the permeabilisation of the cell membranes and thus also transfection efficiency. The same is also valid in vivo. Tissues with more organized structure, such as muscle are more easy to transfect than highly heterogenic tissue, such as tumors [16]. In addition, in tumors with large 124 Maja Čemažar cells higher transfection efficiency was obtained compared to tumors with smaller cells [38-41]. The importance of careful selection of plasmid DNA and electrical parameters for specific application, was recently reinforced by experiments showing that gene electrotransfer of plasmids devoid of therapeutic gene can induced complete regression of tumors and that cytosolic DNA sensors activating innate immune response were upregulated following gene electrotransfer [42]. The inflammation and induction of immune response was demonstrated also for muscle and skin transfection [41-43]. Preclinical and clinical gene electrotransfer of therapeutic genes The preclinical studies using therapeutic genes were mainly dedicated to evaluation of gene electrotransfer for DNA vaccination or treatment of various diseases, such as cancer, where therapies are targeted either directly to tumor cells or aim to increase the immune response of the organism against cancer cells. In general, gene therapy can be performed using two different approaches. The first one is ex vivo gene therapy, where cells, including stem cells, are removed from patient, transfected in vitro with the plasmid or viral vector, selected, amplified, and then reinjected back into the patient. The other approach is in vivo gene therapy, where exogenous DNA is delivered directly into host's target tissue e.g. locally to tumor or peritumorally and for systemic release of the therapeutic molecule into skeletal muscle depending on the type of therapeutic molecules and intent of treatment [45]. Gene electrotransfer was first used for DNA vaccination in 1996 [46]. Currently, numerous studies, using gene electrotransfer mainly to muscle and skin for DNA vaccination against infectious diseases, arthritis, multiple sclerosis, inflammation are undergoing. In addition, several clinical trials, against infectious diseases, such as HIV, hepatitis are going on. Gene electrotransfer of plasmid DNA resulted in stimulation of both arms of adaptive immune system, humoral and cellular [8,9]. In cancer gene therapy, gene electrotransfer of therapeutic genes directly into tumors facilitates local intratumoral production of therapeutic proteins, enabling sufficient therapeutic concentration and thus therapeutic outcome. This is especially important in case of cytokines, where high systemic concentrations are associated with severe toxicity. Gene electrotransfer in vivo 125 The first evaluation of intratumoral electrogene therapy for cancer treatment was performed 3 years after the first DNA vaccination study in 1999 in murine melanoma tumor model [47]. Since then, a variety of therapeutic genes, mostly encoding cytokines, but also tumor suppressor proteins, siRNA molecules against various targets, such as oncogenes, have been tested in a numerous animal tumor models. Overall, results of preclinical studies indicate, that intratumoral therapeutic gene electrotransfer enables efficient transgene expression with sufficient production of therapeutic proteins, which can lead to even complete tumor regression and in some cases to induction of long-term antitumor immunity in treated animals. Some of the most significant antitumor effect to date in cancer gene therapy have been achieved with employment of active nonspecific immunotherapy, i.e. use of cytokines. Gene electrotransfer of genes, encoding different cytokines, has already shown promising results in preclinical trials on different animal tumor models. Cytokine genes, which showed the most potential for cancer therapy, are interleukin (IL)-2, IL-12, IL-18, interferon (IFN) α, and GM-CSF[47–52]. Currently, the most advanced therapy is using IL-12, which plays important role in the induction of cellular immune response through stimulation of T-lymphocyte differentiation and production of IFN-γ and activation of natural killer cells[54]. Antitumor effect of IL-12 gene electrotransfer, has already been established in various tumor models, e.g. melanoma, lymphoma, squamous cell carcinoma, urinary bladder carcinoma, mammary adenocarcinoma and hepatocellular carcinoma[53]. Results of preclinical studies show that beside regression of tumor at primary and distant sites, electrogene therapy with IL-12 also promotes induction of long-term antitumor memory and therapeutic immunity, suppresses metastatic spread and increases survival time of experimental animals[53]. On preclinical level, gene electrotransfer to tumors was also employed in suicide gene therapy of cancer, replacement of oncogenes therapies, introduction of wild type tumor suppressor genes etc [47,54–56]. Another approach in cancer gene therapy, which is currently being widely investigated, is based on inhibition of angiogenesis of tumors. The basic concept of antiangiogenic gene therapy is transfection of cells with genes, encoding inhibitors of tumor angiogenesis. Electrotransfer of plasmids encoding antiangiogenic factors (angiostatin and endostanin) was demonstrated to be effective in inhibition of tumor growth and metastatic spread of different tumors[57–59]. Recently, RNA interference approach was evaluated, using siRNA molecule against endoglin, which is a co-receptor of transforming growth factor β and is overproduced in activated endothelial and also certain tumor cells. Gene electrotransfer of either siRNA or shRNA molecules against endoglin resulted in vascular targeted 126 Maja Čemažar effect in mammary tumors as well as antitumor and antivascular effect in melanoma tumors that are expressing high level of endoglin [60,61]. Muscle tissue is, besides in DNA vaccination, used also as a target tissues due to the possibility of high production and secretion of therapeutic proteins. Gene electrotransfer to muscle was evaluated with the aim to treat various muscle diseases, for local secretion of angiogenic or neurotrophic factors or for systemic secretion of different therapeutic proteins, such as erythropoietin, coagulation factors, cytokines, monoclonal antibodies, etc. [62–64]. In cancer gene therapy, gene electrotransfer of plasmid DNA encoding cytokines IL-12, IL-24, and antiangiogenic factors was evaluated with encouraging results. Clinical studies on gene electrotransfer with plasmid DNA encoding cytokine IL-12 in patients with melanoma, as well as in veterinary patients show great promise for further development of this therapy[65,66]. In human clinical study, 24 patients with malignant melanoma subcutaneous metastases were treated 3 times. The response to therapy was observed in treated as well as in distant non-treated tumor nodules. In 53% of patients a systemic response was observed resulting in either stable disease or an objective response. The major adverse side-effect was transient pain after application of electric pulses. In post-treatment biopsies, tumor necrosis and immune cell infiltration was observed. This first human clinical trial with IL-12 electrogene therapy in metastatic melanoma proved that this therapy is safe and effective[66]. In veterinary oncology, 8 dogs with mastocytoma were treated with IL-12 gene electrotransfer. A good local antitumor effect with significant reduction of treated tumors' size, ranging from 15% to 83% (mean 52%) of the initial tumor volume was obtained. Additionally, a change in the histological structure of treated nodules was seen as reduction in the number of malignant mast cells and inflammatory cell infiltration of treated tumors. Furthermore, systemic release of IL-12 and IFN-γ in treated dogs was detected, without any noticeable local or systemic side-effects[67]. Again, the data suggest that intratumoral IL-12 electrogene therapy could be used for controlling local as well as systemic disease. For example, results of intramuscular IL-12 gene electrotransfer in canine patients indicate that it is a safe procedure, which can result in systemic shedding of hIL-12 and possibly trigger IFN-γ response in treated patients, leading to prolonged disease free period and survival of treated animals [68]. Gene electrotransfer in vivo 127 Perspectives In oncology, local ablative treatments are very effective, however they lack a systemic component. Therefore, much effort is dedicated to development of treatments, that would act systemically or that would add a systemic component to the local treatment. With the progress of knowledge in tumor immunology, new immunomodulating therapies were developed for treatment of cancer and are currently combined with standard treatment with great success. DNA vaccination and immune gene therapies with cytokines aim to stimulated antitumor immunity and are thus good candidates to be combined with local therapies[68,69]. Several studies combining electrochemotherapy or radiotherapy with gene electrotransfer have been evaluated preclinically. The most promising immune-gene therapy that already reached clinical trials in veterinary and human oncology, is gene electrotransfer of IL-12. In the preclinical studies IL-12 gene electrotransfer was combined with electrochemotherapy and radiotherapy in different tumor models. Intramuscular gene electrotransfer of IL-12 combined with electrochemotherapy with cisplatin increased the percentage of complete regression of fibrosarcoma SA-1 tumors to 60% compared to 17% complete regression after electrochemotherapy alone [71]. When combined with radiotherapy even 100% complete response of LPB tumors was obtained [72]. Intratumoral IL-12 gene electrotransfer resulted in ~2.0 radiation dose modifying factor [73]. Clinically, only several studies were performed in client owned dogs, combining electrochemotherapy with either bleomycin or cisplatin and intratumoral or peritumoral application of IL-12 gene electrotransfer [73– 76]. The results of these clinical studies are very promising and further studies, hopefully also in human oncology are foreseen. Gene electrotransfer holds big potential for further development, which might lead to new clinical trials in both DNA vaccination and gene therapy application. 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[58] M. Cemazar and G. Sersa, “Electrotransfer of therapeutic molecules into tissues,” Curr Opin Mol Ther, vol. 9, no. 6. pp. 554–562, 2007. 132 Maja Čemažar [59] T. Cichoń, L. Jamrozy, J. Glogowska, E. Missol-Kolka, and S. Szala, “Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: inhibition of primary tumor growth and metastatic spread.,” Cancer Gene Ther. , vol. 9, no. 9, pp. 771–7, Oct. 2002. [60] M. Uesato, Y. Gunji, T. Tomonaga, S. Miyazaki, T. Shiratori, H. Matsubara, T. Kouzu, H. Shimada, F. Nomura, and T. Ochiai, “Synergistic antitumor effect of antiangiogenic factor genes on colon 26 produced by low-voltage electroporation.,” Cancer Gene Ther. , vol. 11, no. 9, pp. 625–32, Sep. 2004. [61] J. M. Weiss, R. Shivakumar, S. Feller, L.-H. Li, A. Hanson, W. E. Fogler, J. C. Fratantoni, and L. N. 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Attema, and D. van Bekkum, “A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer.,” BMC Mol. Biol. , vol. 3, p. 12, Aug. 2002. [66] N. Perez, P. Bigey, D. Scherman, O. Danos, M. Piechaczyk, and M. Pelegrin, “Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation.,” Genet. Vaccines Ther. , vol. 2, no. 1, p. 2, Mar. 2004. [67] A. I. Daud, R. C. DeConti, S. Andrews, P. Urbas, A. I. Riker, V. K. Sondak, P. N. Munster, D. M. Sullivan, K. E. Ugen, J. L. Messina, and R. Heller, “Phase I Trial of Interleukin-12 Plasmid Electroporation in Patients With Metastatic Melanoma,” J. Clin. Oncol. , vol. 26, no. 36, pp. 5896–5903, 2008. [68] D. Pavlin, M. Cemazar, A. Cor, G. Sersa, A. Pogacnik, and N. Tozon, “Electrogene therapy with interleukin-12 in canine mast cell tumors,” Radiol Oncol, vol. 45, no. 1, pp. 31–39, 2011. [69] D. Pavlin, M. Cemazar, G. Sersa, and N. Tozon, “IL-12 based gene therapy in veterinary medicine.,” J. Transl. Med. , vol. 10, p. 234, 2012. [70] G. Sersa, J. Teissie, M. Cemazar, E. Signori, U. Kamensek, G. Marshall, and D. Miklavcic, “Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer.,” Cancer Immunol. Immunother. , vol. 64, no. 10, pp. 1315– 27, Oct. 2015. [71] C. Y. Calvet and L. M. Mir, “The promising alliance of anti-cancer electrochemotherapy with immunotherapy.,” Cancer Metastasis Rev. , vol. 35, no. 2, pp. 165–77, Jun. 2016. [72] A. Sedlar, T. Dolinsek, B. Markelc, L. Prosen, S. Kranjc, M. Bosnjak, T. Blagus, M. Cemazar, and G. Sersa, “Potentiation of electrochemotherapy by intramuscular IL-12 Gene electrotransfer in vivo 133 gene electrotransfer in murine sarcoma and carcinoma with different immunogenicity,” Radiol. Oncol. , vol. 46, no. 4, 2012. [73] S. Kranjc, G. Tevz, U. Kamensek, S. Vidic, M. Cemazar, and G. Sersa, “Radiosensitizing effect of electrochemotherapy in a fractionated radiation regimen in radiosensitive murine sarcoma and radioresistant adenocarcinoma tumor model,” Radiat Res, vol. 172, no. 6, pp. 677–685, 2009. [74] A. Sedlar, S. Kranjc, T. Dolinsek, M. Cemazar, A. Coer, and G. Sersa, “Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma,” BMC Cancer, vol. 13, 2013. [75] J. Cutrera, M. Torrero, K. Shiomitsu, N. Mauldin, and S. Li, “Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs.,” Methods Mol. Biol. , vol. 423, pp. 319–25, Jan. 2008. [76] J. Cutrera, G. King, P. Jones, K. Kicenuik, E. Gumpel, X. Xia, and S. Li, “Safety and efficacy of tumor-targeted interleukin 12 gene therapy in treated and non-treated, metastatic lesions.,” Curr. Gene Ther. , vol. 15, no. 1, pp. 44–54, Jan. 2015. [77] S. D. Reed, A. Fulmer, J. Buckholz, B. Zhang, J. Cutrera, K. Shiomitsu, and S. Li, “Bleomycin/interleukin-12 electrochemogenetherapy for treating naturally occurring spontaneous neoplasms in dogs,” Cancer Gene Ther, vol. 17, no. 8, pp. 571–578, 2010. [78] M. Cemazar, J. Ambrozic Avgustin, D. Pavlin, G. Sersa, A. Poli, A. Krhac Levacic, N. Tesic, U. Lampreht Tratar, M. Rak, and N. Tozon, “Efficacy and safety of electrochemotherapy combined with peritumoral IL-12 gene electrotransfer of canine mast cell tumours,” Vet Comp Oncol. vol. 15, no. 2, pp. 641-654, Jun 2017. Acknowledgement This research was funded by research grants from Slovenian Research Agency and was conducted in the scope of the EBAM European Associated Laboratory (LEA) and COST Action TD1104. Maja Čemažar received her PhD in basic medical sciences from the Medical Faculty, University of Ljubljana in 1998. She was a postdoctoral fellow and researcher at Gray Cancer Institute, UK from 1999 to 2001. She was an associate researcher at the Institute of Pharmacology and Structural Biology in Toulouse, France in 2004. Currently, she works at the Department of Experimental Oncology, Institute of Oncology Ljubljana and teaches Cell and tumor biology at various courses at the University of Ljubljana and University of Primorska, Slovenia. Her main research interests are in the field of gene electrotransfer employing plasmid DNA encoding different immunomodulatory and antiangiogenic therapeutic genes. In 2006 she received the Award of the Republic of Slovenia for important achievements in scientific research and development in the field of experimental oncology. She is the author of more than 170 articles in peer-reviewed journals. Chapter 8 Electrotransfer of DNA vaccine Véronique Préat and Gaëlle Vandermeulen University of Louvain, Brussels, Belgium DNA vaccines DNA vaccine is an attractive strategy to induce immune memory. Bacterial plasmids are constructed and optimized to express in vivo a protein that will induce an immune response. Preclinical studies have shown that plasmid DNA encoding antigens provides protection in small animals and to a lesser extend in large animals for a wide range of diseases e.g. prophylactic viral and bacterial infections as well as therapeutic cancer vaccines. Several DNA vaccines have been licensed for veterinary use or are under clinical trials for human use. DNA vaccine comprises several key elements. A promoter is inserted upstream the sequence of the antigen of interest to drive expression in mammalian cells and a polyadenylation signal is located downstream. The production of plasmid DNA requires the presence of a replication origin and of a specific marker able to select plasmid-containing bacteria after transformation and during the amplification process. The use of antibiotic resistance genes as selection markers for plasmid production raises safety concerns which are often pointed out by the regulatory authorities and a new generation of plasmid backbones devoid of antibiotic resistance marker has emerged. DNA vaccines are attractive due to their stability, low cost, easy production and ability to induce a broad immune response. Allowing the in vivo expression of the antigen by the transfected cells, DNA vaccines ensure a closer resemblance to the antigen than recombinant proteins, with mammalian glycosylation and other posttranslational modifications. They Electrotransfer of DNA vaccine 135 are able to stimulate all three arms of adaptive immunity: antibodies, helper T cells (Th) and cytotoxic T lymphocytes (CTL) and they contribute to the stimulation of innate immunity. The safety profile of DNA vaccines is generally considered as good and neither observable integration of the DNA in the host genome nor autoimmunity has been reported in human clinical trials. A number of studies demonstrated the robustness of DNA plasmid encoding pathogen and tumor antigens to elicit immune response. DNA vaccines induce a predominantly Th1 response, CTL response and antibodies but both the delivery route and the administration method have been shown to influence the type and the magnitude of the immune response. To elicit CTL responses, the antigen needs to be present in the cytoplasm of antigen presenting cells (APC). The protein is either directly produced by transfected APC or reaches them through endocytosis by APC of the protein produced by other transfected cells (cross presentation). Peptides derived from the protein degradation bind to the major histocompatibility complex (MHC) class I or class II. Peptide association to MHC class I stimulates CTL while binding to MHC class II stimulate Th cells. Although DNA vaccines were initially developed to introduce antigen to MHC class-I processing pathway to induce CTL, they have also been shown to generate protective antibody responses: a transmembrane or secreted protein can activate B cells for antibody production. In the past years, many efforts have been made to improve their immunogenicity and clinical potential relying on the use of electroporation, codon optimisation of the plasmid constructs or co-administration of adjuvants. Electroporation-mediated delivery of DNA vaccines Even if naked plasmid DNA vaccines injected in muscle can induce an immune response, a relatively low magnitude of response is usually induced in large target species. Electroporation addresses two limitations of the poor immunogenicity of DNA vaccines. (i) By inducing a transient membrane permeabilization and by promoting electrophoresis of the negatively charged DNA, it facilitates DNA uptake in the host cells. Thereby the antigen expression is strongly enhanced, usually by two orders of magnitude, in the muscle or the skin. (ii) By creating a low level of inflammation at the site of 136 Véronique Préat and Gaëlle Vandermeulen injection/electroporation, it enhances the recruitment of APC to the injection site. Consequently, electroporation-mediated delivery of DNA vaccines enhances up to100-fold the immune responses elicited compared to simple injection. It is a useful strategy to increase both humoral and cellular responses in small and large animals including primates. A survey of the preclinical studies indicates that electroporation-mediated DNA vaccination induces long-lasting and robust cellular responses characterised by the induction of CTL, interferon γ and interleukin-2 by CD4+ and CD8+ T cells. Antibodies are usually detected. Combination with adjuvant (e.g. TLR-9 stimulation by CpG or interleukin-12) enhances the potency of DNA vaccination. Two major organs have been investigated for DNA immunisation by electroporation. The skin is an immunocompetent organ with many resident APC e.g. Langerhans cells cover approximately 20% of the skin surface. It is easily accessible. Protein expression is limited to a few weeks. In contrast, the muscle induces a long term and stronger expression of the protein but contains few APC. Most of the preclinical studies indicate that a stronger humoral response is observed after intramuscular electrotransfer of the DNA than after intradermal electrotransfer. Several electroporation-mediated DNA vaccinations are currently under clinical trials as therapeutic vaccines against cancers (e.g. melanoma or prostate cancer) and chronic infectious diseases (e.g. HIV, HCV). The uncompleted data suggest that electroporation-mediated vaccination is well tolerated and improves DNA vaccine potency. Devices are also been optimized to enhance immune response and/or improve patient comfort. Recommended papers DNA vaccines [1] Li L, Petrovsky N. Molecular mechanisms for enhanced DNA vaccine immunogenicity. Expert Rev Vaccines. 2016;15(3):313-29. Review [2] Vandermeulen G, Marie C, Scherman D, Préat V. New Generation of Plasmid Backbones Devoid of Antibiotic Resistance Marker for Gene Therapy Trials. Mol Ther. 2011;19(11):1942-9. Review [3] Lopes A, Vandermeulen G, Préat V. Cancer DNA vaccines: current preclinical and clinical developments and future perspectives. J Exp Clin Cancer Res. 2019 Apr 5;38(1):146. Review Electrotransfer of DNA vaccine 137 Electroporation of DNA vaccines [4] Todorova B, Adam L, Culina S, Boisgard R, Martinon F, Cosma A, Ustav M, Kortulewski T, Le Grand R, Chapon C. Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques. Sci Rep. 2017 Jun 23;7(1):4122. [5] Sardesai NY, Weiner DB. Electroporation delivery of DNA vaccines: prospects for success. Curr Opin Immunol. 2011;23(3):421-9. Review. [6] Broderick KE, Humeau LM. Electroporation-enhanced delivery of nucleic acid vaccines. Expert Rev Vaccines. 2015 Feb;14(2):195-204. Review [7] Vandermeulen G, Staes E, Vanderhaeghen ML, Bureau MF, Scherman D, Préat V. Optimisation of intradermal DNA electrotransfer for immunisation. J Control Release. 2007;124(1-2):81-7. [8] Vandermeulen G, Vanvarenberg K, De Beuckelaer A, De Koker S, Lambricht L, Uyttenhove C, Reschner A, Vanderplasschen A, Grooten J, Préat V. The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation. Vaccine. 2015 Jun 22;33(28):3179-85. Clinical trials with DNA vaccines and electroporation [9] Lambricht L, Lopes A, Kos S, Sersa G, Préat V, Vandermeulen G. Clinical potential of electroporation for gene therapy and DNA vaccine delivery. Expert Opin Drug Deliv. 2016;13(2):295-310. [10] El-Kamary SS, Billington M, Deitz S, Colby E, Rhinehart H, Wu Y, Blackwelder W, Edelman R, Lee A, King A. Safety and tolerability of the Easy Vax™ clinical epidermal electroporation system in healthy adults. Mol Ther. 2012;20(1):214-20. [11] Yang FQ, Yu YY, Wang GQ, Chen J, Li JH, Li YQ, Rao GR, Mo GY, Luo XR, Chen GM. A pilot randomized controlled trial of dual-plasmid HBV DNA vaccine mediated by in vivo electroporation in chronic hepatitis B patients under lamivudine chemotherapy. J Viral Hepat. 2012;19(8):581-93. [12] Vasan S, Hurley A, Schlesinger SJ, Hannaman D, Gardiner DF, Dugin DP, Boente-Carrera M, Vittorino R, Caskey M, Andersen J, Huang Y, Cox JH, Tarragona-Fiol T, Gill DK, Cheeseman H, Clark L, Dally L, Smith C, Schmidt C, Park HH, Kopycinski JT, Gilmour J, Fast P, Bernard R, Ho DD. In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers. PLoS One. 2011;6(5):e19252. [13] Chudley L, McCann K, Mander A, Tjelle T, Campos-Perez J, Godeseth R, Creak A, Dobbyn J, Johnson B, Bass P, Heath C, Kerr P, Mathiesen I, Dearnaley D, Stevenson F, Ottensmeier C. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8(+) T-cell responses and increases PSA doubling time. Cancer Immunol Immunother. 2012 May 22. 138 Véronique Préat and Gaëlle Vandermeulen Optimisation of delivery methods [14] Lin F, Shen X, Kichaev G, Mendoza JM, Yang M, Armendi P, Yan J, Kobinger GP, Bello A, Khan AS, Broderick KE, Sardesai NY. Optimization of electroporation-enhanced intradermal delivery of DNA vaccine using a minimally invasive surface device. Hum Gene Ther Methods. 2012;23(3):157-68. [15] Hallengard D, Bråve A, Isaguliants M, Blomberg P, Enger J, Stout R, King A, Wahren B.A combination of intradermal jet-injection and electroporation overcomes in vivo dose restriction of DNA vaccines. Genet Vaccines Ther. 2012;10(1):5. [16] Kos S, Vanvarenberg K, Dolinsek T, Cemazar M, Jelenc J, Préat V, Sersa G, Vandermeulen G. Gene electrotransfer into skin using noninvasive multi-electrode array for vaccination and wound healing. Bioelectrochemistry. 2017 Apr;114:33-41. Chapter 9 Electrochemotherapy from bench to bedside: principles, mechanisms and applications Gregor Serša Institute of Oncology Ljubljana, Slovenia Abstract: Electrochemotherapy consists of administration of the chemotherapeutic drug followed by application of electric pulses to the tumour, in order to facilitate the drug uptake into the cells. Only two chemotherapeutics are currently used in electrochemotherapy, bleomycin, and cisplatin, which both have hampered transport through the plasma membrane without electroporation of tumours. Besides these two drugs also calcium is used and is termed calcium electroporation. Preclinical studies elaborated on the treatment parameters, route of drug administration and proved its effectiveness on several experimental tumour models. Based on the known mechanisms of action, electrochemotherapy has been successfully tested in the clinics and is now in standard treatment of cutaneous tumours and metastases. Electrochemotherapy as a platform technology is now being translated also into the treatment of bigger and deep-seated tumours. With new electrodes and new electric pulse generators, clinical trials are on-going for treatment of liver metastases and primary tumours, of pancreas, bone metastases and soft tissue sarcomas, as well as brain metastases, tumours in oesophagus or in rectum. Introduction Electrochemotherapy consists of administration of the chemotherapeutic drug followed by application of electric pulses to the tumour, in order to facilitate the drug uptake into the cells. Electrochemotherapy protocols were optimized in preclinical studies in vitro and in vivo, and basic mechanisms elucidated, such as electroporation of cells, tumour drug 140 Gregor Serša entrapment (vascular lock), vascular-disrupting effect and involvement of the immune response. Based on all these data, electrochemotherapy with bleomycin and cisplatin was promptly evaluated in clinical trials. Up today, electrochemotherapy has spread in Europe into 160 cancer centers. The timeline of electrochemotherapy development presents the milestones of its development, with the first multicentric study – ESOPE, and the first SOP, to development of new electrodes and inclusion in recommendations for treatment of tumors in different countries in Europe (Fig. 1). Several reviews electroporation technology and its applications in biomedicine and clinical practice [1–4]. Figure 1: Development and timeline of clinical electrochemotherapy. Legend: EP = electroporation; ESOPE = European Standard Operating Procedures of ECT; GET = gene electrotransfer; Tx = treatment. With permission from (4) . PRECLINICAL STUDIES In vitro studies Electroporation proved to be effective in facilitating transport of different molecules across the plasma membrane for different biochemical and pharmacological studies. However, when using chemotherapeutic drugs, this facilitated transport increases intracellular drug accumulation with the aim to increase their cytotoxicity. Since electroporation can facilitate drug transport through the cell membrane only for molecules which are poorly Electrochemotherapy from bench to bedside: principles, mechanisms and applications 141 permeant or non-permeant, suitable candidates for electrochemotherapy are limited to those drugs that are hydrophilic and/or lack a transport system in the membrane. Several chemotherapeutic drugs were tested in vitro for potential application in combination with electroporation of cells. Among the tested drugs, only two were identified as potential candidates for electrochemotherapy of cancer patients. The first is bleomycin, which is hydrophilic and has very restricted transport through the cell membrane, but its cytotoxicity can be potentiated up to several 1000 times by electroporation of cells. A few hundred internalized molecules of bleomycin are sufficient to kill the cell. The second is cisplatin, whose transport through the cell membrane is also hampered. Early studies suggested that cisplatin is transported through the plasma membrane mainly by passive diffusion, while recent studies have demonstrated that transporters controlling intracellular copper homeostasis are significantly involved in influx (Ctr1) and efflux (ATP7A and ATP7B) of cisplatin [5]. Electroporation of the plasma membrane enables greater flux and accumulation of the drug in the cells, which results in an increase of cisplatin cytotoxicity by up to 80-fold [6–8]. This promising preclinical data obtained in vitro on a number of different cell lines has paved the way for testing these two drugs in electrochemotherapy in vivo on different tumor models. Recently calcium has been demonstrated to be suitable drug for electrochemotherapy. Its cytotoxicity is enhanced and the method is called calcium electroporation [9]. In vivo studies Bleomycin and cisplatin were tested in an electrochemotherapy protocol in animal models in vivo (Fig. 2). Extensive studies in different animal models with different types of tumors, either transplantable or spontaneous, were performed [6–8,10]. In these studies, different factors controlling antitumor effectiveness were determined: The drugs can be given by different routes of administration, they can be injected either intravenously or intratumourally. The prerequisite is that, at the time of application of electric pulses to the tumour, a sufficient amount of drug is present in the tumour. Therefore, after intravenous drug administration into small laboratory animals (for example 4 mg/kg of cisplatin or 0.5 mg/kg bleomycin), only a few minutes interval is needed to reach the maximal drug concentration in the tumours. After intratumoural administration, this interval is even 142 Gregor Serša shorter and the application of electric pulses has to follow the administration of the drug as soon as possible (within a minute) [6–8]. Good antitumor effectiveness may be achieved by good tissue electroporation. Electroporation of the plasma membrane is obtained if the cell is exposed to a sufficiently high electric field. This depends on the electric field distribution in the tissue which is controlled by the electrode geometry and tissue composition. The electric field distribution in the tissue and cell electroporation can be improved by rotating the electric field. Surface tumours can be effectively treated by plate electrodes, whereas appropriate electric field distribution in the deeper parts of the tumour is assured by using needle electrodes [11– 13]. The antitumor effectiveness depends on the amplitude, number, frequency, and duration of the electric pulses applied. Several studies in which parallel plate electrodes were used for surface tumours showed that amplitude over distance ratio above 1000 V/cm is needed for tumour electroporation and that above 1500 V/cm, irreversible changes in the normal tissues adjacent to the tumour occurred. For other types of electrodes, the electric field distribution and thus, also the necessary amplitude of electric pulses, need to be determined by numerical calculations. Repetition frequencies of the pulses for electrochemotherapy are either 1 Hz or 5 kHz with equal effect if the concentration of drug present in the tumour is high enough. The minimal number of pulses used is 4; most studies use 8 electric pulses of 100 μs [12,14–16]. All the experiments conducted in vivo in animals provided sufficient data to demonstrate that electrochemotherapy with either bleomycin or cisplatin is effective in the treatment of solid tumours, using drug concentrations which have no or minimal antitumor effect without application of electric pulses. A single treatment by electrochemotherapy already induces partial or complete regression of tumours, whereas treatment with bleomycin or cisplatin alone or application of electric pulses alone has no or minimal antitumour effect. Electrochemotherapy from bench to bedside: principles, mechanisms and applications 143 Figure 2: Protocol of electrochemotherapy of experimental tumours presented schematically. The drug is injected either intravenously or intratumourally at doses which do not usually exert an antitumour effect. After an interval which allows sufficient drug accumulation in the tumours, electric pulses are applied to the tumour either by plate or needle electrodes. The electrodes are placed in such a way that the whole tumour is encompassed between the electrodes, providing good electric field distribution in the tumours for optimal electroporation of cells in the tumours. Mechanisms of action The principal mechanism of electrochemotherapy is electroporation of cells in the tumours, which increases the drug effectiveness by enabling the drug to reach the intracellular target. This was demonstrated in studies which measured the intratumoural drug accumulation and the amount of drug bound to DNA. Basically, the amounts of bleomycin and cisplatin in the electroporated tumours were up to 2-4 fold higher than in those without application of electric pulses [6–8]. Besides membrane electroporation, which facilitates drug transport and its accumulation in the cell, other mechanisms that are involved in the antitumor effectiveness of electrochemotherapy were described. The application of electric pulses to tissues induces a transient, but reversible reduction of blood flow [17,18]. Restoration of the blood flow in normal tissue is much faster than that in tumours [19,20]. The vascular lock in the tumour induces drug entrapment in the tissue, providing more time for the drug to act. The cytotoxic effect of electrochemotherapy is not limited only to tumour cells in the tumours. Electrochemotherapy also acts on stromal cells, including endothelial cells in the lining of tumour blood vessels, which 144 Gregor Serša undergo cell death [20]. Consequently, by vascular-disrupting action of electrochemotherapy, a cascade of tumour cell death occurs due to long-lasting hypoxia in the affected vessels. This represents yet another mechanism involved in the antitumour effectiveness of electrochemotherapy, i.e. a vascular-disrupting effect [21–23]. This vascular-disrupting action of electrochemotherapy is important in clinical situations where haemorrhagic tumour nodules need to be treated [24]. A difference in the antitumor effectiveness of electrochemotherapy was observed between immunocompetent and immunodeficient experimental animals, indicating on involvement of the immune response in antitumour effectiveness [25]. Due to massive tumour antigen shedding in organisms after electrochemotherapy, systemic immunity can be induced and also upregulated by additional treatment with biological response modifiers like IL-2, IL-12, GM-CSF and TNF-α [25–30]. To sum up, the electrochemotherapy protocol was optimized in preclinical studies in vitro and in vivo, and basic mechanisms were elucidated. In addition to the electroporation of cells, vascular lock leading to drug entrapment in tumours, a vascular-disrupting effect and involvement of the immune response were also demonstrated. Based on all this data, electrochemotherapy with bleomycin and cisplatin was promptly evaluated in clinical trials and is now in routine use in human and veterinary oncology. Electrochemotherapy from bench to bedside: principles, mechanisms and applications 145 Figure 3: Basic mechanisms of electrochemotherapy; direct cytotoxic effect and indirect ones, vascular disrupting and immunomodulating. With permission from [3]. 146 Gregor Serša Clinical studies The first clinical study was published in 1991 on head and neck tumour nodules [31], which was thereafter followed by several others [2]. These clinical studies demonstrated the antitumor effectiveness of electrochemotherapy using either bleomycin or cisplatin, given intravenously or intratumourally. In addition to single or multiple cutaneous or subcutaneous melanoma nodules, a response was demonstrated in breast and head and neck cancer nodules, as well as Kaposi’s sarcoma, hypernephroma, chondrosarcoma and basal cell carcinoma. However, these clinical studies were performed with slightly variable treatment protocols, different electrodes and different electric pulse generators. Thus, there was a need for a prospective multi-institutional study, which was conducted by a consortium of four cancer centers gathered in the ESOPE project funded under the European Commission's 5th Framework Programme. In this study, the treatment response after electrochemotherapy according to tumour type, drug used, route of administration and type of electrodes, was tested [32]. The results of this study can be summarized as follows: • An objective response rate of 85% (73.7% complete response rate) was achieved for electrochemotherapy-treated tumour nodules, regardless of tumour histology and drug or route of administration used. • At 150 days after treatment, the local tumour control rate for electrochemotherapy was 88% with bleomycin given intravenously, 73% with bleomycin given intratumourally and 75% with cisplatin given intratumourally, demonstrating that all three approaches were equally effective in local tumour control. • Side effects of electrochemotherapy were minor and tolerable (muscle contractions and pain sensation). The results of the ESOPE study confirmed previously reported results on the effectiveness of electrochemotherapy and Standard Operating Procedures (SOP) for electrochemotherapy were prepared [33]. The ESOPE study set the stage for the introduction of electrochemotherapy in Europe. After the encouraging results of the ESOPE study, several cancer centers have started to use electrochemotherapy and reported the results of their studies. Collectively, the results were again similar as reported in the ESOPE study. However, some advances in the treatment were reported. Predominantly it was reported that tumours bigger than 3 cm in diameter can be successfully treated by electrochemotherapy in successive electrochemotherapy sessions Electrochemotherapy from bench to bedside: principles, mechanisms and applications 147 [34,35]. In general, electrochemotherapy provides a benefit to patients especially in quality of life [35]. Clinical use and treatment procedures for electrochemotherapy Based on all these reports, electrochemotherapy has been recognized as a treatment option for disseminated cutaneous disease in melanoma, and accepted in many national and also international guidelines for treatment of melanoma [36]. Treatment advantages and clinical use for electrochemotherapy can be summarized as follows: Effective in treatment of tumours of different histology in the cutaneous or subcutaneous tissue. Palliative treatment with improvement of patient’s quality of life. Treatment of choice for tumours refractory to conventional treatments. Cytoreductive treatment before surgical resection in an organ sparing effect. Treatment of bleeding metastases. The treatment after a single electrochemotherapy session in most cases results in complete tumour eradication. When necessary, treatment can be repeated at 4-8 week intervals with equal antitumor effectiveness. The treatment has a good cosmetic effect without scarring of the treated tissue. In summary, electrochemotherapy has been recognized as a valid treatment approach; over 160 cancer centers have started to use it and have reported positive results. So far the effectiveness of the therapy is on case based evidence and further controlled and randomized studies are needed for the translation of this technology into broader and standard clinical practice. For further acceptance of electrochemotherapy in medical community, the first important step has been made, since electrochemotherapy for treatment of melanoma skin metastases and for treatment of primary basal cell and primary squamous cell carcinoma was recently listed in NICE guidelines. Recently all published studies up to 2012 on electrochemotherapy in treatment of superficial nodules were reviewed in systematic review and meta-analysis [37]. Data analysis confirmed that electrochemotherapy had 148 Gregor Serša a significantly (p<0.001) higher effectiveness (by more than 50%) than bleomycin or cisplatin alone, where only 8% of the tumours were in CR. After a single electrochemotherapy, the treatment can be repeated with similar effectiveness. The overall effectiveness of electrochemotherapy was 84.1% objective responses (OR), from these 59.4% complete responses (CR). Based on broad acceptance of electrochemotherapy as effective local ablative technique, the group of experts that has been involved in preparation of the first SOP has prepared an updated version of SOP [38]. Described are indications, potential side effects and procedures for safe and effective execution of electrochemotherapy. The choice of the drug administration and anesthesia depends on the size and the number of tumour nodules to be treated (Fig. 4). Figure 4: Decision on treatment strategy based on number and size of the tumors to be treated. With permission from [38]. New clinical applications of electrochemotherapy Based on clinical experience that electrochemotherapy can be effectively used in treatment of cancer with different histology, when appropriately executed, the treatment could be used also for treatment of deep seated tumours. Prerequisite for that is further development of the technology in order to reach and effectively treat the tumours located either in the muscle, liver, bone, oesophagus, rectum, brain or other internal organs. The first reports have already been published in treatment of colorectal liver metastases (Fig. 5) [39], hepatocellular carcinoma [40], pancreatic Electrochemotherapy from bench to bedside: principles, mechanisms and applications 149 tumours, bone metastases, colorectal tumours [4]. These approaches have been undertaken during open surgery, however the future directions are in percutaneous treatment. Some attempts have been already published as case reports [41–43]. New major clinical indication in treatment is head and neck tumours. In this indication is the highest number of recently treated tumours [44,45]. Electrochemotherapy is also gaining importance in treatment of basal cell carcinoma, where the highest (>90%) complete responses are obtained [44]. The future of electrochemotherapy is also in the combined treatment with immunotherapy. Electrochemotherapy induces immunological cell death, that can serve as in situ immunization for the combination with immune checkpoint inhibitors. This concept is already being verified in the clinics [26,46]. Another approach is also to combine it with gene therapy, for instance with gene electrotransfer of plasmid coding for IL12 [47]. This concept has already been tested pre-clinical, but awaits verification in human oncology as well. Figure 5: Electrochemotherapy of liver metastasis. Electrodes were inserted into the tumour and around the tumour in healthy liver tissue and connected to the electric pulse generator. Electric pulses were delivered between the pairs of electrodes according to the treatment plan. Conclusion Electrochemotherapy is one of the biomedical applications of electroporation. Its development has reached clinical application and is an example of successful translational medicine. However, its development is 150 Gregor Serša not finished yet; new technical developments will certainly enable further clinical uses and eventually clinical benefit for the patients. Another application of electroporation is still awaiting such translation, gene therapy based on gene electrotransfer. References [1] Yarmush ML, Golberg A, Serša G, Kotnik T, Miklavčič D. Electroporation-Based Technologies for Medicine: Principles, Applications, and Challenges. Annu Rev Biomed Eng. 2014;16(1):295–320. [2] Miklavcic D, Mali B, Kos B, Heller R, Sersa G. Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online. 2014;13(1):29. [3] Campana LG, Miklavčič D, Bertino G, Marconato R, Valpione S, Imarisio I, et al. Electrochemotherapy of superficial tumors - Current status:: Basic principles, operating procedures, shared indications, and emerging applications. Semin Oncol. 2019 Apr;46(2):173–91. [4] Campana LG, Edhemovic I, Soden D, Perrone AM, Scarpa M, Campanacci L, et al. Electrochemotherapy – Emerging applications technical advances, new indications, combined approaches, and multi-institutional collaboration. Eur J Surg Oncol. 2019 Feb;45(2):92–102. [5] Howell SB, Safaei R, Larson CA, Sailor MJ. Copper Transporters and the Cellular Pharmacology of the Platinum-Containing Cancer Drugs. Mol Pharmacol. 2010 Jun;77(6):887–94. [6] Mir LM. Therapeutic perspectives of in vivo cell electropermeabilization. Bioelectrochemistry. 2001 Jan;53(1):1–10. [7] Gehl J. Electroporation: Theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand. 2003;177(4):437–47. [8] Mir LM. Bases and rationale of the electrochemotherapy. Eur J Cancer Suppl. 2006 Nov 1;4(11):38–44. [9] Frandsen SK, Gissel H, Hojman P, Tramm T, Eriksen J, Gehl J. Direct Therapeutic Applications of Calcium Electroporation to Effectively Induce Tumor Necrosis. Cancer Res. 2012 Mar 15;72(6):1336–41. [10] Vásquez JL, Ibsen P, Lindberg H, Gehl J. In vitro and in vivo experiments on electrochemotherapy for bladder cancer. J Urol. 2015 Mar;193(3):1009–15. [11] Miklavcic D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G, et al. The importance of electric field distribution for effective in vivo electroporation of tissues. Biophys J. 1998;74(5):2152–8. [12] Miklavcic D, Corovic S, Pucihar G, Pavselj N. Importance of tumour coverage by sufficiently high local electric field for effective electrochemotherapy. Eur J Cancer, Suppl. 2006;4(11):45–51. [13] Corovic S, Al Sakere B, Haddad V, Miklavcic D, Mir LM. Importance of contact surface between electrodes and treated tissue in electrochemotherapy. Technol Cancer Res Treat. 2008;7(5):393–400. [14] Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M. Electrochemotherapy Electrochemotherapy from bench to bedside: principles, mechanisms and applications 151 in treatment of tumours. Eur J Surg Oncol. 2008;34(2):232–40. [15] Miklavčič D, Pucihar G, Pavlovec M, Ribarič S, Mali M, MačEk-Lebar A, et al. The effect of high frequency electric pulses on muscle contractions and antitumor efficiency in vivo for a potential use in clinical electrochemotherapy. Bioelectrochemistry. 2005;65(2):121–8. [16] Sersa G, Kranjc S, Scancar J, Krzan M, Cemazar M. Electrochemotherapy of mouse sarcoma tumors using electric pulse trains with repetition frequencies of 1 Hz and 5 kHz. J Membr Biol. 2010;236(1):155–62. [17] Sersa G, Cemazar M, Parkins CS, Chaplin DJ. Tumour blood flow changes induced by application of electric pulses. Eur J Cancer. 1999;35(4):672–7. [18] Bellard E, Markelc B, Pelofy S, Le Guerroué F, Sersa G, Teissié J, et al. Intravital microscopy at the single vessel level brings new insights of vascular modification mechanisms induced by electropermeabilization. J Control Release. 2012 Nov 10;163(3):396–403. [19] Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochim Biophys Acta - Gen Subj. 2002 Jan;1569(1–3):51–8. [20] Cemazar M, Parkins CS, Holder AL, Chaplin DJ, Tozer GM, Sersa G. Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy. Br J Cancer. 2001 Feb 15;84(4):565–70. [21] Jarm T, Cemazar M, Miklavcic D, Sersa G. Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases. Expert Rev Anticancer Ther. 2010;10(5):729–46. [22] Sersa G, Jarm T, Kotnik T, Coer a, Podkrajsek M, Sentjurc M, et al. Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma. Br J Cancer. 2008;98(2):388–98. [23] Markelc B, Bellard E, Sersa G, Pelofy S, Teissie J, Coer A, et al. In vivo molecular imaging and histological analysis of changes induced by electric pulses used for plasmid DNA electrotransfer to the skin: a study in a dorsal window chamber in mice. J Membr Biol. 2012 Sep 27;245(9):545–54. [24] Gehl J, Geertsen PF. Palliation of haemorrhaging and ulcerated cutaneous tumours using electrochemotherapy. Eur J Cancer Suppl. 2006 Nov 1;4(11):35–7. [25] Serša G, Miklavčič D, Čemažar M, Belehradek J, Jarm T, Mir LM. Electrochemotherapy with CDDP on LPB sarcoma: comparison of the anti-tumor effectiveness in immunocompetent and immunodeficient mice. Bioelectrochemistry Bioenerg. 1997;43:279–83. [26] Calvet CY, Mir LM. The promising alliance of anti-cancer electrochemotherapy with immunotherapy [Internet]. Vol. 35, Cancer metastasis reviews. Springer; 2016 [cited 2016 May 19]. p. 165–77. [27] Sersa G, Cemazar M, Menart V, Gaberc-Porekar V, Miklavčič D. Antitumor effectiveness of electrochemotherapy is increased by TNF-a on SA-1 tumors in mice. Cancer Lett. 1997;116:85–92. [28] Mir LM, Roth C, Orlowski S, Quintin-Colonna F, Fradelizi D, Belehradek J, et al. Systemic antitumor effects of electrochemotherapy combined with histoincompatible cells secreting interleukin-2. J Immunother. 1995 Jan;17(1):30–8. [29] Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, Heller R. In vivo electroporation of 152 Gregor Serša plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity. Melanoma Res. 2000;10(6):577–83. [30] Cemazar M, Todorovic V, Scancar J, Lampreht U, Stimac M, Kamensek U, et al. Adjuvant TNF-α therapy to electrochemotherapy with intravenous cisplatin in murine sarcoma exerts synergistic antitumor effectiveness. Radiol Oncol. 2015 Mar;49(1):32– 40. [31] Mir LM, Belehradek M, Domenge C, Orlowski S, Poddevin B, Belehradek J, et al. [Electrochemotherapy, a new antitumor treatment: first clinical trial]. C R Acad Sci III. 1991;313(13):613–8. [32] Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy - An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer, Suppl. 2006;4(11):3–13. [33] Mir LM, Gehl J, Sersa G, Collins CG, Garbay JR, Billard V, et al. Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes. Eur J Cancer, Suppl. 2006;4(11):14–25. [34] Campana LG, Mocellin S, Basso M, Puccetti O, De Salvo GL, Chiarion-Sileni V, et al. Bleomycin-based electrochemotherapy: clinical outcome from a single institution’s experience with 52 patients. Ann Surg Oncol. 2009;16(1):191–9. [35] Quaglino P, Mortera C, Osella-Abate S, Barberis M, Illengo M, Rissone M, et al. Electrochemotherapy with intravenous bleomycin in the local treatment of skin melanoma metastases. Ann Surg Oncol. 2008;15(8):2215–22. [36] Testori a., Rutkowski P, Marsden J, Bastholt L, Chiarion-Sileni V, Hauschild a., et al. Surgery and radiotherapy in the treatment of cutaneous melanoma. Ann Oncol. 2009;20(SUPPL. 4):22–9. [37] Mali B, Jarm T, Snoj M, Sersa G, Miklavcic D. Antitumor effectiveness of electrochemotherapy: a systematic review and meta-analysis. Eur J Surg Oncol. 2013 Jan;39(1):4–16. [38] Gehl J, Sersa G, Matthiessen LW, Muir T, Soden D, Occhini A, et al. Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases. Acta Oncol (Madr). 2018 Jul 3;57(7):874–82. [39] Edhemovic I, Brecelj E, Gasljevic G, Marolt Music M, Gorjup V, Mali B, et al. Intraoperative electrochemotherapy of colorectal liver metastases. 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Cardiovasc Intervent Radiol. 2019 Aug 22; [44] Bertino G, Sersa G, De Terlizzi F, Occhini A, Plaschke CC, Groselj A, et al. European Research on Electrochemotherapy in Head and Neck Cancer (EURECA) project: Results of the treatment of skin cancer. Eur J Cancer. 2016 Aug;63:41–52. [45] Plaschke CC, Bertino G, McCaul JA, Grau JJ, de Bree R, Sersa G, et al. European Research on Electrochemotherapy in Head and Neck Cancer (EURECA) project: Results from the treatment of mucosal cancers. Eur J Cancer. 2017 Dec;87:172–81. [46] Sersa G, Teissie J, Cemazar M, Signori E, Kamensek U, Marshall G, et al. Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer. Cancer Immunol Immunother. 2015 Oct;64(10):1315–27. [47] Milevoj N, Tratar UL, Nemec A, Brožič A, Žnidar K, Serša G, et al. A combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine IL-12 and cytoreductive surgery in the treatment of canine oral malignant melanoma. Res Vet Sci. 2019 Feb;122:40–9. Acknowledgement This research was funded by a research grant from the Research Agency of the Republic of Slovenia and was conducted in the scope of the EBAM European Associated Laboratory (LEA) and resulted from the networking efforts of the COST Action TD1104 (www.electroporation.net). Gregor Sersa, graduated from the Biotechnical Faculty at the University of Ljubljana in 1978, where he is currently a professor of molecular biology. He is employed at the Institute of Oncology Ljubljana as Head of the Department of Experimental Oncology. His specific field of interest is the effect of electric field on tumor cells and tumors as drug and gene delivery system in different therapeutic approaches. Besides experimental work, he is actively involved in the education of undergraduate and postgraduate students at the University of Ljubljana. Chapter 10 Electrochemotherapy in clinical practice; Lessons from development and implementation - and future perspectives Julie Gehl Clinical Oncology at the University of Copenhagen, Denmark. Abstract: In just two decades electrochemotherapy has developed from an experimental treatment to standard therapy. This paper describes this development and also goes into the details of how a new technology can become implemented, to benefit patients. Electrochemotherapy is a technology that involves the use of electric pulses and chemotherapy. Thus the development of this technology has required specialists in biology, engineering and medicine to pull together, in order to achieve this accomplishment. This paper describes the development of equipment, as well as standard operating procedures, for treatment with electrochemotherapy. This chapter also deals with sharing knowledge about the use of the technology, and ensuring access for patients. Development of electrochemotherapy Initial studies on the organization of the cell membrane, and on deformation of this membrane by electric forces, were performed through the particularly the 1960s and 70s. In 1977 rupture of erythrocytes was described in a Nature paper [1], and another highly influential paper was Neumanns study from 1982 [2], demonstrating DNA electrotransfer which is now one of the most frequently used laboratory methods in molecular biology. A very active field in cancer therapy in the 1970s and 80s was resistance to drug therapy, and there was great optimism that understanding resistance Electrochemotherapy in clinical practice; Lessons from development and implementation - and future perspectives 155 to therapy could ultimately lead to a cure for cancer. Different important cellular resistance systems were discovered, e.g. the multidrug transporter p-glycoprotein, that enables cancer cells to export chemotherapy [3]. In this landscape electroporation was a new technology that allowed circumvention of membrane based resistance by simply plowing a channel through cell membrane, allowing non-permeant drugs inside. A number of studies were published about enhancement of cytotoxicity by electroporation [4,5] in vitro, and also in vivo [6], principally from Lluis Mir’s group at Institut Gustave-Roussy. It was also here that, in a remarkable short time-frame, the first clinical study was reported, preliminary results in French in 1991, and the final publication in 1993 [7]. A few years later [8], the first studies from the US came out, as well as studies from Slovenia [9], and Denmark [10]. Out of a wish to create electroporation equipment for clinical use, which would be able to perform both gene therapy and electrochemotherapy, which could be adapted by the user to accommodate developments, and which was a useful instrument for the treating physician, i.e. by showing precise recordings of voltage and current along with the treatment, the Cliniporator consortium was formed. This European consortium developed and tested the Cliniporator [11,12]. A subsequent European consortium, named ESOPE (European Standard Operating Procedures for Electrochemotherapy) set out to get the Cliniporator approved for clinical use, to produce electrodes for it, to test the system in a clinical protocol, as well as to make concluding standard operating procedures. Four groups went into the clinical study of which three had previous experience with electrochemotherapy. And the methods used differed between those three centers. In France, a hexagonal electrode was used, with 7.9 mm between electrodes and a firing sequence allowing each of seven electrodes to be pairwise activated 8 times, a total of 96 pulses delivered at high frequency, with a voltage of 1.3 kV/cm (voltage to electrode distance ratio). Patients were sedated, bleomycin was given iv, and the procedure took place in an operating theatre [7]. In the Slovenian studies, patients were treated with cisplatin intratumorally, and with plate electrodes using 1.3 kV/cm, anesthesia not described. Pulses were administered as two trains of each four pulses [9]. In Denmark we used intratumoral bleomycin, a linear array electrode of two opposing rows of needles activated against each other using 1.2 kV/cm, 8 pulses at 1 Hz. Local anesthesia with lidocaine was used [10]. 155 156 Julie Gehl In other words, there was agreement about the overall purpose, but three different approaches. The ESOPE study [13] brought these three approaches together, and on the technical side, the three different electrodes were manufactured, and the final conclusion of the different methods and electrodes were defined in collaboration. The standard operating procedures [14] are very detailed, allowing a newcomer to the field to immediately implement the procedure. Thus it is described how to administer the drug and pulses, how to make treatment decisions, and how to evaluate response and perform follow-up. The standard operating procedures, together with the availability of certified equipment, marked a dramatic change in the use of electrochemotherapy. Thus when the standard operating procedures were published in 2006 only few European centers were active, and after the publication of the procedures the number of centers quickly rose and is today over 140. It would be estimated that this number will continue to grow, and also that the generators now being placed in various institutions will be increasingly used also for new indications. Implementation In an ideal world, new developments in cancer therapy become immediately available to patients. But experience shows that from the development of the technology, and the emergence of the first results, there is still quite a road to be traveled in order for the individual patient to be able to be referred, if the treatment is relevant to the particular case. First of all, equipment must be present at the individual institution, along with knowledgeable surgeons and oncologists trained to provide the treatment. The logistical set up must be in place, and this includes availability of time in the operating rooms and competent nursing support. Patients need to know that the treatment is an option. As electrochemotherapy is an option for patients suffering from different types of cancer, it requires continuous work to address specialists in the different fields. Information available on the internet can be an important resource for patients, as well as professionals. Various countries have different approval mechanisms for new treatments, and endorsement can be a time-consuming affair. The most renowned national agency is the National Institute of Health and Care Excellence (NICE) in the UK, which has a rigorous scrutinization of new technologies and where central documents are freely available. NICE has guidances for electrochemotherapy for cutaneous metastases, and primary Electrochemotherapy in clinical practice; Lessons from development and implementation - and future perspectives 157 skin cancers respectively [15,16]. These national recommendations, as well as the integration of electrochemotherapy into specific guidelines (see e.g. [17]) are very important for the improving accessibility to treatment. Research A very important point is that the standard operating procedures were a very important foundation – but must be followed up with more detailed experience and further developments. Several groups have published further studies on electrochemotherapy, broadening the knowledge base and answering specific questions of clinical importance [18-26]. Furthermore, electrochemotherapy is now being developed for a number of new indications, including mucosal head and neck cancer, gastro-intestinal cancers, lung cancer (primary and secondary), gynecological cancers, sarcoma, bone metastases, as well as brain metastases. For each of these indications standard operating procedures will need to be developed, in order to allow dissemination of the treatment. References [1] Kinosita K, Tsong TY. Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature 1977;268:438-41. [2] 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(7):841-45. [3] Skovsgaard T, Nissen NI. Membrane transport of anthracyclines. Pharmacol Ther 1982;18(3):293-311. [4] Okino M, Mohri H. Effects of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. JpnJCancer Res 1987;78(0910-5050 SB - M SB - X):1319-21. [5] Orlowski S, Belehradek Jr J, Paoletti C, Mir LM. Transient electropermeabilization of cells in culture. Increase of the cytotoxicity of anticancer drugs. Biochemical Pharmacology 1988;37(24):4727-33. [6] Mir LM, Orlowski S, Belehradek J, Jr., Paoletti C. Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses. Eur J Cancer 1991;27(1):68-72. [7] Belehradek M, Domenge C, Luboinski B, Orlowski S, Belehradek Jr J, Mir LM. Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial. Cancer 1993;72(12):3694-700. [8] Heller R. Treatment of cutaneous nodules using electrochemotherapy. [Review] [32 refs]. Journal of the Florida Medical Association 1995;82(2):147-50. 157 158 Julie Gehl [9] Sersa G, Stabuc B, Cemazar M, Jancar B, Miklavcic D, Rudolf Z. Electrochemotherapy with cisplatin: Potentiation of local cisplatin antitumor effectiveness by application of electric pulses in cancer patients. European Journal of Cancer 1998;34(8):1213-18. [10] Gehl J, Geertsen PF. Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy. Melanoma Res 2000;10(6):585-9. [11] Andre FM, Gehl J, Sersa G, Preat V, Hojman P, Eriksen J, et al. Efficiency of High-and Low-Voltage Pulse Combinations for Gene Electrotransfer in Muscle, Liver, Tumor, and Skin. Human Gene Therapy 2008;19(11):1261-71. [12] Hojman P, Gissel H, Andre F, Cournil-Henrionnet C, Eriksen J, Gehl J, et al. Physiological effect of high and low voltage pulse combinations for gene electrotransfer in muscle. HumGene Ther 2008(1557-7422 (Electronic)). [13] Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M, et al. Electrochemotherapy - An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Ejc Supplements 2006;4(11):3-13. [14] Mir LM, Gehl J, Sersa G, Collins CG, Garbay JR, Billard V, et al. Standard operating procedures of the electrochemotherapy: Instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes. European Journal of Cancer Supplements 2006;4(11):14-25. [15] National Institute for H, Care E. Electrochemotherapy for metastases in the skin from tumours of non-skin origin and melanoma. http://publicationsniceorguk/electrochemotherapy-for-metastases-in-the-skin-from-tumours-of-non-skin-origin-and-melanoma-ipg446 2013. [16] (NICE) NIfHaCE. Electrochemotherapy for primary basal cell carcinoma and primary squamous cell carcinoma. www.nice.org.uk2014. [17] Stratigos A, Garbe C, Lebbe C, Malvehy J, Del Marmol V, Pehamberger H, et al. Diagnosis and treatment of invasive squamous cell carcinoma of the skin: European consensus-based interdisciplinary guideline. Eur J Cancer 2015;51(14):1989-2007. [18] Matthiessen LW, Chalmers RL, Sainsbury DC, Veeramani S, Kessell G, Humphreys AC, et al. Management of cutaneous metastases using electrochemotherapy. Acta Oncol 2011;50:621-29. [19] Matthiessen LW, Johannesen HH, Hendel HW, Moss T, Kamby C, Gehl J. Electrochemotherapy for large cutaneous recurrence of breast cancer: A phase II clinical trial. Acta Oncologica 2012;51(6):713-21. [20] Campana LG, Valpione S, Falci C, Mocellin S, Basso M, Corti L, et al. The activity and safety of electrochemotherapy in persistent chest wall recurrence from breast cancer after mastectomy: a phase-II study. Breast Cancer ResTreat 2012;134:1169-78. [21] Campana LG, Bianchi G, Mocellin S, Valpione S, Campanacci L, Brunello A, et al. Electrochemotherapy treatment of locally advanced and metastatic soft tissue sarcomas: results of a non-comparative phase II study. World JSurg 2014;38:813-22. [22] Campana LG, Mali B, Sersa G, Valpione S, Giorgi CA, Strojan P, et al. Electrochemotherapy in non-melanoma head and neck cancers: a retrospective analysis of the treated cases. BrJOral MaxillofacSurg 2014. [23] Curatolo P, Mancini M, Clerico R, Ruggiero A, Frascione P, Di Marco P, et al. Remission of extensive merkel cell carcinoma after electrochemotherapy. Arch Dermatol 2009;145(4):494-5. Electrochemotherapy in clinical practice; Lessons from development and implementation - and future perspectives 159 [24] Curatolo P, Quaglino P, Marenco F, Mancini M, Nardo T, Mortera C, et al. Electrochemotherapy in the treatment of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial. Ann Surg Oncol 2012;19(1):192-8. [25] Quaglino P, Mortera C, Osella-Abate S, Barberis M, Illengo M, Rissone M, et al. Electrochemotherapy with intravenous bleomycin in the local treatment of skin melanoma metastases. AnnSurgOncol 2008;15:2215-22. [26] Quaglino P, Matthiessen LW, Curatolo P, Muir T, Bertino G, Kunte C, et al. Predicting patients at risk for pain associated with electrochemotherapy. Acta Oncol 2015;54(3):298-306. Julie Gehl heads the Center for Experimental drug and gene Electrotransfer at Department of Oncology, Herlev Hospital at the University of Copenhagen. The center undertakes both preclinical and clinical investigations of the use of electrotransfer in drug and gene delivery. Julie Gehl is an MD, and specialist in Oncology. Dr. Gehl has an extensive publication record, is an experienced principal investigator and has guided numerous ph.d. students and students. 159 Chapter 11 Development of devices and electrodes Damijan Miklavčič, Matej Reberšek University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Abstract: Since first reports on electroporation, numerous electroporation based biotechnological and biomedical applications have emerged. The necessary pulse generators are characterized by the shape of the pulses and their characteristics: pulse amplitude and duration. In addition, the electrodes are the important “connection” between the cells/tissue and pulse generator. The geometry of the electrodes together with the cell/tissue sample properties determine the necessary output power and energy that the electroporators need to provide. The choice of electroporator – the pulse generator depends on biotechnological and biomedical application but is inherently linked also to the electrodes choice. Introduction Since first reports on electroporation (both irreversible and reversible), a number of applications has been developed and list of applications which are based on electroporation is constantly increasing. First pulse generators have been simple in construction and have provided an exponentially decaying pulse of up to several thousands of volts. Also the electrodes were very simple in their design – usually parallel plate electrodes with couple of millimeters distance between them was used, and cells in suspension were placed in-between [1]. Later, new pulse generators were developed which were/are able to provide almost every shape of pulse, and also electrodes which can be bought are extremely diverse [2–6]. It is important to note that most often nowadays devices that generate rectangular pulses are being used. Development of devices and electrodes 161 The amplitude of pulses and their duration depend strongly on biotechnological/biomedical application. For electrochemotherapy most often a number of 1000 V pulses of 100 μs duration are needed. For effective gene transfer longer pulses 5-20 ms pulses but of lower amplitude, or a combination of short high- and longer low-voltage pulses are used. For other applications like tissue ablation by means of irreversible electroporation, or liquid-food or water sterilization, thousands of volts pulses are needed. In addition to the pulse amplitude and duration, an important parameter to be taken into account is also the power and energy that need to be provided by the generator. The energy that needs to be provided is governed by the voltage, current and pulse duration and/or number of pulses. The current if the voltage is set is governed by the load, and this is determined by the geometry of the load, and the load is determined by geometry of the tissue/cell sample and its electrical conductivity. The geometry of the tissue to be exposed to electric pulses are predominantly determined by the shape of the electrodes, the distance between them, depth of electrode penetration/immersion into the sample. Tissue/cell suspension electrical conductivity depends on tissue type or cell sample properties and can be considerably increased while tissue/cells are being exposed to electrical pulses of sufficient amplitude. Based on the above considerations not a single pulse generator will fit all applications and all needs of a researcher [7]. One can either seek for a specialized pulse generator which will only provide the pulses for this specific biotechnological or biomedical application, or for a general purpose pulse generator which will allow to generate “almost” all what researcher may find interesting in his/her research. Irrespective of the choice, it has to be linked also to the electrodes choice [8–10]. Therapeutic and technological applications of electroporation Nowadays electroporation is widely used in various biological, medical, and biotechnological applications [11–16]. Tissue ablation relying on irreversible electroporation is less than a decade old, but its efficacy is promising especially in treating non-malignant tissue, in the field of water treatment where efficacy of chemical treatment is enhanced with electroporation, in food preservation where electroporation has proven, in some cases, to be as effective as pasteurization [17]. In contrast, applications based on reversible electroporation are currently more widespread and established in different experimental and/or practical 162 Damijan Miklavčič, Matej Reberšek protocols. Probably the most important of them is the introduction of definite amount of small or large molecules to cytoplasm through the plasma membrane. Furthermore, slight variation of electric field parameters results in an application where molecules can be directly inserted into the plasma membrane or cells can be effectively fused. Figure 1: Exposure of a cell to an electric field may result either in permeabilization of cell membrane or its destruction. In this process the electric field parameters play a major role. If these parameters are within certain range, the permeabilization is reversible; therefore it can be used in applications such as introduction of small or large molecules into the cytoplasm, insertion of proteins into cell membrane or cell fusion. Electrochemotherapy The most representative application of delivery of small molecules through electroporated membrane is electrochemotherapy. It was demonstrated in several preclinical and clinical studies, both on humans and animals, that electrochemotherapy can be used as treatment of choice in local cancer treatment [18]. Most often a number of short rectangular 100 μs long pulses with amplitudes up to 1000 V, are applied. Number of pulses that are usualy delivered is 8. These can be delivered at pulse repetition frequency of 1 Hz or 5 kHz [19]. New technological developments were made available for in treating deep seated tumours, where 3000 V, 50 A and 100 μs pulses are being delivered [20]. Recent advances in treating liver metastasis, bone Development of devices and electrodes 163 metastasis and soft tissue sarcoma have been reported [20–23]. Recently it was shown that in vitro electrochemotherapy is possible with bipolar high-frequency electroporation pulses that are 1-1-1-1 μs long (positive pulse – pause - negative pulse - pause) and have 2.5 times higher amplitude than 100 μs pulses [23]. Tissue Ablation by Non-thermal IREversible electroporation The ablation of undesirable tissue through the use of irreversible electroporation has recently been suggested as a minimally invasive method for tumor removal but could also be used in cardiac tissue ablation instead of RF heating tissue ablation or other tissue ablation techniques [12], [24], [25]. Similarly as in electrochemotherapy pulses of 50 or 100 μs with amplitudes up to 3000 V are used [26]. The number of pulses delivered to the target tissue is however considerably higher. If in electrochemotherapy 8 pulses are delivered, here 90 or more pulses are used. Pulse repetition frequency needs to be low 1 or 4 Hz in order to avoid excessive heating [27]. To avoid muscle contraction during IRE it was suggested to use high-frequency electroporation pulses H-FIRE [28]. The duration of H-FIRE pulses is around 1 μs and around 2 times higher amplitudes are used than for 50 or 100 μs pulses. Gene ElectroTransfer Exogenous genetic material can be delivered to cells by using non-viral methods such as electropermeabilization [29]. Electrotransfection can be achieved using: exponentially decaying pulses; square wave pulses with superimposed RF signals; or only long square wave pulses up 20 ms and with amplitudes ranging from 200 to 400 V [30]. Although no consensus is reached yet, it can however be stated that longer pulses are generally used in gene transfection than in electrochemotherapy with few exceptions [31]. Furthermore, two distinct roles of electric pulses were described. In experiments where several short high voltage pulses (e.g. 8 × 100 μs of 1000 V) were followed by long low voltage pulses (e.g. 1 × 100 ms of 80 V) [32]. It was demonstrated that short high voltage pulses are permeabilizing the membrane while the longer lower voltage pulses have an electrophoretic effect on DNA itself facilitating interaction of plasmid with the membrane [33]. 164 Damijan Miklavčič, Matej Reberšek Electrofusion So far we have presented applications of electroporation that are used to introduce different molecules either to the cytosol or to the cell plasma membrane. But electroporation of cell plasma membrane can also result in fusion of cells. This process has been termed electrofusion. First reports of in vitro electrofusion of cells date back into 1980s. In these reports it has been shown that fusion between two cells can proceed only if the cells are in contact prior or immediately after electroporation. The contact between the cells can be achieved either by dielectrophoretically connecting neighboring cells, which is followed by electroporation or by centrifugation of cell suspension after exposure to electric field. In both cases cells must be reversibly permeabilized, otherwise they lose viability and there is no electrofusion [34]. Electrofusion in in vitro environment is possible due to high possibility of cell movement while cells in tissues are more or less fixed, nevertheless in vivo electrofusion has been observed in B16 melanoma tumors as well as cells to tissue fusion [35, 36]. Electrofusion of cells of different sizes can be achieved by nanosecond pulsed electric fields [37]. Electroextraction Electroporation can be used to extract substances (e.g. juice, sugar, pigments, lipid and proteins) from biological tissue or cells (e.g. fruits, sugar beets, microalgae, wine and yeast). Electroextraction can be more energy and extraction efficient, and faster than classical extraction methods (pressure, thermal denaturation and fermentation) [38–42]. An economic assessment of microalgae-based bioenergy production was recently made [43]. Recommendations guidelines on the key information to be reported in biotechnological studies because of variability in results obtained in different laboratories [44]. Electro- pasteurization and sterilization Irreversible electroporation can be used in applications where destruction of microorganisms is required, i.e. food processing and water treatment [45]. Still, using irreversible electroporation in these applications means that substance under treatment is exposed to a limited electric field since it is desirable that changes in treated substance do not occur (e.g. change of Development of devices and electrodes 165 food flavor) and that no by-products emerge due to electric field exposure (e.g. by-products caused by electrolysis). This is one of the reasons why short (in comparison to medical applications) in the range of 1-3 μs are used. Especially industrial scale batch or flowthrough exposure systems may require huge power generators with amplitudes up to 40 kV and peak currents up to 500 A. Although batch and flow-through processes are both found on industrial scale, flow-through is considered to be superior as it allows treatment of large volumes. Such mode of operation requires constant operation requiring higher output power of pulse generators [13], [46]. Electric field distribution in vivo In most applications of tissue permeabilization it is required to expose the volume of tissue to E intensities between the two “thresholds” i.e. to choose in advance a suitable electrode configuration and pulse parameters for the effective tissue electroporation [47]. Therefore electric field distribution in tissue has to be estimated before the treatment, which can be achieved by combining results of rapid tests or in situ monitoring [48] with models of electric field distribution [49–53]. However, modeling of electric field distribution in tissue is demanding due to heterogeneous tissue properties and usually complex geometry. Analytical models can be employed only for simple geometries. Usually they are developed for 2D problems and tissue with homogenous electrical properties. Therefore in most cases numerical modeling techniques are still more acceptable as they can be used for modeling 3D geometries and complex tissue properties. For that purpose mostly finite element method and finite difference method are applied. Both numerical methods have been successfully applied and validated by comparison of computed and measured electric field distribution. Furthermore, advanced numerical models were build, which take into consideration also tissue conductivity increase due to tissue or cell electroporation. These advanced models describe E distribution as a function of conductivity σ(E). In this way models represent electroporation tissue conductivity changes according to distribution of electric field intensities [54, 55]. 166 Damijan Miklavčič, Matej Reberšek Electrodes for in vitro and in vivo applications Effectiveness of electroporation in in vitro, in vivo or clinical environment depends on the distribution of electric field inside the treated sample. Namely, the most important parameter governing cell membrane permeabilization is the local electric field to which the cell is exposed [47]. To achieve this we have to use an appropriate set of electrodes and an electroporation device – electroporator that generates required voltage or current signals. Although both parts of the mentioned equipment are important and necessary for effective electroporation, electroporator has a substantially more important role since it has to be able to deliver the required signal to its output loaded by impedance of the sample between electrodes. Nowadays there are numerous types of electrodes that can be used for electroporation in any of the existing applications [56–60]. According to the geometry, electrodes can be classified into several groups, i.e. parallel plate electrodes, needle arrays, wire electrodes, tweezers electrodes, coaxial electrodes, etc (Fig. 2). Each group comprises several types of electrodes that can be further divided according to the applications, dimensions, electrode material etc. In any case selection of electrode type plays an important role in characterization of the load that is connected to the output of the electroporator. During the design of the electroporator load characterization represents the starting point and represents a considerable engineering problem, because electrical characteristics of substance between electrodes (e.g. cell suspension, tissue, etc.) vary from experiment to experiment and even during the course of experiment. In general the load between electrodes has both a resistive and a capacitive component. The value of each component is defined by geometry and material of electrodes and by electrical and chemical properties of the treated sample. In in vitro conditions these parameters that influence the impedance of the load can be well controlled since size and geometry of sample are known especially if cuvettes are used. Furthermore, by using specially prepared cell media, electrical and chemical properties are defined or can be measured. On the other hand, in in vivo conditions, size and geometry can still be controlled to a certain extent but electrical and chemical properties can only be estimated, especially if needle electrodes are used that penetrate through different tissues. However, even if we manage to reliably define these properties during the development of the device, it is practically impossible to predict changes in the electrical and chemical properties of the sample due to exposure to high-voltage electric pulses [61–63]. Besides electropermeabilization of cell membranes which increases electrical Development of devices and electrodes 167 conductivity of the sample, electric pulses also cause side effects like Joule heating and electrolytic contamination of the sample [64], which further leads to increased sample conductivity [65]. Electric pulses For better understanding and critical reading of various reports on electroporation phenomenon and electroporation based applications, complete disclosure of pulse parameters needs to be given. Electric pulses are never “square” or “rectangular”, but they are characterized by their rise time, duration/width, fall time, pulse repetition frequency. Rise time and fall time are determined as time needed to rise from 10% to 90% of the amplitude, drop from 90% to 10% of amplitude, respectively. Pulse width is most often defined as time between 50% amplitude on the rise and 50% amplitude on the fall. Pulse repetition frequency is the inverse of the sum of pulse width and pause between two consecutive pulses. These may seem trivial when discussing pulses of 1 ms, but become an issue when discussing ns or even ps pulses [66]. The cell membrane damage and uptake of ions can be significantly reduced when using bipolar ns pulses instead of monopolar [67]. Shapes other than “rectangular” have been investigated with respect to electroporation efficiency [68]. It was suggested exposure of cells to pulse amplitudes above given critical amplitude and duration of exposure to this above critical value seem to be determining level of membrane electroporation irrespective of pulse shape. Exponentially decaying pulses are difficult to be considered as such but were predominantly used in 80s for gene electrotransfer. Their shape was convenient as the first part of the pulse i.e. the peak acts as the permeabilizing part, and the tail of the pulse acts as electrophoretic part pushing DNA as towards and potentially through the cell membrane [32]. 168 Damijan Miklavčič, Matej Reberšek Figure 2: Examples of electrodes commercially available for electropermeabilization. The electrode belongs to the following groups: (a), parallel plaques electrodes, (b): arrays of needles, (c): finger electrodes, (d): array of needles, (e): endoluminal electrode, and (f), group of independent needle electrodes. The electrodes (a), (b),(c), and (d) are produced by IGEA, Italy, and are used in clinical applications of electrochemotherapy and gene electrotransfer [31]. Electroporators – the necessary pulse generators Electroporator is an electronic device that generates signals, usually square wave or exponentially decaying pulses, required for electroporation [1]. Parameters of the signal delivered to electrodes with the treated sample vary from application to application. Therefore, in investigating of electroporation phenomenon and development of electroporation based technologies and tretaments it is important that electroporator is able to deliver signals with the widest possible range of electrical parameters if used in research. If however used for a specific application only, e.g. clinical treatment such as electrochemotherapy, pulse generator has to provide Development of devices and electrodes 169 exactly required pulse parameters in a reliable manner. Moreover, electroporator must be safe and easy to operate and should offer some possibilities of functional improvements. Clinical electroporators used in electrochemotherapy of deep-seated tumors or in non-thermal tissue ablation are also equipped with ECG synchronization algorithms which minimasizes possible influence of electric pulse delivery on heart function [69]. Figure 3: Areas of amplitude and duration of electrical pulses which are used in the research of electroporation and related effects (a). Five different areas of electroporation pulse generation (b). To amplify or to generate very-high-voltage electroporation pulses (over a few kV) spark gaps and similar elements are used, for high-voltage (a few V to a few kV) transistors and for low-voltage operational amplifiers are used. Nanosecond (short) pulses are generated with different techniques than pulses longer than 1 μs. Originally published in Advanced electroporation techniques in biology and medicine by Reberšek and Miklavčič 2010 [3]. In principle, electroporators can be divided in several groups depending on biological applications, but from the electrical point of view only two types of electroporators exist: devices with voltage output (output is voltage 170 Damijan Miklavčič, Matej Reberšek signal U(t)) and devices with current output (output is current signal I(t)). Both types of devices have their advantages and disadvantages, but one point definitely speaks in favor of devices with voltage output. For example, if we perform in vitro experiments with parallel plate electrodes with plate sides substantially larger than the distance between them, the electric field strength E that is applied to the sample can be approximated by the voltage-to-distance ratio U/ d, where d is the electrode distance and U the amplitude of applied signal obtained from an electroporator with voltage output. On the other hand, if an electroporator with current output is used, the same approximation could be used only if additional measurement of voltage difference between electrodes is performed or if the impedance Z of the sample is known, measured or approximated and voltage difference between electrodes is estimated using Ohm’s law U = I • Z. Nevertheless, there are several commercially available electroporator that fulfill different ranges of parameters and can be used in different applications. A list of commercially available electrodes and electroporators has been presented in 2004 by Puc and colleagues [70], updated in 2010 [3] and in 2017 [71]. To be sure the applied pulses are adequate we have to measure the applied voltage and current during the pulse delivery. In nanosecond applications rise time of the pulse is sometimes shorter than the electrical length (the time in which an electrical signal travels through the line) between the source and the load. In this case, the impedance of the load and the transmission line has to match the impedance of the generator, so that there are no strong pulse reflections and consequently pulse prolongations. Based on the studies reported in the literature it is very difficult to extract a general advice how to design experiments or treatments with electroporation. In principle we can say that pulse amplitude (voltage-to-distance ratio) should typically be in the range from 200 V/cm up to 2000 V/cm. Pulse durations should be in the range of hundreds of microseconds for smaller molecules and from several milliseconds up to several tens of milliseconds for macromolecules such as plasmid DNA (in the latter case, due to the very long pulse duration, optimal pulse amplitude can even be lower than 100 V/cm). If there is any possibility to obtain the equipment that generates bipolar pulses or have a possibility to change electric field orientation in the sample, these types of pulses/electroporators should be used because bipolar pulses yield a lower poration threshold, higher uptake, reduce electrolyte wear and electrolytic contamination of the sample, and an unaffected viability compared to unipolar pulses of the same amplitude and duration. Better permeabilisation or gene transfection efficiency and survival can also be obtained by changing field orientation in the sample using special commutation circuits that commute electroporation pulses Development of devices and electrodes 171 between the electrodes [56, 58, 71]. Short bipolar high-frequency electroporation pulses HF-EP were also investigated as they mitigate nerve/muscle stimulation and electric field distribution of such pulses in tissue is more homogeneous [72–74], but these pulses may already fall into vicinity of “cancellation effect” [74]. However, development of high-frequency electroporators is much more challenging and commercial high-frequency electroporators are not available, therefore unfortunately, research of HF-EP is limited to only a few groups [75], [76]. This general overview of electrical parameters should however only be considered as a starting point for a design of experiments or treatments. Optimal values of parameters namely also strongly depend on the cell type used, on the molecule to be introduced, and on specific experimental conditions. The pulse characteristics determined as optimal or at least efficient and the tissue/sample will than determine the architecture of the pulse generator, whether it will be a Marx generator, Blumlein, or... [7]. Conclusions Electroporation has been studied extensively until now, and a number of applications has been developed. Electrochemotherapy has been demonstrated as an effective local treatment of solid tumors and is the most mature therapeutic application right now. Electroporation for gene transfection however has been long used in in vitro situation. With a hold on viral vectors electroporation represents a viable non-viral alternative also for in vivo gene transfection. Clinical applications and expansion of electrochemotherapy and tissue ablation have been hindered by the lack of adequate electroporators and their certification in Europe (CE Medical Device) and limited approval by FDA in USA [1]. Cliniporator (IGEA, s.r.l. Carpi, Italy) was certified in EU (CE mark) as a medical device and is offered on the market along with standard operating procedures for electrochemotherapy of cutaneous and subcutaneous tumors. NanoKnife (AngioDynamics, Queensbury, USA) was certified in EU and approved by the FDA for surgical ablation of soft tissue. Some electroporators are now available under the license for clinical evaluation purpuses: Cellectra, Elgen, Medpulser, Cliniporator VITAE, BetaTech, DermaVax, EasyVax, Ellisphere, TriGrid [4]. Development of new applications warrants further development of pulse generators and electrodes. Based on the above considerations however, a single pulse generator will not fit all applications and all needs of researchers. 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Miklavčič, “Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli,” The Journal of Membrane Biology, Jul. 2013. [43] A. L. Gonçalves, M. C. M. Alvim-Ferraz, F. G. Martins, M. Simões, and J. C. M. Pires, “Integration of Microalgae-Based Bioenergy Production into a Petrochemical Complex: Techno-Economic Assessment,” Energies, vol. 9, no. 4, p. 224, Mar. 2016. Development of devices and electrodes 175 [44] J. Raso et al. , “Recommendations guidelines on the key information to be reported in studies of application of PEF technology in food and biotechnological processes,” Innovative Food Science & Emerging Technologies, Aug. 2016. [45] J. R. Beveridge, S. J. MacGregor, L. Marsili, J. G. Anderson, N. J. Rowan, and O. Farish, “Comparison of the effectiveness of biphase and monophase rectangular pulses for the inactivation of micro-organisms using pulsed electric fields,” IEEE Transactions on Plasma Science, vol. 30, no. 4, pp. 1525–1531, Aug. 2002. [46] S. Toepfl, “Pulsed electric field food processing industrial equipment design and commercial applications,” Stewart Postharvest Review, vol. 8, no. 2, pp. 1–7, 2012. [47] T. Kotnik, P. Kramar, G. Pucihar, D. Miklavcic, and M. Tarek, “Cell membrane electroporation- Part 1: The phenomenon,” IEEE Electrical Insulation Magazine, vol. 28, no. 5, pp. 14–23, Oct. 2012. [48] M. Kranjc et al. , “In Situ Monitoring of Electric Field Distribution in Mouse Tumor during Electroporation,” Radiology, vol. 274, no. 1, pp. 115–123, Jan. 2015. [49] D. Miklavcic, K. Beravs, D. Semrov, M. Cemazar, F. Demsar, and G. Sersa, “The importance of electric field distribution for effective in vivo electroporation of tissues.,” Biophys J, vol. 74, no. 5, pp. 2152–2158, May 1998. [50] N. Pavselj, Z. Bregar, D. Cukjati, D. Batiuskaite, L. M. Mir, and D. Miklavcic, “The Course of Tissue Permeabilization Studied on a Mathematical Model of a Subcutaneous Tumor in Small Animals,” IEEE Transactions on Biomedical Engineering, vol. 52, no. 8, pp. 1373–1381, Aug. 2005. [51] D. Sel, D. Cukjati, D. Batiuskaite, T. Slivnik, L. M. Mir, and D. Miklavcic, “Sequential Finite Element Model of Tissue Electropermeabilization,” IEEE Transactions on Biomedical Engineering, vol. 52, no. 5, pp. 816–827, May 2005. [52] D. Miklavcic, S. Corovic, G. Pucihar, and N. Pavselj, “Importance of tumour coverage by sufficiently high local electric field for effective electrochemotherapy,” European Journal of Cancer Supplements, vol. 4, no. 11, pp. 45–51, Nov. 2006. [53] D. Miklavcic et al. , “Towards treatment planning and treatment of deep-seated solid tumors by electrochemotherapy,” Biomed Eng Online, vol. 9, no. 10, pp. 1–12, 2010. [54] S. Corovic, I. Lackovic, P. Sustaric, T. Sustar, T. Rodic, and D. Miklavcic, “Modeling of electric field distribution in tissues during electroporation,” Biomedical engineering online, vol. 12, no. 1, p. 16, 2013. [55] J. Langus, M. Kranjc, B. Kos, T. Šuštar, and D. Miklavčič, “Dynamic finite-element model for efficient modelling of electric currents in electroporated tissue,” Sci Rep, vol. 6, p. 26409, May 2016. [56] R. A. Gilbert, M. J. Jaroszeski, and R. Heller, “Novel electrode designs for electrochemotherapy,” Biochim. Biophys. Acta, vol. 1334, no. 1, pp. 9–14, Feb. 1997. [57] S. Mazères et al. , “Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery,” J Control Release, vol. 134, no. 2, pp. 125–131, Mar. 2009. [58] M. Reberšek, S. Čorović, G. Serša, and D. Miklavčič, “Electrode commutation sequence for honeycomb arrangement of electrodes in electrochemotherapy and corresponding electric field distribution,” Bioelectrochemistry, vol. 74, no. 1, pp. 26– 31, Nov. 2008. [59] J. Čemažar, D. Miklavčič, and T. Kotnik, “Microfluidic devices for manipulation, modification and characterization of biological cells in electric fields - a review,” Informacije MIDEM, vol. 43, no. 3, pp. 143–161, Sep. 2013. 176 Damijan Miklavčič, Matej Reberšek [60] P. F. Forde et al. , “Preclinical evaluation of an endoscopic electroporation system,” Endoscopy, vol. 48, no. 5, pp. 477–483, May 2016. [61] M. Pavlin et al. , “Effect of Cell Electroporation on the Conductivity of a Cell Suspension,” Biophysical Journal, vol. 88, no. 6, pp. 4378–4390, Jun. 2005. [62] D. Cukjati, D. Batiuskaite, F. André, D. Miklavčič, and L. M. Mir, “Real time electroporation control for accurate and safe in vivo non-viral gene therapy,” Bioelectrochemistry, vol. 70, no. 2, pp. 501–507, May 2007. [63] M. Kranjc, F. Bajd, I. Serša, and D. Miklavčič, “Magnetic resonance electrical impedance tomography for measuring electrical conductivity during electroporation,” Physiological Measurement, vol. 35, no. 6, pp. 985–996, Jun. 2014. [64] M. Phillips, L. Rubinsky, A. Meir, N. Raju, and B. Rubinsky, “Combining Electrolysis and Electroporation for Tissue Ablation,” Technol. Cancer Res. Treat. , vol. 14, no. 4, pp. 395–410, Aug. 2015. [65] I. Lackovic, R. Magjarevic, and D. Miklavcic, “Three-dimensional finite-element analysis of joule heating in electrochemotherapy and in vivo gene electrotransfer,” Dielectrics and Electrical Insulation, IEEE Transactions on, vol. 16, no. 5, pp. 1338– 1347, 2009. [66] K. Mitsutake, A. Satoh, S. Mine, K. Abe, S. Katsuki, and H. Akiyama, “Effect of pulsing sequence of nanosecond pulsed electric fields on viability of HeLa S3 cells,” Dielectrics and Electrical Insulation, IEEE Transactions on, vol. 19, no. 1, pp. 337– 342, 2012. [67] B. L. Ibey et al. , “Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses,” Biochem. Biophys. Res. Commun. , vol. 443, no. 2, pp. 568–573, Jan. 2014. [68] T. Kotnik, D. Miklavčič, and L. M. Mir, “Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses: Part II. Reduced electrolytic contamination,” Bioelectrochemistry, vol. 54, no. 1, pp. 91–95, Aug. 2001. [69] B. Mali et al. , “Electrochemotherapy of colorectal liver metastases-an observational study of its effects on the electrocardiogram,” Biomedical engineering online, vol. 14, no. Suppl 3, p. S5, 2015. [70] M. Puc, S. Čorović, K. Flisar, M. Petkovšek, J. Nastran, and D. Miklavčič, “Techniques of signal generation required for electropermeabilization: Survey of electropermeabilization devices,” Bioelectrochemistry, vol. 64, no. 2, pp. 113–124, Sep. 2004. [71] E. Pirc, M. Reberšek, and D. Miklavčič, “Dosimetry in Electroporation-Based Technologies and Treatments,” in Dosimetry in Bioelectromagnetics, M. Markov, Ed. 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487–2742: CRC Press, 2017, pp. 233–268. [72] M. Reberšek, M. Kandušer, and D. Miklavčič, “Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells,” Radiology and Oncology, vol. 45, no. 3, pp. 204–208, 2011. [73] M. B. Sano et al. , “Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth,” Scientific Reports, vol. 5, p. 14999, Oct. 2015. [74] D. C. Sweeney, M. Reberšek, J. Dermol, L. Rems, D. Miklavčič, and R. V. Davalos, “Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses,” Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 1858, no. 11, pp. 2689–2698, Nov. 2016. Development of devices and electrodes 177 [75] V. Novickij et al. , “High-frequency submicrosecond electroporator,” Biotechnology & Biotechnological Equipment, pp. 1–7, Feb. 2016. [76] A. Grainys, V. Novickij, and J. Novickij, “High-power bipolar multilevel pulsed electroporator,” Instrumentation Science & Technology, vol. 44, no. 1, pp. 65–72, Jan. 2016. Acknowledgement This research was in part supported by Slovenian Research Agency, and by Framework Programs of European Commission through various grants. Research was conducted in the scope of the EBAM European Associated Laboratory (LEA). Damijan Miklavčič was born in Ljubljana, Slovenia, in 1963. He received a Masters and a Doctoral degree in Electrical Engineering from University of Ljubljana in 1991 and 1993, respectively. He is currently Professor and the Head of the Laboratory of Biocybernetics at the Faculty of Electrical Engineering, University of Ljubljana. His research areas are biomedical engineering and study of the interaction of electromagnetic fields with biological systems. In the last years he has focused on the engineering aspects of electroporation as the basis of drug delivery into cells in tumor models in vitro and in vivo. His research includes biological experimentation, numerical modeling and hardware development for electrochemotherapy, irreversible electroporation, transdermal drug delivery and gene electrotransfer. Matej Reberšek, was born in Ljubljana, Slovenia, in 1979. He received the Ph.D. degree in electrical engineering from the University of Ljubljana, Slovenia. He is an Assistant Professor and a Research Associate in the Laboratory of Biocybernetics, at the Faculty of Electrical Engineering, University of Ljubljana. His main research interests are in the field of electroporation, especially design of electroporation devices and investigation of biological responses to different electric pulse parameters. He is an Assistant Professor and a Research Associate in the Laboratory of Biocybernetics, at the Faculty of Electrical Engineering, University of Ljubljana. Chapter 12 Electroporation and electropermeabilisation - pieces of puzzle put together Lluis M Mir Vectorology and Anticancer Therapies, UMR 8203, CNRS, Univ. Paris-Sud, Université Paris-Saclay, Gustave-Roussy, 114,Rue Edouard Vaillant, F-94805 Villejuif Cédex, France; European Associated Laboratory (LEA) on the pulsed Electric fields in Biology And Medicine (LEA EBAM). Until now, two main generic approaches have been used to detect the cell permeabilization after the application of electric pulses to cells or tissues. They are based either on the detection of electrical changes of the tissue/cells (bioimpedance measurements, or simply conductance determinations) or on molecular exchanges across the membrane (diffusion or electrotransfer of markers, like fluorescent small molecules, radioactive compounds, plasmids coding for reporter genes, etc.). The second approach, based on the transport of a given molecular species, is very depending on the physic-chemical characteristics of the marker used (molecular weight, net charge, fluorescence yield, merker-target interactions (if any), mode of transport [1], …) The models built to describe the phenomena occurring at the cell membrane (even at artificial membranes, whether these artificial membranes were planar membranes or membranes of vesicles of different sizes and compositions) have been mainly based on the physical principles that could explain the transport of molecules across the membrane. The input of the bioimpedance measurements, while very useful in practical Electroporation and electropermeabilisation - pieces of puzzle put together 179 terms, has brought a limited contribution to the understanding of these phenomena. However, in the transport phenomena there are parameters not related to the structural features of the membrane before, during and after the pulses. Indeed, as already mentioned, there is an impact of the size of the molecules, their charge, the gradient of concentration between the inside and the outside, the sensitivity of their detection inside the cells, etc. There are a number of examples, whatever the duration of the pulses, nanosecond pulses or microseconds pulses, that can be reported. In this context, it is important to highlight that penetration of Calcium ions can be detected at electric field amplitudes for which many other electropermeabilization markers do not yet reveal the electropermeabilization of the cells. This allows manipulating cytosolic calcium content in conditions where cell survival is fairly well protected [2,3]. Several new techniques have been recently applied to explore the changes in the membrane itself, independently of any transport phenomenon. Some of these techniques come from technologies that were not previously used to analyse the effects of the electric pulses on the lipid bilayers or the cell membranes. On the one hand, the use of Giant Unilamellar Vesicles (composed of a defined lipid species and having the size of an animal cell) has allowed analysing chemical changes occurring in the lipid bilayers during the delivery of the pulses [4]; molecular dynamics has started to bring the explanations for these reactions to occur. It is important to note that these two approaches (experimental and in silico) restrain their analysis to the lipid part of the complex cell membranes. On the other hand, using cells in culture, non linear optical methods are producing new elements of the puzzle. Spontaneous Raman microspectroscopy has brought new information about modifications of proteins that could occur during (or, maybe, after) the delivery of the electric pulses [5]. Confocal Raman microscopes has brought spatial as well as dynamical information on the changes in the Raman spectra that reflects these changes in the proteins [6]. Because biological objects are immersed in water-based media, Confocal Raman microscopes must be used to eliminate the non-resonant Raman contribution of the water. Coherent Raman microspectroscopy, like the Coherent AntiStockes Raman Scattering microspectroscopy, seems more attractive because of the enhancement of the signal caused by the “coherence” provided by the use of two lasers accordingly tuned. Enhancement of the signal with respect to spontaneous Raman signal can reach 108 times. Coherent AntiStockes Raman Scattering microspectroscopy has recently provided us with information on changes in 180 Lluis M Mir the interfacial water (the few layers of water molecules organized at the surface of the membranes) and even of the interstitial water. After the pulses delivery, an important loss of the interfacial water signal has been recorded, which means that the alterations of the membrane structure consecutive to the pulses application also affects the water surrounding the membrane (to be submitted). We are thus acquiring information on the changes occurring in the membranes independently of any transport phenomenon. This information has now to be introduced into the models that tentatively describe the phenomena occurring at the membranes, to continue improving the knowledge of the electroporation/electropermeabilization of cells as well as of even much smaller biological objects [7]. However, there is another level of perturbations that has also to be taken into account, for which information is rapidly accumulating: the cell reactions to the stress caused by the electric pulses delivery. It corresponds to the ensemble of the biological aspects linked to the electric pulses delivery, with kinetics that can be orders of magnitude longer than the duration of the electric pulses and even of the duration of the recovery of the cells impermeability to classical electropermeabilization markers. The construction of any new model is therefore becoming incredibly complex. This just reflects the complexity of the phenomena that have been presented in the Electroporation-Based Technologies and Treatments school. The viscous, elastic and viscoelastic models of membranes electrical breakdown are far behind us. The models describing the generation of stable pores are also insufficient nowadays. Models including several terms to explain the evolution of the permeability and the conductivity of the cell membranes are arising [8]. It is a hope that they will be able to give clues about the many questions that are still unsolved. For example, considering the “irreversible electroporation”, it is still unknown what the “irreversible” event is … All the aspects developed here above will be discussed in the frame of a new model of the phenomena occurring in the membranes of the cells exposed to the electric pulses. This model will be presented, and terminology will be delivered for a correct use of terms that have been used indistinctly until now. Therefore a distinction between “electroporation” and “electropermeabilization” will be brought in the context of the cells “electropulsation”, as parts of a puzzle that collectively we want to put together. Recent references (former references can be found in these papers): [1] A. Azan, F. Gailliègue, L.M. Mir and M. Breton. Cell Membrane Electropulsation: Chemical Analysis of Cell Membrane Modifications and Associated Transport Electroporation and electropermeabilisation - pieces of puzzle put together 181 Mechanisms. In: Advs Anatomy, Vol. 227, Transport Across Natural and Modified Biological Membranes and its Implications in Physiology and Therapy, eds. J. Kulbaka and S. Satkauskas. ISBN : 978-3-319-56894-2 [2] H. Hanna, A. Denzi, M. Liberti, F.M. Andre and L.M. Mir. Electropermeabilization of inner and outer membranes of cells with microsecond pulsed electric fields: Quantitative study with calcium ions. Scientific Reports in press 2017 [3] H. Hanna, F.M. Andre and L.M. Mir. Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields. Stem cell research and therapy, vol 8, art 91, 2017, DOI: 10.1186/s13287-017-0536-z [4] M. Breton and L. M. Mir. Investigation of the Chemical Mechanisms Involved in the Electropulsation of Membranes at the Molecular Level. Bioelectrochemistry 119 (2018) e7966; doi:10.1016/j.bioelechem.2017.09.005 [5] A. Azan, V. Untereiner, C. Gobinet, G. D. Sockalingum, M. Breton, O. Piot and L. M. Mir. Demonstration of Protein Involvement in Living Cell Electropulsation using Confocal Raman Microspectroscopy. Scientific Reports 7. 297–306, 2017. doi:10.1038/srep40448. [6] A. Azan, V. Untereiner, L. Descamps, C. Merla, C. Gobinet, M. Breton, O. Piot and L. M. Mir. Comprehensive Characterization of the Interaction between Pulsed Electric Fields and Live Cells by Confocal Raman Microspectroscopy. Analytical Chemistry in press 2017. [7] A. Denzi, E. della Valle, G. Esposito, L. M. Mir, F. Apollonio and Micaela Liberti. Technological and Theoretical Aspects for Testing Electroporation on Liposomes. BioMed Research International, vol. 2017, Article ID 5092704, 10 pages, 2017. doi:10.1155/2017/5092704. [8] D.Voyer, A. Silve, L. M. Mir, R. Scorretti and C. Poignard. Dynamic modeling of tissue electroporation. Bioelectrochemistry in press 2017 Lluis M. Mir was born in Barcelona, Spain, in 1954. He received a Masters in Biochemistry in 1976 from Ecole Normale Supérieure, Paris, and a Doctorate (D.Sc.) in Cell Biology in 1983. In 1978 he entered CNRS as Attaché de Recherches in the Laboratory of Basic Pharmacology and Toxicology, Toulouse. In 1983 he was promoted to Chargé de Recherches at CNRS, and in 1985 he moved to the Laboratory of Molecular Oncology CNRS-Institute Gustave-Roussy and Univ. Paris Sud, Villejuif). In 1989 he moved to the Laboratory of Molecular Pharmacology (Villejuif), and in 2002 to the Laboratory of Vectorology and Gene Transfer (Villejuif). In 1999, he was promoted to Directeur de Recherches at CNRS. Lluis M. Mir was one of the pioneers of the research of electropermea-bilization (electroporation) and the applications of this technique for antitumor electrochemotherapy and DNA electrotransfer. He is the author of 193 articles in peer-reviewed journals, 21 chapters in books, and over 500 presentations at national and international meetings, invited lectures at international meetings and seminars. He received the Award for the medical applications of electricity of the Institut Electricité Santé in 1994, the Annual Award of 182 Lluis M Mir Cancerology of the Ligue contre le Cancer (committee Val-de-Marne) in 1996, the Award of the Research of Rhône-Poulenc-Rorer in 1998, the medal of the CNFRS under the auspices of the French Sciences Academy in 2012, the Frank Reidy Award in Bioelectrics in 2015 and the Balthazar van der Pol Gold Medal of the International Union of Radio Sciences in 2017. He is an Honorary Senator of the University of Ljubljana (2004). He is also fellow of the American Institute of Biological and Medical Engineering. He has been visiting professor of the Universities of Berkeley (USA), Bielefeld (Germany) and Jerusalem (Israel). He is the director of the laboratory of Vectorology (UMR 8203 of CNRS, Universiy Paris-Sud and Institut Gustave-Roussy), and he is also the founder and co-director of the European Associated Laboratory on Electroporation in Biology and Medicine of the CNRS, the Universities of Ljubljana, Primorska, Toulouse and Limoges, the Institute of Oncology Ljubljana and the Institut Gustave-Roussy. This book covers the latest and most to date information on electroporation, electropermeabilisation and pulsed power fields. It provides concise introduction to the topic covering basic knowledge and biomedical applications that anyone in the field needs to understand in order to be successful. Document Outline The cell and its plasma membrane Resting transmembrane voltage Induced transmembrane voltage Spherical cells Spheroidal and ellipsoidal cells Irregularly shaped cells Cells in dense suspensions High field frequencies and very short pulses References Acknowledgement Introduction Biological materials in the electric field Measurements of dielectric properties of tissues Inhomogeneity of tissues Anisotropy of tissues Physiological factors and changes of tissue Electrode polarization Electrical response of tissue to electric field Tissue properties during high frequency electroporation Conclusions References Acknowledgement Introduction Preambule: what is a biological membrane? A- A biophysical description and a biological validation B- Structural Investigations C-Practical aspects of electropermeabilization Conclusion Acknowledgement References APPENDIX - Transmembrane transport Introduction Membrane electropermeabization Mechanisms of electrotransfer of dna molecules into cells Conclusions References Acknowledgement Introduction MD simulations of lipid membranes Modeling membranes electroporation Electroporation induced by dirrect effect of an electric field Electroporation induced by ionic salt concentration gradients Internal electric field distribution and origin of membranes electroporaiton COMPLEX BILYAER MODELS: EP THRESHOLDS AND PORE FEATURES TRANSPORT OF MOLECULES Discussion and perspectives References Acknowledgement Introduction Nanosecond bioelectrics Nanosecond experiments, models Nanosecond excitation Acknowledgment References Introduction Preclinical gene electrotransfer of reporter genes Preclinical and clinical gene electrotransfer of therapeutic genes Perspectives References Acknowledgement DNA vaccines Electroporation-mediated delivery of DNA vaccines Recommended papers Introduction PRECLINICAL STUDIES Clinical studies Clinical use and treatment procedures for electrochemotherapy New clinical applications of electrochemotherapy Conclusion References Acknowledgement Development of electrochemotherapy Implementation Research References Introduction Therapeutic and technological applications of electroporation Electrochemotherapy Tissue Ablation by Non-thermal IREversible electroporation Gene ElectroTransfer Electrofusion Electroextraction Electro- pasteurization and sterilization Electric field distribution in vivo Electrodes for in vitro and in vivo applications Electric pulses Electroporators – the necessary pulse generators Conclusions REFERENCES Acknowledgement Recent references