Electrical Modeling of the Influence of Medium Conductivity on Electroporation Antoni Ivorra *,1 , Julien Villemejane 2, 3, 4 and Lluis M. Mir 2,3 1 Dept. of Information and Communication Technologies, Universitat Pompeu Fabra, Barcelona, Spain 2 CNRS, UMR 8203, Institut Gustave-Roussy, Villejuif, France 3 Univ Paris-Sud, UMR 8203 4 CNRS, SATIE, Institut d'Alembert, ENS Cachan, Cachan, France * Correspondig author E-mail address: [email protected] Electroporation is the phenomenon in which cell membrane permeability is increased by exposing the cell to short high electric field pulses. Experimental data shows that the amount of permeabilization depends on the conductivity of the extracellular medium. If medium conductivity decreases then it is necessary to deliver a pulse of larger field amplitude in order to achieve the same effect. Models that do not take into account the permeabilization effect on the membrane conductivity cannot reproduce qualitatively the experimental observations. Here we employ an exponential function for describing the strong dependence of membrane conductivity on transmembrane potential. Combining that model with numerical methods we demonstrate that the dependence on medium conductivity can be explained as being the result of increased membrane conductance due to electroporation. As experimentally observed, extracellular conductivities of about 1 S/m and 0.1 S/m yield very similar results, however, for lower conductivities ( < 0.01 S/m) the model predicts that significantly higher field magnitudes will be required to achieve the same amount of permeabilization. 1. Introduction Electroporation, or electropermeabilization, is the phenomenon in which cell membrane permeability to ions and macromolecules is increased by exposing the cell to short (microseconds to milliseconds) high electric field pulses. The permeabilization can be either temporary (reversible electroporation) or permanent (irreversible electroporation) as a function of the electrical field magnitude and duration, pulse repetition frequency and number of the pulses. In vitro, reversible electroporation is now commonly used for gene transfection of cells in culture 1 whereas irreversible electroporation is considered as a potential viable method for cold sterilization of liquid media 2, 3. In addition, reversible electroporation is also used in living tissues for in vivo gene therapy 4 and to enhance the penetration of anti-cancer drugs into undesirable cells (electrochemotherapy, ECT) 5. Recently, irreversible electroporation (IRE) has also found a use in tissues as a minimally invasive surgical procedure to ablate undesirable tissue without the use of adjuvant drugs 6. Multiple in vitro experimental studies demonstrate that the extracellular medium conductivity (σ ) has an influence on the electroporation phenomenon 1, 7-12. In five out of seven of e these cited studies, it was observed that electroporation efficiency decreases when medium conductivity decreases but in the other two the opposite effect was observed, that is, efficiency decreased when medium conductivity increased 7, 9. In those two singular cases the pulse generator consisted of a capacitance charged to a high voltage that was allowed to discharge through the medium in which cells where contained. That is the oldest method used to generate electroporation pulses 13 and it has a well identified drawback when compared to modern square pulse generators: in these capacitance-discharge generators the applied pulse length depends on medium conductivity. And such dependence would explain the exceptional observation for those two cases: as medium conductivity increases the pulse length decreases and as a consequence the intensity of the electroporation phenomenon diminishes 14. Observations in all the other studies can be summarized as follows: 1) electroporation efficiency decreases as σ decreases, 2) e decrease rate in efficiency as a function of σ is higher for lower σ values and 3) electroporation e e efficiency sensitivity to σ varies significantly between experiments and depending on the way e permeabilization is assessed. Here it is convenient to note that in the cited studies the depletion of ions from the medium was osmotically counterbalanced with neutral compounds such as sucrose. Otherwise, it could be reasonably argued that it is the osmolarity, and not the electrical conductivity, the main factor that influences electroporation.15 If we define a "critical" electric field magnitude, E , at which the electroporation effect is c quantitatively the same for different extracellular conductivities, then it is possible to plot data from some of above cited studies 1, 10, 11 so that the above observations are illustrated in a comprehensible way (Fig. 1). The other two studies 8, 12 show that electroporation efficiency diminishes as σ decreases but do not provide data for representing E at different extracellular e c conductivities. The cases B and D on Fig. 1 are particularly illustrative. Both cases correspond to the same study and followed the same experimental procedures apart from the method employed for assessing electroporation efficiency. In D cell reversible permeabilization was evaluated by quantifying cell survival rate after electroporation in a medium containing bleomycin, which is a powerful cytotoxic drug barely able to penetrate the cells under normal conditions. In B direct cell death (necrosis) caused by irreversible permeabilization was quantified. As it can be observed in D, which is the case that requires a lower level of permeabilization, the permeabilization sensitivity to σ is almost insignificant whereas in B, which requires a higher e level of permeabilization, the required field magnitude increases as σ decreases. Moreover, such e field sensitivity to σ seems to become larger as σ decreases. The motivation of the present study e e was to analyze whether it was possible to justify such patterns in electrical terms. Fig.1 Results from experimental studies showing that the electric field magnitude required to achieve a specific electroporation effect, E, depends on the extracellular conductivity, σ. A: 50 % green algae cells permeabilization c e after a single square pulse of 200 µs as assessed by Serva Blue dye uptake 1, B: 50% fibroblasts survival after a train of eight 100 µs pulses 10, C: definite level of gene expression (beta-galactosidase activity of 0.002 mU/ng DNA) in mesenchymal cells after a train of eight 100 µs square pulses in a medium containing a plasmid encoding the lacZ reporter gene 11, D: 50% fibroblasts permeabilization after a train of eight 100 µs square pulses as assessed by bleomycin uptake 10. There are numerous evidences from experiments on cell suspensions 16-18, on isolated cells 19-21 and on artificial membranes 22-24 that electroporation occurs when the transmembrane potential (V ) induced by the electric field reaches a specific threshold. The value of such m threshold depends on the characteristics of the applied pulses (number, duration and shape) and also on how electroporation is assessed (e.g. by noticing an increase of membrane conductance, by detecting intracellular contents release or by observing cell lysis). Nevertheless most authors report V threshold values in the range from 200 mV to 1 V. m Therefore, a plausible hypothesis that was proposed in the past25, 26 for explaining the dependence of E on σ can be phrased as follows: when an external electric field is applied, the c e induced transmembrane potential (∆V ) depends on the conductivity of the extracellular medium m so that the threshold for the manifestation of the electroporation phenomenon can only be reached under some specific conditions. As it is reported in the appendix, in the case of a spherical cell it is possible to obtain analytical expressions for ∆V when an external electric is applied. And, m taking into account realistic values for the geometry and for the dielectric parameters of the cells, such analytical expressions indeed show that the extracellular conductivity has an effect on the maximum value that ∆V can reach. However, the magnitude of such σ effect on the calculated m e ∆V is insufficient for justifying the large dependences of E on σ that are observed m c e experimentally (e.g. trace A in Fig. 1). Moreover, such analytical expressions are not able to predict the fact that E sensitivity to σ depends on the permeabilization level to be achieved, as c e illustrated in the cases B and D of Fig. 1. In other words, if it is assumed that σ only has an e impact on the electroporation phenomenon by modifying the minimum electric field at which electroporation can be manifested, then traces B and D should be proportional. Hence we believe that the model described by such analytical expressions needs to be improved with other phenomena in order to predict the experimentally observed behaviors. In particular, we propose to include the electroporation phenomenon (i.e. the formation of pores) in the model. Here we hypothesize that the fact that membrane conductivity increases when electroporation occurs explains the observed phenomena. Reasoning behind this hypothesis is: 1) when an external voltage is applied, part of it drops at the membrane but the rest drops in the extracellular and intracellular media; 2) thus, voltage drop at the membrane, V , not only depends m on its conductivity, σ , but also on the conductivity of extracellular and intracellular media (σ m e and σ respectively); 3) when no electroporation occurs, membrane conductivity is very low and i voltage drop at the membrane is almost independent on σ and on σ, as described by the e i equations in the appendix; 4) however, when electroporation occurs because V reaches the m required threshold, the conductivity of the membrane increases abruptly 27 and with a strong dependence on V 24; 5) as a consequence, voltage drop at the membrane is now significantly m dependent on σ and on σ and in turn the membrane conductivity increase is also dependent on e i both conductivities. Then, since membrane conductivity reflects the permeabilization state at the time of the pulse, if is reasonable to expect that post-pulse effects of electroporation (e.g. uptake of molecular dyes, cell lysis, gene electrotransfer) will also be dependent on σ and on σ. e i The purpose of the numerical study presented here is to demonstrate that the above hypothesis can indeed justify the dependence of electroporation efficiency on σ that is e experimentally observed in the case of cells in suspension. In the present report, the methods and the results sections are organized in two subsections. In both sections, the first subsection deals with the description and validation of a membrane conductivity model. The second subsection describes how that membrane conductivity model is employed in order to demonstrate that σ can indeed have a significant effect on the e amount of permeabilization. 2. Materials and methods 2.1 Membrane conductivity model As a preliminary step, before addressing how extracellular conductivity influences permeabilization, we have selected a membrane conductivity model and we have assessed its performance by comparing simulated results with data from an experimental study in which the conductance of a dense cell suspension is measured during electroporation pulses 28. Details about the model and its justification can be found in the results and discussion section. Here we simply specify some aspects concerning the numerical method we employed for validating the performance of the model. We have performed numerical modeling of the conductance of a cell in suspension in a similar way to what is described in 29. In particular, we have used a commercial finite element (FEM) software platform, COMSOL Multiphysics 3.5 (Stockholm, Sweden), to solve the steady- state problem depicted in Fig. 2. The parameter values and the dimensions of the FEM model are summarized in Table 1. We selected the values for w and h (dimensions of the measurement chamber) so that the volume of the cell is equivalent to 19 % of the total volume of the chamber, as in the experimental study 28. The membrane thickness in the model is unrealistically large (50 nm instead of 5 nm) in order to facilitate FEM analysis. We verified that in the much slower simulations with a membrane thickness of 5nm the results do not differ significantly from the results obtained with a 50 nm membrane. Since the membrane conductivity depends on the electric field and, in turn, the electric field depends on the membrane conductivity, an iterative process is required to perform the simulation. In this iterative process, it is computed first the electric field distribution assuming σ m = σ , and then the equivalent V at each point of the membrane is obtained as V =|E |.50 m0 m m membrane nm, a new value for σ is obtained according to Eq. 1 and the process is repeated until a stable m solution is found. If in this iterative process it is modeled that the voltage at the electrodes is present since the first step, in some cases, the solution does not converge easily and oscillations in conductivity appear. Therefore, an alternative approach was tried here in order to facilitate convergence of the solution: the COMSOL simulation was in fact configured as a time transient simulation in which the applied voltage increased smoothly from 0 V to the final voltage and then remained at that final value until the end of the simulation. More specifically, the voltage raise consisted of a ramp function from time 0 to time 0.1 and the simulation finished at time 2. It must be noted that here the time points are indicated without units (e.g. seconds) as they are in fact arbitrary; time has no physical meaning in the simulation, it is employed for facilitating the solving process. For each simulation it was verified that the solution was indeed stable and that no oscillations remained during the phase in which the applied voltage reached its final value. Table 1 Model parameters for simulation of conductance of a single cell in suspension Symbol Value Definition, justification or source ( ) σm s m =s m0 +K eb Vm - 1 Membrane conductivity model Membrane conductivity when V =0, form 30 if membrane σ 2.5· 10-7 S/m m m0 thickness = 5nm σ 1.58 S/m Extracellular conductivity, isotonic NaCl in 28 e1 Extracellular conductivity, isotonic 10% NaCl/90% sucrose 28, see σ 0.3 S/m e2 text for further justification σ 0.5 S/m Intracellular conductivity 31 i δ 50 nm Simulation membrane thickness, see text for justification R 2.8 µm Cell radius 28 V 0 to 6 V Applied potential, to produce fields from 0 to ~6 kV/cm as in 28 p w 4.2 µm Simulation region width h 8.8 µm Simulation region height n 13320 Number of elements in the mesh (FEM parameter) Fig. 2 Geometrical features of a model for a single cell in suspension. This model was solved by means of FEM and an axial symmetry was introduced in order to reduce computation time. The conductivities of the extracellular and intracellular media are constant whereas the conductivity of the membrane (σ ) depends on the local electric field m which in turn depends on σ . The volume fraction of the cell is approximately 20%, as in 28. m 2.2 Modeling membrane conductivity increase dependence on extracellular conductivity For studying the influence of extracellular conductivity on membrane conductivity increase during electroporation, we have employed the previous numerical model with a minor modification of the geometrical dimensions (see Table 2); only the width, w, and the height, h, of the chamber are slightly larger in order to ensure electric field uniformity at distant points from the cell. Further details about the model and its justification, particularly the reason why we change the value of the constant K in the membrane conductivity model for each extracellular conductivity value, are given in the results and discussion section. Table 2 Model parameters for simulation of membrane conductivity dependence on extracellular conductivity in the case of a single cell in suspension Symbol Value Definition, justification or source ( ) σm s m =s m0 +K eb Vm - 1 Membrane conductivity β 16 Constant for σ m 10-9 · : 3.75 for σ , 1.5 for e1 K σ , 1.275 for σ and Constant for σ e2 e3 m 1.2525 for σ e4 σ 2.5· 10-7 S/m Membrane conductivity when V =0, see Table 1 m0 m σ 1 S/m Extracellular conductivity e1 σ 0.1 S/m Extracellular conductivity e2 σ 0.01 S/m Extracellular conductivity e3 σ 0.001 S/m Extracellular conductivity e4 σ 0.5 S/m Intracellular conductivity 31 i δ 50 nm Simulation membrane thickness, see text for justification R 2.8 µm Cell radius 28 V 0 to 5 V Applied potential, to produce fields from 0 to 5 kV/cm p w 5 µm Simulation region width h 10 µm Simulation region height n 16212 Number of elements in the mesh (FEM parameter) 3. Results and discussion 3.1 Membrane conductivity model Membrane conductivity models for electroporation range from simple voltage-sensitive switches 32 to complicated models that are supposed to be able to quantify the total number of pores and their dimensions 31, 33. Here we have decided to minimize assumptions regarding the electroporation process and the nature of the pores and we have chosen to employ a simple model that can be described with a single continuous function: ( ) s =s +K eb Vm - 1 (1) m m0 where σ is the conductivity of the membrane for V =0 (before electroporation occurs) and K m0 m and β are two constants that describe how membrane conductivity increases as the transmembrane potential increases. Under the hypothesis that membrane conductivity rise is due to pores, we can rewrite Eq. 1 as: ( ( )) s =s +s l eb Vm - 1 (2) m m0 pores where σ would be the conductivity of the solution filling the pores, λ would be another pores constant and the term ( ) S =l eb Vm - 1 (3) p would represent the relative area of the pores. Therefore, the constant K in Eq. 1 would depend on σ . pores For determining the value of the constants K and β we have made use of data reported in an experimental study in which the conductivity of a dense cell suspension was measured during electroporation for multiple pulse amplitudes 28. In particular, we manually adjusted the value of the constants K and β so that our model for a cell in suspension (see paragraphs below and Table 1 and Fig. 2) provided conductance measurements that matched the experimental measurements at 2 µs after the beginning of the pulse. This initial adjustment was done for data obtained in isotonic saline (NaCl 0.9%, σ =1.58 S/m 28). Then, we tried to use the same membrane e conductivity model for fitting experimental results obtained in isotonic 10% NaCl/90% sucrose (σ =0.3 S/m) and we discovered that experimental data seemed better approximated if K was e halved (Fig. 3); which would be in agreement with Eq. 2 under the reasonable assumption that σ depends on σ , as justified below. pores e Ionic species such as Na+, Cl- and K+ have diffusion coefficients in water of about 1.5· 10- 9 m2/s. 34 This implies that in a pore with a length of 5 nm (cell membrane thickness) the ionic content will be a good mixture of the ionic contents of both compartments in less than 2 µs after pore creation (further discussed and justified some paragraphs below). Therefore, the conductivity of the pores can be assumed to be: s +s s = e i (4) pores 2 And the value of K for σ , K , relative to the value of K for σ , K , is: e2 2 e1 1 s s +s K = pores2 K = e2 i K (5) 2 s 1 s +s 1 pores1 e1 i In the above case (σ =0.5 S/m , σ =1.58 S/m, σ =0.3 S/m) the value of K would be i e1 e2 2 ~0.4K which is reasonably close to the value obtained by fitting the experimental data 1 (K =0.5K ). As a consequence, for modeling the membrane conductivity increase dependence on 2 1 extracellular conductivity we have decided to follow the model described in Eq. 1 with a K value that depends on the extracellular conductivity as described in Eq. 5. Actually, we have rounded σ to 1.5 S/m so that the expression we used for K is simplified: e1 s +s K =5· 10- 9 e i (6) 2 As it can be observed in Fig. 3, with the exponential model for σ (V ) described by Eq. 1 m m it is possible to reproduce very accurately the conductance measurements experimentally obtained from a cell suspension subjected to electroporation. 28 A remarkable phenomenon that should be pointed out first is that whereas membrane conductivity rise as a function of V is extremely abrupt (model plotted in a logarithmic scale in m the insert of Fig. 3), suspension conductance increase as a function of the applied field magnitude is much smoother. This behavior, which is also observed in living tissues 35, is linked to the main hypothesis of the present study: as |E| increases V also tends to rise which causes an increase of m σ which in turn tends to lessen the increase of V in a sort of negative feedback fashion. m m As it has been mentioned, we manually adjusted the constants K and β of Eq. 1 for matching the simulated data (bottom thick continuous line in Fig. 3) to the experimental data measured in saline (σ = 1.58 S/m). This adjustment yielded: K= 5· 10-9 and β= 16. Then, by using e the same model and constants for σ , we tried to fit the experimental data obtained in isotonic m 10% NaCl/90% sucrose with the extracellular conductivity value reported by the authors of the experimental study 28, σ = 0.158 S/m. However, such fitting was only partially successful, even e when we tried by modifying the values of the constants K and β, we were not able to reproduce exactly the shape of the cell suspension conductance as a function of |E| (data not reported here). On the other hand, by modifying the value σ used in the simulations we noticed that we were e able to get curves that matched the pattern of the experimental data. In particular, a value for σ of e 0.3 S/m yielded an almost perfect matching. Therefore, we are inclined to assume that the actual conductivity of the extracellular medium in 28 was 0.3 S/m (instead of 0.158 S/m). In favor of this assumption we can point out at least another fact besides our simulations results: according to 28, media conductivities were measured before the cell suspensions were prepared, not afterwards, and then, since an outflow of intracellular ions would be favorable during suspension storage because of the concentration gradient (time not indicated in 28), it is reasonable to expect lower values for σ and higher values for σ than the initially measured ones (σ = 0.5 S/m, σ = 0.158 i e i e S/m). As a matter of fact, we verified that by decreasing the value of σ we also obtained almost i perfect fittings without requiring values of σ as high as 0.3 S/m (data not reported here). e Moreover, dense concentrations of microorganisms in suspension, as it is the case, are known to increase significantly the conductivity of the medium due to metabolic byproducts 36. Hence the assumption that σ is larger than 0.158 S/m is quite plausible, together with a possible decrease of e σ. However, for the sake of simplicity, in our simulations we have decided to keep σ constant i i and to assume that σ for isotonic 10% NaCl/90% sucrose is 0.3 S/m. With these values, the e fitting was further improved when K was 2.5· 10-9 instead of 5· 10-9 (see Fig. 3). As pointed out above, this improvement by halving K is in agreement with the model described by Eq. 4 in which the conductivity of the pores, σ , is the average value of σ and σ. pores e i Fig. 3 Experimental and simulated cell suspension relative conductance (conductance at 2 µs / conductance before pulse). Experimental data is from 28 when the extracellular medium is isotonic saline (○) and when the extracellular medium is isotonic 10% NaCl/90% sucrose (●). The simulated results (continuous lines) are based on the model presented in Fig. 2 when the membrane conductivity behaves as it is described in Eq. 3 (see insert). The parameters σ and β (adjusted for isotonic saline experimental data) are equal for all simulated results whereas K has two m0 different values (see text). The two thin dashed lines are sixth order polynomial approximations of the experimental data. Fig. 4 shows that simulated transmembrane potential along the cell behaves according to Eq. 9 in the appendix (i.e. V proportional to cos (θ)) when the electric field magnitude is 1500 m V/cm. On the other hand, for larger field magnitudes (3000 V/cm and 6000 V/cm), V saturates m at values around 1 V at the cell poles facing the hypothetical location of the electrodes. This phenomenon is the consequence of a large increase in membrane conductivity at the areas where V is above a specific threshold (around 1 V in this case) and it has been experimentally observed m in actual cells by employing voltage-sensitive dyes 21.