Peter Kramar

Cell membrane electroporation- Part 1: The phenomenon

Each biological cell, trillions of which build our bodies, is enveloped by its plasma membrane. Composed largely of a bilayer (double layer) of lipids just two molecules thick (about 5 nm), and behaving partly as a liquid and partly as a gel, the cell plasma membrane nonetheless separates and protects the cell from its surrounding environment very reliably and stably. Embedded within the lipid bilayer, also quite stably, are a number of different proteins, some of which act as channels and pumps, providing a pathway for transporting specific molecules across the membrane. Without these proteins, the membrane would be a largely impenetrable barrier. Electrically, the cell plasma membrane can be viewed as a thin insulating sheet surrounded on both sides by aqueous electrolyte solutions. When exposed to a sufficiently strong electric field, the membrane will undergo electrical breakdown, which renders it permeable to molecules that are otherwise unable to cross it. The process of rendering the membrane permeable is called membrane electroporation. Unlike solid insulators, in which an electrical breakdown generally causes permanent structural change, the membrane, with its lipids behaving as a two-dimensional liquid, can spontaneously return to its prebreakdown state. If the exposure is sufficiently short and the membrane recovery sufficiently rapid for the cell to remain viable, electroporation is termed reversible; otherwise, it is termed irreversible. Since its discovery [1]���[3], electroporation has steadily gained ground as a useful tool in various areas of medicine and biotechnology. Today, reversible electroporation is an established method for introducing chemotherapeutic drugs into tumor cells (electrochemotherapy) [4]. It also offers great promise as a technique for gene therapy without the risks caused by viral vectors (DNA electrotransfer) [5]. In clinical medicine, irreversible electroporation is being investigated as a method for tissue ablation (nonthermal electroablation) [6], whereas in biotechnology, it is useful for extraction of biomolecules [7] and for microbial deactivation, particularly in food preservation [8]. This article, the first in a series of three focusing on electroporation, describes the phenomenon at the molecular level of the lipid bilayer, and then proceeds to the cellular level, explaining how exposure of a cell as a whole to an external electric field results in an inducement of voltage on its plasma membrane, its electroporation, and transport thorough the electroporated membrane. The second article will review the most important and promising applications of electroporation, and the third article will focus on the hardware for electroporation (pulse generators and electrodes) and on the need for standards, safety, and certification.

On the electroporation thresholds of lipid bilayers: molecular dynamics simulation investigations.

Electroporation relates to the cascade of events that follows the application of high electric fields and that leads to cell membrane permeabilization. Despite a wide range of applications, little is known about the electroporation threshold, which varies with membrane lipid composition. Here, using molecular dynamics simulations, we studied the response of dipalmitoyl-phosphatidylcholine, diphytanoyl-phosphocholine-ester and diphytanoyl-phosphocholine-ether lipid bilayers to an applied electric field. Comparing between lipids with acyl chains and methyl branched chains and between lipids with ether and ester linkages, which change drastically the membrane dipole potential, we found that in both cases the electroporation threshold differed substantially. We show, for the first time, that the electroporation threshold of a lipid bilayer depends not only on the “electrical” properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails.

Chapter Seven Electroporation of Planar Lipid Bilayers and Membranes

Abstract Strong external electric field can destabilize membranes and induce formation of pores thus increasing membrane permeability. The phenomenon is known as membrane electroporation, sometimes referred to also as dielectric breakdown or electropermeabilization. The structural changes involving rearrangement of the phospholipid bilayer presumably lead to the formation of aqueous pores, which increases the conductivity of the membrane and its permeability to water-soluble molecules which otherwise are deprived of membrane transport mechanisms. This was shown in variety of experimental conditions, on artificial membranes such as planar lipid bilayers and vesicles, as well as on biological cells in vitro and in vivo. While studies of electroporation on artificial lipid bilayers enabled characterization of the biophysical processes, electroporation of biological cells led to the development of numerous biomedical applications. Namely, cell electroporation increases membrane permeability to otherwise nonpermeant molecules, which allows different biological and medical applications including transfer of genes (electrogene transfer), transdermal drug delivery and electrochemotherapy of tumors. In general, the key parameter for electroporation is the induced transmembrane voltage generated by an external electric field due to the difference in the electric properties of the membrane and the external medium, known as Maxwell–Wagner polarization. It was also shown that pore formation and the effectiveness of cell electroporation depend on parameters of electric pulses like number, duration, repetition frequency and electric field strength, where the later is the crucial parameter since increased transmembrane transport due to electroporation is only observed above a certain threshold field. Two main theoretical approaches were developed to describe electroporation. The electromechanical approach considers membranes as elastic or viscoelastic bodies, and applying principles of electrostatics and elasticity predict membrane rupture above critical membrane voltage. A conceptually different approach describing formation and expansion of pores is based on energy consideration; it is assumed that external electric field reduces the free energy barrier for formation of hydrophilic pores due to lower polarization energy of water in the pores compared to the membrane. Combined with stochastic mechanism of pores expansion it can describe experimental data of bilayer membranes. Still, the molecular mechanisms of pore formation and stabilization during electroporation are not fully understood and rigorous experimental conformation of different theories is still lacking. The focus of this chapter is to review experimental and theoretical data in the field of electroporation and to connect biophysical aspects of the process with the phenomenological experimental observations obtained on planar lipid bilayers, vesicles and cells.

Chapter two - Voltage- and Current-Clamp Methods for Determination of Planar Lipid Bilayer Properties

Biological membranes, the barriers that envelope the cell and its inner organelles, play a crucial role in the normal functioning of cells. The simplest model of these biological membranes is the planar lipid bilayer. Because its geometry allows chemical and electrical access to both sides of the bilayer, the physical properties of this model membrane can be easily measured. Usually, a thin bimolecular film composed of specified phospholipids and organic solvent is formed on a small aperture in a hydrophobic partition separating two compartments containing aqueous solutions. From the electrical point of view, a planar lipid bilayer can be considered as an imperfect capacitor; therefore, two electrical properties, capacitance (C) and resistance (R), determine most of its behavior. Electrodes placed in the aqueous compartments on each side of the planar lipid bilayer permit the measurement of current and voltage across the model membrane. The two measuring techniques most commonly used to measure the properties of planar lipid bilayers are voltage-clamp methods and current-clamp methods. The focus of this chapter is to review measurement systems and methods for the determination of the physical properties of planar lipid bilayers.

Monovalent Ions and Water Dipoles in Contact with Dipolar Zwitterionic Lipid Headgroups-Theory and MD Simulations

The lipid bilayer is a basic building block of biological membranes and can be pictured as a barrier separating two compartments filled with electrolyte solution. Artificial planar lipid bilayers are therefore commonly used as model systems to study the physical and electrical properties of the cell membranes in contact with electrolyte solution. Among them the glycerol-based polar phospholipids which have dipolar, but electrically neutral head groups, are most frequently used in formation of artificial lipid bilayers. In this work the electrical properties of the lipid layer composed of zwitterionic lipids with non-zero dipole moments are studied theoretically. In the model, the zwitterionic lipid bilayer is assumed to be in contact with aqueous solution of monovalent salt ions. The orientational ordering of water, resulting in spatial variation of permittivity, is explicitly taken into account. It is shown that due to saturation effect in orientational ordering of water dipoles the relative permittivity in the zwitterionic headgroup region is decreased, while the corresponding electric potential becomes strongly negative. Some of the predictions of the presented mean-field theoretical consideration are critically evaluated using the results of molecular dynamics (MD) simulation.

A System for the Determination of Planar Lipid Bilayer Breakdown Voltage and Its Applications

In this paper, we focus on measurement principles used in electroporation studies on planar lipid bilayers. In particular, we point out the voltage-clamp measurement principle that has great importance when the breakdown voltage of a planar lipid bilayer is under consideration; however, it is also appropriate for the determination of other planar lipid bilayer electrical properties such as resistance and capacitance. A new experimental system that is based on the voltage-clamp measurement principle is described. With the use of a generator that can generate arbitrary-type signals, many specific shapes of a voltage signal could be generated, and therefore, the experimental system is appropriate for a broad spectrum of measurements.

Measurement protocol for planar lipid bilayer viscoelastic properties

This paper describes how to estimate planar lipid bilayers elasticity module E and surface tension sigma by means of measuring its breakdown voltage and using Dimitrovs viscoelastic model of electric field-induced breakdown of lipid bilayers. Planar lipid bilayers (BLMs) were made of two components: 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) and 1-palmitoyl 2-oleoyl phosphatidylserine (POPS) in five different compositions. Folding method for forming planar lipid bilayers in the salt solution of 100 mM KCl was used. Breakdown voltages Ubr and membrane life times tbr were measured by means of applying linear rising voltage signals of seven different slopes. Specific capacitances cBLM of bilayers were measured with charge pulse method. Then Dimitrovs viscoelastic model was fitted to measured data allowing for estimation of elasticity module and surface tension of the lipid bilayer. Our results show that onecomponent bilayers composed from POPS were more stable and thus having higher breakdown voltages and elasticity moduli then bilayers composed of POPC. Surface tension values were similar regardless of the membrane composition. Values of the elasticity (E) and surface tension (sigma) are comparable to those published in the literature. We conclude that the protocol used, though time consuming, is an alternative to other methods used for determination of bilayers mechanical properties.

Electroporation Threshold of POPC Lipid Bilayers with Incorporated Polyoxyethylene Glycol (C12E8)

Electroporation relates to a phenomenon in which cell membranes are permeabilized after being exposed to high electric fields. On the molecular level, the mechanism is not yet fully elucidated, although a considerable body of experiments and molecular dynamic (MD) simulations were performed on model membranes. Here we present the results of a combined theoretical and experimental investigation of electroporation of palmitoy-oleoyl-phosphatidylcholine (POPC) bilayers with incorporated polyoxyethylene glycol (C12E8) surfactants. The experimental results show a slight increase of the capacitance and a 22% decrease of the voltage breakdown upon addition of C12E8 to pure POPC bilayers. These results were qualitatively confirmed by the MD simulations. They later revealed that the polyoxyethylene glycol molecules play a major role in the formation of hydrophilic pores in the bilayers above the electroporation threshold. The headgroup moieties of the latter are indeed embedded in the interior of the bilayer, which favors formation of water wires that protrude into its hydrophobic core. When the water wires extend across the whole bilayer, they form channels stabilized by the C12E8 head groups. These hydrophilic channels can transport ions across the membrane without the need of major lipid head-group rearrangements.

Internal configuration and electric potential in planar negatively charged lipid head group region in contact with ionic solution.

The lipid bilayer composed of negatively charged lipid 1-palmitoyl-3-oleoyl-sn-glycero-3-phosphatidylserine (POPS) in contact with an aqueous solution of monovalent salt ions was studied theoretically by using the mean-field modified Langevin-Poisson-Boltzmann (MLPB) model. The MLPB results were tested by using molecular dynamic (MD) simulations. In the MLPB model the charge distribution of POPS head groups is theoretically described by the negatively charged surface which accounts for negatively charged phosphate groups, while the positively charged amino groups and negatively charged carboxylate groups are assumed to be fixed on the rod-like structures with rotational degree of freedom. The spatial variation of relative permittivity, which is not considered in the well-known Gouy-Chapman (GC) model or in MD simulations, is thoroughly derived within a strict statistical mechanical approach. Therefore, the spatial dependence and magnitude of electric potential within the lipid head group region and its close vicinity are considerably different in the MLPB model from the GC model. The influence of the bulk salt concentration and temperature on the number density profiles of counter-ions and co-ions in the lipid head group region and aqueous solution along with the probability density function for the lipid head group orientation angle was compared and found to be in qualitative agreement in the MLPB and MD models.

Specific electrical capacitance and voltage breakdown as a function of temperature for different planar lipid bilayers

The breakdown voltage and specific electrical capacitance of planar lipid bilayers formed from lipids isolated from the membrane of archaeon Aeropyrum pernix K1 as a function of temperature were studied and compared with data obtained previously in MD simulation studies. Temperature dependence of breakdown voltage and specific electrical capacitance was measured also for dipalmitoylphosphatidylcholine (DPPC) bilayers and bilayers formed from mixture of diphytanoylphosphocholine (DPhPC) and DPPC in ratio 80:20. The breakdown voltage of archaeal lipids planar lipid bilayers is more or less constant until 50°C, while at higher temperatures a considerable drop is observed, which is in line with the results from MD simulations. The breakdown voltage of DPPC planar lipid bilayer at melting temperature is considerably higher than in the gel phase. Specific electrical capacitance of planar lipid bilayers formed from archaeal lipids is approximately constant for temperatures up to 40°C and then gradually decreases. The difference with MD simulation predictions is discussed. Specific electrical capacitance of DPPC planar lipid bilayers in fluid phase is 1.75 times larger than that of the gel phase and it follows intermediated phases before phase transition. Increase in specific electrical capacitance while approaching melting point of DPPC is visible also for DPhPC:DPPC mixture.