Osmotically induced membrane tension facilitates the triggering of living cell electropermeabilization

Very little is known about the molecular mechanisms supporting living cell membrane electropermeabilization. This concept is based on the local membrane permeability induced by cell exposure to brief and intense external electric field pulses. During the electric field application, an electro-induced membrane electric potential difference is created that is locally associated with the dielectric properties of the plasma membrane. When the new membrane electric potential difference locally reaches a critical value, a local alteration of the membrane structure is induced and leads to reversible permeabilization. In our study, we attempted to determine whether mechanical tension could modulate the triggering of membrane electropermeabilization. Change in lateral tension of Chinese Hamster Ovary cell membrane has been osmotically induced. Cell electropermeabilization was performed in the minute time range after the osmotic stress, i.e., before the regulatory volume decrease being activated by the cell. Living cell electropermeabilization was analyzed on cell population using flow cytometry. We observed that electropermeabilization triggering was significantly facilitated when the lateral membrane tension was increased. The main conclusion is that the critical value of transmembrane potential needed to trigger membrane electropermeabilization, is smaller when the membrane is under lateral mechanical constraint. This supports the hypothesis that both mechanical and electrical constraints play a key role in transient membrane destabilization.     

Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part I. Increased efficiency of permeabilization

The paper presents a comparative study of electropermeabilization of cells in suspension by unipolar and symmetrical bipolar rectangular electric pulses. While the parameters of electropermeabilization by unipolar pulses have been investigated extensively both in cell suspensions and in tissues, studies using bipolar pulses have been rare, partly due to the lack of commercially available bipolar pulse generators with pulse parameters suitable for electropermeabilization. We have developed a high-frequency amplifier and coupled it to a function generator to deliver high-voltage pulses of programmable shapes. With symmetrical bipolar pulses, the pulse amplitude required for the permeabilization of 50% of the cells was found to be approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing the death of 50% of the cells. Bipolar pulses also led to more than 20% increase in the uptake of lucifer yellow. We show that these results have a theoretical background, because bipolar pulses (i) counterbalance the asymmetry of the permeabilized areas at the poles of the cell which is introduced by the resting transmembrane voltage, and (ii) increase the odds of permeabilization of cells having a nonspherical shape or a nonhomogeneous membrane. If similar results are also obtained in tissues, bipolar pulse generators could in due course gain a wide, or even a predominant use in cell membrane electropermeabilization.     

The influence of medium conductivity on electropermeabilization and survival of cells in vitro.

Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.     

The sensitivity of cells and tissues to exogenous fields: effects of target system initial state

The effect of the initial biochemical or metabolic state of a cell membrane target pathway on its sensitivity to exogenous electromagnetic (EMF) fields is considered. It is shown that the resting or initial transmembrane voltage can affect the frequency response of the membrane pathway and substantially alter the signal to thermal noise threshold (SNR) of the target. EMF sensitivity is examined using a model which describes the response to applied fields of both single cells and cells in gap junction contact via a distributed parameter electrical circuit analog, wherein a voltage-dependent membrane impedance, relating to the initial biochemical state of the target cell(s), is considered. Application of the Hodgkin-Huxley K(+)-conduction pathway membrane to this model results, at a given transmembrane voltage, in a preferential array response to applied field frequencies in the 1-100 Hz range, centered at approximately 16 Hz for 1-10 mm array lengths. Extension of the model to consider the voltage dependence of the Hodgkin-Huxley K+ pathway results in a significant modulation of array frequency response with changing membrane resting potential. The result is EMF sensitivity (SNR) depends upon the initial state of the target tissue, providing a possible explanation of why, e.g., repairing, rather than resting, bone exhibits a physiologically relevant response to certain weak EMF signals.     

The temperature effect during pulse application on cell membrane fluidity and permeabilization

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.     

Techniques of signal generation required for electropermeabilization. Survey of electropermeabilization devices

Electropermeabilization is a phenomenon that transiently increases permeability of the cell plasma membrane. In the state of high permeability, the plasma membrane allows ions, small and large molecules to be introduced into the cytoplasm, although the cell plasma membrane represents a considerable barrier for them in its normal state. Besides introduction of various substances to cell cytoplasm, permeabilized cell membrane allows cell fusion or insertion of proteins to the cell membrane. Efficiency of all these applications strongly depends on parameters of electric pulses that are delivered to the treated object using specially developed electrodes and electronic devices--electroporators. In this paper we present and compare most commonly used techniques of signal generation required for electropermeabilization. In addition, we present an overview of commercially available electroporators and electroporation systems that were described in accessible literature.     

Nonlinear current-voltage relationship of the plasma membrane of single CHO cells

The application of electric field pulses to Chinese Hamster Ovary (CHO) cells causes membrane electroporation (MEP). If a voltage or current ramp is applied to the cellular membrane of a single CHO cell, the membrane conductance increases nonlinearly with field strength reaching saturation. In particular, the kinetics of the induced conductance changes represents the data basis for the interpretation in terms of underlying structural changes. The current/voltage characteristic is found to be continuous, but displays occasionally a sharp increase in the conductance. The step-like increases are interpreted to reflect the formation of one (or more) larger pore(s). The analysis of current clamp data yields pores of radius (r(p)) in the range of 2.5< or =r(p)/nm< or =20; the pores of the voltage clamp data are in the range of 2.5< or =r(p)/nm< or =55. The larger pores occur predominantly during hyperpolarising and less frequently during depolarising conditions, respectively. The different kinetics of pore formation in the hyperpolarising condition, where the inward field increases, and the depolarising condition, where the inward field first decreases and then increases in the opposite direction, suggests structural asymmetry with respect to the direction of the electric membrane field. At the required higher voltage, the effect of the resting potential is negligibly small.     

The in-plane switching of homogeneously aligned nematic liquid crystals

We have investigated the electro-optical effects and physical switching principle of homogeneously aligned nematic liquid crystals when applying an in-plane electric field with interdigital electrodes. By using the in-plane switching (IPS) of the liquid crystals which is achieved by the in-plane electric field, the viewing angle characteristics of the electro-optical effects were confirmed to be far superior to those of the conventional twisted nematic mode in which the electric field is applied along the direction perpendicular to the substrates. The non-reversal region of grey scales was extremely wide in which a high contrast ratio was kept, even along quite an oblique direction in the IPS mode. In order to clarify the switching principle of the liquid crystals in the IPS mode, a simplified expression describing the threshold behaviour of the device was derived with the assumption that a uniform in-plane electric field was applied along a direction perpendicular to the director and parallel to the homogeneously aligned nematic slab, and found to be sufficiently able to explain the experimental results. First, a critical field at which the liquid crystals just began to twist, was found to be proportional to the reciprocal of the cell gap. Second, it was the electric field and not the voltage that drives the liquid crystals. This relationship was due to the independence of the electric field regarding the liquid crystal layer normal direction. So the threshold voltage in the IPS mode was strongly dependent on the variation of the cell gap. For the dynamical response mechanism of the liquid crystals to the in-plane electric field, the switching on and off processes of the liquid crystals were analysed quantitatively. The relaxation time of the liquid crystals when removing the electric field could be described as proportional to the square of the cell gap. A thinner cell gap also proved to be effective in obtaining a fast response time in the IPS mode. In contrast, the switching on time when applying the in-plane electric field was found to be inversely proportional to the difference between the square of the electric field strength and the square of the critical electric field strength at which the liquid crystals began to deform.     

Modeling the induction of lipid membrane electropermeabilization

Experiments show significant effects of an electric field on lipid membrane, leading to a pore formation when a high intensity field is applied. The phenomenon of electroporation is preceded by the induction and expansion of defects, responsible for the pre-pore excitation. We examine the mechanism of the induction of the field-driven defects by Monte Carlo simulations. The study is based on the improved Pink's model, which includes explicit interactions between the polar heads and energy of interactions between the heads and the field. No anomalous deformation of the molecules is considered. The study, provided for bilayer dipalmitoyl-phosphatidylcholine (DPPC) membrane in the gel (300 K) and fluid (330 K) phases, shows dependence of the membrane conformational and energetical state on the value of the electric field. We observe that the electric field affects the number of molecules in the gel and in the fluid states. In the layer at the negative potential, when the transmembrane voltage is above U(c) approximately 280 mV, lipid heads abruptly reorient and the number of local spots with fluid conformation increases. The other layer slightly tends to tighten its structure, producing additional mechanical stress between layers. Lipids showed complete insensitivity to the electric field within physiological limits, U<70 mV.     

Double-sided anodic titania nanotube arrays: a lopsided growth process

In the past decade, the pore diameter of anodic titania nanotubes was reported to be influenced by a number of factors in organic electrolyte, for example, applied potential, working distance, water content, and temperature. All these were closely related to potential drop in the organic electrolyte. In this work, the essential role of electric field originating from the potential drop was directly revealed for the first time using a simple two-electrode anodizing method. Anodic titania nanotube arrays were grown simultaneously at both sides of a titanium foil, with tube length being longer at the front side than that at the back side. This lopsided growth was attributed to the higher ionic flux induced by electric field at the front side. Accordingly, the nanotube length was further tailored to be comparable at both sides by modulating the electric field. These results are promising to be used in parallel configuration dye-sensitized solar cells, water splitting, and gas sensors, as a result of high surface area produced by the double-sided architecture.     

Electrochromism of merocyanine dyes as detector of internal charges in sandwich arrangements with molecular crystals

The photosensitized charge transfer reaction from a merocyanine dye laser to a p-chloranil single crystal is studied. It was found that the maximum of the action spectrum of the photocurrent shifts by several nanometers when the externally applied voltage or the illuminating light level is increased. A model is proposed which interprets these observations as being due to the induced electric field of a layer of ionized dye molecules at the very surface of the single crystal.     

Electropermeabilization of human cultured cells grown in monolayers: Incorporation of monoclonal antibodies

Cultured cells grown in monolayers were permeabilized by applying low-voltage electric pulses. The permeabilized cells were able to incorporate extracellular nucleotides into their DNA, as well as various monoclonal antibodies as determined by indirect immunofluorescence. Complete reversibility and long term viability were retained. Both applied voltage and pulse duration were important parameters for this method and the results were different for different cell lines.     

Passivity of titanium,part 1: film growth model diagnostics

Diagnostic criteria for the growth of the anodic oxide film on titanium in H₂SO₄ are reported. The criteria apply to the generalized high field model, which postulates that the electric field within the film is dependent upon the film thickness, and the point defect model, which describes the electric field as being constant during film growth. The diagnostic criteria show that the PDM more realistically models film growth than does the HFM, and we conclude that in this system the electric field strength is invariant with applied voltage and film thickness. The constancy of the electric field in the passive film on titanium, as demonstrated in this work, is attributed to band-to-band Esaki tunneling, which buffers the electric field against changes in the applied voltage and film thickness.     

Effects of Applied Electric Fields on Drop—Interface and Drop—Drop Coalescence*

In this work, coalescence of a single organic or aqueous drop with its homophase at a horizontal liquid interface was investigated under applied electric fields. The coalescence time was found to decrease for aqueous drops as the applied voltage was increased, regardless of the polarity of the voltage. For organic drops, the coalescence time increased with increasing applied voltage of positive polarity and decreased with increasing applied voltage of negative polarity. Under an electric field, the coalescence time of aqueous drops decreases due to polarization of both the drop and the flat interface. The dependency of organic drop-interface coalescence on the polarity of the electric field may be a result of the negatively charged organic surface in the aqueous phase. Due to the formation of a double layer, organic drops are subjected to an electrostatic force under an electric field, which, depending on the field polarity, can be attractive or repulsive. Pair-drop coalescence of aqueous drops in the organic phase was also studied. Aqueous drop-drop coalescence is facilitated by polarization and drop deformation under applied electric fields. Without applied electric fields, drop deformation increases the drainage time of the liquid film between two approaching drops. Therefore, a decrease in the interfacial tension, which causes drop deformation, accelerates drop-drop coalescence under an electric field and inhibits drop coalescence in the absence of an electric field.     

Modelling the internal field distribution in human erythrocytes exposed to MW radiation

This paper studies the internal electric field distribution in human erythrocytes exposed to MW radiation. For this purpose, an erythrocyte cell model is exposed to linearly polarized electromagnetic (EM) plane waves of frequency 900 MHz and the electric field within the cell is calculated by using a finite element (FE) technique with adaptive meshing. The results obtained show the dependence of the induced electric field distribution on the main modelling parameters, i.e., the electrical properties (permittivity and conductivity) of the membrane and cytoplasm and the orientation of the cell with respect to the applied field. It is found that for certain orientations, the field amplification within the membrane of the erythrocyte shape cell can be higher than the one observed in an equivalent simple spheroidal geometry cell, commonly used in bioelectromagnetism. The present work shows that a better insight of the interaction of electromagnetic fields with basic biological structures is obtained when the most possible realistic cell shape is used.     

Model of a confined spherical cell in uniform and heterogeneous applied electric fields

Cells exposed to electric fields are often confined to a small volume within a solid tissue or within or near a device. Here we report on an approach to describing the frequency and time domain electrical responses of a spatially confined spherical cell by using a transport lattice system model. Two cases are considered: (1) a uniform applied field created by parallel plane electrodes, and (2) a heterogeneous applied field created by a planar electrode and a sharp microelectrode. Here fixed conductivities and dielectric permittivities of the extra- and intracellular media and of the membrane are used to create local transport models that are interconnected to create the system model. Consistent with traditional analytical solutions for spherical cells in an electrolyte of infinite extent, in the frequency domain the field amplification, G(m) (f) is large at low frequencies, f<1 MHz. G(m) (f) gradually decreases above 1 MHz and reaches a lower plateau at about 300 MHz, with the cell becoming almost "electrically invisible". In the time domain the application of a field pulse can result in altered localized transmembrane voltage changes due to a single microelectrode. The transport lattice approach provides modular, multiscale modeling capability that here ranges from cell membranes (5 nm scale) to the cell confinement volume ( approximately 40 microm scale).     

PHOTOINITIATED VECTORIAL TRANSMEMBRANE ELECTRON TRANSFER IN BILAYERS SENSITIZED BY A FACE TO FACE TRIPORPHYRIN ACTING AS A MOLECULAR ELECTRONIC DEVICE. AMPLIFICATION DUE TO IONIC COUPLING

Under light excitation transmembrane electron transfer is observed when a stacked Zn-Cu-Zn triporphyrin is incorporated in a bilayer between aqueous redox phases. The electric polarization of the membrane due to the photoinduced transmembrane charge flux drives ion transport. This effect increases the net charge transfer across the system, giving rise to an amplification similar to a field effect transistor. Thus this system can be considered an organic phototransistor.     

TiO2 photoanodes prepared by cathodic electrophoretic deposition in 2-propanol: effect of the electric field and deposition time

This paper evaluates the influence of electric field and deposition time applied on cathodic electrophoretic formation of TiO₂ films in organic medium (2-propanol). The film morphology was tracked by measuring the deposited mass and film thickness. The variation in film porosity was correlated with the apparition of surface states distribution in the cyclic voltammetric characterization in the dark, due to grain boundaries defects generated in the contact of the TiO₂ particles. The open-circuit voltage decay curves showed that there is no formation of deep energy states inside the band gap of the TiO₂. The photopotential of the films increased until a critical thickness but the photocurrents showed to be dependent on operational variables, due to the fact that anodic polarization in thin films increases the electric field generated by the illumination at the ITO/TiO₂ interface, favoring the transport of the photogenerated electrons to the rear contact.