A theoretical analysis of the feasibility of a singularity-induced micro-electroporation system

Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1) insulator thickness and (2) electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids.     

Galvanic apparent internal impedance: An intrinsic tissue property

Using basic galvanic cell principles, the ability of tissues to generate electrical current through electrolysis was characterized. Studying Zn/Cu electrolysis in animal organs revealed a fundamental and measurable tissue-specific property - the galvanic apparent internal impedance (GAII), that is most likely related to the salt bridge function of tissues delineated by electrodes. Further to the fundamental knowledge acquired, GAII enables a new diagnostic method to distinguish between tissue types and to determine their health status without a need for expensive calibration, as often required when external power source is used. We demonstrated the GAII sensitivity in detecting tissue ablation with microwave heating or irreversible electroporation. The results open the way for a novel, inexpensive self-powered tissue diagnostic system for a wide range of applications such as minimally invasive tissue health status, ischemia, hydration, real time intra-operative control of minimally invasive surgery, medical imaging, virtual biopsy and many others.     

Variable electric fields for high throughput electroporation protocol design in curvilinear coordinates

The mathematical solution to the electric field equation in cylindrical coordinates, has suggested to us a new experimental methodology and device for reducing experimental effort in designing electroporation protocols. Using a new cylindrical electroporation system, we show, with an Escherichia coli cell model, how key electroporation parameters emerge precisely from single experiments rather than through interpolation from numerous experiments in the conventional Cartesian electroporation system.     

Dependence of the efficacy of transfecting muscle fibers in vivo on electroporation conditions

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electroporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Three dimensional electrode array for cell lysis via electroporation

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.     

Influence of Electroporation Conditions on Transfection of Muscle Fibers in Vivo

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electoporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

Previous studies have shown greater fluorophore uptake during electroporation on the anode-facing side of the cell than on the cathode-facing side. Based on these observations, we hypothesized that hyperpolarizing a cell before electroporation would decrease the requisite pulsed electric field intensity for electroporation outcomes, thereby yielding a higher probability of reversible electroporation at lower electric field strengths and a higher probability of irreversible electroporation (IRE) at higher electric field strengths. In this study, we tested this hypothesis by hyperpolarizing HL-60 cells using ionomycin before electroporation. These cells were then electroporated in a solution containing propidium iodide, a membrane integrity indicator. After 20 min, we added trypan blue to identify IRE cells. Our results showed that hyperpolarizing cells before electroporation alters the pulsed electric field intensity thresholds for reversible electroporation and IRE, allowing for greater control and selectivity of electroporation outcomes.     

Numerical modeling of in vivo plate electroporation thermal dose assessment

Electroporation is an approach used to enhance the transport of large molecules to the cell cytosol in which a targeted tissue region is exposed to a series of electric pulses. The cell membrane, which normally acts as a barrier to large molecule transport into the cell interior, is temporarily destabilized due to the development of pores in the cell membrane. Consequently, agents that are ordinarily unable enter the cell are able to pass through the cell membrane. Of possible concern when exposing biological tissue to an electric field is thermal tissue damage associated with joule heating. This paper explores the thermal effects of various geometric, biological, and electroporation pulse parameters including the blood vessel presence and size, plate electrode configuration, and pulse duration and frequency. A three-dimensional transient finite volume model of in vivo parallel plate electroporation of liver tissue is used to develop a better understanding of the underlying relationships between the physical parameters involved with tissue electroporation and resulting thermal damage potential.     

高压电场不可逆电穿孔诱发A549肺癌细胞凋亡的实验研究

目的：不可逆电穿孔是治疗肿瘤的新兴技术,本文探讨高压电场引起的不可逆电穿孔诱发A549肺癌细胞凋亡的特点。方法：选择处于生长周期的A549细胞,共分为A—G7个组进行研究,其中A组为不施加电场的空白对照组,B-G组为实验组,B组施加500V／cm强度高压电场,G组施加1750V／cm的高压电场,BG组之间各组的高压电场强度间隔为250V／cm。采取细胞抑制实验、不可逆电穿孔示踪实验、细胞凋亡实验,检验A549细胞细胞凋亡与电场强度的关系。结果：①各实验组与对照组、各实验组之间的细胞抑制率,均存在显著性差异（P〈0．05）;②电场强度≥1000V／cm时,细胞不可逆电穿孔率明显增加,有统计学意义（P〈0．05）;电场强度≥1500V／cm时,细胞不可逆电穿孔率增加不明显,无统计学意义（P〉0．05）;③电场强度≥1250V／cm时,细胞早期凋亡率明显增加,有统计学意义（P〈0．05）。结论：高压电场不可逆电穿孔诱发A549肺癌细胞发生早期凋亡的强度为1250V／cm,发生晚期凋亡的强度为1500V／cm,且凋亡率随着电场强度的增加持续升高。这对于高压电场不可逆电穿孔效应引起的肿瘤细胞凋亡机制的研究具有重要意义。     

Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment.     

Electroporation of the photosynthetic membrane: A study by intrinsic and external optical probes

The study examines the relationship between electric field-induced conductivity and permeability changes in a biological membrane (electroporation) and the amplitude-duration parameters of the externally applied electric field. These reversible changes were characterized in giant photosynthetic membrane vesicles by means of the calibrated response of an intrinsic voltage-sensitive optical probe (electrophotoluminescence) and by the uptake studies of dextran-FITC fluorescent probes of different molecular weights. We quantitatively monitored electric field-induced conductivity changes by translating the electrophotoluminescence changes into conductivity changes. This was carried out by measuring the attenuation of the electrophotoluminescent signal after the addition of known amounts of gramicidin. The results demonstrate that electroporation involves the reversible formation of discrete holes in the membrane having radii <5.8 nm. The total area of the electric field-induced holes was 0.075% of the total surface of the vesicle. The formation of the electropores was affected differently by the electric field strength than by its duration. Increase in electric field strength caused increase in the total area of the vesicle that undergoes electroporation. Increase in the duration of the electric field increases the area of single electropores. Each of the two electric parameters can be rate limiting for the dynamics of electropore formation. These results are in accordance with the model of electroporation based on electric field-induced expansion of transient aqueous holes.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

Self-electroporation as a model for fusion pore formation

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237-1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than approximately 3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.     

The importance of electric field distribution for effective in vivo electroporation of tissues.

Cells exposed to short and intense electric pulses become permeable to a number of various ionic molecules. This phenomenon was termed electroporation or electropermeabilization and is widely used for in vitro drug delivery into the cells and gene transfection. Tissues can also be permeabilized. These new approaches based on electroporation are used for cancer treatment, i.e., electrochemotherapy, and in vivo gene transfection. In vivo electroporation is thus gaining even wider interest. However, electrode geometry and distribution were not yet adequately addressed. Most of the electrodes used so far were determined empirically. In our study we 1) designed two electrode sets that produce notably different distribution of electric field in tumor, 2) qualitatively evaluated current density distribution for both electrode sets by means of magnetic resonance current density imaging, 3) used three-dimensional finite element model to calculate values of electric field for both electrode sets, and 4) demonstrated the difference in electrochemotherapy effectiveness in mouse tumor model between the two electrode sets. The results of our study clearly demonstrate that numerical model is reliable and can be very useful in the additional search for electrodes that would make electrochemotherapy and in vivo electroporation in general more efficient. Our study also shows that better coverage of tumors with sufficiently high electric field is necessary for improved effectiveness of electrochemotherapy.     

Electroporation of extraneous proteins into CHO cells: Increased efficacy by utilizing centrifugal force and microsecond electrical pulses

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.     

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.     

A multi-channel electroporation microchip for gene transfection in mammalian cells

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.     

Changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy.

Cells can be transiently permeabilized by exposing them briefly to an intense electric field (a process called "electroporation"), but it is not clear what structural changes the electric field induces in the cell membrane. To determine whether membrane pores are actually created in the electropermeabilized cells, rapid-freezing electron microscopy was used to examine human red blood cells which were exposed to a radio-frequency electric field. Volcano-shaped membrane openings appeared in the freeze-fracture faces of electropermeabilized cell membranes at intervals as short as 3 ms after the electrical pulse. We suggest that these openings represent the membrane pathways which allow entry of macromolecules (such as DNA) during electroporation. The pore structures rapidly expand to 20-120 nm in diameter during the first 20 ms of electroporation, and after several seconds begin to shrink and reseal. The distribution of pore sizes and pore dynamics suggests that interactions between the membrane and the submembrane cytoskeleton may have an important role in the formation and resealing of pores.     

Stochastic model for electric field-induced membrane pores. Electroporation

Electric impulses (1-20 kV cm-1, 1-5 microseconds) cause transient structural changes in biological membranes and lipid bilayers, leading to apparently reversible pore formation ( electroporation ) with cross-membrane material flow and, if two membranes are in contact, to irreversible membrane fusion ( electrofusion ). The fundamental process operative in electroporation and electrofusion is treated in terms of a periodic lipid block model, a block being a nearest-neighbour pair of lipid molecules in either of two states: (i) the polar head group in the bilayer plane or (ii) facing the centre of a pore (or defect site). The number of blocks in the pore wall is the stochastic variable of the model describing pore size and stability. The Helmholtz free energy function characterizing the transition probabilities of the various pore states contains the surface energies of the pore wall and the planar bilayer and, if an electric field is present, also a dielectric polarization term (dominated by the polarization of the water layer adjacent to the pore wall). Assuming a Poisson process the average number of blocks in a pore wall is given by the solution of a non-linear differential equation. At subcritical electric fields the average pore size is stationary and very small. At supercritical field strengths the pore radius increases and, reaching a critical pore size, the membrane ruptures (dielectric breakdown). If, however, the electric field is switched off, before the critical pore radius is reached, the pore apparently completely reseals to the closed bilayer configuration (reversible electroporation ).     

Optimization of an electroporation protocol using the K562 cell line as a model: role of cell cycle phase and cytoplasmic DNAses

The improvement of gene therapy protocols is intimately related to the establishment of efficient gene transfer methods. Electroporation has been increasingly employed in in vitro and in vivo protocols, and much attention has been given to increasing its transfection potential. The method is based on the application of an electric field of short duration and high voltage to the cells, forming reversible pores through which molecules can enter the cell. In this work, we describe the optimization of a protocol for the electroporation of K562 cells involving the combination of electric field, resistance and capacitance values. Using RPMI 1640 as pulsing buffer and 30 μg of pEGFP-N1 plasmid, 875 V cm⁻¹, 500 μF and infinite resistance, we achieved transfection rates of 82.41 ± 3.03%, with 62.89 ± 2.93% cell viability, values higher than those reported in the literature. Analyzing cell cycle after electroporation, with three different electric field conditions, we observed that in a heterogeneous population of cells, viability of G₁ cells is less affected by electroporation than that of cells in late S and G₂/M phases. We also observed that efficiency of electroporation can be improved using the DNAse inhibitor Zn, immediately after the pulse. These results can represent a significant improvement of current methods of electroporation of animal and plant cells.