电穿孔基因导入技术在临床前与临床试验中的应用

电穿孔基因作为非病毒递送手段之一,可安全、有效地将外源核酸导入靶组织或器官。近年来用电穿孔法进行基因治疗的报道不断增多,优势日趋显著。本文重点阐述了电穿孔在基因治疗中的优势、影响因素及在临床前和临床试验中应用。     

脉冲电场在医学中的基础研究及电穿孔的临床应用

脉冲电场利用方波直流脉冲发生器改变细胞膜的通透性,并在细胞膜上形成纳米级细孔,其被称为电穿孔是一种新型微创技术,分为可逆电穿孔(reversible electroporation)及不可逆电穿孔(irreversible electroporation)。在过去的四十年,电穿孔大量的实验研究及其自身的优点及先进性,使电穿孔相关的技术已被允许应用与临床。目前临床和实验中应用电穿孔的化疗药物已有十余种,通过电穿孔进行基因转染及DNA疫苗的研发已取得巨大成功。尤其近几年发展的非热能的不可逆电穿孔对实体肿瘤的消融作用,为肿瘤治疗提供新的思路,因其比其他局部治疗方法:具有治疗时间短,减少间接热损伤,对毗邻主要血管的肿瘤组织有消融能力等优点引起了对不可逆电穿孔巨大的临床研究兴趣。本文就电穿孔的基本理论,电化学治疗,基因电转染及不可逆电穿孔的临床应用进行探讨。     

Actin networks regulate the cell membrane permeability during electroporation

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.     

Network for Development of Electroporation-Based Technologies and Treatments: COST TD1104

Exposure of biological cells to a sufficiently strong external electric field results in increased permeability of cell membranes, referred to as “electroporation.” Since all types of cells (animal, plant and microorganism) can be effectively electroporated, electroporation is considered to be a universal method and a platform technology. Electroporation has become a widely used technology applicable to, e.g., cancer treatment, gene transfection, food and biomass processing and microbial inactivation. However, despite significant progress in electroporation-based applications, there is a lack of coordination and interdisciplinary exchange of knowledge between researchers from different scientific domains. Thus, critical mass for new major breakthroughs is missing. This is why we decided to establish cooperation between research groups working in different fields of electroporation. Cooperation in Science and Technology (COST), which funds networking and capacity-building activities, presents a perfect framework for such scientific cooperation. This COST action aims at (1) providing necessary steps toward EU cooperation of science and technology to foster basic understanding of electroporation; (2) improving communication between research groups, resulting in streamlining European research and development activities; and (3) enabling development of new and further development of existing electroporation-based applications by integrating multidisciplinary research teams, as well as providing comprehensive training for early-stage researchers. Results of this COST action will provide multiple societal, scientific and technological benefits from improving existing electroporation-based applications to adding new ones in the fields of medicine, biotechnology and environmental preservation.     

Molecular-Level Characterization of Lipid Membrane Electroporation using Linearly Rising Current

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-μm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.     

Electroporation in Food Processing and Biorefinery

Electroporation is a method of treatment of plant tissue that due to its nonthermal nature enables preservation of the natural quality, colour and vitamin composition of food products. The range of processes where electroporation was shown to preserve quality, increase extract yield or optimize energy input into the process is overwhelming, though not exhausted; e.g. extraction of valuable compounds and juices, dehydration, cryopreservation, etc. Electroporation is—due to its antimicrobial action—a subject of research as one stage of the pasteurization or sterilization process, as well as a method of plant metabolism stimulation. This paper provides an overview of electroporation as applied to plant materials and electroporation applications in food processing, a quick summary of the basic technical aspects on the topic, and a brief discussion on perspectives for future research and development in the field. The paper is a review in the very broadest sense of the word, written with the purpose of orienting the interested newcomer to the field of electroporation applications in food technology towards the pertinent, highly relevant and more in-depth literature from the respective subdomains of electroporation research.     

'Shocking' developments in chick embryology: electroporation and in ovo gene expression

Efficient gene transfer by electroporation of chick embryos in ovo has allowed the development of new approaches to the analysis of gene regulation, function and expression, creating an exciting opportunity to build upon the classical manipulative advantages of the chick embryonic system. This method is applicable to other vertebrate embryos and is an important tool with which to address cell and developmental biology questions. Here we describe the technical aspects of in ovo electroporation, its different applications and future perspectives.     

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.     

Electroporation: A general phenomenon for manipulating cells and tissues

Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (μs to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5–1.5 V. In the case of isolated cells, the pulse magnitude is 10³–10⁴ V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.     

Recovery of adherent cells after in situ electroporation monitored electrically

Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.     

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.     

Diverse effects of nanosecond pulsed electric fields on cells and tissues

The application of pulsed electric fields to cells is extended to include nonthermal pulses with shorter durations (10-300 ns), higher electric fields (< or =350 kV/cm), higher power (gigawatts), and distinct effects (nsPEF) compared to classical electroporation. Here we define effects and explore potential application for nsPEF in biology and medicine. As the pulse duration is decreased below the plasma membrane charging time constant, plasma membrane effects decrease and intracellular effects predominate. NsPEFs induced apoptosis and caspase activation that was calcium-dependent (Jurkat cells) and calcium-independent (HL-60 and Jurkat cells). In mouse B10-2 fibrosarcoma tumors, nsPEFs induced caspase activation and DNA fragmentation ex vivo, and reduced tumor size in vivo. With conditions below thresholds for classical electroporation and apoptosis, nsPEF induced calcium release from intracellular stores and subsequent calcium influx through store-operated channels in the plasma membrane that mimicked purinergic receptor-mediated calcium mobilization. When nsPEF were applied after classical electroporation pulses, GFP reporter gene expression was enhanced above that observed for classical electroporation. These findings indicate that nsPEF extend classical electroporation to include events that primarily affect intracellular structures and functions. Potential applications for nsPEF include inducing apoptosis in cells and tumors, probing signal transduction mechanisms that determine cell fate, and enhancing gene expression.     

Effects and applications of sub-lethal ultrasound, electroporation and UV radiations in bioprocessing

Advances in bioprocess technology involving microbial cells have led to increased and improved production of beneficial new products and bioactive compounds. However, the semipermeable barrier of the cell membrane often retards the efficient productivity or reaction rate of the cells. Physical treatments such as ultrasound, electroporation and UV radiation provide an efficient approach to increase membrane permeability, leading to enhanced productivity of microbial cells. It is important to note that extensive membrane permeabilization by these physical treatments could be detrimental to cell viability leading to lower yield. An appropriate selection of sublethal dosage and intensity of these physical treatments are critical to preserve the viability of cells and at the same time maintain their bioprocess applications. Despite the promising applications of these physical treatments, safety issues related to possible genotoxicity or mutation of cells upon treatments have been raised. This genotoxic effect of physical treatments could be prevented if appropriate measures are taken, without compromising their bioprocess potentials. The current review highlights the effect of sublethal physical treatments such as ultrasound, electroporation and UV radiation on the viability of cells, their potential bioprocess applications, and the possibility of mutations.     

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study

Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI? and Lipofectamine? 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI? was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.     

Modeling the electromobility of ions in a target tissue

Electroporation is a clinical and laboratory technique for the delivery of molecules to cells. This method imposes electric fields onto cells or tissues through the use of electrodes and a set of electrical parameters to ultimately incorporate molecules into the cells. Clinical applications may include using directional fields to bring therapeutics to the target tissues before triggering an electroporation event. The choice of applicator may also have a significant influence on this molecular flow. Modeling ionic flow in tissues will yield insight into selecting the appropriate parameters or electroporation signature for a desired target application. In this paper, the motion of tissue injected ions was modeled for two common electroporation applicator configurations-the parallel plate, and the four needle electrodes. This electric field induced fluid flow model predicts that the parallel plate applicator ultimately directs the movement of an ionic therapeutic in a forward manner with side motion due only to obstruction, while the four-needle applicator directs anisotropic flow within the field ultimately forcing the therapeutic into a mound at the fringes of the induced electric field.     

In vivo DNA electrotransfer into muscle

Naked plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed. Among the non-viral techniques for gene transfer in vivo , this method is especially simple, inexpensive, and safe. However, the relatively low expression levels attained by this method have limited its applications for uses other than as a DNA vaccine. We and other groups investigated the applicability of in vivo electroporation for gene transfer into muscle, using plasmid DNA vector. The results demonstrated that gene transfer into muscle by in vivo electroporation is far more efficient than simple intramuscular DNA injection and provides a potential approach to systemically delivering cytokines, growth factors, and other serum proteins for basic research and human gene therapy.     

Electroporating Fields Target Oxidatively Damaged Areas in the Cell Membrane

Reversible electropermeabilization (electroporation) is widely used to facilitate the introduction of genetic material and pharmaceutical agents into living cells. Although considerable knowledge has been gained from the study of real and simulated model membranes in electric fields, efforts to optimize electroporation protocols are limited by a lack of detailed understanding of the molecular basis for the electropermeabilization of the complex biomolecular assembly that forms the plasma membrane. We show here, with results from both molecular dynamics simulations and experiments with living cells, that the oxidation of membrane components enhances the susceptibility of the membrane to electropermeabilization. Manipulation of the level of oxidative stress in cell suspensions and in tissues may lead to more efficient permeabilization procedures in the laboratory and in clinical applications such as electrochemotherapy and electrotransfection-mediated gene therapy.     

Transient Transfection of Oligodendrocyte Progenitors by Electroporation

The transient transfection of transgenes into oligodendrocytes offers an important tool for studying the function of proteins during myelin formation. Currently established procedures, however, have generally resulted in low survival rates and low levels of uptake of the transgene into primary oligodendrocyte progenitors. We describe an electroporation method which yields transient transfection of oligodendrocyte progenitors of up to 10–15% of the surviving cells, and provides approximately 10⁴ surviving, transfected cells per electroporation reaction. In recent applications transgene expression persisted as the transfected progenitors progressed through subsequent stages of the oligodendrocyte lineage. This technique is expected to facilitate the study of the function of key proteins and lipids during the development of primary cultured oligodendrocytes.     

The application of gene transfer techniques to marine resource management: recent advances, problems and future directions

Recent advantages in transgenic fish research are reviewed, with special reference to the methods for gene transfer. These include microinjection, electroporation, particle bombardment, and lipofection. The success and problems associated with each of these methods, and the possible applications of transgenic fish research to aquaculture are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electric Field-Driven Water Dipoles: Nanoscale Architecture of Electroporation

Electroporation is the formation of permeabilizing structures in the cell membrane under the influence of an externally imposed electric field. The resulting increased permeability of the membrane enables a wide range of biological applications, including the delivery of normally excluded substances into cells. While electroporation is used extensively in biology, biotechnology, and medicine, its molecular mechanism is not well understood. This lack of knowledge limits the ability to control and fine-tune the process. In this article we propose a novel molecular mechanism for the electroporation of a lipid bilayer based on energetics analysis. Using molecular dynamics simulations we demonstrate that pore formation is driven by the reorganization of the interfacial water molecules. Our energetics analysis and comparisons of simulations with and without the lipid bilayer show that the process of poration is driven by field-induced reorganization of water dipoles at the water-lipid or water-vacuum interfaces into more energetically favorable configurations, with their molecular dipoles oriented in the external field. Although the contributing role of water in electroporation has been noted previously, here we propose that interfacial water molecules are the main players in the process, its initiators and drivers. The role of the lipid layer, to a first-order approximation, is then reduced to a relatively passive barrier. This new view of electroporation simplifies the study of the problem, and opens up new opportunities in both theoretical modeling of the process and experimental research to better control or to use it in new, innovative ways.