Electroporation
Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-voltage electric field induces a temporary destabilization of the lipid bilayer, resulting in the formation of nanoscale pores that permit the entry or exit of macromolecules.
This method is widely employed to introduce molecules—including small molecules, DNA, RNA, and proteins—into cells. Electroporation can be performed on cells in suspension using electroporation cuvettes, or directly on adherent cells in situ within their culture vessels.
In microbiology, electroporation is frequently utilized for the transformation of bacteria or yeast cells, often with plasmid DNA. It is also used in the transfection of plant protoplasts and mammalian cells. Notably, electroporation plays a critical role in the ex vivo manipulation of immune cells for the development of cell-based therapies, such as CAR T-cell therapy. Moreover, in vivo applications of electroporation have been successfully demonstrated in various tissue types.
Bulk electroporation confers advantages over other physical delivery methods, including microinjection and gene gun techniques. However, it is limited by reduced cell viability. To address these issues, researchers have developed miniaturized approaches such as micro-electroporation and nanotransfection. These techniques utilize nanochannel-mediated electroporation to deliver molecular cargo to cells in a more controlled and less invasive manner.
Alternative methods for intracellular delivery include the use of cell-penetrating peptides, cell squeezing techniques, and chemical transformation, with selection depending on the specific cell type and cargo characteristics.
Electroporation is also employed to induce cell fusion. A prominent application of cell fusion is hybridoma technology, where antibody-producing B lymphocytes are fused with immortal myeloma cell lines to produce monoclonal antibodies.
Laboratory research
Electroporation is widely utilized in laboratory settings due to its ability to achieve high transformation efficiencies, particularly for plasmid DNA, with reported yields approaching 1010 colony-forming units per microgram of DNA. Electroporation is generally more costly than chemical transformation methods due to the specialized equipment required. This includes electroporators—devices designed to generate controlled electrostatic fields for cell suspension—and electroporation cuvettes, which are typically constructed from glass or plastic and contain parallel aluminum electrodes.A standard bacterial transformation protocol involves several steps. First, electro-competent cells are prepared by washing to remove ions that could cause arcing. These cells are then mixed with plasmid DNA and transferred into an electroporation cuvette. A high-voltage electric pulse is applied, with specific parameters such as voltage and pulse duration tailored to the particular cell type being used. Following electroporation, recovery medium is added, and the cells are incubated at an appropriate temperature to allow for outgrowth. Finally, the cells are plated onto selective agar plates to assess transformation efficiency.
The success of electroporation depends on several factors, including the purity of the plasmid DNA solution, salt concentration, and electroporation parameters. High salt concentrations can lead to arcing, significantly reducing the viability of electroporated cells. Therefore, the electroporation conditions must be optimized for each cell type to achieve an effective balance between cell viability and DNA uptake.
In addition to in vitro applications, electroporation is employed in vivo to enhance cell membrane permeability during injections and surgical procedures. The effectiveness of in vivo electroporation depends greatly on selected parameters such as voltage, pulse duration, and number of pulses. Developing central nervous systems are particularly suitable for in vivo electroporation, as ventricles provide clear visibility for nucleic acid injections, and dividing cells exhibit increased permeability. Electroporation of embryos injected in utero is performed through the uterine wall, often using forceps-type electrodes to minimize embryo damage.
Low-cost and portable electroporation devices have also been described, including a piezoelectric lighter-derived handheld electroporator and a microneedle-array skin electroporation system designed for portable DNA vaccination studies.
History
Researchers in the 1960s discovered that applying an external electric field would create a large membrane potential at the two poles of a cell.History
Early studies of pulsed electric fields in biology examined membrane damage and microbial inactivation.In the 1970s, it was found that when a critical membrane potential is reached, the cellular membrane would break down and subsequently recover. By the 1980s, this temporary membrane breakdown was exploited to introduce various molecules into cells.
Sale and Hamilton reported that high-intensity electric field pulses could kill bacteria and yeasts and proposed membrane disruption as a key mechanism.
In the mid-1970s, Zimmermann and colleagues described reversible electric-field–induced membrane permeabilization using erythrocytes and bacteria, including experiments using an aperture-based Coulter-counter setup and follow-up measurements with electrodes and capacitor-discharge pulses.
Work in erythrocytes helped establish that pores could form and reseal under controlled conditions. In parallel, Auer, Brandner, and Bodemer reported uptake of SV40 DNA and mammalian RNA in red blood cells during dielectric breakdown.
The term electroporation entered common use with work demonstrating electric-field–mediated gene transfer in mammalian cells.
In vivo gene electroporation was first described in 1991. This method delivers a large variety of therapeutic genes for the potential treatment of several diseases, including immune disorders, tumors, metabolic disorders, monogenetic diseases, cardiovascular diseases, and analgesia.
Regarding irreversible electroporation, the first successful treatment of malignant cutaneous tumors implanted in mice was accomplished in 2007 by a group of scientists who achieved complete tumor ablation in 12 of 13 mice. They accomplished this by sending 80 pulses of 100 microseconds at 0.3 Hz with an electrical field magnitude of 2500 V/cm to treat the cutaneous tumors.
The first group to apply electroporation used a reversible procedure in conjunction with impermeable macromolecules. The first research on how nanosecond pulses might be used on human cells was published in 2003.
Medical applications
The first medical application of electroporation was used for introducing poorly permeant anti-cancer drugs into tumor nodules. Gene electro-transfer soon became of interest because of its low cost, ease of implementation, and alleged safety. Viral vectors have since been found to have limitations in terms of immunogenicity and pathogenicity when used for DNA transfer.Irreversible electroporation is being used and evaluated as cardiac ablation therapy to kill specific areas of heart muscle. This is done to treat irregularities of heart rhythm. A cardiac catheter delivers trains of high-voltage, ultra-rapid electrical pulses that form irreversible pores in cell membranes, resulting in cell death.
N-TIRE
Non-thermal irreversible electroporation is a technique that treats many different types of tumors and other unwanted tissue. This procedure is done using small electrodes, placed either inside or surrounding the target tissue to apply short, repetitive bursts of electricity at a predetermined voltage and frequency. These bursts of electricity increase the resting transmembrane potential so that nanopores form in the plasma membrane. When the electricity applied to the tissue is above the electric field threshold of the target tissue, the cells become permanently permeable from the formation of nanopores. As a result, the cells are unable to repair the damage and die due to a loss of homeostasis. N-TIRE is unique to other tumor ablation techniques in that it does not create thermal damage to the tissue around it.Reversible electroporation
In contrast, reversible electroporation occurs when the electricity applied with the electrodes is below the target tissue's electric field threshold. Because the electricity applied is below the cells' threshold, it allows the cells to repair their phospholipid bilayer and continue with their normal cell functions. Reversible electroporation is typically done with treatments that involve inserting a drug or gene into the cell. Not all tissues have the same electric field threshold; therefore, to improve safety and efficacy, careful calculations need to be made prior to a treatment.N-TIRE, when done correctly, only affects the target tissue. Proteins, the extracellular matrix, and critical structures such as blood vessels and nerves are all unaffected and left healthy by this treatment. This facilitates a more rapid replacement of dead tumor cells and a faster recovery.
Imaging technology such as CT scans and MRIs are commonly used to create a 3D image of the tumor. Computed tomography is used to help with the placement of electrodes during the procedure, particularly when the electrodes are being used to treat tumors in the brain.
The procedure takes five minutes with a high success rate. It may be used for future treatment in humans. One disadvantage of using N-TIRE is that the electricity delivered from the electrodes can stimulate muscle cells to contract, which could have lethal consequences, depending on the situation. Therefore, a paralytic agent must be used when performing the procedure. The paralytic agents that have been used in such research have risks when using anesthetics.