# Electroporation

Electroporation, also known as electropermeabilization, is a technique in which an electric field is applied to cells to increase the permeability of the cell membrane. Subjecting membranes to a high-voltage electric field results in their temporary breakdown, resulting in pores that are large enough to allow macromolecules to enter or leave the cell. This process allows the introduction of small molecules, DNA, RNA or proteins into cells.
In microbiology, electroporation is routinely used to transform bacteria or yeast with DNA, often in the form of plasmid DNA. Electroporation is also used for the transfection of plant protoplasts and mammalian cells. For example, it is used in the ex vivo production of cell-based therapies, such as CAR T cells. Additionally, electroporation has been successfully used on tissues in vivo.
Cells are typically electroporated in suspension using electroporation cuvettes. Alternatively, adherent cells can also be transfected in situ.
Although bulk electroporation offers some advantages over physical delivery methods such as microinjections and gene guns, it has certain limitations, including low cell viability. To address these issues, researchers have explored miniaturization, leading to microelectroporation and nanotransfection, which use electroporation-based techniques via nanochannels to deliver cargo to cells in a minimally invasive manner. Depending on the cell type and material being transferred, alternative methods such as cell-penetrating peptides, cell squeezing and chemical transformation may also be used.
Electroporation has also been employed as a mechanism for inducing cell fusion. The most well-known application of cell fusion is hybridoma technology, which is used for the generation of immortal cell lines producing monoclonal antibodies by fusing specific antibody-producing B lymphocytes with myeloma (B lymphocyte cancer) cell lines.

## Laboratory research

For the transformation of bacterial cells, electroporation is a simple and rapid method. It routinely yields high transformation efficiencies for plasmid DNA (~10$^{10}$ cfu/μg DNA) but is more expensive than chemical transformation, due to the need for a dedicated electroporation device (electroporators) and the cost for electroporation cuvettes. Electroporators are purpose-built appliances that create an electrostatic field in a cell suspension. Electroporation cuvettes are made of glass or plastic and have two aluminum electrodes on their sides. Briefly, a bacterial transformation protocol may follow the following steps:
* Preparation of electro-competent cells. For this, cells are typically washed 2-3 times using 10% glycerol in water.
* Mixing of suspended cells (~100 μL) with plasmid DNA (~1 μg) and pipetting into the electroporation cuvette.
* Application of the electroporation pulse with parameters depending on the particular cell type (as an example, ~18 kV/cm, 100 Ω, 25 μF and 5 ms pulse time may be suitable for most E. coli strains)
* Addition of recovery media and outgrowth at the optimal growth temperature for 1-2 h.
* Plating of cells on selective agar plates.
The success of electroporation can depend on several factors, including the purity of the plasmid solution, its salt content and the parameters used for electroporation. Notably, high salt concentrations might cause an electrical discharge (known as arcing), which may severely reduce the viability of the electroporated cells. Electroporation parameters typically need to be optimized for each cell type to balance cell viability and DNA uptake.
Additionally, electroporation can be used to increase permeability of cells during in vivo injections and surgeries. The success of in vivo electroporation depends greatly on the chosen electroporation parameters (voltage and pulse time, number of pulses). Developing central nervous systems are effective for in vivo electroporation due to the visibility of ventricles for injections of nucleic acids, as well as the increased permeability of dividing cells. Electroporation of injected in utero embryos is performed through the uterus wall, often with forceps-type electrodes to limit damage to the embryo.

### History

In the 1960s, it was known that by applying an external electric field, a large membrane potential at the two poles of a cell could be created. In the 1970s, it was discovered that when a membrane potential reached a critical level, the membrane would break down and that it could recover. By the 1980s, this opening was being used to introduce various molecules into the cells.
In vivo gene electroporation was first described in 1991. The method is used to deliver large variety of therapeutic genes for potential treatment of several diseases, such as disorders in immune system, tumors, metabolic disorders, monogenetic diseases, cardiovascular diseases, and analgesia, among others.
With regards to irreversible electroporation, the first successful treatment of malignant cutaneous tumors implanted in mice was completed in 2007 by a group of scientists who achieved complete tumor ablation in 12 out 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 of 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 anticancer drugs into tumor nodules. Soon also gene ElectroTransfer became of interest because of its low cost, easiness of realization and alleged safety. Namely, viral vectors can have serious 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

A technique called non-thermal irreversible electroporation (N-TIRE) has proven successful in treating many different types of tumors and other unwanted tissue. This procedure is done using small electrodes (about 1mm in diameter), 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 (TMP), 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

Contrastingly, reversible electroporation occurs when the electricity applied with the electrodes is below the electric field threshold of the target tissue. Because the electricity applied is below the cells' threshold, it allows the cells to repair their phospholipid bilayer and continue on with their normal cell functions. Reversible electroporation is typically done with treatments that involve getting a drug or gene (or other molecule that is not normally permeable to the cell membrane) into the cell. Not all tissue has the same electric field threshold; therefore, careful calculations need to be made prior to a treatment to improve safety and efficacy.
One advantage of using N-TIRE is that, when done correctly according to careful calculations, it 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 allows for a quicker recovery and facilitates a more rapid replacement of dead tumor cells with healthy cells.
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 entire procedure typically takes about five minutes. The success rate of these procedures is high and may be used for future treatment in humans. One disadvantage to 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 risk when using anesthetics.

### H-FIRE

High-frequency irreversible electroporation (H-FIRE) uses electrodes to apply bipolar bursts of electricity at a high frequency, as opposed to unipolar bursts of electricity at a low frequency. This type of procedure has the same tumor ablation success as N-TIRE. However, it has one distinct advantage, H-FIRE does not cause muscle contraction in the patient and therefore there is no need for a paralytic agent. Furthermore, H-FIRE has been demonstrated to produce more predictable ablations due to the lesser difference in the electrical properties of tissues at higher frequencies.

### Drug and gene delivery

Electroporation can also be used to help deliver drugs or genes into the cell by applying short and intense electric pulses that transiently permeabilize cell membrane, thus allowing transport of molecules otherwise not transported through a cellular membrane. This procedure is referred to as electrochemotherapy when the molecules to be transported are chemotherapeutic agents or gene ElectroTransfer when the molecule to be transported is DNA. Scientists from Karolinska Institute and the University of Oxford use electroporation of exosomes to deliver siRNAs, antisense oligonucleotides, chemotherapeutic agents and proteins specifically to neurons after inject them systemically (in blood). Because these exosomes are able to cross the blood brain barrier, this protocol could solve the problem of poor delivery of medications to the central nervous system and may potentially treat Alzheimer's disease, Parkinson's disease, and brain cancer, among other conditions.
Recent research has shown that shock waves could be used for pre-treating the cell membrane prior to electroporation. This synergistic strategy has shown to reduce external voltage requirement and create larger pores. Also, application of shock waves allow scope to target desired membrane site. This procedure allows to control the size of the pore.

## Physical mechanism

Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. Electropores were optically imaged in lipid bilayer models like droplet interface bilayers and giant unilamellar vesicles, while addition of cytoskeletal proteins such as actin networks to the giant unilamellar vesicles seem to prevent the formation of visible electropores. Experimental evidences for actin networks in regulating the cell membrane permeability has also emerged. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore which acts as the conductive pathway through the bilayer as it is filled with water.
Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each point on the cell membrane. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 V). This leads to the definition of an electric field magnitude threshold for electroporation (E$_{th}$). That is, only the cells within areas where E≧E$_{th}$ are electroporated. If a second threshold (E$_{ir}$) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation (IRE).
Electroporation is a process with several distinct phases. A short electrical pulse is be applied. Typical parameters would be 300–400 mV for < 1 ms across the membrane (note- the voltages used in cell experiments are typically much larger because they are being applied across large distances to the bulk solution so the resulting field across the actual membrane is only a small fraction of the applied bias). Upon application of this potential the membrane charges like a capacitor through the migration of ions from the surrounding solution. Once the critical field is achieved there is a rapid localized rearrangement in lipid morphology. The resulting structure is believed to be a "pre-pore" since it is not electrically conductive but leads rapidly to the creation of a conductive pore. Evidence for the existence of such pre-pores comes mostly from the "flickering" of pores, which suggests a transition between conductive and insulating states. It has been suggested that these pre-pores are small (~3 Å) hydrophobic defects. If this theory is correct, then the transition to a conductive state could be explained by a rearrangement at the pore edge, in which the lipid heads fold over to create a hydrophilic interface. Finally, these conductive pores can either heal, resealing the bilayer or expand, eventually rupturing it. The resultant fate depends on whether the critical defect size was exceeded which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

### Gene electroporation

Application of electric pulses of sufficient strength to the cell causes an increase in the trans-membrane potential difference, which provokes the membrane destabilization. Cell membrane permeability is increased, and otherwise non-permeant molecules enter the cell. Although the mechanisms of gene ElectroTransfer are not yet fully understood, it was shown that the introduction of DNA only occurs in the part of the membrane facing the cathode and that several steps are needed for successful transfection: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the membrane, migration of DNA towards the nucleus, transfer of DNA across the nuclear envelope and finally gene expression. There are a number of factors that can influence the efficiency of gene ElectroTransfer, such as: temperature, parameters of electric pulses, DNA concentration, electroporation buffer used, cell size and the ability of cells to express transfected genes. In in vivo gene ElectroTransfer, DNA diffusion through extracellular matrix, properties of tissue, and overall tissue conductivity may be crucial.