Electroporation

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Cuvettes for in-vitro electroporation. These are plastic with aluminium electrodes and a blue lid. They hold a maximum of 400 ml. Electroporation Cuvettes.jpg
Cuvettes for in-vitro electroporation. These are plastic with aluminium electrodes and a blue lid. They hold a maximum of 400 μl.

Electroporation, or electropermeabilization, is a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane. This may allow chemicals, drugs, electrode arrays or DNA to be introduced into the cell (also called electrotransfer). [1] [2] [3] [4]

Contents

In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred, cell-penetrating peptides or cell squeeze could also be used. Electroporation works by passing thousands of volts (~8 kV/cm) across suspended cells in an electroporation cuvette. [2] Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective in increasing cell membrane's permeability than chemical transformation, although many laboratories lack the specialized equipment needed for electroporation. [5] [6] [ verification needed ]

Electroporation is also highly efficient for the introduction of foreign genes into tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNA into eukaryotic cells is known as transfection. Electroporation is highly effective for transfecting cells in suspension using electroporation cuvettes. Electroporation has proven efficient for use on tissues in vivo, for in utero applications as well as in ovo transfection. Adherent cells can also be transfected using electroporation, providing researchers with an alternative to trypsinizing their cells prior to transfection. One downside to electroporation, however, is that after the process the gene expression of over 7,000 genes can be affected. [7] This can cause problems in studies where gene expression has to be controlled to ensure accurate and precise results.

Although bulk electroporation has many benefits over physical delivery methods such as microinjections and gene guns, it still has limitations, including low cell viability. Miniaturization of electroporation has been studied, leading to microelectroporation and nanotransfection of tissue utilizing electroporation-based techniques via nanochannels to minimally invasively deliver cargo to the cells. [8] [9]

Electroporation has also been used as a mechanism to trigger cell fusion. Artificially induced cell fusion can be used to investigate and treat different diseases, like diabetes, [10] [11] [12] regenerate axons of the central nerve system, [13] and produce cells with desired properties, such as in cell vaccines for cancer immunotherapy. [14] However, the first and most known application of cell fusion is production of monoclonal antibodies in hybridoma technology, where hybrid cell lines (hybridomas) are formed by fusing specific antibody-producing B lymphocytes with a myeloma (B lymphocyte cancer) cell line. [15]

Laboratory practice

Electroporation is performed with electroporators, purpose-built appliances that create an electrostatic field in a cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminium electrodes on its sides. For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation, this suspension of bacteria is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance are set, and the cuvette is inserted into the electroporator. The process requires direct contact between the electrodes and the suspension. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in an Eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of the plasmid, followed by bacterial culture on agar plates.

The success of the electroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria. For a further detailed investigation of the process, more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. salt content).

Since the cell membrane is not able to pass current (except in ion channels), it acts as an electrical capacitor. Subjecting membranes to a high-voltage electric field results in their temporary breakdown, resulting in pores that are large enough to allow macromolecules (such as DNA) to enter or leave the cell. [16]

Additionally, electroporation can be used to increase permeability of cells during in Utero injections and surgeries. Particularly, the electroporation allows for a more efficient transfection of DNA, RNA, shRNA, and all nucleic acids into the cells of mice and rats. The success of in vivo electroporation depends greatly on voltage, repetition, pulses, and duration. Developing central nervous systems are most 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. [17]

In vitro and animal studies

In vivo gene electrotransfer was first described in 1991 [18] and today there are many preclinical studies of gene electrotransfer. 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, analgesia.... [19] [20] [21]

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. [22] Currently, a number of companies, including AngioDynamics, Inc. and VoltMed, Inc., are continuing to develop and deploy irreversible electroporation-based technologies within clinical environments.

The first group to look at electroporation for medical applications was led by Lluis M Mir at the Institute Gustave Roussy. In this case, they looked at the use of reversible electroporation in conjunction with impermeable macromolecules. The first research looking at how nanosecond pulses might be used on human cells was conducted by researchers at Eastern Virginia Medical School and Old Dominion University, and published in 2003. [23]

Medical applications

The first medical application of electroporation was used for introducing poorly permeant anticancer drugs into tumor nodules. [24] Soon also gene electrotransfer became of special interest because of its low cost, easiness of realization and safety. Namely, viral vectors can have serious limitations in terms of immunogenicity and pathogenicity when used for DNA transfer. [25]

Irreversible electroporation is being used and evaluated as cardiac ablation therapy to kill very small 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. It is thought to allow better selectivity than the previous techniques, which used heat or cold to kill larger volumes of muscle. [26]

A higher voltage of electroporation was found in pigs to irreversibly destroy target cells within a narrow range while leaving neighboring cells unaffected, and thus represents a promising new treatment for cancer, heart disease and other disease states that require removal of tissue. [27] Irreversible electroporation (IRE) has since proven effective in treating human cancer, with surgeons at Johns Hopkins and other institutions now using the technology to treat pancreatic cancer previously thought to be unresectable. [28]

Also first phase I clinical trial of gene electrotransfer in patients with metastatic melanoma was reported. [29] [30] Electroporation mediated delivery of a plasmid coding gene for interleukin-12 (pIL-12) was performed and safety, tolerability and therapeutic effect were monitored. Study concluded, that gene electrotransfer with pIL-12 is safe and well tolerated. In addition partial or complete response was observed also in distant non treated metastases, suggesting the systemic treatment effect. Based on these results they are already planning to move to Phase II clinical study. There are currently several ongoing clinical studies of gene electrotransfer [31] where safety, tolerability and effectiveness of immunization with DNA vaccine, which is administered by the electric pulses is monitored.

Although the method is not systemic, but strictly local one, it is still the most efficient non-viral strategy for gene delivery.

N-TIRE

A recent 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. [32] 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 ensure safety and efficacy. [33]

One major 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. [34]

Before doing the procedure, scientists must carefully calculate exactly what needs to be done and treat each patient on an individual case-by-case basis. To do this, imaging technology such as CT scans and MRI's are commonly used to create a 3D image of the tumor. From this information, they can approximate the volume of the tumor and decide on the best course of action including the insertion site of electrodes, the angle they are inserted in, the voltage needed, and more, using software technology. Often, a CT machine will be used to help with the placement of electrodes during the procedure, particularly when the electrodes are being used to treat tumors in the brain. [35]

The entire procedure is very quick, typically taking about five minutes. The success rate of these procedures is high [16] and is very promising 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 are successful[ citation needed ]; however, there is always some risk, albeit slight, when using anesthetics.

H-FIRE

A more recent technique has been developed called high-frequency irreversible electroporation (H-FIRE). This technique 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. [36] 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. [37]

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 Institutet 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 potentially treat Alzheimer's disease, Parkinson's disease, and brain cancer, among other conditions. [38]

Bacterial transformation is generally the easiest way to make large amounts of a particular protein needed for biotechnology purposes or in medicine. Since gene electrotransfer is very simple, rapid and highly effective technique it first became very convenient replacement for other transformation procedures. [39]

Recent research has shown that shock waves could be used for pre-treating the cell membrane prior to electroporation. [40] [41] 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

Schematic cross-section showing the theoretical arrangement of lipids in a hydrophobic pore (top) and a hydrophilic pore (bottom). Pore schematic.svg
Schematic cross-section showing the theoretical arrangement of lipids in a hydrophobic pore (top) and a hydrophilic pore (bottom).

Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. [2] This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane. [42] Electropores were optically imaged in lipid bilayer models like droplet interface bilayers [43] and giant unilamellar vesicles, [44] while addition of cytoskeletal proteins such as actin networks to the giant unilamellar vesicles seem to prevent the formation of visible electropores. [45] Experimental evidences for actin networks in regulating the cell membrane permeability has also emerged. [46] 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 (Eth). That is, only the cells within areas where E≧Eth are electroporated. If a second threshold (Eir) is reached or surpassed, electroporation will compromise the viability of the cells, i.e., irreversible electroporation (IRE). [47]

Electroporation is a multi-step process with several distinct phases. [48] [49] First, a short electrical pulse must 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. [50] Evidence for the existence of such pre-pores comes mostly from the "flickering" of pores, which suggests a transition between conductive and insulating states. [51] 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 [52] which in turn depends on the applied field, local mechanical stress and bilayer edge energy.

Gene electroporation

Electrogenetransfer.JPG

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 nonpermeant molecules enter the cell. [53] [54] 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. [55] 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. [56] In in vivo gene electrotransfer, DNA diffusion through extracellular matrix, properties of tissue and overall tissue conductivity are also crucial. [57]

History

In the 1960s it was known that by applying an external electric field, a large membrane potential at the two pole of a cell can 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. [58] By the 1980s, this opening was being used to introduce various materials/molecules into the cells. [59]

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<span class="mw-page-title-main">Lipid bilayer</span> Membrane of two layers of lipid molecules

The lipid bilayer is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many viruses are made of a lipid bilayer, as are the nuclear membrane surrounding the cell nucleus, and membranes of the membrane-bound organelles in the cell. The lipid bilayer is the barrier that keeps ions, proteins and other molecules where they are needed and prevents them from diffusing into areas where they should not be. Lipid bilayers are ideally suited to this role, even though they are only a few nanometers in width, because they are impermeable to most water-soluble (hydrophilic) molecules. Bilayers are particularly impermeable to ions, which allows cells to regulate salt concentrations and pH by transporting ions across their membranes using proteins called ion pumps.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<span class="mw-page-title-main">Small interfering RNA</span> Biomolecule

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. It was discovered in 1998, by Andrew Fire at Carnegie Institution for Science in Washington DC and Craig Mello at University of Massachusetts in Worcester.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

<span class="mw-page-title-main">Transduction (genetics)</span> Transfer process in genetics

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Sonophoresis also known as phonophoresis, is a method that utilizes ultrasound to enhance the delivery of topical medications through the stratum corneum, to the epidermis and dermis. Sonophoresis allows for the enhancement of the permeability of the skin along with other modalities, such as iontophoresis, to deliver drugs with lesser side effects. Currently, sonophoresis is used widely in transdermal drug delivery, but has potential applications in other sectors of drug delivery, such as the delivery of drugs to the eye and brain.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

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Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection systems, and viruses. This method offers advantages such as high transfection efficiency and biocompatibility which are balanced with limitations.

Electrochemotherapy (ECT) is a type of chemotherapy that allows delivery of non-permeant drugs to the cell interior. It is based on the local application of short and intense electric pulses that transiently permeabilize the cell membrane, thus allowing transport of molecules otherwise not permitted by the membrane. Applications for treatment of cutaneous and subcutaneous tumors have reached clinical use by utilizing drugs such as bleomycin or cisplatin). Electrochemotherapy with bleomycin was used to treat a patient for the first time in 1991 at the Institute Gustave Roussy in France, while electrochemotherapy with cisplatin was used to treat for the first time in 1995 at the Institute of Oncology, Ljubljana, Slovenia. Since then, more than 4000 patients were treated with electrochemotherapy all over the world. Recently, new electrochemotherapy modalities have been developed for treatment of internal tumors using surgical procedures, endoscopic routes, or percutaneous approaches to gain access to the treatment area.

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<span class="mw-page-title-main">Sonoporation</span> Technique in molecular biology

Sonoporation, or cellular sonication, is the use of sound in the ultrasonic range for increasing the permeability of the cell plasma membrane. This technique is usually used in molecular biology and non-viral gene therapy in order to allow uptake of large molecules such as DNA into the cell, in a cell disruption process called transfection or transformation. Sonoporation employs the acoustic cavitation of microbubbles to enhance delivery of these large molecules. The exact mechanism of sonoporation-mediated membrane translocation remains unclear, with a few different hypotheses currently being explored.

<span class="mw-page-title-main">Vectors in gene therapy</span>

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

Irreversible electroporation is a soft tissue ablation technique using short but strong electrical fields to create permanent and hence lethal nanopores in the cell membrane, to disrupt cellular homeostasis. The resulting cell death results from induced apoptosis or necrosis induced by either membrane disruption or secondary breakdown of the membrane due to transmembrane transfer of electrolytes and adenosine triphosphate. The main use of IRE lies in tumor ablation in regions where precision and conservation of the extracellular matrix, blood flow and nerves are of importance. The first generation of IRE for clinical use, in the form of the NanoKnife System, became commercially available for research purposes in 2009, solely for the surgical ablation of soft tissue tumors. Cancerous tissue ablation via IRE appears to show significant cancer specific immunological responses which are currently being evaluated alone and in combination with cancer immunotherapy.

Tissue nanotransfection (TNT) is an electroporation-based technique capable of gene and drug cargo delivery or transfection at the nanoscale. Furthermore, TNT is a scaffold-less tissue engineering (TE) technique that can be considered cell-only or tissue inducing depending on cellular or tissue level applications. The transfection method makes use of nanochannels to deliver cargo to tissues topically. 

Donald Choy Chang is a founding professor of the Hong Kong University of Science and Technology (HKUST). He was also the founding President of the Biophysical Society of Hong Kong. He is currently Professor Emeritus and Adjunct Professor in HKUST. Chang has wide research interests. He was an experimental physicist by training; but his publication ranges from nuclear magnetic resonance, biophysics and quantum physics. He was elected American Physical Society Fellow in 2023.

<span class="mw-page-title-main">Intracellular delivery</span> Scientific research area

Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.

<span class="mw-page-title-main">Hydrodynamic delivery</span> Gene Transfer Method

Hydrodynamic Delivery (HD) is a method of DNA insertion in rodent models. Genes are delivered via injection into the bloodstream of the animal, and are expressed in the liver. This protocol is helpful to determine gene function, regulate gene expression, and develop pharmaceuticals in vivo.

References

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