**3. History and definition of electroporation**

Electroporation was introduced in the 1960s and comprises the application of controlled electric fields to facilitate cell permeabilization. The success of *in vitro* delivery by electropo‐ ration has led to the development of *in vivo* applications [Takei et al., 2008]. The first *in vitro* and *in vivo* attempts to use electroporation in gene transfer were demonstrated in 1982 and 1991, respectively [Al-Dosari and Gao, 2009]. *In vivo* electroporation depends on electric pulses to drive gene transfer. These pulses generated transient pores in cell membranes followed by intracellular electrophoretic DNA movement. Typically, *in vivo* electroporation is performed by first injecting DNA to the target tissue followed by electric pulses, with varied voltage, pulse duration and number of cycles, from two applied electrodes [Al-Dosari and Gao, 2009, Hao et al., 2012]. This technique is generally safe, efficient and can produce good reproducibility compared to other non-viral methods. When its parameters are optimized, this method can generate transfection efficiency equal to that in viral vectors [Al-Dosari and Gao, 2009]. The initial study of *in vivo* EP was the delivery of chemotherapeutic agents to solid tumors. In the mid-to late 1990s, the efficacy of this approach for drug delivery was demonstrated in a variety of different animal and human tumors. This technique was then tested for enhanced plasmid DNA delivery and subsequently, the initiation of the first clinical trials [Heller and Heller, 2006]. Furthermore, the expression of reporter genes was used to optimize *in vivo* EP param‐ eters, to explore the mechanism of EP and to show delivery in a new tissue. The use of *in vivo* EP for gene delivery including immune modulators, cell cycle regulators, suicide genes, anti-angiogenic genes and genes encoding toxins has established its potential for many therapeutic applications [Heller and Heller, 2006]. *In vivo* electroporation as compared to other gene transfer methods, such as viral vectors, has several advantages: **a)** various types of DNA constructs (or RNAi vectors) are readily introduced to the cells without limitation of DNA size; **b)** more than two different DNA constructs can be introduced into the same cells [Matsuda and Cepko, 2007]. Altogether, delivery by electroporation has been performed to a number of tissues including skin, muscle, liver, testes and tumors employing a wide range of electrical conditions and electrodes. While this preclinical research is promising, further optimization of electrical conditions and electrodes would be necessary for clinical use [Fioretti et al., 2013; Heller and Lucas, 2000].

broaden topical delivery to drugs not suitable for delivery by passive diffusion (i.e., hydro‐ philic, charged, and/or large molecular drugs). The use of high-voltage pulses could also enhance the permeability of viable cells as demonstrated by the electrochemotherapy of tumors (e.g., bleomycin) or DNA transfection [Escobar-Chávez et al., 2009]. Indeed, the application of electrical pulses to a cell creates a transient permeability that allows entry of hydrophilic molecules such as drugs and plasmid DNA. The exact mechanism by which the plasmid enters the cell following electroporation is unclear. Although, small molecules such as drugs can enter cells via transient pores, it seems that macromolecules such as plasmid DNA enter by a more complex interaction with the cell membrane. This interaction is enhanced by the application of repeated pulses that brings the plasmid into closer contact with the cell membrane. The voltage required for electroporation varies considerably and is dependent on cell size and shape [Wells, 2010]. It ranges from values of approximately 100 V/cm in large cells up to 1-2 kV/cm in small cells such as bacteria. Plasmid electrotransfer is a multistep process from interaction with the cell membrane, movement into the cell, intracellular trafficking and passage across the nuclear membrane [Wells, 2010; Nakamura and Funahashi, 2013]. A variety of different electrodes could be used depending on the cells to be treated. For *in vitro* studies, electrode patterns vary from a cuvette figure for cells in suspension to complex electrode arrays for adherent cells. An equal variety of electrodes have been developed for *in vivo* use, based on the nature of the tissue being treated [Wells, 2010]. A wide range of pulse patterns have been used both *in vitro* and *in vivo*. Repeated pulses appear better than single pulses. Some authors suggest a combination of one high-voltage pulse with a series of low-voltage pulses. Pulse magnitude and duration also has an effect on the damage caused to the cells. Pretreatment of skeletal muscle *in vivo* with hyaluronidase allows the use of a decreased voltage and so reduces damage while maintaining efficiency. Plasmid size has a significant effect on the efficiency of electroporation with a decreasing efficiency observed with increasing plasmid size using the same expression cassette [Wells, 2010]. The *in vitro* and *in vivo* studies using

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**a.** *In vitro* electroporation: Electroporation can be used to transfer a range of genetic materials into cells including DNA, RNA and oligonucleotides. In addition, *in vitro* electroporation is useful for synthetic oligonucleotides which have an uncharged backbone such as the phosphorodiamidate morpholino oligomers [Wells, 2010]. The effects of electrical treatment with high field intensity (200-1000 V/cm) were evaluated on two breast cancer cells (MDA-MB-231 and MCF-7) and one fibroblast cell line 3T3. The degree of electro‐ permeabilization of the adherent cells elevated steadily with the increasing of the field intensity. Furthermore, cell replication of both cancer cell lines was disturbed after electropermeabilization. Altogether, the use of suitable electric pulses could trigger changes in the cytoskeleton organization and cell adhesiveness, led to the enhancement

**b.** *In vivo* electroporation: *In vivo* electroporation has been shown to be effective for a wide range of tissues, including tumors, skin, liver, lung, kidney, thymus, bladder, adipose tissue, vasculature, retina, cornea, ciliary muscle, brain, spinal cord, skeletal muscle and testis, for delivering a range of genetic material such as DNA, RNA and oligonucleotides

electroporation have been further described as following:

of anti-tumor effects [Pehlivanova et al., 2012].
