**1. Introduction**

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368 Application of Nanotechnology in Drug Delivery

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Lack of potent drug and gene delivery is one of the major problems of cancer chemotherapy and biotherapy. Different non-viral approaches have been proposed for drug and gene delivery such as physical and chemical methods. Physical delivery systems are one of the efficient non-viral methods including electroporation, micro-injection, gene gun, tattooing, laser and ultrasound [Bolhassani and Rafati, 2011]. Electroporation (EP) is the formation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) high-voltage pulse to overcome the barrier of the cell membrane. This transient, permeabilized state can be used to load cells with a variety of different molecules including ions, drugs, dyes, tracers, antibodies, oligonucleotides, RNA and DNA [Faurie et al., 2005]. Electroporation has proven useful both *in vitro*, *in vivo* and in patients, where drug delivery to malignant tumors has been performed. In addition, the data show that electroporation of DNA vaccines *in vivo* is an effective method to increase cellular uptake of DNA and gene expression in tissue leading to marked improvement in immune responses. Electroporation represents a way of increasing the number of DNA-transfected cells and enhancing the magnitude of gene expression, while reducing intersubject variability and requiring less time to reach a maximal immune response compared to conventional intramuscular injection of the vaccine [Monie et al., 2010].

Delivery of DNA vaccines using electroporation has already been tested successfully in a wide range of disease models. Electroporation has been used to enhance immune responses using DNA vaccines directed against infectious diseases such as influenza, HIV, hepatitis C, malaria, anthrax or to treat or prevent the development of tumors including breast cancer, prostate cancer and melanoma [Daemi et al., 2012; Best et al., 2009]. The studies have shown that *in vivo* EP mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has potential to be used as a neo-adjuvant or adjuvant therapy [Ahmad et al.,

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2010]. Electroporation has been successfully used to administer HPV DNA vaccine to mice as well as rhesus macaques, which has prompted its use in an ongoing phase I clinical trial such as VGX-3100, a vaccine that includes plasmids targeting E6 and E7 proteins of both HPV subtypes 16 and 18, for treatment of patients with CIN 2 or 3. In addition, electroporation has been used as an effective vaccination technique for the treatment of HPV induced cancers using the pNGVL4a-CRT/E7 (detox) DNA vaccine [Monie et al., 2010]. The application of *in vivo* electroporation to the sites receiving injected plasmid DNA has allowed for dramatic increases in immune responses compared with plasmid DNA injection alone. Among the tissues targeted for *in vivo* electroporation have been skin, liver, tumors and muscle [Widera et al., 2000]. Regarding to *in vivo* EP is predominantly carried out intramuscularly (i.m.), currently, skin EP is used as an attractive and less invasive option that is able to induce robust adaptive immune responses. To date, studies of DNA EP in skin have mainly focused on antigen expression, antigen specific humoral immunity, induction of IFN-γ-producing T cells and protective efficacy to infection [Daemi et al., 2012; Brave et al., 2011]. Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8+Tcell immune responses while limiting invasiveness of delivery [Brave et al., 2011].

and Préat, 2005]. Various cell types of the skin are involved in the development of immune response. Langerhans cells (LC) due to their long dendritics and their horizontal orientation, create an almost continuous network that enables them to capture most antigens that enter through the skin. Delivery of DNA into the skin could induce direct-presentation of the encoded antigen by APC or cross-presentation after uptake by keratinocytes. Some studies have indicated that EP induces IgG and Th-cell responses higher than Intramuscular (IM) delivery [Lee et al., 2011]. This chapter is further focused on the use of electroporation-induced delivery of anti-cancer drugs, gene and vaccines in human cancer cells along with description

Electroporation – Advantages and Drawbacks for Delivery of Drug, Gene and Vaccine

http://dx.doi.org/10.5772/58376

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Generally, the methods of delivering a gene, vaccine and drug are divided into: **a)** Physical/ non-viral approaches such as tattooing, gene gun, ultrasound, electroporation, laser; **b)** Chemical/ non-viral systems such as: cationic lipids/liposomes, polysaccharides, cationic polymers, cationic peptides, micro-/ nano-particles and **c)** Biological/ viral vectors [Bolhassani et al., 2011]. Non-viral vectors are safe in human body and easy for use. Among them, electroporation can be used to distribute nucleic acid fragments, oligonucleotides, siRNA and plasmids to cells. Studies using electroporation were performed *in vivo*; however electropo‐ ration is sometimes harmful to differentiated adult cells [Anwer, 2011; Wang et al., 2012]. Nonviral vectors are attractive tools in gene therapy and vaccine delivery [Draghia-Akli et al., 2005].

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‐

of its advantages and disadvantages.

**2. Non-viral delivery systems**

**3. History and definition of electroporation**

However, this method sometimes leads to cell death, primarily when the electrical fields cause permanent permeabilization of the membrane and the consequent loss of cell homeostasis, in a process known as irreversible electroporation [Rubinsky, 2007]. This is an unusual mode of cell death that is not understood yet. The electroporation procedures used in many laboratories could be optimized with limited effort. Moreover, electroporation, used alone or in combina‐ tion with other enhancement methods, expands the range of drugs (small to macromolecules, lipophilic or hydrophilic, charged or neutral molecules) that can be delivered transdermally [Escobar-Chávez et al., 2009; Denet et al., 2004]. The efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. The *in vivo* application of high-voltage pulses is well tolerated, but muscle contractions are usually induced. The electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans [Denet et al., 2004]. It was shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. These techniques show a potential for drug and gene delivery. However, site-specific and efficient delivery still remains a difficult problem [Tsoneva et al., 2010]. The voltages generally used for electropo‐ ration in animals range from 100 to 1200 V/cm. The investigators have shown that low-voltage electroporation can induce immunity and protect mice effectively [Daemi et al., 2012; Zhou et al., 2008]. In addition, intradermal DNA electroporation is one of the most efficient non-viral methods for the delivery of gene into the skin [Lin et al., 2012]. Previous studies have demon‐ strated that a combination of a short high voltage pulse (HV) and a long duration low-voltage pulse (LV) was efficient for DNA electroporation in the skin and that intradermal electropo‐ ration was suitable to deliver DNA vaccine when a Th1-oriented response is desired [Pavselj and Préat, 2005]. Various cell types of the skin are involved in the development of immune response. Langerhans cells (LC) due to their long dendritics and their horizontal orientation, create an almost continuous network that enables them to capture most antigens that enter through the skin. Delivery of DNA into the skin could induce direct-presentation of the encoded antigen by APC or cross-presentation after uptake by keratinocytes. Some studies have indicated that EP induces IgG and Th-cell responses higher than Intramuscular (IM) delivery [Lee et al., 2011]. This chapter is further focused on the use of electroporation-induced delivery of anti-cancer drugs, gene and vaccines in human cancer cells along with description of its advantages and disadvantages.
