**2. Non-viral delivery systems**

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+T-

370 Application of Nanotechnology in Drug Delivery

cell immune responses while limiting invasiveness of delivery [Brave et al., 2011].

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

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].
