**5. Applications of electroporation**

Skin electroporation could be particularly appropriate for topical drug delivery. Skin electro‐ poration temporarily permeabilizes the barrier to drug permeation and therefore could 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 electroporation have been further described as following:

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;

The development of theoretical models has developed our understanding of electroporation mechanism. Electropermeabilization of cells mainly involves the interaction of the electric field with the lipid domains of the cell membrane. Experimentally measured quantities consist of the membrane lifetimes, the current, the membrane conductance and transmembrane voltage. Regarding to the accumulated evidence, the pores are formed because of the electric field. The transient aqueous pore theory describes the main features of electropermeabilization, which is one major consequence of electroporation. Molecular transport of charged molecules appears to be predominantly due to electrical flow through pores, such that the elevated transmembrane voltage plays two roles: (a) creation of pores and (b) provision of a local driving force [Weaver and Chizmadzhev, 1996]. Electrochemotherapy (ECT) is a cancer therapy that conjugates the administration of a chemotherapy agent to the delivery of permeabilizing pulses released singularly or as bursts. This approach results in higher number of anticancer mole‐ cules delivered to their biological targets, but is also associated to undesirable side effects such as pain and muscular spasms. A new electroporator delivering eight biphasic pulses at the voltage of 1,300 V/cm lasting + 50 μsec each, with a frequency of 1 Hz, and with 10-μsec

human lung cancer cell line (A549) and both in mice xenografts and rabbits with spontaneous tumors. The tumor cell line treated with electroporation showed efficient drug delivery suggesting further cell death. In addition, *in vivo* data demonstrated that the new permeabi‐ lizing protocol adopting biphasic electric pulses displays a significant higher efficacy com‐ pared to previous ECT treatments and consequently, substantial reduction of the morbidity

Skin electroporation could be particularly appropriate for topical drug delivery. Skin electro‐ poration temporarily permeabilizes the barrier to drug permeation and therefore could

of treated area) was tested on the

Heller and Lucas, 2000].

372 Application of Nanotechnology in Drug Delivery

[Spugnini et al., 2014].

**5. Applications of electroporation**

**4. Electroporation mechanisms**

interpulse intervals (total treatment time: 870 μsec/cm<sup>2</sup>


(e.g. siRNA, antisense oligonucleotides). *In vivo* plasmid electroporation has also been used as either a primary or booster vaccination strategy that enhances cell-mediated immune responses. Most of the studies using electroporation have involved local delivery into the target organ but a few have studied local electroporation following systemic (intravenous) delivery of the plasmid. For example, local electroporation targeted plasmid delivery to the liver was effective for liver, kidney and spleen but was not successful for skeletal muscle or skin [Wells, 2010]. Taken together, electroporation has been applied to efficient delivery of drugs, genes and vaccines as described below. Figure 1 shows common application of electroporation.

V. For a cell with a 10 μm diameter, the field strength needed to reach and exceed a potential

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375

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection [Escobar-Chávez et al., 2009]. The subcutaneous layer forms the major barrier to most water-soluble and many hydrophobic drugs and contributes the major portion of the electric resistance of the skin. Electroporation is one of the approaches to improve the transdermal delivery by transiently permeabilizing the skin to facilitate drug transport. Transdermal drug delivery has several potential advantages over other parenteral delivery methods. Apart from the convenience and non-invasiveness, the skin also provides a "reservoir" that sustains delivery over a period of days [Hui, 2013]. The authors have shown that if the voltage of the pulses exceeds a voltage threshold at 75–100 V (equivalent to the breakdown threshold of 8–10 lipid bilayers in the SC), microchannels or "local transport regions" are created through the breakdown sites of the SC [Hui, 2013]. Many small-molecule drugs have been successfully delivered through the skin by electroporation. Transport efficiency for small charged molecules (MW ≤ 1000, e.g., protoporphyrin IX), using the same polarity pulses, was higher than that for uncharged molecules (e.g., protoporphyrin IX methyl ester) or charged molecules with opposite polarity pulses. The results indicated that, besides passive diffusion through electropores, electrophoretic force of the pulses also contributes to the electroporation-enhanced transport of these charged molecules [Hui, 2013]. Therefore, the efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. Some studies indicated that the *in vivo* application of high-voltage pulses is well tolerated, but muscle contractions are generally induced. Furthermore, the electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans [Escobar-Chávez et al., 2009]. The electroporation has been first used to enhance the delivery of chemotherapeutic drugs like cisplatin and bleomycin in cancer cells and solid tumors, respectively. This application has been termed electrochemotherapy [Tsoneva et al., 2007;

Electrochemotherapy, via cell membrane permeabilizing electric pulses, potentiates the cytotoxicity of non-permeant or poorly permeant anticancer drugs with high intrinsic cyto‐ toxicity, such as bleomycin or cisplatin, at the site of electric pulse. Its advantages are high efficacy on tumors with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment. In clinical studies, electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumor nodules. The treatment response for various tumors (predominantly melanoma) was approximately 75% complete and 10% partial response of the treated nodules [Escobar-Chávez

A consistent finding is that lipo-or amphiphilic drugs traverse the cell membrane without electroporation, while an enhancement in cytotoxicity is found with drugs that, under normal

of 0.5 V at each end is about 1,000 V/cm [Hui, 2013].

Gehl, 2008].

et al., 2009].

*5.1.1.1. Bleomycin*

*5.1.1. Anti-cancer drugs*

**Figure 1.** Common applications of electroporation

#### **5.1. Drug delivery**

Several studies have investigated the use of electroporation to enhance the efficacy of the drugs especially used for the treatment of various cancer types. Current electroporation protocols are based on preclinical studies. The authors reported the use of 1,000 V/cm (voltage/electrode distance ratio) up to approximately 1,300 V/ cm for electrochemotherapy. One simple way of lowering the applied voltage was to decrease the gap between electrodes, e.g., 0.4 cm [Gehl, 2008]. The threshold potential for transient electric breakdown of cell membranes is about 0.5 V. For a cell with a 10 μm diameter, the field strength needed to reach and exceed a potential of 0.5 V at each end is about 1,000 V/cm [Hui, 2013].

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection [Escobar-Chávez et al., 2009]. The subcutaneous layer forms the major barrier to most water-soluble and many hydrophobic drugs and contributes the major portion of the electric resistance of the skin. Electroporation is one of the approaches to improve the transdermal delivery by transiently permeabilizing the skin to facilitate drug transport. Transdermal drug delivery has several potential advantages over other parenteral delivery methods. Apart from the convenience and non-invasiveness, the skin also provides a "reservoir" that sustains delivery over a period of days [Hui, 2013]. The authors have shown that if the voltage of the pulses exceeds a voltage threshold at 75–100 V (equivalent to the breakdown threshold of 8–10 lipid bilayers in the SC), microchannels or "local transport regions" are created through the breakdown sites of the SC [Hui, 2013]. Many small-molecule drugs have been successfully delivered through the skin by electroporation. Transport efficiency for small charged molecules (MW ≤ 1000, e.g., protoporphyrin IX), using the same polarity pulses, was higher than that for uncharged molecules (e.g., protoporphyrin IX methyl ester) or charged molecules with opposite polarity pulses. The results indicated that, besides passive diffusion through electropores, electrophoretic force of the pulses also contributes to the electroporation-enhanced transport of these charged molecules [Hui, 2013]. Therefore, the efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. Some studies indicated that the *in vivo* application of high-voltage pulses is well tolerated, but muscle contractions are generally induced. Furthermore, the electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans [Escobar-Chávez et al., 2009]. The electroporation has been first used to enhance the delivery of chemotherapeutic drugs like cisplatin and bleomycin in cancer cells and solid tumors, respectively. This application has been termed electrochemotherapy [Tsoneva et al., 2007; Gehl, 2008].

#### *5.1.1. Anti-cancer drugs*

(e.g. siRNA, antisense oligonucleotides). *In vivo* plasmid electroporation has also been used as either a primary or booster vaccination strategy that enhances cell-mediated immune responses. Most of the studies using electroporation have involved local delivery into the target organ but a few have studied local electroporation following systemic (intravenous) delivery of the plasmid. For example, local electroporation targeted plasmid delivery to the liver was effective for liver, kidney and spleen but was not successful for skeletal muscle or skin [Wells, 2010]. Taken together, electroporation has been applied to efficient delivery of drugs, genes and vaccines as described below. Figure 1 shows

Several studies have investigated the use of electroporation to enhance the efficacy of the drugs especially used for the treatment of various cancer types. Current electroporation protocols are based on preclinical studies. The authors reported the use of 1,000 V/cm (voltage/electrode distance ratio) up to approximately 1,300 V/ cm for electrochemotherapy. One simple way of lowering the applied voltage was to decrease the gap between electrodes, e.g., 0.4 cm [Gehl, 2008]. The threshold potential for transient electric breakdown of cell membranes is about 0.5

common application of electroporation.

374 Application of Nanotechnology in Drug Delivery

**Figure 1.** Common applications of electroporation

**5.1. Drug delivery**

Electrochemotherapy, via cell membrane permeabilizing electric pulses, potentiates the cytotoxicity of non-permeant or poorly permeant anticancer drugs with high intrinsic cyto‐ toxicity, such as bleomycin or cisplatin, at the site of electric pulse. Its advantages are high efficacy on tumors with different histologies, simple application, minimal side effects and the possibility of effective repetitive treatment. In clinical studies, electrochemotherapy has proved to be a highly efficient and safe approach for treating cutaneous and subcutaneous tumor nodules. The treatment response for various tumors (predominantly melanoma) was approximately 75% complete and 10% partial response of the treated nodules [Escobar-Chávez et al., 2009].

#### *5.1.1.1. Bleomycin*

A consistent finding is that lipo-or amphiphilic drugs traverse the cell membrane without electroporation, while an enhancement in cytotoxicity is found with drugs that, under normal circumstances, do not pass the cell membrane easily. The most prominent example is bleo‐ mycin, which is a well-known drug. One bleomycin molecule can cause several DNA strand breaks and is highly toxic inside the cell [Gehl, 2008]. Drug doses used in bleomycin-based electrochemotherapy have been variable. Some groups have used intratumoral injection with relatively high doses, while others have applied its lower doses. Also, for *i.v.* administration, bleomycin is generally given in the doses used in standard treatment protocols. The results of the different regimens are comparable, but there may be more necrosis with the higher doses and a better chance to conserve normal tissue with the lower doses [Gehl, 2008]. In bleomycin chemotherapy, treatment was more than 1000 times more effective with electroporation than without electroporation. In comparison with bleomycin, other drugs such as daunorubicin, doxorubincin, 5-fluorouracil and paclitaxel had no electroporation benefits [Hui, 2008]. Bleomycin electrochemotherapy has been successfully applied to treat melanomas, head and neck squamous cell carcinomas, Kaposi's sarcomas, as well as lung, breast, kidney, and bladder cancers. Its cytotoxicity is higher in cancer tissues than in normal tissues, including arteries and nerves. In certain stage II and III clinical trials, 100% complete recovery has been reported. Bleomycin electrochemotherapy induces temporary vasoconstriction, which helps to retain the drug in the tumor tissue [Hui, 2008].

*5.1.2. Analgesic and anti-inflammatory drugs*

*5.1.3. Anti-diuretic drugs*

Chávez et al., 2009].

*5.1.4. Anti-viral drugs*

*5.1.5. Beta-blocker agents*

*5.1.6. Insulin*

important parameters [Escobar-Chávez et al., 2009].

insulin in rats [Escobar-Chávez et al., 2009].

Electroporation increased the permeation of h-cyclodextrin (BCD) and hydroxy propyl hcyclodextrin (HPCD), relative to passive transport. The presence of BCD and HPCD enhanced the total transport of the permeants piroxicam and carboxyfluorescein (CF), respectively, from both permeant solutions and suspensions. Another studies demonstrated that electroporation may enhance and control transdermal permeation of nalbuphine (NA) and its prodrugs including nalbuphine benzoate (NAB) and sebacoyl dinalbuphine ester (SDN). The results indicated that the use of iontophoresis or electroporation significantly enhanced the *in vitro* permeation of NA and its prodrugs. In addition, lipophilicity and molecular size had signifi‐ cant effects on skin permeation of NA, NAB, and SDN via passive diffusion or under the electric field. The permeation amounts of NA and its prodrugs may be increased by application of higher pulse voltage, pulse duration and pulse number [Escobar-Chávez et al., 2009].

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

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377

Macromolecules were investigated as chemical enhancers of transdermal transport by skin electroporation. Skin electroporation increased transdermal mannitol delivery [Escobar-

The use of electroporation pulses enhancing the skin permeability to deliver anti-viral drugs is in the early stages of development. A systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to rat skin was previously reported. It was found that voltage, pulse length and number of pulses were the three most

The studies have shown the effects of electroporation on iontophoretic transport of 2 betablockers, timolol (lipophilic) and atenolol (hydrophilic). The iontophoretic transport of timolol was decreased by electroporation because the high accumulation of the lipophilic cation timolol in the *s.c.* resulted in a decrease of electroosmosis. In contrast, electroosmosis was not affected by atenolol, and the iontophoretic transport of atenolol was increased by electropo‐ ration. Using two different beta-blockers, the researchers showed that lipophilicity and

The data represented that *in vivo*, non-invasive insulin delivery to therapeutic levels and glucose extraction may be achieved by combining electroporation with anionic lipids and electroosmosis [Escobar-Chávez et al., 2009]. These studies confirmed the synergistic effects of electroporation (EP) and iontophoresis (IP) on the *in vivo* percutaneous absorption of human

positive charges affected the electrotransport of drugs [Escobar-Chávez et al., 2009].

As described above, the bleomycin is used with electroporation (electrochemotherapy) for treatment of tumors in the clinical setting. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions mentioned about cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing electrochemotherapy [Frandsen et al., 2014]. Calcium electroporation can induce ATP depletion-associated cellular death. The effects of calcium and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma) [Frandsen et al., 2014]. Further‐ more, the effects of electrical pulsing parameters and calcium compounds on treatment efficacy were determined. The results showed that electroporation with either calcium or bleomycin significantly reduced cell survival, without a synergistic effect at similar voltage parameters. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability. Indeed, the effect of calcium electroporation is independent of calcium compound [Frandsen et al., 2014]. Briefly, the calcium electroporation can be sug‐ gested as a potential cancer therapy in future clinical trial.

#### *5.1.1.2. Poloxamer 188*

Poloxamer 188, added before or immediately after an electrical pulse, decreased the number of dead cells as well as it did not reduce the number of reversible electropores. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Its advantage is the increased uptake and accumulation into reversibly electroporated tumor cells [Tsoneva et al., 2010].

#### *5.1.2. Analgesic and anti-inflammatory drugs*

Electroporation increased the permeation of h-cyclodextrin (BCD) and hydroxy propyl hcyclodextrin (HPCD), relative to passive transport. The presence of BCD and HPCD enhanced the total transport of the permeants piroxicam and carboxyfluorescein (CF), respectively, from both permeant solutions and suspensions. Another studies demonstrated that electroporation may enhance and control transdermal permeation of nalbuphine (NA) and its prodrugs including nalbuphine benzoate (NAB) and sebacoyl dinalbuphine ester (SDN). The results indicated that the use of iontophoresis or electroporation significantly enhanced the *in vitro* permeation of NA and its prodrugs. In addition, lipophilicity and molecular size had signifi‐ cant effects on skin permeation of NA, NAB, and SDN via passive diffusion or under the electric field. The permeation amounts of NA and its prodrugs may be increased by application of higher pulse voltage, pulse duration and pulse number [Escobar-Chávez et al., 2009].

#### *5.1.3. Anti-diuretic drugs*

circumstances, do not pass the cell membrane easily. The most prominent example is bleo‐ mycin, which is a well-known drug. One bleomycin molecule can cause several DNA strand breaks and is highly toxic inside the cell [Gehl, 2008]. Drug doses used in bleomycin-based electrochemotherapy have been variable. Some groups have used intratumoral injection with relatively high doses, while others have applied its lower doses. Also, for *i.v.* administration, bleomycin is generally given in the doses used in standard treatment protocols. The results of the different regimens are comparable, but there may be more necrosis with the higher doses and a better chance to conserve normal tissue with the lower doses [Gehl, 2008]. In bleomycin chemotherapy, treatment was more than 1000 times more effective with electroporation than without electroporation. In comparison with bleomycin, other drugs such as daunorubicin, doxorubincin, 5-fluorouracil and paclitaxel had no electroporation benefits [Hui, 2008]. Bleomycin electrochemotherapy has been successfully applied to treat melanomas, head and neck squamous cell carcinomas, Kaposi's sarcomas, as well as lung, breast, kidney, and bladder cancers. Its cytotoxicity is higher in cancer tissues than in normal tissues, including arteries and nerves. In certain stage II and III clinical trials, 100% complete recovery has been reported. Bleomycin electrochemotherapy induces temporary vasoconstriction, which helps to retain

As described above, the bleomycin is used with electroporation (electrochemotherapy) for treatment of tumors in the clinical setting. Calcium electroporation offers several advantages over standard treatment options: calcium is inexpensive and may readily be applied without special precautions mentioned about cytostatic drugs. Therefore, details on the use of calcium electroporation are essential for carrying out clinical trials comparing electrochemotherapy [Frandsen et al., 2014]. Calcium electroporation can induce ATP depletion-associated cellular death. The effects of calcium and bleomycin electroporation (alone or in combination) were compared in three different cell lines (DC-3F, transformed Chinese hamster lung fibroblast; K-562, human leukemia; and murine Lewis Lung Carcinoma) [Frandsen et al., 2014]. Further‐ more, the effects of electrical pulsing parameters and calcium compounds on treatment efficacy were determined. The results showed that electroporation with either calcium or bleomycin significantly reduced cell survival, without a synergistic effect at similar voltage parameters. At equimolar concentrations, calcium chloride and calcium glubionate resulted in comparable decreases in cell viability. Indeed, the effect of calcium electroporation is independent of calcium compound [Frandsen et al., 2014]. Briefly, the calcium electroporation can be sug‐

Poloxamer 188, added before or immediately after an electrical pulse, decreased the number of dead cells as well as it did not reduce the number of reversible electropores. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Its advantage is the increased uptake and accumulation into reversibly electroporated tumor cells [Tsoneva et

the drug in the tumor tissue [Hui, 2008].

376 Application of Nanotechnology in Drug Delivery

gested as a potential cancer therapy in future clinical trial.

*5.1.1.2. Poloxamer 188*

al., 2010].

Macromolecules were investigated as chemical enhancers of transdermal transport by skin electroporation. Skin electroporation increased transdermal mannitol delivery [Escobar-Chávez et al., 2009].

#### *5.1.4. Anti-viral drugs*

The use of electroporation pulses enhancing the skin permeability to deliver anti-viral drugs is in the early stages of development. A systematic study examining the parameters influencing electroporative transdermal delivery of terazosin hydrochloride to rat skin was previously reported. It was found that voltage, pulse length and number of pulses were the three most important parameters [Escobar-Chávez et al., 2009].

#### *5.1.5. Beta-blocker agents*

The studies have shown the effects of electroporation on iontophoretic transport of 2 betablockers, timolol (lipophilic) and atenolol (hydrophilic). The iontophoretic transport of timolol was decreased by electroporation because the high accumulation of the lipophilic cation timolol in the *s.c.* resulted in a decrease of electroosmosis. In contrast, electroosmosis was not affected by atenolol, and the iontophoretic transport of atenolol was increased by electropo‐ ration. Using two different beta-blockers, the researchers showed that lipophilicity and positive charges affected the electrotransport of drugs [Escobar-Chávez et al., 2009].

#### *5.1.6. Insulin*

The data represented that *in vivo*, non-invasive insulin delivery to therapeutic levels and glucose extraction may be achieved by combining electroporation with anionic lipids and electroosmosis [Escobar-Chávez et al., 2009]. These studies confirmed the synergistic effects of electroporation (EP) and iontophoresis (IP) on the *in vivo* percutaneous absorption of human insulin in rats [Escobar-Chávez et al., 2009].

### *5.1.7. Photosensitizers*

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the *s.c*. The researchers used the electric pulses to increase transdermal transport of D-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX)-precursor for the photodynamic therapy of superficial skin cancer and cutaneous metastases of internal malignancies. A two-fold enhancement of PpIX produc‐ tion with electroporative delivery was observed compared to passive delivery. The application of iontophoresis also increased the ALA permeation by approximately 15-fold [Escobar-Chávez et al., 2009].

effectively activate tumor specific immune responses. Optimization of the approach indicated that a four-dose regimen provided highest tumor protection. Furthermore, the four-dose regimen showed optimal and further tumor protection using co-administration of synthetic oligo CpG. Thus, the *in vivo* EP-mediated vaccination has potential to be use as a neo-adjuvant or adjuvant therapy in cancer treatment [Ahmad et al., 2010]. The effect of electroporation on DNA vaccine potency and gene delivery was studied using skin as a target tissue in larger animal species such as pig, macaque and sheep. In a macaque model, the higher cellular and humoral responses were observed to an HIV DNA vaccine harboring IL-12 gene, with electroporation compared to intradermal DNA injection alone [Hirao et al., 2008]. Further‐ more, the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine was studied using a combination of the delivery methods jet-injection and intradermal electroporation. The data showed that plasmids persist in the skin at the site of injection for at least four months after immunization [Brave, et al., 2010]. The researchers demonstrated that mice and guinea pigs vaccinated with single-and multi-gene DNA via EP and then with recombinant gp120 protein (i.e., the synthetic DNA prime-protein boost protocol) induced significantly higher antibody binding titers [Muthumani et al., 2013]. Recently, Minicircle DNA (a new form of DNA containing only gene expression cassette but lacking backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery improv‐ ing the levels and the duration of transgene expression *in vivo*. A novel vaccine delivery system, including the combined *in vivo* EP and the minicircle DNA carrying codon-optimized HIV-1 gag gene was prepared to evaluate the immunogenicity of this system. The use of EP delivery further increased minicircle-based gag gene expression led to the augmentation of humoral and cellular immune responses. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, up-regulating inflammatory genes and recruiting immune cells [Wang et al., 2014]. In sheep, the significantly higher antibody responses to plasmid-encoded HBsAg were observed after IM delivery followed by electro‐ poration in comparison with conventional IM or ID injection. Importantly, these antibody responses were sustained for 25 weeks after vaccination [van Drunen Littel-van den Hurk et al., 2008]. Moreover, various reports have illustrated that cytokine adjuvants have significant effects on modulating the immune responses to DNA vaccination. Indeed, the co-delivery of plasmid encoded cytokines is able to quantitatively and qualitatively modulate the immune responses in a large animal following *in vivo* electroporation of a DNA vaccine [Yen and Scheerlinck, 2007]. Although, intra-tumor delivery does not generally result in detectable serum transgene expression, intramuscular electroporation does result in serum expression. However, intratumor delivery is more successful than intramuscular delivery in eradicating primary tumors and in generating systemic immunity. For instance, a number of studies have demonstrated long-term, complete tumor regression, using delivery of plasmids encoding IL-12 or IFN-γ as a single agent in melanoma and squamous cell carcinoma (SCC) [Heller and Heller, 2006]. Complete regression after IL-12 gene therapy in combination with herpes simplex virus (HSV) thymidine kinase, bleomycin, or recombinant bacillus Calmette-Guérin (rBCG) has been observed in several experimental models. Electrically mediated bleomycin delivery combined with IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy also induced long-term complete regression in a small percentage of mice with

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379

#### *5.1.8. Folic acid antagonists*

The topical administration of methotrexate (MTX) for the treatment of psoriasis and neoplastic diseases is restricted by the poor diffusion of MTX across the *s.c*. Some studies showed that electroporation is an efficient method to increase the transdermal transport of MTX. Further‐ more, electroporation of MTX with an anion lipid enhancer under a mild hyperthermic environment provided a significant transdermal delivery within a short time [Escobar-Chávez et al., 2009].

#### **5.2. Vaccine delivery**

Electroporation-based immunization (especially, EP-mediated DNA vaccine) has been effective in a number of species including mice, rats, rabbits, non-human primates, pigs and sheep.

#### *5.2.1. DNA vaccine*

DNA immunization has known as an efficient strategy for vaccination [Bolhassani and Rafati, 2009]. The main disadvantage of plasmid DNA vaccines is their poor immunogenicity when administered as an unformulated intramuscular injection [Anderson and Schneider, 2007]. A number of approaches for enhancing the potency of DNA vaccines have developed over the past few years such as: **a)** Optimization of DNA constructs; **b)** Development of new DNA manufacturing processes and formulations; **c)** Augmentation of immune responses with novel encoded molecular adjuvants; and **d)** Improvement of *in vivo* DNA delivery strategies including electroporation [Sardesai and Weiner, 2011].

Among them, EP-mediated delivery has generated considerable attention and appeared to have a great impact in vaccine immunogenicity and efficacy by increasing antigen delivery up to a 1000 fold versus naked DNA delivery alone [van Drunen Littel-van den Hurk and Hannaman, 2010; Sardesai and Weiner, 2011]. In many cases, the immune responses and protection rates observed following DNA administration via EP were comparable or superior to other vaccine strategies including viral vectors and live/attenuated/inactivated virus vaccines [Sardesai and Weiner, 2011, Daemi et al., 2012; Hosseinzadeh et al., 2013]. An electroporation driven plasmid DNA vaccination strategy was studied in animal models for treatment of prostate cancer. This phPSA plasmid electroporation vaccine strategy could effectively activate tumor specific immune responses. Optimization of the approach indicated that a four-dose regimen provided highest tumor protection. Furthermore, the four-dose regimen showed optimal and further tumor protection using co-administration of synthetic oligo CpG. Thus, the *in vivo* EP-mediated vaccination has potential to be use as a neo-adjuvant or adjuvant therapy in cancer treatment [Ahmad et al., 2010]. The effect of electroporation on DNA vaccine potency and gene delivery was studied using skin as a target tissue in larger animal species such as pig, macaque and sheep. In a macaque model, the higher cellular and humoral responses were observed to an HIV DNA vaccine harboring IL-12 gene, with electroporation compared to intradermal DNA injection alone [Hirao et al., 2008]. Further‐ more, the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine was studied using a combination of the delivery methods jet-injection and intradermal electroporation. The data showed that plasmids persist in the skin at the site of injection for at least four months after immunization [Brave, et al., 2010]. The researchers demonstrated that mice and guinea pigs vaccinated with single-and multi-gene DNA via EP and then with recombinant gp120 protein (i.e., the synthetic DNA prime-protein boost protocol) induced significantly higher antibody binding titers [Muthumani et al., 2013]. Recently, Minicircle DNA (a new form of DNA containing only gene expression cassette but lacking backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery improv‐ ing the levels and the duration of transgene expression *in vivo*. A novel vaccine delivery system, including the combined *in vivo* EP and the minicircle DNA carrying codon-optimized HIV-1 gag gene was prepared to evaluate the immunogenicity of this system. The use of EP delivery further increased minicircle-based gag gene expression led to the augmentation of humoral and cellular immune responses. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, up-regulating inflammatory genes and recruiting immune cells [Wang et al., 2014]. In sheep, the significantly higher antibody responses to plasmid-encoded HBsAg were observed after IM delivery followed by electro‐ poration in comparison with conventional IM or ID injection. Importantly, these antibody responses were sustained for 25 weeks after vaccination [van Drunen Littel-van den Hurk et al., 2008]. Moreover, various reports have illustrated that cytokine adjuvants have significant effects on modulating the immune responses to DNA vaccination. Indeed, the co-delivery of plasmid encoded cytokines is able to quantitatively and qualitatively modulate the immune responses in a large animal following *in vivo* electroporation of a DNA vaccine [Yen and Scheerlinck, 2007]. Although, intra-tumor delivery does not generally result in detectable serum transgene expression, intramuscular electroporation does result in serum expression. However, intratumor delivery is more successful than intramuscular delivery in eradicating primary tumors and in generating systemic immunity. For instance, a number of studies have demonstrated long-term, complete tumor regression, using delivery of plasmids encoding IL-12 or IFN-γ as a single agent in melanoma and squamous cell carcinoma (SCC) [Heller and Heller, 2006]. Complete regression after IL-12 gene therapy in combination with herpes simplex virus (HSV) thymidine kinase, bleomycin, or recombinant bacillus Calmette-Guérin (rBCG) has been observed in several experimental models. Electrically mediated bleomycin delivery combined with IL-2 or granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy also induced long-term complete regression in a small percentage of mice with

*5.1.7. Photosensitizers*

378 Application of Nanotechnology in Drug Delivery

Chávez et al., 2009].

et al., 2009].

sheep.

**5.2. Vaccine delivery**

*5.2.1. DNA vaccine*

*5.1.8. Folic acid antagonists*

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the *s.c*. The researchers used the electric pulses to increase transdermal transport of D-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX)-precursor for the photodynamic therapy of superficial skin cancer and cutaneous metastases of internal malignancies. A two-fold enhancement of PpIX produc‐ tion with electroporative delivery was observed compared to passive delivery. The application of iontophoresis also increased the ALA permeation by approximately 15-fold [Escobar-

The topical administration of methotrexate (MTX) for the treatment of psoriasis and neoplastic diseases is restricted by the poor diffusion of MTX across the *s.c*. Some studies showed that electroporation is an efficient method to increase the transdermal transport of MTX. Further‐ more, electroporation of MTX with an anion lipid enhancer under a mild hyperthermic environment provided a significant transdermal delivery within a short time [Escobar-Chávez

Electroporation-based immunization (especially, EP-mediated DNA vaccine) has been effective in a number of species including mice, rats, rabbits, non-human primates, pigs and

DNA immunization has known as an efficient strategy for vaccination [Bolhassani and Rafati, 2009]. The main disadvantage of plasmid DNA vaccines is their poor immunogenicity when administered as an unformulated intramuscular injection [Anderson and Schneider, 2007]. A number of approaches for enhancing the potency of DNA vaccines have developed over the past few years such as: **a)** Optimization of DNA constructs; **b)** Development of new DNA manufacturing processes and formulations; **c)** Augmentation of immune responses with novel encoded molecular adjuvants; and **d)** Improvement of *in vivo* DNA delivery strategies

Among them, EP-mediated delivery has generated considerable attention and appeared to have a great impact in vaccine immunogenicity and efficacy by increasing antigen delivery up to a 1000 fold versus naked DNA delivery alone [van Drunen Littel-van den Hurk and Hannaman, 2010; Sardesai and Weiner, 2011]. In many cases, the immune responses and protection rates observed following DNA administration via EP were comparable or superior to other vaccine strategies including viral vectors and live/attenuated/inactivated virus vaccines [Sardesai and Weiner, 2011, Daemi et al., 2012; Hosseinzadeh et al., 2013]. An electroporation driven plasmid DNA vaccination strategy was studied in animal models for treatment of prostate cancer. This phPSA plasmid electroporation vaccine strategy could

including electroporation [Sardesai and Weiner, 2011].

melanomas. Furthermore, complete responses have been observed in a fibrosarcoma model after delivery of a plasmid encoding GM-CSF and B7.1 [Heller and Heller, 2006]. One of the main challenges for efficient electroporation in larger animals is to ensure correct match between the electrical field and the injected DNA. Intramuscular injection of plasmid DNA followed by electrical stimulation (electroporation) is an efficient method for achieving therapeutic levels of encoded proteins or eliciting efficient immune responses in smaller animals such as mice and rats [Tjelle et al., 2006]. Application of short electrical pulses can be used to enhance gene delivery and DNA vaccination in large animals led to improved cellular and humoral immune responses. In addition, lowering the electrical field will therefore be important for reducing electroporation-induced pain. Increasing the number of electrodes and/or injection volume, could enhance the transfection efficiency of the conventional electroporation devices [Tjelle et al., 2006]. It will be interesting to electroporate different plasmids that were mixed together, plasmids mixed with proteins, or mixed proteins to understand the immune response intensity. It was reported that there is no interference with two different DNA vaccines, implying that it is possible to co-administrate vaccines directed against different pathogens at one time [Yuan, 2008; Yuan, 2008]. Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer's disease (AD) patients. The potency of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP and intradermally by gene gun (GG) was evaluated for treatment and prevention of AD. The results indicated that both delivery methods are effective at promoting potent antibodies specific for Aβ [Davtyan et al., 2012].

class I pathway and increase cytotoxic CD8+T cell production. However, even with these strategies, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration [Best et al., 2009]. Intramuscular administration of HPV DNA vaccines followed by electroporation increased the number of antigen-loaded dendritic cells resulting in the enhancement of gene expression. In a compar‐ ison study of the HPV DNA vaccine administered by different methods, electroporation has been shown to elicit the highest number of E7-specific cytotoxic CD8+T cells and greatest antitumor immune response compared to intramuscular injection and intradermal gene gun delivery [Best et al., 2009; Monie et al., 2010]. Generally, electroporation can be considered as a promising method for delivery of HPV DNA vaccines in human clinical trials [Best et al., 2009]. For instance, electroporation has been successfully used to administer several HPV DNA vaccines to mice as well as rhesus macaques, which has prompted its use in an ongoing Phase I clinical trial of 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 [Monie et al., 2010].

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

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381

Regarding to *in vivo* EP is predominantly carried out intramuscularly, 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. Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8+T-cell immune responses while limiting invasiveness of delivery [Daemi et al., 2012]. It was shown that subcutaneous administration of HPV16 E7 DNA linked to C-terminal fragment of gp96 followed by electroporation can significantly enhance the potency of DNA vaccines [Daemi et al., 2012, Bolhassani et al.,

Larger molecules, including heparin, polylysine, antisense polynucleotides, lactalbumin, and IgG, have been delivered by transdermal electroporation with proper enhancers. The transport of calcium-regulating hormones was found to be increased by applying electroporation and iontophoresis. Anionic lipid formulation has shown significant synergistic effect with electro‐ poration on delivering insulin *in vitro* and *in vivo* and has the potential to lower the voltage threshold to a small level [Hui, 2008]. The reports showed an enhanced transport of human luteinizing hormone releasing hormone through heat-stripped human epidermis by electro‐ poration. Furthermore, the presence of an ionic surfactant such as sodium dodecyl sulfate (SDS) reduced the electroporation threshold and significantly improved the transdermal transport of molecules by electroporation. Indeed, saturated anionic lipids tend to be prefer‐ entially retained in the epidermis during electroporation and result in disrupting the lamellar structure of the *sc* lipids, leading to prolonged lifetime of electropores. Using this method, the transport of both charged and neutral macromolecules was enhanced [Hui, 2008]. Recently, peptides and mini-gene vaccines are of particular interest since several epitopes of tumorassociated antigens have been employed as therapeutic and prophylactic cancer vaccines. Although, small molecular size antigens may be delivered into and through the skin by

2011; Bolhassani et al., 2009].

*5.2.2. Peptide/ protein vaccine*

Gene delivery into solid tumors after direct injection of formulated or naked DNA preparations is generally low due to a large number of delivery barriers e.g., tumor complexity. Tumor electroporation significantly enhanced DNA delivery into solid tumors. Electroporation of *luciferase* DNA into mouse and human tumors produced 10-to 1200-fold increases in luciferase expression compared to tumors injected with *luciferase* DNA alone [Anwer, 2008]. Tumor electroporation by six-needle electrodes (100-μs pulses, 1,500 V/cm) produced a 21-fold enhancement over control while tumor electroporation by caliper electrodes (5,000-μs pulses, 800 V/cm) produced a 42-fold increase. The transfection efficiency of DNA electroporation was compared with that of non-electroporation methods including, liposome-DNA complexes and integrin-liposome-DNA complexes in different tumors [Anwer, 2008]. The electroporation delivery was found to be superior to all other test methods. The maximal enhancement in transfection efficiency by electroporation was up to 30-fold over naked DNA, 5-to 10-fold over liposome-DNA complexes, and over 100-fold over integrin-liposome-DNA complexes. Electroporation produced detectable gene expression in every tumor type while non-electro‐ porated methods were effective only in some tumors [Anwer, 2008]. Moreover, electroporation enhancement of luciferase transfection was up to 16-fold in mouse skin and up to 83-fold in pig skin, as compared to that in non-electroporated groups. In another study, the delivery and anticancer efficacy of MBD2 antisense DNA in electroporated tumors were comparable to the *adenovirus*-treated groups [Anwer, 2008].

Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC class I pathway and increase cytotoxic CD8+T cell production. However, even with these strategies, the efficacy of such immunotherapeutic strategies is dependent on the identification of an effective route and method of DNA administration [Best et al., 2009]. Intramuscular administration of HPV DNA vaccines followed by electroporation increased the number of antigen-loaded dendritic cells resulting in the enhancement of gene expression. In a compar‐ ison study of the HPV DNA vaccine administered by different methods, electroporation has been shown to elicit the highest number of E7-specific cytotoxic CD8+T cells and greatest antitumor immune response compared to intramuscular injection and intradermal gene gun delivery [Best et al., 2009; Monie et al., 2010]. Generally, electroporation can be considered as a promising method for delivery of HPV DNA vaccines in human clinical trials [Best et al., 2009]. For instance, electroporation has been successfully used to administer several HPV DNA vaccines to mice as well as rhesus macaques, which has prompted its use in an ongoing Phase I clinical trial of 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 [Monie et al., 2010].

Regarding to *in vivo* EP is predominantly carried out intramuscularly, 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. Plasmid DNA vaccination using skin electroporation (EP) is a promising method able to elicit robust humoral and CD8+T-cell immune responses while limiting invasiveness of delivery [Daemi et al., 2012]. It was shown that subcutaneous administration of HPV16 E7 DNA linked to C-terminal fragment of gp96 followed by electroporation can significantly enhance the potency of DNA vaccines [Daemi et al., 2012, Bolhassani et al., 2011; Bolhassani et al., 2009].

#### *5.2.2. Peptide/ protein vaccine*

melanomas. Furthermore, complete responses have been observed in a fibrosarcoma model after delivery of a plasmid encoding GM-CSF and B7.1 [Heller and Heller, 2006]. One of the main challenges for efficient electroporation in larger animals is to ensure correct match between the electrical field and the injected DNA. Intramuscular injection of plasmid DNA followed by electrical stimulation (electroporation) is an efficient method for achieving therapeutic levels of encoded proteins or eliciting efficient immune responses in smaller animals such as mice and rats [Tjelle et al., 2006]. Application of short electrical pulses can be used to enhance gene delivery and DNA vaccination in large animals led to improved cellular and humoral immune responses. In addition, lowering the electrical field will therefore be important for reducing electroporation-induced pain. Increasing the number of electrodes and/or injection volume, could enhance the transfection efficiency of the conventional electroporation devices [Tjelle et al., 2006]. It will be interesting to electroporate different plasmids that were mixed together, plasmids mixed with proteins, or mixed proteins to understand the immune response intensity. It was reported that there is no interference with two different DNA vaccines, implying that it is possible to co-administrate vaccines directed against different pathogens at one time [Yuan, 2008; Yuan, 2008]. Induction of a humoral response against amyloid-β peptide may be beneficial for Alzheimer's disease (AD) patients. The potency of an AD DNA epitope vaccine (DepVac) delivered intramuscularly by EP and intradermally by gene gun (GG) was evaluated for treatment and prevention of AD. The results indicated that both delivery methods are effective at promoting potent antibodies specific for

Gene delivery into solid tumors after direct injection of formulated or naked DNA preparations is generally low due to a large number of delivery barriers e.g., tumor complexity. Tumor electroporation significantly enhanced DNA delivery into solid tumors. Electroporation of *luciferase* DNA into mouse and human tumors produced 10-to 1200-fold increases in luciferase expression compared to tumors injected with *luciferase* DNA alone [Anwer, 2008]. Tumor electroporation by six-needle electrodes (100-μs pulses, 1,500 V/cm) produced a 21-fold enhancement over control while tumor electroporation by caliper electrodes (5,000-μs pulses, 800 V/cm) produced a 42-fold increase. The transfection efficiency of DNA electroporation was compared with that of non-electroporation methods including, liposome-DNA complexes and integrin-liposome-DNA complexes in different tumors [Anwer, 2008]. The electroporation delivery was found to be superior to all other test methods. The maximal enhancement in transfection efficiency by electroporation was up to 30-fold over naked DNA, 5-to 10-fold over liposome-DNA complexes, and over 100-fold over integrin-liposome-DNA complexes. Electroporation produced detectable gene expression in every tumor type while non-electro‐ porated methods were effective only in some tumors [Anwer, 2008]. Moreover, electroporation enhancement of luciferase transfection was up to 16-fold in mouse skin and up to 83-fold in pig skin, as compared to that in non-electroporated groups. In another study, the delivery and anticancer efficacy of MBD2 antisense DNA in electroporated tumors were comparable to the

Intracellular targeting of tumor antigens through its linkage to immunostimulatory molecules such as calreticulin (CRT) can improve antigen processing and presentation through the MHC

Aβ [Davtyan et al., 2012].

380 Application of Nanotechnology in Drug Delivery

*adenovirus*-treated groups [Anwer, 2008].

Larger molecules, including heparin, polylysine, antisense polynucleotides, lactalbumin, and IgG, have been delivered by transdermal electroporation with proper enhancers. The transport of calcium-regulating hormones was found to be increased by applying electroporation and iontophoresis. Anionic lipid formulation has shown significant synergistic effect with electro‐ poration on delivering insulin *in vitro* and *in vivo* and has the potential to lower the voltage threshold to a small level [Hui, 2008]. The reports showed an enhanced transport of human luteinizing hormone releasing hormone through heat-stripped human epidermis by electro‐ poration. Furthermore, the presence of an ionic surfactant such as sodium dodecyl sulfate (SDS) reduced the electroporation threshold and significantly improved the transdermal transport of molecules by electroporation. Indeed, saturated anionic lipids tend to be prefer‐ entially retained in the epidermis during electroporation and result in disrupting the lamellar structure of the *sc* lipids, leading to prolonged lifetime of electropores. Using this method, the transport of both charged and neutral macromolecules was enhanced [Hui, 2008]. Recently, peptides and mini-gene vaccines are of particular interest since several epitopes of tumorassociated antigens have been employed as therapeutic and prophylactic cancer vaccines. Although, small molecular size antigens may be delivered into and through the skin by diffusion or by iontophoresis methods, but, higher molecular weight antigens (>1 kDa), such as peptides, DNA, carbohydrates, as well as vaccine adjuvants need to deliver using an efficient rout of administration. Needle-free non-adjuvant skin immunization by electroporation has been reported [Hui, 2008]. For example, delivering the antigenic peptide MYR to mice by electroporation resulted in mucosal immunity and specific lymph node cell proliferation. Also, the others indicated that antigen-specific CTL response to the peptide vaccine delivered by needle-free electroporation/electroosmosis was equivalent to that delivered by intradermal injection with Freund's Complete Adjuvant. In this experiment, the Kb-binding OVA peptide SIINFEKL was used as an example to induce the peptide-specific cytotoxic T lymphocyte (CTL) response in mice [Hui, 2008; Escobar-Chávez et al., 2009].

induced a strikingly lower cell toxicity. Next, mRNA electroporation was used for non-viral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD341 progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types [Van Tendeloo et al., 2001]. In addition, mRNA-electroporated DCs retained their phenotype and maturational potential. Strikingly, a non-specific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrated that Mo-DCs electropo‐ rated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens was a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-

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

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

383

Much intensive research has gone into the development of safe and efficient methods for the delivery of therapeutic genes [Tamura and Sakata, 2003]. Recently, an improved electropora‐ tion protocol was established by optimizing the electroporation parameters including plasmid concentration, voltage and pulse duration, to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption [Yao et al., 2009]. Using this approach, highly efficient gene transfer has already been achieved in muscle and liver as well as in tumors [Tamura and Sakata, 2003]. Electroporation of mouse muscle with secretory *alkaline phospha‐ tase* (*SEAP*) plasmid produced systemic levels of SEAP that were up to 120-fold higher than those achieved with *SEAP* plasmid alone. Intramuscular injection of *erythropoietin* plasmid in mouse leg produced systemic levels of erythropoietin that were 100-fold higher than those from *erythropoietin* plasmid alone. Electroporation of IL-5 plasmid DNA into mouse tibialis muscle produced 20 ng IL-5/mL while the non-electroporated delivery generated only 0.2 ng IL-5/mL in the blood. Electroporation of mouse muscle with IL*-*12 plasmid produced 1500 pg of IL-12 per injected muscle and 170 pg IL-12/ mL in the blood. The huge improvement in muscle delivery (up to 10,000-fold over naked DNA) compared with other non-viral gene delivery systems (10-fold over naked DNA) opens new opportunities for muscle-based gene

Numerous studies on gene transfer have been published in a wide variety of tissues from animal models [Gehl, 2008]. Most of the studies investigated the treatment of protein defi‐ ciencies and cancers using cytokines. DNA formulations were designed to minimize tissue damage or enhance expression at weaker electric pulses. These formulations were prepared with the addition of transfection reagents, membrane permeating agents, tissue matrix modifiers, targeted ligands, or agents modifying electrical conductivity or membrane stability to enhance delivery efficiency or reduce tissue damage. These advancements in DNA formu‐ lation could prove to be useful in improving the safety of electroporation protocols for human applications [Anwer, 2008]. In addition, several DNA formulations have been described for *in vivo* gene electroporation. DNA electroporation in saline were shown to enhance transfection

based tumor vaccines [Van Tendeloo et al., 2001].

**5.3. Gene therapy**

therapy [Anwer, 2008].

*5.3.1. DNA delivery*

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for novel approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the use of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses [Kang et al., 2011]. Another approach involves employing a method capable of improving the intramuscularly delivery of protein-based vaccine led to the slow release of the protein. The studies showed that intramuscular injection of protein (OVA)-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+T cell immune responses and antitumor effects in vaccinated mice. Similar results were observed using the HPV-16 E7 protein-based vaccination system [Kang et al., 2011].

#### *5.2.3. RNA-based vaccines*

RNA-based vaccines represent an interesting immunization modality, but suffer from poor stability and a lack of efficient and clinically feasible delivery technologies. A study evaluated the immunogenic potential of naked *in vitro* transcribed Semliki Forest virus replicon RNA (RREP) delivered intradermally in combination with electroporation [Johansson et al., 2012]. Replicon-immunized mice showed a strong cellular and humoral response, compared to mice immunized with regular mRNA. RREP-elicited induction of interferon-γ secreting CD8+T cells and antibody responses were significantly increased by electroporation. The immune response during the contraction phase was further in‐ creased by a booster immunization, and the proportion of effector memory cells in‐ creased significantly. These results demonstrated that naked RREP delivered via intradermal electroporation can constitute an immunogenic, safe and attractive alternative immuniza‐ tion strategy to DNA-based vaccines [Johansson et al., 2012].

#### *5.2.4. DC-based vaccine*

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. In a study, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs was described. Preliminary experiments in K562 cells revealed that mRNA electroporation compared to plasmid DNA electroporation showed improved transfection efficiency and induced a strikingly lower cell toxicity. Next, mRNA electroporation was used for non-viral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD341 progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types [Van Tendeloo et al., 2001]. In addition, mRNA-electroporated DCs retained their phenotype and maturational potential. Strikingly, a non-specific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrated that Mo-DCs electropo‐ rated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens was a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DCbased tumor vaccines [Van Tendeloo et al., 2001].

#### **5.3. Gene therapy**

diffusion or by iontophoresis methods, but, higher molecular weight antigens (>1 kDa), such as peptides, DNA, carbohydrates, as well as vaccine adjuvants need to deliver using an efficient rout of administration. Needle-free non-adjuvant skin immunization by electroporation has been reported [Hui, 2008]. For example, delivering the antigenic peptide MYR to mice by electroporation resulted in mucosal immunity and specific lymph node cell proliferation. Also, the others indicated that antigen-specific CTL response to the peptide vaccine delivered by needle-free electroporation/electroosmosis was equivalent to that delivered by intradermal injection with Freund's Complete Adjuvant. In this experiment, the Kb-binding OVA peptide SIINFEKL was used as an example to induce the peptide-specific cytotoxic T lymphocyte (CTL)

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for novel approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the use of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses [Kang et al., 2011]. Another approach involves employing a method capable of improving the intramuscularly delivery of protein-based vaccine led to the slow release of the protein. The studies showed that intramuscular injection of protein (OVA)-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+T cell immune responses and antitumor effects in vaccinated mice. Similar results were observed using the

RNA-based vaccines represent an interesting immunization modality, but suffer from poor stability and a lack of efficient and clinically feasible delivery technologies. A study evaluated the immunogenic potential of naked *in vitro* transcribed Semliki Forest virus replicon RNA (RREP) delivered intradermally in combination with electroporation [Johansson et al., 2012]. Replicon-immunized mice showed a strong cellular and humoral response, compared to mice immunized with regular mRNA. RREP-elicited induction of interferon-γ secreting CD8+T cells and antibody responses were significantly increased by electroporation. The immune response during the contraction phase was further in‐ creased by a booster immunization, and the proportion of effector memory cells in‐ creased significantly. These results demonstrated that naked RREP delivered via intradermal electroporation can constitute an immunogenic, safe and attractive alternative immuniza‐

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. In a study, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs was described. Preliminary experiments in K562 cells revealed that mRNA electroporation compared to plasmid DNA electroporation showed improved transfection efficiency and

response in mice [Hui, 2008; Escobar-Chávez et al., 2009].

HPV-16 E7 protein-based vaccination system [Kang et al., 2011].

tion strategy to DNA-based vaccines [Johansson et al., 2012].

*5.2.3. RNA-based vaccines*

382 Application of Nanotechnology in Drug Delivery

*5.2.4. DC-based vaccine*

Much intensive research has gone into the development of safe and efficient methods for the delivery of therapeutic genes [Tamura and Sakata, 2003]. Recently, an improved electropora‐ tion protocol was established by optimizing the electroporation parameters including plasmid concentration, voltage and pulse duration, to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption [Yao et al., 2009]. Using this approach, highly efficient gene transfer has already been achieved in muscle and liver as well as in tumors [Tamura and Sakata, 2003]. Electroporation of mouse muscle with secretory *alkaline phospha‐ tase* (*SEAP*) plasmid produced systemic levels of SEAP that were up to 120-fold higher than those achieved with *SEAP* plasmid alone. Intramuscular injection of *erythropoietin* plasmid in mouse leg produced systemic levels of erythropoietin that were 100-fold higher than those from *erythropoietin* plasmid alone. Electroporation of IL-5 plasmid DNA into mouse tibialis muscle produced 20 ng IL-5/mL while the non-electroporated delivery generated only 0.2 ng IL-5/mL in the blood. Electroporation of mouse muscle with IL*-*12 plasmid produced 1500 pg of IL-12 per injected muscle and 170 pg IL-12/ mL in the blood. The huge improvement in muscle delivery (up to 10,000-fold over naked DNA) compared with other non-viral gene delivery systems (10-fold over naked DNA) opens new opportunities for muscle-based gene therapy [Anwer, 2008].

#### *5.3.1. DNA delivery*

Numerous studies on gene transfer have been published in a wide variety of tissues from animal models [Gehl, 2008]. Most of the studies investigated the treatment of protein defi‐ ciencies and cancers using cytokines. DNA formulations were designed to minimize tissue damage or enhance expression at weaker electric pulses. These formulations were prepared with the addition of transfection reagents, membrane permeating agents, tissue matrix modifiers, targeted ligands, or agents modifying electrical conductivity or membrane stability to enhance delivery efficiency or reduce tissue damage. These advancements in DNA formu‐ lation could prove to be useful in improving the safety of electroporation protocols for human applications [Anwer, 2008]. In addition, several DNA formulations have been described for *in vivo* gene electroporation. DNA electroporation in saline were shown to enhance transfection efficiency in several tissues, producing both local and systemic levels of therapeutic proteins. The enhancement of gene electroporation is associated with significant tissue damage directly related to electroporation intensity. Milder electroporation conditions, although less toxic, are transfectionally inefficient. Several formulation strategies have been examined to reduce electroporation toxicity without affecting transfection activity [Anwer, 2008]. Naked DNA in saline is the most commonly used formulation for *in vivo* gene electroporation. In skeletal muscle, electroporation enhancement of *luciferase* gene transfer was 10,000-fold over nonelectroporated control. The enhancement of luciferase activity was observed in both small and large animal species. Histochemical analysis of *b*-*galactosidase* plasmid electroporated muscle showed a larger transfection area per muscle and a higher plasmid copy number per muscle cell when compared with non-electroporated muscle. Muscle electroporation with *FGF*1 plasmid also indicated significantly larger transfection area in electroporated muscle as compared to non-electroporated muscle [Anwer, 2008]. Also, electroporation enhanced intraarterial administration of a transgenic construct in rats resulted in expression in mesengial cells [Stokman et al., 2010]. A report demonstrated the feasibility of electroporating genes into intact nerve to modify Schwann cell gene expression [Aspalter et al., 2009]. Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. *In vivo* electropo‐ ration with an optimized protocol was also a safe and effective tool for non-viral gene delivery to the beating heart [Ayuni et al., 2010]. This method was used to examine whether introduction and expression of PPARγ gene could differentiate skeletal muscle satellite cells to adipocytes *in vivo* [Bonamassa and Liu, 2010]. The studies indicated that the cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains was studied using *in vivo* electroporation. Plasmid DNA harboring an enhanced green fluorescent protein (EGFP) gene was injected into adult cricket brains, followed by electroporation at a sufficient voltage. Expression of EGFP was observed within the brain tissue [Matsumoto et al., 2013]. Gene therapies for cancer utilizing *in vivo* electroporation have been proved effective in a number of experimental murine tumor models. The therapeutic genes delivered in those cases were diverse including cytokine genes (IL-12) and cytotoxic genes (TRAIL), making a wide range of therapeutic strategies [Tamura and Sakata, 2003]. Generally, cancer gene therapy has been studied using *in vivo* electroporation including suicide genes (e.g., combination of HSV-TK and prodrug GCV: TK-GCV), apoptosis inducing genes (e.g., TRAIL), immuno-stimulatory genes (e.g., IFN-gamma, IL-12 and IL-18) and anti-angiogenic genes (e.g., Endostatin) [Tamura and Sakata, 2003].

*5.3.3. SiRNA delivery*

There are increasing interests in physical methods for delivery of siRNA [Oh and Park, 2009]. Among physical methods, electroporation has been frequently studied to stimulate the cellular and *in vivo* localized delivery of siRNA by electric pulses [Oh and Park, 2009]. An electropo‐ ration method was established to involve a constant voltage and ''plate and fork'' type electrodes and use it for *in vivo* delivery of siRNA. The electric current correlated to the microvascular density and vascular endothelial growth factor (VEGF) expression and exhib‐ ited a threshold that assures efficient delivery. VEGF siRNA electroporation suppressed the growth of tumors exhibiting high VEGF expression to less than 10% of the control level, but it had no effect on low VEGF-expressing tumors. Notably, a long interval (20 days) of electro‐ poration was enough to obtain a satisfactory effect. Systemically injected siRNA could also be delivered into tumors by this method [Valero et al., 2008]. In atopic dermatitis mouse model, the intradermal delivery of cyclooxygenase specific siRNA into the skin by electroporation resulted in the silencing of the target gene in the skin, and reduced the scratching behavior of mice [Oh and Park, 2009]. The delivery of tumor necrosis factor α-specific siRNA via electro‐ poration was shown to inhibit inflammation in mice with collagen-induced arthritis. More‐ over, the *in vivo* silencing of target genes by electrically mediated siRNA delivery was reported in mice bearing solid tumors [Oh and Park, 2009]. Some studies reported the successful use of electroporation of siRNA delivery to renal tissue. In rats, injection of siRNA into the renal artery followed by electroporation led to predominant knockdown of the target protein in the glomeruli [Stokman et al., 2010]. A number of studies have demonstrated the feasibility of targeted delivery of oligonucleotides, small interfering RNA (siRNA), plasmid DNA, and viral vectors to the corneal cells *in vivo*, specifically stromal keratocytes and corneal epithelial cells, via intrastromal injection, iontophoresis, electroporation, and gene gun. The combination of iontophoresis and electroporation was found to be effective in delivering siRNA but not plasmid DNA into the corneal epithelium [Hao et al., 2010]. Altogether, there is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissues [Broderick et al., 2012]. However, the *in vivo*

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

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

385

delivery of RNA enhanced by EP has not been extensively characterized.

The type of a nucleic acid and the type of the transfected cell generally affect the efficiency of electroporation [Stroh et al., 2010]. Skeletal muscle is a preferable target tissue for a number of reasons including long-term secretion of therapeutic proteins for systemic distribution and promotion of strong humoral and cellular immune responses post-vaccination. Numerous factors impact plasmid uptake and expression after intramuscular injection followed by EP. Briefly, they include: species, targeted muscle, age, plasmid formulation, plasmid concentra‐ tion and dose, pulse pattern, electric field intensity (current, voltage and resistance), pulse length, lag time, electrode configuration and orientation. These improvements in the condi‐ tions of EP can increase the efficacy of plasmid transfer and lower the total amount of plasmid

**6. Efficient agents involved in electroporation**

#### *5.3.2. Protein delivery*

A substantial improvement in muscle delivery with the use of electroporation has renewed interest in muscle tissue for systemic protein therapy. Several therapeutic proteins have been expressed from skeletal muscle and secreted into systemic circulation at substantial concen‐ trations with the use of electroporation [Tamura and Sakata, 2003].

#### *5.3.3. SiRNA delivery*

efficiency in several tissues, producing both local and systemic levels of therapeutic proteins. The enhancement of gene electroporation is associated with significant tissue damage directly related to electroporation intensity. Milder electroporation conditions, although less toxic, are transfectionally inefficient. Several formulation strategies have been examined to reduce electroporation toxicity without affecting transfection activity [Anwer, 2008]. Naked DNA in saline is the most commonly used formulation for *in vivo* gene electroporation. In skeletal muscle, electroporation enhancement of *luciferase* gene transfer was 10,000-fold over nonelectroporated control. The enhancement of luciferase activity was observed in both small and large animal species. Histochemical analysis of *b*-*galactosidase* plasmid electroporated muscle showed a larger transfection area per muscle and a higher plasmid copy number per muscle cell when compared with non-electroporated muscle. Muscle electroporation with *FGF*1 plasmid also indicated significantly larger transfection area in electroporated muscle as compared to non-electroporated muscle [Anwer, 2008]. Also, electroporation enhanced intraarterial administration of a transgenic construct in rats resulted in expression in mesengial cells [Stokman et al., 2010]. A report demonstrated the feasibility of electroporating genes into intact nerve to modify Schwann cell gene expression [Aspalter et al., 2009]. Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. *In vivo* electropo‐ ration with an optimized protocol was also a safe and effective tool for non-viral gene delivery to the beating heart [Ayuni et al., 2010]. This method was used to examine whether introduction and expression of PPARγ gene could differentiate skeletal muscle satellite cells to adipocytes *in vivo* [Bonamassa and Liu, 2010]. The studies indicated that the cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains was studied using *in vivo* electroporation. Plasmid DNA harboring an enhanced green fluorescent protein (EGFP) gene was injected into adult cricket brains, followed by electroporation at a sufficient voltage. Expression of EGFP was observed within the brain tissue [Matsumoto et al., 2013]. Gene therapies for cancer utilizing *in vivo* electroporation have been proved effective in a number of experimental murine tumor models. The therapeutic genes delivered in those cases were diverse including cytokine genes (IL-12) and cytotoxic genes (TRAIL), making a wide range of therapeutic strategies [Tamura and Sakata, 2003]. Generally, cancer gene therapy has been studied using *in vivo* electroporation including suicide genes (e.g., combination of HSV-TK and prodrug GCV: TK-GCV), apoptosis inducing genes (e.g., TRAIL), immuno-stimulatory genes (e.g., IFN-gamma, IL-12 and IL-18) and anti-angiogenic genes (e.g., Endostatin) [Tamura

A substantial improvement in muscle delivery with the use of electroporation has renewed interest in muscle tissue for systemic protein therapy. Several therapeutic proteins have been expressed from skeletal muscle and secreted into systemic circulation at substantial concen‐

trations with the use of electroporation [Tamura and Sakata, 2003].

and Sakata, 2003].

384 Application of Nanotechnology in Drug Delivery

*5.3.2. Protein delivery*

There are increasing interests in physical methods for delivery of siRNA [Oh and Park, 2009]. Among physical methods, electroporation has been frequently studied to stimulate the cellular and *in vivo* localized delivery of siRNA by electric pulses [Oh and Park, 2009]. An electropo‐ ration method was established to involve a constant voltage and ''plate and fork'' type electrodes and use it for *in vivo* delivery of siRNA. The electric current correlated to the microvascular density and vascular endothelial growth factor (VEGF) expression and exhib‐ ited a threshold that assures efficient delivery. VEGF siRNA electroporation suppressed the growth of tumors exhibiting high VEGF expression to less than 10% of the control level, but it had no effect on low VEGF-expressing tumors. Notably, a long interval (20 days) of electro‐ poration was enough to obtain a satisfactory effect. Systemically injected siRNA could also be delivered into tumors by this method [Valero et al., 2008]. In atopic dermatitis mouse model, the intradermal delivery of cyclooxygenase specific siRNA into the skin by electroporation resulted in the silencing of the target gene in the skin, and reduced the scratching behavior of mice [Oh and Park, 2009]. The delivery of tumor necrosis factor α-specific siRNA via electro‐ poration was shown to inhibit inflammation in mice with collagen-induced arthritis. More‐ over, the *in vivo* silencing of target genes by electrically mediated siRNA delivery was reported in mice bearing solid tumors [Oh and Park, 2009]. Some studies reported the successful use of electroporation of siRNA delivery to renal tissue. In rats, injection of siRNA into the renal artery followed by electroporation led to predominant knockdown of the target protein in the glomeruli [Stokman et al., 2010]. A number of studies have demonstrated the feasibility of targeted delivery of oligonucleotides, small interfering RNA (siRNA), plasmid DNA, and viral vectors to the corneal cells *in vivo*, specifically stromal keratocytes and corneal epithelial cells, via intrastromal injection, iontophoresis, electroporation, and gene gun. The combination of iontophoresis and electroporation was found to be effective in delivering siRNA but not plasmid DNA into the corneal epithelium [Hao et al., 2010]. Altogether, there is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissues [Broderick et al., 2012]. However, the *in vivo* delivery of RNA enhanced by EP has not been extensively characterized.
