**6. Efficient agents involved in electroporation**

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 and DNA vaccines required to generate targeted levels of biologically active proteins or antibodies [Draghia-Akli et al., 2005].

*in vivo* could be attributed to poor stability of the targeted complexes in extracellular milieu, altered integrin receptor affinity for integrin ligand or suboptimal transfection conditions. Hence, the use of targeted ligands is an attractive approach to improve target specificity of

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DNA dispersion in muscle is highly restricted because of the rigid collagen-and hyaluronanrich matrix surrounding muscle fibers. Pretreatment of tissue with hyaluronidase has been shown to improve gene delivery into liver and skeletal muscles [Anwer, 2008]. Hyaluroni‐ dase treatment prior to electroporation in skeletal muscle produced a substantial increase both in levels and extent of gene transfer in skeletal muscle. Hyaluronidase treatment enhanced transfection efficiency at low electric pulses without significantly damaging the muscle structure or function. This tissue-protective effect of hyaluronidase has been observed in ischemic myocardium and tissue edema. These results demonstrated that hyaluronidase treatment is a useful approach to improve electrogene transfer in higher species where rigid interstitium is a major limitation to plasmid delivery [Anwer, 2008]. Application of electromigration field (3 V for 30 s) has been shown to enhance the uptake of DNA-modified gold nanoparticles during cell electroporation. Gold nanoparticles devoid of DNA coating were not taken up by cells during electroporation. Formulations that can enhance DNA binding to cell surface *in vivo* may also enhance electroporation efficiency at

Currently used methods to introduce foreign DNA into mammalian cells are based on bulk procedures in which large cell numbers are simultaneously transfected, electroporated or virally infected. All of these methods have a number of specific limitations, such as limited control over the amount of DNA uptake, the intracellular half-life and fate of the introduced DNA, and site of genomic integration [Valero et al., 2008]. These limitations represent a serious drawback in situations where genetically modified stem cells have to be produced for therapeutic application, including gene therapy and regenerative medicine, especially when these cells are hard to isolate in large enough numbers. Recently, microfluidic devices have shown great benefits for studying a variety of cell processes. Of particular importance is the use of such devices for electroporation, enabling high efficiency transfer of a variety of macromolecules into cells [Valero et al., 2008]. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans [Hallengärd et al., 2012]. The "plate and fork" electrodes were used for the transfer of a plasmid vector for erythropoietin expression into rat skin and were compared with needle-type and disc-type electrodes. Therefore, the electroporation conditions for significant efficacy vary with the

In general, there are differences in effective variables between a drug and a gene for delivery by electroporation. High field strength and a short pulse length gave good results, at least with some of the drugs investigated (e.g., bleomycin), whereas electroporation for genes benefits

Membrane poration methods, such as electroporation and sonoporation, are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA and RNA delivery applications for previously difficult-to-transfect primary

from a combination of a low electric field and a long pulse length [Takei et al., 2008].

electroporation, but its *in vivo* application has not been fully established.

weak electric pulses [Anwer, 2008].

molecule to be delivered [Takei et al., 2008].

#### **7. Advantages, disadvantages and solutions**

The electroporation can be applied equally to all cell types and at all stages of the cell cycle [Escobar-Chávez et al., 2009]. Collateral damage by electroporation can be serious, compared with some other physical methods. When electroporation field is applied through the skin using surface plate electrodes, the major potential drop develops across the skin instead of across the targeted subcutaneous tissues. Skin edema is a common consequence. Most electroporation protocols aim to permeate only the plasma membranes. Electroporation of the nucleus requires a further step, using higher threshold voltage and shorter pulse length (nucleoporation) [Hui, 2008]. Although the principle of electroporation is applicable to all cell types, its efficiency depends on the electrical properties of the cells. Smaller cells require higher field to permeate. This is an important consideration for *ex vivo* gene delivery especially to hematopoietic cells. Cells with less conductive contents (such as adipocytes) are less suscep‐ tible. The thresholds for different cells in a heterogeneous tissue would thus vary [Hui, 2008].

DNA formulation with certain types of polymers has been found to enhance electroporation efficiency and, in some cases, reduce treatment-related toxicity. Anionic polymers, including poly-L-glutamate, polyacrylic acid, poly-L-aspartate, dextran sulfate, and pectin have been examined for their ability to enhance electroporation mediated gene transfer in skeletal muscle. In addition, DNA complexes of cationic liposomes were electroporated into several histolog‐ ically distinct mouse subcutaneous tumors, and the efficiency of gene transfer was compared with that of naked DNA electroporation [Anwer, 2008, Lai et al., 2008]. Liposomal formulations were transfectionally superior to naked DNA in B16 melanoma, P22 carcinoma, and SaF sarcoma but not in T24 human bladder carcinoma or MC2 mammary carcinoma. This variation in tumor response could be due to differences in the state of tumor necrosis, tumor conduc‐ tivity, or matrix complexity between the different tumors [Anwer, 2008]. A higher interaction of positively charged lipid-DNA complexes with negatively charged cell surfaces could be one of the underlying mechanisms in the lipid enhancement of the electroporation. Addition of anionic liposomes into the electroporation medium has been found to enhance the delivery of macromolecules into cells. For example, dextran uptake during electroporation was enhanced by 80-fold with the addition of phosphatidylglycerol and phosphatidylcholine into the transfection medium. The magnitude of liposome enhancement was dependent on the degree of lipid saturation but independent of polar head group [Anwer, 2008]. DNA delivery by electroporation is not target-specific. Several attempts have been made to improve tissuespecific targeting of electroporated DNA with the use of cell-specific ligands. Antibodies and other molecular entities that recognize specific cell surface receptors have been conjugated to delivery vehicles to achieve high cell specificity during electroporation. The technical feasi‐ bility of *in vivo* DNA targeting by electroporation has not been fully established. For example, electroporation of integrin conjugated liposome-DNA complexes yields much lower transfec‐ tion efficiency than do the non-targeted systems [Anwer, 2008]. This failure of tumor targeting *in vivo* could be attributed to poor stability of the targeted complexes in extracellular milieu, altered integrin receptor affinity for integrin ligand or suboptimal transfection conditions. Hence, the use of targeted ligands is an attractive approach to improve target specificity of electroporation, but its *in vivo* application has not been fully established.

and DNA vaccines required to generate targeted levels of biologically active proteins or

The electroporation can be applied equally to all cell types and at all stages of the cell cycle [Escobar-Chávez et al., 2009]. Collateral damage by electroporation can be serious, compared with some other physical methods. When electroporation field is applied through the skin using surface plate electrodes, the major potential drop develops across the skin instead of across the targeted subcutaneous tissues. Skin edema is a common consequence. Most electroporation protocols aim to permeate only the plasma membranes. Electroporation of the nucleus requires a further step, using higher threshold voltage and shorter pulse length (nucleoporation) [Hui, 2008]. Although the principle of electroporation is applicable to all cell types, its efficiency depends on the electrical properties of the cells. Smaller cells require higher field to permeate. This is an important consideration for *ex vivo* gene delivery especially to hematopoietic cells. Cells with less conductive contents (such as adipocytes) are less suscep‐ tible. The thresholds for different cells in a heterogeneous tissue would thus vary [Hui, 2008]. DNA formulation with certain types of polymers has been found to enhance electroporation efficiency and, in some cases, reduce treatment-related toxicity. Anionic polymers, including poly-L-glutamate, polyacrylic acid, poly-L-aspartate, dextran sulfate, and pectin have been examined for their ability to enhance electroporation mediated gene transfer in skeletal muscle. In addition, DNA complexes of cationic liposomes were electroporated into several histolog‐ ically distinct mouse subcutaneous tumors, and the efficiency of gene transfer was compared with that of naked DNA electroporation [Anwer, 2008, Lai et al., 2008]. Liposomal formulations were transfectionally superior to naked DNA in B16 melanoma, P22 carcinoma, and SaF sarcoma but not in T24 human bladder carcinoma or MC2 mammary carcinoma. This variation in tumor response could be due to differences in the state of tumor necrosis, tumor conduc‐ tivity, or matrix complexity between the different tumors [Anwer, 2008]. A higher interaction of positively charged lipid-DNA complexes with negatively charged cell surfaces could be one of the underlying mechanisms in the lipid enhancement of the electroporation. Addition of anionic liposomes into the electroporation medium has been found to enhance the delivery of macromolecules into cells. For example, dextran uptake during electroporation was enhanced by 80-fold with the addition of phosphatidylglycerol and phosphatidylcholine into the transfection medium. The magnitude of liposome enhancement was dependent on the degree of lipid saturation but independent of polar head group [Anwer, 2008]. DNA delivery by electroporation is not target-specific. Several attempts have been made to improve tissuespecific targeting of electroporated DNA with the use of cell-specific ligands. Antibodies and other molecular entities that recognize specific cell surface receptors have been conjugated to delivery vehicles to achieve high cell specificity during electroporation. The technical feasi‐ bility of *in vivo* DNA targeting by electroporation has not been fully established. For example, electroporation of integrin conjugated liposome-DNA complexes yields much lower transfec‐ tion efficiency than do the non-targeted systems [Anwer, 2008]. This failure of tumor targeting

antibodies [Draghia-Akli et al., 2005].

386 Application of Nanotechnology in Drug Delivery

**7. Advantages, disadvantages and solutions**

DNA dispersion in muscle is highly restricted because of the rigid collagen-and hyaluronanrich matrix surrounding muscle fibers. Pretreatment of tissue with hyaluronidase has been shown to improve gene delivery into liver and skeletal muscles [Anwer, 2008]. Hyaluroni‐ dase treatment prior to electroporation in skeletal muscle produced a substantial increase both in levels and extent of gene transfer in skeletal muscle. Hyaluronidase treatment enhanced transfection efficiency at low electric pulses without significantly damaging the muscle structure or function. This tissue-protective effect of hyaluronidase has been observed in ischemic myocardium and tissue edema. These results demonstrated that hyaluronidase treatment is a useful approach to improve electrogene transfer in higher species where rigid interstitium is a major limitation to plasmid delivery [Anwer, 2008]. Application of electromigration field (3 V for 30 s) has been shown to enhance the uptake of DNA-modified gold nanoparticles during cell electroporation. Gold nanoparticles devoid of DNA coating were not taken up by cells during electroporation. Formulations that can enhance DNA binding to cell surface *in vivo* may also enhance electroporation efficiency at weak electric pulses [Anwer, 2008].

Currently used methods to introduce foreign DNA into mammalian cells are based on bulk procedures in which large cell numbers are simultaneously transfected, electroporated or virally infected. All of these methods have a number of specific limitations, such as limited control over the amount of DNA uptake, the intracellular half-life and fate of the introduced DNA, and site of genomic integration [Valero et al., 2008]. These limitations represent a serious drawback in situations where genetically modified stem cells have to be produced for therapeutic application, including gene therapy and regenerative medicine, especially when these cells are hard to isolate in large enough numbers. Recently, microfluidic devices have shown great benefits for studying a variety of cell processes. Of particular importance is the use of such devices for electroporation, enabling high efficiency transfer of a variety of macromolecules into cells [Valero et al., 2008]. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans [Hallengärd et al., 2012]. The "plate and fork" electrodes were used for the transfer of a plasmid vector for erythropoietin expression into rat skin and were compared with needle-type and disc-type electrodes. Therefore, the electroporation conditions for significant efficacy vary with the molecule to be delivered [Takei et al., 2008].

In general, there are differences in effective variables between a drug and a gene for delivery by electroporation. High field strength and a short pulse length gave good results, at least with some of the drugs investigated (e.g., bleomycin), whereas electroporation for genes benefits from a combination of a low electric field and a long pulse length [Takei et al., 2008].

Membrane poration methods, such as electroporation and sonoporation, are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA and RNA delivery applications for previously difficult-to-transfect primary cells. However, this method can cause cell death and has been shown to damage sensi‐ tive materials such as quantum dots, which aggregate due to exposure to electric fields. There have also been limited reports of successful protein delivery by this mechanism [Sharei et al., 2013].

cell death by initiating apoptosis in patients with Hepatocellular carcinoma (HCC) [Narayanan

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One of the methods that improve DNA penetration of the cell is electroporation [Bolhassani and Rafati, 2011]. EP itself works as an adjuvant to enhance the necessary "danger signals" that become detectable by the immune system. The tissue damage caused by the application of EP causes inflammation and recruits DCs, macrophages and lymphocytes to the injection site inducing significant immune responses, including antibody and T-cell responses [Fioretti et al., 2014; Saade and Petrovsky, 2012]. *In vivo* use of electroporation is done by injecting naked DNA followed by electric pulses from electrodes that are located *in situ* in the target tissues. Successful use of electroporation was observed in transfecting muscles, brain, skin, liver, and tumors. Since every tissue is specific and has its own characteristics, there are no generally accepted optimal conditions of electroporation that are suitable for effective transfection. These are dependent both on the amplitude and duration of the electric pulses and on the amount and concentration of DNA [Bolhassani and Rafati, 2011]. Up to now, several clinical trials have been planned using the electroporation with DNA vaccines for cancer therapy such as: a) Intratumoral IL-12 DNA plasmid (pDNA) [ID: NCT00323206, phase I clinical trials in patients with malignant melanoma, Heller and Heller, 2006; Daud et al., 2008]; 2) Intratumoral VCL-IM01 (encoding IL-2) [ID: NCT00223899; phase I clinical trials in patients with metastatic melanoma]; 3) Xenogeneic tyrosinase DNA vaccine [ID: NCT00471133, phase I clinical trials in patients with melanoma]; 4) VGX-3100 [ID: NCT00685412, phase I clinical trials for HPV infections], and 5) IM injection prostate-specific membrane antigen (PSMA)/ pDOM fusion gene [ID: UK-112, phase I/II clinical trials for prostate cancer, Low et al., 2009; Fioretti et al., 2010] [Saade and Petrovsky, 2012; Bolhassani and Rafati, 2011]. Furthermore, Hepatitis C virus DNA vaccine showed acceptable safety when delivered by Inovio Biomedical's electroporation delivery system in phase I/II clinical study at Karolinska University Hospital. ChronVac-C is a therapeutic DNA vaccine being given to individuals already infected with hepatitis C virus with the aim to clear the infection by boosting a cell-mediated immune response against the virus. This vaccination was among the first infectious disease DNA vaccine to be delivered in humans using electroporation based DNA delivery [Bolhassani and Rafati, 2011]. Recent patents have been focused on the use of genetic immunomodulators, such as "universal" T helper epitopes derived from tetanus toxin, *E. coli* heat labile enterotoxin and vegetable proteins, as well as cytokines, chemokines or co-stimulatory molecules such as IL-6, IL-15, IL-21 to amplify immunity against cancer. Electroporation-based DNA delivery technology dramatically enhances cellular uptake of DNA vaccines [Fioretti et al., 2014]. Preliminary data from an ongoing clinical trial showed electroporation enhanced the frequency and the

magnitude of the anti-HIV-1 T-cell response [Saade and Petrovsky, 2012].

Hemorrhagic fever with renal syndrome (HFRS) is endemic in Asia, Europe and Scandinavia, and is caused by infection with the hantaviruses Hantaan (HTNV), Seoul (SEOV), Puumala (PUUV), or Dobrava (DOBV) viruses. The candidate DNA vaccines were developed for HFRS

et al., 2013].

**8. Clinical trials**

Electroporation is a technique that increases the permeability of cell membranes by changing the transmembrane potential and subsequently disrupting the lipid bilayer integrity to allow transportation of molecules across the cell membrane *via* nano-size pores. This process when used in a *reversible* fashion has been used in medicine and research for drug or macromolecule delivery into cells [Guo et al., 2010; Heish et al., 2011; Phillips et al., 2012; Li et al., 2012; Niessen et al., 2013; Narayanan et al., 2013]. Irreversible electroporation (IRE) is a new minimally invasive tumor ablation technique which induces irreversible disruption of cell membrane integrity by changing the transmembrane potential resulting in cell death. Irreversible electroporation is currently undergoing clinical investigation as local tumor therapy for malignant liver and lung lesions [Niessen et al., 2013].

The use of *irreversible* electroporation (IRE) has been introduced by Rubinsky's group as a method to induce irreversible disruption of cell membrane integrity subsequently causing cell death. IE can effectively create tissue death in micro-to millisecond ranges of treatment time compared to conventional ablation techniques, which require at least 30 minutes to hours. Additionally, it is possible to treat a considerably larger lesion with shorter treatment times than available with current techniques [Guo et al., 2010; Heish et al., 2011; Phillips et al., 2012; Li et al., 2012; Niessen et al., 2013; Narayanan et al., 2013]. A higher electric voltage leading to a larger potential gradient to create irreversible electroporation has been studied using *in vitro* and *in vivo* studies. Irreversible electroporation is technically simple to use and suitable for minimally invasive surgery [Rubinsky, 2007]. Irreversible electroporation is an innovative local-regional therapy that involves delivery of intense electrical pulses to induce nano-scale cell membrane defects for tissue ablation. The purpose of this study was to investigate the feasibility of using irreversible electroporation as a liver-directed ablation technique for the treatment of hepatocellular carcinoma (HCC) in the N1-S1 rodent model. The findings suggested that IRE was effective for targeted ablation of liver tumors in the N1-S1 rodent model; IRE may offer a promising new approach for liver-directed treatment of HCC [Guo et al., 2010]. The advantage of this technique is that it is drug-free and is targeted [Heish et al., 2011]. In an experiment, it was shown that direct IRE completely ablated the tumor cells in osteosarcoma-bearing rats. A significant increase in peripheral lymphocytes, especially CD3+and CD4+cells, as well as an increased ratio of CD4+/CD8+were detectable after the IE application. As compared to the surgical resection group, the IRE group exhibited a stronger cellular immune response. These findings indicated that IRE could not only locally destroy the tumor but also change the status of cellular immunity in osteosarcoma-bearing rats [Li et al., 2012]. Some reports indicate that this novel procedure can be used for abdominal cancer treatment while minimising collateral damage to adjacent tissues because of the unique ability of the ablation method to target the cell membrane [Phillips et al., 2012]. Irreversible electro‐ poration (IRE) is a new ablative technology that uses high-voltage, low-energy DC current to create nanopores in the cell membrane, disrupting the homeostasis mechanism and inducing cell death by initiating apoptosis in patients with Hepatocellular carcinoma (HCC) [Narayanan et al., 2013].
