**8. Clinical trials**

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

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

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

[Sharei et al., 2013].

388 Application of Nanotechnology in Drug Delivery

malignant liver and lung lesions [Niessen et al., 2013].

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 expressing Gn and Gc genes of HTNV or PUUV and evaluated in a Phase I study. Three groups of nine subjects each were vaccinated on days 0, 28 and 56 with the DNA vaccines for HTNV, PUUV, or mixture of both vaccines using the Ichor Medical Systems TriGrid™ Intramuscular Delivery System (TDS-IM) [Hooper et al., 2012]. All vaccinations consisted of a total dose of 2.0 mg DNA in an injected volume of 1 mL saline. For the combined vaccine, the mixture contained equal amounts (1.0 mg) of each DNA vaccine. There were no study-related serious adverse events (SAEs). Neutralizing antibody responses were detected in 5/9 and 7/9 of individuals who completed all three vaccinations with the HTNV or PUUV DNA vaccines, respectively. In the combined vaccine group, 7/9 of the volunteers receiving all three vaccina‐ tions developed neutralizing antibodies to PUUV. The three strongest responders to the PUUV vaccine also had strong neutralizing antibody responses to HTNV. These results demonstrated that the HTNV and PUUV DNA vaccines delivered by electroporation separately or as a mixture are safe. In addition, both vaccines were immunogenic, although when mixed together, more subjects responded to the PUUV than to the HTNV DNA vaccine [Hooper et al., 2012]. Figue 2 shows several important EP-mediated DNA vaccines used in clinical trials.

**9. Conclusions**

contraction.

**Author details**

Azam Bolhassani\*

, Afshin Khavari and Zahra Orafa

Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran

\*Address all correspondence to: azam.bolhassani@yahoo.com; A\_bolhasani@pasteur.ac.ir

Electroporation is a widely recognized method of gene delivery into mammalian tissues. It is a highly efficient method, with delivery efficiency better than many non-viral vectors. The preclinical development of electroporation in vivo is focused on tissues that are easily accessible to electroporation and can resist to electric pulsation. The standard DNA formula‐ tion for electroporation is DNA in physiological saline. Under optimal conditions, DNA electroporation in saline yields a 10- to 10,000-fold enhancement in gene delivery efficiency over non-electroporated controls. This enormous increase in transfection activity, however, accompanies significant tissue damage and local inflammation, which might not be a disad‐ vantage, if the target is cancer. However, for applications in which expression from normal tissues is desired, tissue damage and inflammatory response are not favorable to therapeutic objectives and, therefore, must be minimized. Several formulation strategies have been designed to enhance electroporation efficiency and minimize toxicity. Hopeful results have been obtained with some approaches, which must be further developed into clinically viable formulations for non-cancer applications. Some progresses, such as HIV vaccine, West Nile virus vaccine have been made; however, these also propose some questions: What are the differences for best parameters when conduct electroporation on various muscle cells with distinct morphology and membrane properties that are also different among species? How to reduce the pain during electroporation? How long can gene expression be maintained after electrotransfer? Many experiments showed that electroporation is a safe and potent method, thus electroporation-mediated anticancer gene therapy represents a great therapeutic poten‐ tial. The further improvements of electrodes including shape or arrangement of electrodes and electric conditions, by which more efficient and reliable gene transfer is achieved, are important especially in clinical trials. Furthermore, electroporation is an efficient method for enhancing transdermal drug delivery in vitro and in vivo and expands the range of compounds delivered transdermally. The combined use of electroporation with other physical enhancers such as iontophoresis is likely to yield useful and interesting data, to further explore electroporation as an efficient method of transdermal drug delivery. The technique of electroporation to enhance anticancer drug (such as bleomycin) delivery to tumor cells, so-called as electroche‐ motherapy, is already being applied clinically against head and neck cancers with little or no side effects. In summary, electroporation is one of the physicochemical methods for gene and drug delivery. It is superior in some aspects but also has several drawbacks. Pulse protocol and electrode design need to be optimized to reduce the main side effects e.g., muscle

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

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

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**Figure 2.** EP-mediated DNA vaccines used in cancer clinical trials

Drug delivery by electroporation has been in experimental use for cancer treatment since 1991 as shown in 11 studies of electrochemotherapy (ECT) of malignant cutaneous or subcutaneous lesions, e.g., metastases from melanoma, breast or head-and neck cancer. The treatment was well tolerated and could be performed on an out-patient basis [Gothelf et al., 2003]. At the Institut Gustave-Roussy, France, the fist clinical trial of ECT with bleomycin in eight patients with recurrent or progressive head and neck squamous cell carcinoma was published in 1991. After that, several clinical studies have been performed in different tumors. Clinical trials have been performed in the treatment of basal cell carcinoma, head and neck cancer (squamous cell carcinoma, adenocarcinoma and adenoid cystic carcinoma), adenocarcinoma of the breast, and malignant melanoma. In addition, a case report was published in which metastatic lesions from a bladder cancer have been successfully treated [Gothelf et al., 2003].
