**9. Conclusions**

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.

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].

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

390 Application of Nanotechnology in Drug Delivery

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 contraction.
