*5.1.1. Electroporation for DNA transfer*

**Ref. Year Authors Recipient**

**112** 2000 Dujardin et al.

**113** 2004 Yamauchi et al.

**114** 2006 Yamaoka et al.

**116** 2010 Kaufman et al.

**cells**

Human embryonic kidney cells, HEK293

Male Japanese white rabbits (2.5–3.0 kg body

A549 cells (ATCC, Manassas, VA,USA) a human

adenocarcinoma cell line

lung

**115** 2008 Takei et al. MKN-1, PC-3, F12 VEGF Si RNA Square Electro Porator

wt; Kyudo, Tosu, Saga, Japan)

**Plasmid /gene**

78 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

Rat keratinocytes pEGFP-N1 with CMV promoter

cloned into pRc/ CMV plasmid

pEGFP-C1 and pDsRed-C1

**Pulsing CD:E0;τ**

Cytopulse PA-4000 (Cyto Pulse Sciences, Inc., Maryland, USA), 10 pulses of 1000V and 100µs duration

ElectroSquare-

100v/cm, 10ms

Plasmid DNA Electric pulse generator (model CUY 201 BTX)

10

Plasmid DNA BTX ECM 830 ,

Porator T820, BTX, San Diego,

Pon=5ms, Poff=95 ms, No of pulse

(CUY21; Nepagene).

Electroporation coupled with a

Petri-Pulser PP35–2P electrode (Harvard Apparatus,

Holliston, MA, USA) using a single 10 ms 160 V

square wave

**Results**

A localized expression of GFP was observed for at least 7 days in the epidermis. Skin viability was not compromised by electroporation

Efficient to transfer multiple genes, in parallel, into cultured mammalian cells for highthroughput reverse genetics

Optimal gene transfer efficiency

in situ jugular veins of rabbits, and transgene expression was

primarily in endothelial cells.

The delivery efficiency correlated to the electric current. The electric current correlated to the microvascular

density and vascular endothelial growth factor (VEGF) expression and exhibited a threshold that guaranteed

efficient delivery.

and transthoracic

mice that were

lung using

cyclic stretching of the murine

ventilation immediately after endotracheal administration

electroporation of plasmid DNA increases exogenous gene expression up to fourfold in

not ventilated after plasmid administrationand transfection by electroporation in vivo

research.

in the

observed

The first reversible electroporation with DNA electrotransfer has been investigated in 1982 [7]. After application of an external electric pulse, cell membrane can permeabilize and DNA will move towards the cell membrane by electrophoretic force and finally it can enter into cyto‐ plasm of the cell. It has been reported that, small molecules can diffuse into the cell before membrane reseals but DNA cannot transfer inside the cell, if DNA is added immediately after the pulse applications [124]. For better DNA electrotransfer, electric field pulses are important. The electroporating pulse can stimulate a vascular lock (i.e., a transient hypoperfusion) as well as affects the blood circulation to the electropulsed tissues, caused by histamine dependent physiological reaction [125]. For better electrotransfer, electric field pulses have three steps which includes,

(a) Molecules can increase the electrophoretic displacement of the charged molecules due to application of electric filed pulses (b) Cell membrane can enhance the permeabilization (c) Exposed tissues can stimulate the vascular lock [126].

Moreover to deliver the electric pulse for DNA is electrotransfer, just short or high amplitude pulse (e.g. six pulses, 100µs and 1.4 kV cm-1) required to deliver small molecules [127]. For better electrophoretic effect, longer pulses with low amplitude (e.g. eight pulses, 20ms, 200 kV cm-1) are required to increase the transfection rates [124]. However short, high amplitude pulse can follow the long low amplitude pulse. From these two pulses, high amplitude pulse can permeabilize the cell membrane, then long duration low voltage pulse can play the role to drive the DNA into destabilized membrane of the cell [128]. The transfection threshold values are the same for cell electropermeabilization [39]. The transfection efficiency maintains the following equation as mentioned below

$$\text{Transfection Efficiency} = \text{KNT}^{2.3} \text{(1} \cdot \text{E}\_{\text{P}} / \text{E)f} \text{(ADN)} \tag{15}$$

where plasmid concentration f(AND) is complex and high level of plasmid is toxic [129] and K is constant. As results, for DNA electrotransfer, the pulse effect (Field strength, short high amplitude pulse, long low amplitude pulse) are very important and which is the major parameters for efficient gene expression into cell and tissues.

### *5.1.2. Electrotransfer for clinical developments*

The electroporation technique has been used widely for transfection of plasmid in vitro and in vivo. Recently this technique has been used for application of DNA vaccine and gene therapies for clinical trials. Electroporation technology are not only the basis for human studies, but also it influence veterinary medical for animals, which can make the bridge between human and animal studies [130-134]. In this section, different clinical trials with electroporation techniques are mentiond below.

### *5.1.2.1. DNA vaccine*

DNA vaccines have excellent potential as preventive or therapeutic agents against can‐ cers and infectious diseases. For a successful DNA delivery into the cell or tissues, DNA must need to subsequently achieve gene expression of the encoded protein at desired level or for the desired duration of time. In vivo electroporation, which can enhance the delivery efficiency and the cellular uptake of an agent by 1,000 times and it can increase the levels of gene expression (i.e. production of the coded protein) by 100 times or more compared to plasmid DNA delivered without other delivery enhancements. DNA vaccination by electroporation technique has been developed in last several years [134-140]. For DNA vaccination by electroporation, preclinical trials for mouse studies revealed that xenogene‐ ic DNA vaccination with gene encoding tyrosinase family membrane can induced anti‐ body and cytotoxic T cell responses resulted in tumor rejection [141-142]. DNA vaccine, p.DOM-PSMA encoded a domain (DOM) of fragment C of tetanus toxin to induced CD4+ T cell helps to fuse to a tumor-derived epitope from prostate-specific membrane antigen (PSMA) for use in HLA-A2+ patients with recurrent prostate cancer [139]. For this open level phase I/II work, DNA was delivered by intracellular injection followed by electropo‐ ration with five patients per dose level. Plasmid DNA vaccination using electroporation able to elicited robust humoral and CD8+ T-cell immune responses, while limited invasive‐ ness of delivery [140]. DNA delivered method which included phase I clinical trial investigated safety and immunogenicity of xenogenic tyrosinase DNA vaccine, adminis‐ tered intramuscularly with electroporation to patient with stage IIB, IIC,III or IV melano‐ ma(Clinical Trials. Gov ID NCT00471133). Electroporation with xenogeneic tyrosinase DNA vaccine can increase the human response and anti-tumor effects compared to the vaccine alone [143].
