## 1 Introduction  
The importance of gene therapy in various biomedical applications has been on an increase in the recent years. Over the last few decades, viral vectors have been widely used for the effective gene transfer to the target cells or tissues [1]. However, viral vectors pose serious safety concerns due to their immunogenicity, oncogenicity and/or inflammatory potential [2]. Hence, a non-viral gene delivery method is a promising alternative that bypasses the inherent problems of the viral gene vectors [3]. Electroporation is one of the techniques that is widely exploited as a non-viral gene delivery method. It relies on electric field induced transient alteration of cell membrane permeability to introduce various molecules, including plasmid DNA, to the interior of the cell [4]. To trigger electroporation, the cell must be treated with electric field of over-threshold intensity [5], which, in turn, induces a change in the transmembrane potential of the cell [6]. According to the hydrophilic pore theory, once the electric field induced transmembrane potential exceeds the threshold value, which is reported to be in the range between 0.25-1.0 V, hydrophilic pores occur in the cell plasma membrane [7; 8; 9]. The side of the cell facing the positive electrode is permeabilized first due to the negative interior of the cell, and later, another side of the cell facing the negative electrode gets permeabilized. [10]. Depending on the electric field parameters, electroporation of the cell membrane can be reversible or irreversible [11]. As suggested by the name, irreversible electroporation is permanent and can be utilized for non-thermal ablation of malignant cells [12]. On the contrary, reversible electroporation is transient and, if the electric field parameters are properly chosen, membranes reseal and cells remain viable [11]. One of the applications for reversible electroporation is the transfer of hydrophilic or amphiphilic molecules from extracellular matrix into the cell through the electropores [3], which are estimated to have radii of 1-60 nm [13]. Therefore, it is believed that the mechanism of action that drives small molecules (less than electropore size) into the cells during the process of electrotransfer is mainly gradient diffusion. However, it is observed that molecules exceeding the size of the electropores also enter the cell after electroporation, yet different pathways are being utilized [14]. Plasmid DNA, considered as a large molecule, is dragged towards the cell via electrophoresis during the electric pulses, forms a complex with electroporated part of the membrane [15] and later is translocated through the plasma membrane by various types of endocytosis [16; 17]. However, to achieve protein expression, the translocation of the plasmid DNA is not enough since the plasmid must migrates to the nucleus where it undergoes transcription, after which mRNA translation can begin [9]. Although the feasibility of electrotransfection has been shown numerous times [17; 18; 19], the widespread implementation of this method in clinical and industrial applications is still, at least partially, limited as a result of poor transfection efficiency [20]. Numerous studies have been performed on the optimization of the electric field [21; 22; 23] and electroporation medium parameters [24; 25] to achieve efficient electrotransfection. However, the consistent and detailed research is neccessary to be performed regarding other electroporation parameters, such as plasmid concentration. A few research works show an improvement in the electrotransfection efficiency with an increase in plasmid concentration. However, they do not report cell viability data [18; 19; 26]. Others reported an increase in cell electrotransfection efficiency with higher plasmid concentration and noted the decrease in cell viability to ~70-80%. However, these works did not provide thorough research on the dependence of cell viability in the wide range of plasmid DNA concentration [27; 28; 29].