**2. Electroporation conditions**

To achieve excellent gene delivery into the cells, several electroporation conditions will be accomplished during experiment. Also these electroporation conditions depend upon cell to cell variation. Generally these conditions can be divided into three categories (a) cellular factors (b) physiochemical factors and (c) electrical parameters.

### **2.1. Cellular factors**

The gene delivery by electroporation into living cells should take place with high transfection efficiency and high cells viability in a physiological unperturbed state, so that, the effect of gene on a specific cellular function can be measured. The transformation efficiency can be influenced by growth phase of the cells, cell density, cell diameter, cell rigidity etc. The growth period of the cells in higher transformation success can be achieved from early to middle phase [20]. For electroporation, two main parameters needed to be optimized, one is electric field strength and the other is the pulse duration of electric field. When we apply voltage between two electrodes (this two electrodes maintain some distance), the pulse is generally an expo‐ nentially decayed signal with a time constant given by the product of the capacitance and resistance of the buffer solution. For any kind of field strength and pulse duration, the extent of macromolecular entry and degree of mortality will vary among different cell lines [21]. If transmembrane potential (TMP) difference is proportional to the cell size, the electric field strength will be more sensitive for larger cells compared to smaller cells [22]. Also it has been reported that, transmembrane potential difference is related to cell angles and the directions of applied electric fields, where TMP values linearly proportional to the external applied electric field and cell diameter [23]. For the detection of specific effect of electroporated antibody, cellular function can depends on many variables, such as (a) concentration and affinity of introduced antibody into the target cell (b) restriction of antibodies to bind by target molecules (c) antibody can effect by intracellular concentration of target molecules (d) target molecules cellular factor such as epitopes(s) which can recognize the antibodies are unable to bind with target molecules (e) the cellular distribution of target molecules is accessible or not for antibody [21]. The cell viability during electroporation is also an important factor. Several literatures reported that nucleic acid molecules can be delivered in a highly efficient manner by optimizing the electroporation parameters, and the optimized electroporation conditions using a fluorescently labeled transfection control siRNA resulted in 75% transfection efficiency for Neuro-2A, 93% for human primary fibroblasts, and 94% for HUVEC cells, as analyzed by flow cytometry [24]. Saunders et al. have shown the successful uptake of trypan blue and FDA in cells, protoplasts and pollen from different plants using variety of pulse generator for optimizing the electroporation conditions [25].

### **2.2. Physiochemical factors**

endocytosis, liposomes, and biological vectors [10-16]. But electroporation have some advantages when compared to other gene transfer methods such as, (a) easy and rapid operation with high reproducibility due to control of electrical parameters (b) higher transformation efficiency when compared to CaCl2 and PEG mediated chemical transforma‐ tion (c) controllable pore size with variation of electrical pulse and minimizing effect of cytosolic components, and (d) easy to uptake DNA into cells with smaller amount, when compared to other techniques [17-19]. For bulk electroporation, drug delivery can be performed in homogeneous electric field, whereas as single cell electroporation (SCEP), can introduce an inhomogeneous electric field focused on targeted single adherent or suspend‐ ed cell without affecting other neighboring cells. Both techniques can deliver molecules such as DNA, RNA, anticancer drugs into cells in–vitro and in-vivo. However SCEP is more advanced technique compared to the bulk electroporation technique. Recently researchers are concentrating on more advanced research area, such as localized single cell membrane electroporation (LSCMEP), which is an efficient and fast method to deliver drugs into single cell by selective and localized way from millions of cells. This LSCMEP can judge cell to cell variation precisely with their organelles and intracellular biochemical effect. This process can deliver more controllable drug delivery inside the single cell with application of different pulse duration. Both single cell electroporation (SCEP) and localized single cell membrane electroporation (LSCMEP) can provide high cell viability rate, high transfection efficiency, lower sample contamination, and smaller Joule heating effect in comparison with bulk

62 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

To achieve excellent gene delivery into the cells, several electroporation conditions will be accomplished during experiment. Also these electroporation conditions depend upon cell to cell variation. Generally these conditions can be divided into three categories (a) cellular factors

The gene delivery by electroporation into living cells should take place with high transfection efficiency and high cells viability in a physiological unperturbed state, so that, the effect of gene on a specific cellular function can be measured. The transformation efficiency can be influenced by growth phase of the cells, cell density, cell diameter, cell rigidity etc. The growth period of the cells in higher transformation success can be achieved from early to middle phase [20]. For electroporation, two main parameters needed to be optimized, one is electric field strength and the other is the pulse duration of electric field. When we apply voltage between two electrodes (this two electrodes maintain some distance), the pulse is generally an expo‐ nentially decayed signal with a time constant given by the product of the capacitance and resistance of the buffer solution. For any kind of field strength and pulse duration, the extent of macromolecular entry and degree of mortality will vary among different cell lines [21]. If transmembrane potential (TMP) difference is proportional to the cell size, the electric field

electroporation (BEP) process.

**2.1. Cellular factors**

**2. Electroporation conditions**

(b) physiochemical factors and (c) electrical parameters.

Physiochemical factors are more important for electroporation. This phenomena can occur during tissue development which contain the transportation, consumption of nutrients and oxygen, waste generated by cells, mechanical loading of tissue or cells, electromechanical phenomena (piezoelectricity), chemomachanical phenomena (swelling), electrochemical phenomena (Debye length) or osmotic phenomena (transport through the cell membrane). During cell culture stage, cells have to proliferate, colonize homogeneously in porous scaffolds and synthesized extracellular matrixes [26]. Different type of molecules or elements can interact with cells during cell culture [27]. Among all of the soluble elements, oxygen molecules possess the major importance for tissue growth particularly for osteoarticular system [28-29]. The magnitude of cell local oxygen consumption could be affected by cell concentration and temperature. The oxygen molecules passes through the cell membrane subject to enzymatic chemical, which is familiar as fundamental in enzymology. The oxygen consumption (Rs) per unit area of cell layer with surface density σcell can be described as the following expression

$$R\_S = \sigma\_{cell} \times V\_{\text{max}} \times \frac{\text{C}}{\text{C} + K\_M} = -R\_{\text{max}} \times \frac{\text{C}}{\text{C} + K\_M} \tag{1}$$

Where KM is the Michaelis constant, C is the nutrient molecular concentration and the negative sign indicated that all cell layers have a sink effect. The maximum oxygen consumption rates Vmax depends upon cell types and it can vary several order of magnitude. The oxygen con‐ sumption in unit volume such as porous substrate as written as

$$R\_V = \mathbf{S}\_V \times \mathbf{R}\_S \tag{2}$$

This law also can be utilized for other biological phenomena such as cell population growth, drug uptake by tumor cells or absorption of biochemical molecules within kidney [26]. The electroporation efficiency can be affected by ionic composition of buffer solution. The resis‐ tivity and RC time constant of the electric pulse can be determined by ionic concentration of the buffer as written as [20]

$$V = V\_0 \exp(\frac{-t}{RC})\tag{3}$$

$$
\pi = RC \tag{4}
$$

where, V is the voltage across the pulsing chamber, V0 is initial voltage, t is the time after starting of the pulse, R is the resistance of suspension, C is the capacitor of the capacitance, and τ is the time constant. The salt concentration of the electroporation buffer as well as pH of the buffer solution can affect the electroporation efficiency [30-31]. Generally the pH value 7.2 can be considered as an appropriate value for electroporation condition. The permeability of the cell membrane depends upon the solubility properties (such as salt composition, pH), charges or chemistry and solute size. The water molecule can transport inside and outside by osmotic balance. Osmosis can maintain the turgor pressure of the cells, across the cell mem‐ brane between the cell interior and relatively hypotonic environment [32]. The swelling properties of biological tissues can be explained by osmotic disjoining pressure [33]. Also the electroporation efficiency is much better, by introducing gene into cells at (0-4 °C) compared to elevated temperature during electroporation experiment [34-35]. This low temperature helps to protect the rapid resealing of the pores and enhance the uptake efficiency of gene inside the cell [17]. It has been reported that high transformation efficiency can be achieved by cell suspension of slow growing mycobacteria at elevated temperature [36]. Regarding the transfer of DNA into cells , it has been shown that cooling at the time of permeabilization and subsequent incubation (37 °C), can enhance the transformation efficiencies and cell viability [37]. Some of the authors has reported that, the use of low conductivity medium for DNA transfer, can increase the cell viability and transformation efficiency [37]. Increasing the amount of DNA into the pulse chamber can increase linearly transfection level [38-39]. However the toxic effect can be observed for high DNA concentration [39-40]. It is generally considered the use of calcium in the medium during electroporation for not causing high intracellular level of electrolyte. However some researchers use calcium and magnesium into the buffer solution for performing DNA transformation into the cell. In such a condition, DNA with calcium ions can act as positively charged 'glue' and attracted by the negatively charged ions on the exterior cell membrane, as a result, DNA molecules are approximating to the membrane before the electroporation process [41-42].

### **2.3. Electrical parameters**

*RSR VVS* = ´ (2)


= *RC* (4)

This law also can be utilized for other biological phenomena such as cell population growth, drug uptake by tumor cells or absorption of biochemical molecules within kidney [26]. The electroporation efficiency can be affected by ionic composition of buffer solution. The resis‐ tivity and RC time constant of the electric pulse can be determined by ionic concentration of

64 Advances in Micro/Nano Electromechanical Systems and Fabrication Technologies

<sup>0</sup> exp( ) *<sup>t</sup> V V*

t

membrane before the electroporation process [41-42].

*RC*

where, V is the voltage across the pulsing chamber, V0 is initial voltage, t is the time after starting of the pulse, R is the resistance of suspension, C is the capacitor of the capacitance, and τ is the time constant. The salt concentration of the electroporation buffer as well as pH of the buffer solution can affect the electroporation efficiency [30-31]. Generally the pH value 7.2 can be considered as an appropriate value for electroporation condition. The permeability of the cell membrane depends upon the solubility properties (such as salt composition, pH), charges or chemistry and solute size. The water molecule can transport inside and outside by osmotic balance. Osmosis can maintain the turgor pressure of the cells, across the cell mem‐ brane between the cell interior and relatively hypotonic environment [32]. The swelling properties of biological tissues can be explained by osmotic disjoining pressure [33]. Also the electroporation efficiency is much better, by introducing gene into cells at (0-4 °C) compared to elevated temperature during electroporation experiment [34-35]. This low temperature helps to protect the rapid resealing of the pores and enhance the uptake efficiency of gene inside the cell [17]. It has been reported that high transformation efficiency can be achieved by cell suspension of slow growing mycobacteria at elevated temperature [36]. Regarding the transfer of DNA into cells , it has been shown that cooling at the time of permeabilization and subsequent incubation (37 °C), can enhance the transformation efficiencies and cell viability [37]. Some of the authors has reported that, the use of low conductivity medium for DNA transfer, can increase the cell viability and transformation efficiency [37]. Increasing the amount of DNA into the pulse chamber can increase linearly transfection level [38-39]. However the toxic effect can be observed for high DNA concentration [39-40]. It is generally considered the use of calcium in the medium during electroporation for not causing high intracellular level of electrolyte. However some researchers use calcium and magnesium into the buffer solution for performing DNA transformation into the cell. In such a condition, DNA with calcium ions can act as positively charged 'glue' and attracted by the negatively charged ions on the exterior cell membrane, as a result, DNA molecules are approximating to the

the buffer as written as [20]

Electrical parameters are the most important factors to achieve high transformation efficien‐ cy and high cell viability during successful gene transfer into living cells. The electrical parameters mainly depend upon electric field strength, pulse length, number of pulses, time between two pulses and etc. Cell plasma membrane always have a tendency to protect the cytoplasmic volume from outside of any exogenous molecules. Cell membrane also continu‐ ously prevent cell to cell fusion. However, if we apply external electric field pulses and if this electric field just surpasses the capacitance of the cell membrane, then transient electro‐ permeabilized state can occur, which allow the delivery of various extracellular molecules, such as drugs, antibodies, DNA, RNA, dyes, tracers and oligonucleotides from outside of the cell to inside of the cell. If the molecular size is small, it can enter inside the cell membrane by diffusion after electropermeabilization. However if the size is large, the molecules can enter into the cell through electrophoretically driven process as like DNA transferring into the cell membrane. Previously it was reported that, short and strong electric field pulses can make the membrane permeable in a spontaneously reversible way [43]. Also, it was reported, an extremely short pulse in nanosecond range with very high voltages, cellular organelles can be electroporated without cell membrane permeabilization [44]. The cell membrane permeabilization area can be controlled by pulse amplitude. By this permeabilization area, diffusion can take place into the cell membrane [45]. The degree of permeabilization can be controlled by the pulse duration and pulse number, where the longer the pulse, the greater the perturbation of the membrane in a given area [46]. Also it has been reported that area of the membrane being permeabilized is larger on the pole facing positive electrode, but degree of permeabilization is greater on the cell, where pole facing negative electrode [47]. Howev‐ er high transformation efficiency can be obtained, when three successive pulses with two intermittent cooling steps of one minute in each or single pulse without cooling for transfor‐ mation of *Enterococcus faealits*, *E. coli* and *Pseudomonas putida* [38]. Kinetic study of electroper‐ meabilization leads to 5 steps.


**Table 1.** Time dependence of electropermeabilization. Permission to reprint obtained from Elsevier [50].

Table-1, illustrates the five steps where "Induction step" describes the field induced mem‐ brane potential increase which provides local defects, when it reached to a certain critical value (above 200mV). Here mechanical strength of the cell membrane depends upon buffer composi‐ tion.The"Expansionstep"comeswhenfieldpresentswithastrengthlargerthanacriticalvalue. Inthiscaseelectromechanicalstresspresent."Stabilizationstep"indicates,fieldintensityislower than threshold value, a stabilization process will take place in a few milliseconds. As a result membrane will be permeabilized for small molecules. "Resealing step" demonstrates a slow resealing on a scale of seconds and minutes. The "Memory effect" comes due to some changes of the membrane properties for longer time, such as an hours, but cell behavior is still normal [48-50].Table-2demonstrate electroporationconditions ofvarious celltypes [51], where electric field strength, pulse length, no of pulses, time between two pulses vary in each different type of cells.


**Table 2.** Electroporation conditions for various cell types. Permission to reprint obtained from RNA society [51].
