Electroporation Based Drug Delivery and Its Applications

When a certain strong electrical pulse applied across a cell or tissue, the structures of the cell or tissue would be rearranged to cause the permeabilization of the cell membrane, named in early 1980’s “electroporation”[1]. The theoretical and experimental studies of electric field effects on living cells with their bilayer lipid membrane has been studies in 1960’s to 1970’s century [1-6]. During these years, the researches were primarily dealt with reversible and irreversible membrane breakdown in vitro. Based on these research, the first gene transfer by custom-built electroporation chamber on murine cells was performed by Neumann et al. in 1982 [7]. When electric field (E≈0.2V, Usually 0.5-1V) applied across the cell membrane, a significant amount of electrical conductivity can increase on the cell plasma membrane. As a result, this electric field can create primary membrane “nanopores” with minimum 1 nm radius, which can transport small amount of ions such as Na+ and Clthrough this mem‐ brane “nanopores”. The essential features of electroporation included (a) short electric pulse application (b) lipid bilayer charging (c) structural rearrangements within the cell mem‐ brane (d) water-filled membrane structures, which can perforate the membrane (“aqueous pathways” or pores) and (e) increment of molecular and ionic transportation [8]. In conven‐ tional electroporation (Bulk electroporation) technique, an external high electric field pulses were applied to millions of cells in suspension together in-between two large electrodes. When this electric field was above the critical breakdown potential of the cell, a strong polarization of the cell membrane occur due to the high external electric field. Applying a very high electric field could be resulted in the formation of millions of pores into the cell membrane simultaneously without reversibility [9]. Several methods other than electropora‐ tion can be used for gene transfer like microprecipitates, microinjection, sonoporation,


Introduction
When a certain strong electrical pulse applied across a cell or tissue, the structures of the cell or tissue would be rearranged to cause the permeabilization of the cell membrane, named in early 1980's "electroporation" [1].The theoretical and experimental studies of electric field effects on living cells with their bilayer lipid membrane has been studies in 1960's to 1970's century [1][2][3][4][5][6].During these years, the researches were primarily dealt with reversible and irreversible membrane breakdown in vitro.Based on these research, the first gene transfer by custom-built electroporation chamber on murine cells was performed by Neumann et al. in 1982 [7].When electric field (E≈0.2V,Usually 0.5-1V) applied across the cell membrane, a significant amount of electrical conductivity can increase on the cell plasma membrane.As a result, this electric field can create primary membrane "nanopores" with minimum 1 nm radius, which can transport small amount of ions such as Na + and Cl -through this membrane "nanopores".The essential features of electroporation included (a) short electric pulse application (b) lipid bilayer charging (c) structural rearrangements within the cell membrane (d) water-filled membrane structures, which can perforate the membrane ("aqueous pathways" or pores) and (e) increment of molecular and ionic transportation [8].In conventional electroporation (Bulk electroporation) technique, an external high electric field pulses were applied to millions of cells in suspension together in-between two large electrodes.When this electric field was above the critical breakdown potential of the cell, a strong polarization of the cell membrane occur due to the high external electric field.Applying a very high electric field could be resulted in the formation of millions of pores into the cell membrane simultaneously without reversibility [9].Several methods other than electroporation can be used for gene transfer like microprecipitates, microinjection, sonoporation, endocytosis, liposomes, and biological vectors [10][11][12][13][14][15][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 CaCl 2 and PEG mediated chemical transformation (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][18][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 suspended 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 electroporation (BEP) process.

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.

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 exponentially 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].

Physiochemical factors
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 (R s ) per unit area of cell layer with surface density σ cell can be described as the following expression 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 V max depends upon cell types and it can vary several order of magnitude.The oxygen consumption in unit volume such as porous substrate as written as 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 resistivity and RC time constant of the electric pulse can be determined by ionic concentration of the buffer as written as [20] 0 exp( where, V is the voltage across the pulsing chamber, V 0 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 membrane 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].

Electrical parameters
Electrical parameters are the most important factors to achieve high transformation efficiency 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 continuously 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 electropermeabilized 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].However 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 transformation of Enterococcus faealits, E. coli and Pseudomonas putida [38].Kinetic study of electropermeabilization leads to 5 steps.

Trigger
The external field induces an increase in the transmembrane potential up to the critical permeabilizing threshold Table-1, illustrates the five steps where "Induction step" describes the field induced membrane 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 composition.The "Expansion step" comes when field presents with a strength larger than a critical value.
In this case electromechanical stress present."Stabilization step" indicates, field intensity is lower 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][49][50].

Prospect of SCEP over Bulk Electroporation (BEP)
For single cell electroporation (SCEP), the electric field parameters can be controlled to avoid cell death.In SCEP, where an inhomogeneous electric field is applied locally surrounding the single cell adhesion or suspension, whereas in bulk electroporation (BEP), a homogeneous electric field is applied to suspension of millions of cells together.Fig. 1. shows two types of conventional bulk electroporation(BE) chamber, to apply electric field with suspension of millions of cells together for vitro experiment.Both figures has shown the cross sectional view with two metal electrodes.When an external electric field beyond the certain threshold value of the cell membrane, then cell membrane can permeabilized to deliver biomolecules inside the single cell.The success rate like surviving cell for single cell electroporation is far better compared with bulk electroporation (BEP).This technique is faster and easy to perform with less toxicity and technical difficulty for application of wider tissues and cells.By this electroporation technique, the specific cell membrane region with small volume can be targeted to deliver the drugs, which can help to preserve expansive gene or molecules.Due to small volume of electroporation, different gene can be transferred in different electroporated time without cell damage.SCEP technique can provide precise temporal and spatial gene or dye delivery inside the cell.These processes are affordable methods for fluorescently labeled and genetically manipulated individual cells [52].This level of electroporation study is more convenient to understand molecular and genetic mechanisms with their biological functions and SCEP has ability to control temporally molecular biology of the cell, which was challenging task for transgenic model systems [52].For bulk electroporation the required voltages are very high (10 3 V) and this technique has little control of individual cell resulting in suboptimal parameters [53], as a result it is difficult to achieve reversible electroporation of all cells [54].Moreover in single cell electroporation, there is good opportunity to observe the single cell response with specific cell size, shape, status and orientation of the electric field.SCE is useful for primary culture and heterogeneous culture such as brain tissue culture [55].
The first single cell electroporation has been demonstrated by using two carbon fiber microelectrodes [46], where the electrodes (2 µm to 5 µm) was positioned from the boundary of the cell surface at an 0-20° angle and 160-180° angle with respect to the objective plane.The patch clamp technique demonstrated the single cell electroporation (SCE), where patch-clamp pipette was sealed on the cell at a 90 0 angle with respect to the microelectrodes [56].Using this technique, from transmembrane current response, it was possible to determined electric field strength for ion permeable pore formation and kinetics of pore opening, closing as well as pore opening times [56].The electrolyte-filled capillary (EFC) coupled with a high-voltage power supply has been used for single cell experiment [57].For application of a large voltages across EFC, It causes the formation of pores in the cell membranes which induces an electroosmotic flow of electrolyte.Micropipettes filled with DNA or other molecules stimulated by electric field have been electroporate the single cell at the tip of the pipette and successfully deliver the molecules inside single cell [58].Microfabricated chip was used to incorporated the biomolecules into live biological cells for single cell experiment [59].To achieve successful single cell electroporation, cell must be isolated from its population or inhomogeneous electric field must be focused on a particular cell, leaving neighboring cells unaffected [60].Microfabricated devices can fulfil both isolated single cell and focused the electric field on particular single cell.Also this technology can offer other functionalities into the chip.Nowadays, SCEP research is growing on rapidly for biomedical application in vivo and in vitro.However to allow selective manipulation of single organelles within a cell, the electrode size must be reduced to nanoscale level.Nanoelectrode can provide less toxicity with high cell viability during electroporation experiment.Thus the localized single cell membrane electroporation concept has come in several years [61].Fig. 3. shows the localized single cell membrane electroporation (LSCMEP) process, where electric field is applied very short region of the cell membrane.
As a result, due to permeabilization of the cell membrane, drug/biomolecules can be delivered precisely   Chen et al. demonstrated localized single cell membrane electroporation (LSCMEP) by using microfluidic device.Where ITO thin film was used as microelectrode with 1 µm gap between two micro-electrodes.The ITO microelectrode with 100 nm thickness and 2 µm width intense electric field much more in between two microelectrode gap [63].Fig. 5. shows the device fabrication for localized electroporation experiment.According to the results, 0.93 µm electroporation regions were achieved successfully with 60% cell viability for 20 microsecond pulse.Fig. 6. demonstrates the cell survival fluorescence image of HeLa cell at different time scale during LSCMEP process.

Pore formation on SCEP
In single cell electroporation technique, electroporation occurs in adherent cell and tissue.However single cell electroporation can be visualized for cell in suspension.In BEP, mostly the cells are in suspension as spheres, in which homogeneous electric field can be applied.But for single cell electroporation, electric field is in inhomogeneous form, which targets on a particular cell without effecting neighboring cells.Generally cell membrane described in terms of fluid mosaic membrane model [64].Due to application of an electric electric field, the formation of pores into the cell membrane depends upon field strength with low conductance, which is approximated as electrical capacitors with infinite resistance.The pore as liquid capacitor which converts to the electrical force associated with transmembrane potential U into an expanding pressure within the aqueous pore interior [65][66][67][68].The pore creation energy ΔE can be calculated with pressure balance by removal of planar area πr 2 and creation of a cylindrical pore edge of length 2πr, can be written as where surface energy approximately Г= 1× 10 J/m 2 and the edge energy approximately Υ= 1 to 6 × 10 -11 J/m [69][70][71].Here Υ is constant even it is a function of r [70,[72][73].To expand the pore radius from zero radious to r can be written as The first term is energy related stressed pore edge with length 2πr.The second term is energy to remove a circular flat lipid membrane having energy per unit area Г and the third term is steric repulsion of the lipids with constant A. Fourth term arises when transmembrane potential V m is nonzero, which is related to, The transmembrane potential ΔΨ E , in a uniform electric field E at a point M with time t can be written as where f is the shape of the spheroidal cell [74] and τ is the charging time of the cell membrane, g depends upon the conductivities and R is the radius of the spherical cell.E is the field strength and θ(M) is the angle between normal to the membrane at the position M and direction of the field [55].The exponential term can be ignored if the pulse length is longer than a few microseconds.Because induction time τ<1µs, the value f is generally 3/2, which is for completely insulating membrane [75].

Bulk electroporation 4.1. Electric field effect on cell membrane
Biological systems are mainly heterogeneous from electrical point of view [76][77].When a high electric field pulses is applied across the cell membrane, due to rapid polarization, cell membrane can deform mechanically (e.g., suspended vesicles and cells) and is allowed to redis tribute ionic charges due to electrolyte conductivities and distributed capacitance.Initially every bilayer cell membrane structure is dielectric in nature.After application of electric field pulses, membrane conductivity can increase due to structural change of the cell membrane cause the formation of hydrophilic pores from initially formatted hydrophobic pores [78].
Generally the breakdown potential of lipid bilayer is 100-300 mv, which depends upon the lipid compositions [79].If the pulse electric field (PEF) decreases, then breakdown voltage can increase [80][81].
To consider a cell as a sphere with a small volume of V and current is flow of charges.Both current and charges have relationship between them.If we consider the total current flow through small volume of cell V, then the current must be equal to the net flow of charges with in volume V or equal to the rate of decrease of charge with in volume or net flow of current into volume V must be accompanied by an increase of charge with in volume V.This is the principle of conservation of charge, which can be mathematically expressed as Using divergence theorem, ∫ S A.nds = ∫ V ∇ .AdV then the equation can be written as .
where J is the current density and ρ is the volume charge density.Now we can write equation [11] as .0 Since equation [12] must be true irrespective of the volume, so we can write equation [12] as This is equation of continuity, which is the principle of conservation of charge where steady current involve ∂ ρ ∂ t = 0 and if charges are not generated into the cell during application of electric field pulses, then ∇ .J = 0. Now electric field is the gradient of electric potential.So Maxwell equation becomes Δ 2 ψ = 0, where Ψ denotes the electrical potential.If the conductivity of cytoplasm and external medium of the cell is higher than the cell membrane conductivity, then ΔΨ, the field induced transmembrane potential can be written as: where a cell is the outer radius of the cell, E e is the applied electric field strength and θ is angle between field line and normal to the point of interest in the membrane which can be either 0 0 or 180 0 [82][83][84][85].Under the ideal experimental conditions like pulse width, electric field, number of pulses, removal of external electric field for resealing of the pore membrane, pulse duration and rearrangement of the membrane protein can be preserved the cell viability.If the membrane is not spherical, then equation [14] may not be right explanation.If we consider that the cell has ellipsoidal structure, then equation [14] will not be applicable.But for any practical purpose this equation can be used to evaluate the field induced transmembrane potential.

Reversible electroporation
When a strong external electric field applied across cell and tissue, then membrane conductance and permeability can increase significantly due to strong polarization of the cell membrane, as a result membrane can form nano scale defects (called nanopores).But when we switched off the external electric field, membrane can return from its conducting state to its normal state.This phenomenon is called reversible electric breakdown or reversible electroporation [86][87].The reversible electroporation generally involves reversible electric breakdown (REB), which is generally a temporary high conducting state.This reversible electroporation influences both cell membrane as well as artificial planner bilayer lipid membrane.Reversible electroporation involve with rapid creation of many small pores, where membrane discharge occur before any critical pores can evolve from the small pores.To understand the method of electroporation of bilayer lipid membrane, it is necessary to use the method of voltage clamp [65,71,88] and charge relaxation [80,89] techniques, where for charge relaxation, kinetics of voltage decreases across the membrane after the application of short pulses (20 nsec to 10 µsec).It was also fact that originally membrane breakdown can occur before the start of membrane discharge.From the charge relaxation method, it used to show that, when membrane of oxidized cholesterol are rapidly (~ 500nsec) charged ( approximately 1 V), then membrane resistance reversibly decreased by almost nine orders of magnitude [80,89].By this way it was first observed that reversible breakdown of planner lipid bilayer membrane and the charged could not be exceeded beyond 1.2 V, even pulse amplitude was increased further.After first electroporation, it was able to recharge again.This same phenomenon was investigated later with azolectin bilayers modified UO 2 2+ ions and the membranes of lecithin and cholesterol in the presence of alkaloid holoturin A [90][91][92].The different types of behavior of planer oxidized chelosterol membrane are shown in table III [78,80,93].

Characteristic electrical behavior
Pulse magnitude For voltage clam method, the time resolution is 5-10 µs to monitor continuously charge at specific conductance of membrane from 10 -8 to 10 -1 Ω -1 /cm -2 .Thus voltage clam technique and charge relaxation technique are complement to each other [94].

Irreversible electroporation
In our earlier discussion of reversible electroporation, external electric field can permeabilize the cell membrane temporarily by which, the cell membrane can survive and the process known as "reversible electroporation" whereas, some of strong external electric field can cause t the cell membrane to permanently permeabilize (membrane becomes weak effect on conductance), by which the cell can die and the process is refer to as "irreversible electroporation".This irreversible electroporation was observed in early 1754 due to discharge of static electrical generator of the skin [95][96].The main phenomenon of irreversible breakdown was stochastic quantities by which mean life time of membrane can abruptly decreased with increased of voltage.The pores of the bilayer membrane can be hydrophilic or hydrophobic [65].For hydrophobic cases, the pores can be formed by hydrocarbon lipid tails.Whereas the inner surface of the pores can be covered by polar tails.The hydrophobic pores which can fill by water are energetically unfavorable [66] and thus should be short -lived.The formation of the pores during reversible electroporation can exist for longer periods of time due to hydrophilic pores.The accumulation of pores during reversible electroporation is due to membrane containing lysolecithin, which can decrease the linear tension of hydrophilic pores [97][98].The hydrophilic pores can cause the reversible and irreversible breakdown of lipid membrane.Also every electrical field can produce the thermal effect as familiar as Joule effect is disputed, where as certain electric field is undisputed, which can provide irreversible electroporation [95].Irreversible electroporation can affect only the membrane of living cells and spares of tissues scaffold.During irreversible electroporation, the membrane survives in two stages as (a) steady state current stage and (b) fluctuating current stage.The phenomena of irreversible electroporation can cause by charge pulse technique [80] in which membrane is charged at U=0.1 V (with pulse width 400 ns) and discharged was very slow.The large pulse of the same width, can charge the membrane towards 0.4 V, but after 300-400µs, charges can be decreased as a sigmoidal manner up to zero because of membrane rupture [78].

Bulk electroporation
From the last decade, the application of electroporation has been increasing rapidly.Nowadays, the electroporation technique can be applied in many way to deliver drugs, antibodies, oligonucleotides, proteins, RNA, DNA and plasmid in vivo for clinical, biotechnological and biomedical applications [42,[99][100][101].In vivo electroporation is a special kind of interest for all researchers because it is nonviral gene delivery with low cost, safety and ease of realization.Recently nucleic acid based gene transfer has been investigated successfully which could be helpful for more clinical trials in human body [118].T This technique can be applied for food industry [119].For cancer treatment, electrochemotherapy has emerged and this therapy successfully used for clinical trials [42,99,[120][121][122][123].The different types of application of electroporation has mention below.

Electroporation for DNA transfer
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 cytoplasm 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 Transfection Efficiency = KNT 2.3 (1 -E P / E)f(ADN) (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.

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][131][132][133][134]. In this section, different clinical trials with electroporation techniques are mentiond below.

DNA vaccine
DNA vaccines have excellent potential as preventive or therapeutic agents against cancers 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][135][136][137][138][139][140].For DNA vaccination by electroporation, preclinical trials for mouse studies revealed that xenogeneic DNA vaccination with gene encoding tyrosinase family membrane can induced antibody 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 CD 4+ 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 electroporation with five patients per dose level.Plasmid DNA vaccination using electroporation able to elicited robust humoral and CD 8+ T-cell immune responses, while limited invasiveness of delivery [140].DNA delivered method which included phase I clinical trial investigated safety and immunogenicity of xenogenic tyrosinase DNA vaccine, administered intramuscularly with electroporation to patient with stage IIB, IIC,III or IV melanoma(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].

RNA electroporation
The RNA transfer by electroporation technique has been increases continuously recently.RNA can enter inside the cell alone or be used for transfection of dendritic cells, showing several advantages as a vaccine including feasibility, applicability, safeness, and effectiveness, when it comes to the generation of immune responses.In vitro experiment, dendritic cells (DC) pulsed with whole tumor RNA or RNA encoding specific antigen like TAAs induced the generation of specific positive cytotoxic T lymphocytes (CTLs) into the cell [144].Electroporated monocyte-derived DCs with whole RNA from LP-1, U266 cell lines and induced specific CTLs that lysed LP-1 and U266 myeloma cells [145].The RNA delivery into the dendritic cells (DCs) can be achieved by using electroporation of dendritic cells in the presence of RNA [146].
In vivo study was performed by TriMix dendritic cells (DCs) [146].The transfection of Dendritic cells (DCs) were performed by electroporation technique with mRNA encoding CD40L, CD70, and a constitutively actived TLR4 as enhancing elements.Additionally the cells were electroporated with either Mage-A3, Mage-C2, tyrosinase, or gp100 mRNA.The intradermal injections at four different sites of 1.25 × 10 7 TriMix DCs per antigen were provided to two melanoma patient in four times per week.While antigen-specific CD8 T cell responses was detected in both patients after finished treatment, but no data published for this trials [147].

HIV vaccine
Since early 1980s, for causative agent of acquired immudeficiency syndrome (AIDS), an effective vaccine has been continuously tried to find to recover AIDS.Nowadays, the HIV vaccine is introduced by electroporation technique.In vivo experiment on mice, electroporation technique can amplify cellular and humoral immune response to a HIV type 1 EnvDNA vaccine, capable of tenfold reduction in vaccine dose and resulting in an increased recruitment of inflammatory cells [148].The plasmid HIV vaccine, ADVAX env/gag+ADVAX pol/nef-tat (ADVAX), ongoing to examine in phase I trials for uninfected adults (Clinical Trials.govIdentifier: NCT00249106) combination with electroporation as a potential protective vaccine against HIV (Clinical Trials.govIdentifier:NCT00545987). Now more recent study was going on for safety and immunogenicity of an IM injection of two dose of ADVAX using Electroporation TriGrid Delivery Systems (Inchor Medical Systems, Clinical Tials.govIdentifier: NCT00545987) [143].

Cancer treatment
Electroporation technique for cancertreatment (Electrochemotherapy) have been increasing rapidly after first reported of clinical use of electroporation [122].Electrochemotherapy can combine electroporation and chemotherapeutic agents [149][150].The treatment of cutaneous and subcutaneous tumors has reached for clinical trials using bleomycin or cisplatin by antitumor electrochemotherapy process [120,[151][152][153][154][155][156][157].For localized therapy to avoid systematic drug delivery, bleomycin can be injected directly into the tumors by using electrochemotherapy process.Bleomycin is hydrophilic in nature, which can be internalized in limited amounts only in normal condition [158].The use of bleomycin for electroporation process can directly enter into cytosol and its cytotoxicity can be increased up to 300-5000 fold [159][160][161].Different types of cancer can be treated by electroporation technique.The prostate cancer is one of the most common cancer, which is increasing day to day.For this cancer prostate specific antigen (PSA), targeted to the prostate cancer cell for immunotherapeutic approach.The phase I clinical trials with PSA DNA vaccine for human prostate cancer is safe and which can include cellular and humoral immune responses against PSA protein [162][163].The PSA-DNA vaccine has been investigated by electroporation technique [164][165].Electroporation treated with CD 4+ , CD 8+ cells and antibodies were detected in patient successfully with safe and tolerated mode.Electrochemotherapy has also been investigated for treatment of human colorectal cell line and liver tumours [166][167].
The local treatment of electrochemotherapy (ECT) with master cell tumours of Dog has been experimented in where size of the tumors was 5.2 cm 3 and 2.9 cm 3 treated by surgery and ECT.The ECT treatment was easy, effective and safe local treatment for master cell tumors of Dogs [168].Recently, electrochemotherapy has been developed in more advancement for treat ment of internal tumors using surgical procedures, endoscopic routes or percutaneous approaches to gain access to the treatment area [169][170].

Skin electroporation
Molecule or DNA vaccine can transport across targeted tissue of human skin is of great interest for transdermal drug delivery and non-invasive chemical sensing.Skin has capability to produce therapeutic molecules, which not only acts as a systematically or locally, but it can create immunological response, when antigen presenting cells will be targeted.The skin containing antigen presenting cells like dendritic cells, langerhans cells, and mononuclear cells.The gene delivery through the skin electroporation is feasible, efficient and comparable to other tissues [171].The first skin electroporation study was observed in Newborn mice which transfected with a plasmid coding for a neomycin resistance gene [172].The transfection efficiency can depend upon the age of the skin, where the higher transfection efficiency can be achieve for younger mice compare to the older mice [173].Skin electroporation, only clinical study has been reported belonging to metastatic melanoma [118].To date, the skin electroporation has been studied broadly for animal infectious diseases.For most cases Hapatitis B has been investigated for animals through skin electroporation [137,[174][175][176]. Also experiments have been performed vaccine against HIV, smallpox, malaria [177][178][179].

Single cell electroporation
By using single cell electroporation technique, it is possible to deliver the molecules such as drugs, DNA, RNA, peptide, nucleic acid into the cell membrane in vivo and vitro for single cell analysis.The plasmid delivery inside the cell membrane with high efficiency in adherent cells and tissues has been studied in vitro [180][181][182][183][184] and in vivo [52,[183][184][185][186].Fig. 7. show the different applications of single cell electroporation, where membrane can permeabilized to transport protein, small and large molecules inside the single cell.
When two single cells are closed to each other, then cell fusion can occur.Due to high electric field strength, which exceeds the critical value of cell membrane, irreversible electroporation can occur, resulting in cell membrane rapture and finally cell death.This electroporation successfully investigated cell to cell intracellular biochemical variation from millions of cells.However this technique needs a lot of research in the future for more improvement because this technology is in underdeveloped stage.For intracellular targeting, single cell electroporation based systems can be developed for genomic characterization, where a tagged antisense oligonucleotide is introduced to block expression and proteins can be profile by tagged markers [188].To reduce the electrode size in nanoscale label, selective manipulation of single organelles within a cell can be possible.Thus the localized single cell membrane electroporation (LSCMEP) concept has come in frontier research in last several years [61][62][63].This technique can control spatial-temporal process successfully and its have ability to monitor the transfection results in real time situation.To reduce the electrode size in nanoscale label, effective electroporation region should be reduce.As results transfection efficiency should be increase with high cell viability.Florescent markers with single cell electroporation permits direct visualization of cell morphology, cell growth, and intracellular events over timescales ranging from seconds to days.Fluorescent dye or plasmid DNA can enter the neurons with the intact brain of albino Xenopus tadpoles [189].Individual neurons can be elctroporated by this technique in vivo and in vitro including mature and fully differentiated neurons.The transfection of neurons into brain slices and in intact brains of living animals is possible to use this technique.The neuron transfection achievable up to 1 mm dip into a tissue and electrophysiological recording of individual neuron was possible by use of SCEP [190]

Conclusions
This chapter described the detailed concepts about bulk electroporation(BEP) as well as single cell electroporation (SCEP) techniques.In both electroporation technique different types of exogenous molecules such as DNA, RNA, proteins, anticancer drugs, ions, oligonucleotides can be transported into the cell cytosol in vivo or in vitro.For bulk electroporation, the clinical development of DNA based vaccine and immunotherapeutic delivery is progressing.As a nonviral gene transfer, this technique is important for clinical gene transfer regarding efficacy and safety issue compared to other gene transfer techniques.The new technique such as single cell electroporation (SCEP) makes the possibility to judge cell to cell variations with their organelles and intracellular biochemical effect.The development of SCEP technique at clinical level and for biomedical application needs more research in the future.In SCEP, there still lacks the are lack of understanding of theory and molecular delivery inside the cell.But this technique can initiate new root of research, such as single cell biophysics and drug delivery inside single cell.To reduce the electrode gap at nanoscale level, it is possible to do localized single cell mambrane electroporation (LSCMEP) by which selective specific single cell organelles can be manipulated with higher transfection rate and high cell viability.

Figure 1 .
Figure 1.Bulk electroporation apparatus for vitro experiment.Two types of electroporation chamber, to apply an external electric field into the suspension of millions of cells together.Each chamber (a.b) consists cross sectional view of cuvette with two metal electrodes.Figure has redrawn with reprint permission [8].

Fig. 2 .
Fig.2.demonstrates the single cell electroporation technique, where an external electric field is applied across the single cell membrane surface.

Figure 2 .
Figure 2. Single cell electroporation (SCEP) (a) Electric field was applied outside of the cell membrane (b) When external electric field reaches to a certain threshold value of the cell membrane, then cell membrane can permeabilized to deliver drug/biomolecules inside the single cell.After electroporation cell membrane reseal again.
(through sub micrometer to nanometer region of the cell membrane surface) inside the single cell.By this technique selective manipulation of organelles and biochemical effects can be analyze more precisely of the individual cell and this technique have more advantage compared to SCEP.Also the cell rapture and cell death can be minimize because electric field can intense in localized region of the cell membrane compared to SCEP.But this technology is now in underdeveloped stage.Recently Boukany et al. suggested nanochannel electropo-ration with precise amount of biomolecules delivery by LSCMEP process.Where single cell has been positioned in one microchannel by optical tweezers and transfection agent was loaded to another microchannel.Two microchannel were connected by one nanochannel.Due to application of voltage between two microchannels, transfection agent was delivered through nanochannel using electrophoretically driven process and finally drugs delivered inside single cell through a very small area of the cell membrane[62].Nawarathna et al. demonstrated localized electroporation technique using atomic force microscopy (AFM).Where modified AFM tip (0.5 µm) was used as a nanoelectrode, which was produced localized electric field into the cell membrane[61].Fig 4.(a-h) shows the results of LSCMEP technique using AFM tip for electroporation process and Fig.4(i) demonstrated the AFM tip, which was positioned on top of the single cell for LSCMEP process.

Figure 4 .
Figure 4. (a) Bright field image of AFM tip where the cell in the electroporation medium (cell A is electroporated while cell B and C are about 20 µm away from cell A).(b) Fluorescence image of rat fibroblast cell after electroporation.(c) Confocal fluorescence image of an electroporated cell.(d)-(h) Sequence of real time confocal fluorescence images of rat fibroblast cell after electroporation.(i) Calculated spatial distribution of electric field in the vicinity of the cell being electroporated.Permission to reprint obtained from American Institute of Physics (AIP) [61].

Figure 3 .
Figure 3. Localized single cell membrane electroporation (LSCMEP) technique, where drug/biomolecules can deliver precisely inside the single cell (a) Electric field was applied in a very small region of the cell membrane area (Localized way) (b) After electric field application, due to permeabilization of the cell membrane, drug/biomolecules can successfully deliver inside the single cell.Permission to reprint obtained from Springer [63].

Figure 5 .
Figure 5. Fabrication process of ITO microelectrode based localized single cell electroporation chip.(a) Fabrication process step (b) Optical microscope image of patterned ITO microelectrodes.(c) SEM image of ITO microelectrodes with micro channel (FIB etch), Permission to reprint obtained from Springer [63].

Figure 6 .
Figure 6.After application of 8Vpp 20 ms pulse, cell survival fluorescence image of HeLa cell at different time scale, Permission to reprint obtained from Springer [63].

Figure 7 .
Figure 7. Different application of single cell electroporation.When external applied electric field reaches to the threshold values of the cell membrane, then cell membrane can permeabilized to deliver protein, small and large molecules inside the cell.If two single cells are close to each other, then cell fusion can occur.To apply an intense electric field, which exceeds certain critical value, irreversible electroporation can occur resulting cell membrane rapture and finally cell death.Figure has redrawn from reference.Figure has redrawn with reprint permission obtained from Springer [187].

Table 2 .
Table-2 demonstrate electroporation conditions of various cell types [51], where electric field strength, pulse length, no of pulses, time between two pulses vary in each different type of cells.Electroporation [51]itions for various cell types.Permission to reprint obtained from RNA society[51].
Table 4 described details about gene transfer by electroporation technique with the variation of molecules/gene, targeted cells, different types of electric pulses [7, 102-117].

Table 4 .
[94]fied table of gene delivery by electroporation technique.Permission to reprint obtained from Springer book series[94]