Cell membrane, electroporation

Nanda, G.S., Bedekar, V.W., and Mishra, K.P., Biomedical potential of cell membrane electroporation. Proceedings of 2nd Biomedical Symposium, BARC, Mumbai, 1994. 

Several enveloped viruses, and some physical gene transfer techniques such as electroporation, deliver the nucleic acid into the cell by direct crossing of the cell membrane. Lipid-based, enveloped systems can do this by a physiological, selfsealing membrane fusion process, avoiding physical damage of the cell membrane. For cationic lipid-mediated delivery of siRNA, most material is taken up by endo-cytotic processes. Recently, direct transfer into the cytosol has been demonstrated to be the bioactive delivery principle for certain siRNA lipid formulations [151]. 

FIGURE 13.7 Electroporation occurs when an applied external field exceeds the capacity of the cell membrane. 

Recently another electrically assisted drug delivery technology, electroporation, was proposed as an alternative or adjuvant to iontophoresis. Electroporation comprises the use of electric pulses to induce transient changes in the cell membrane architecture that turn it into more permeable barrier. Beside the permeabilization effect on cell membrane, it was postulated that this technique induces electrophoretic effect on charged macromolecules and drives them to move across the destabilized membrane [205]. 

Application of an electric field to lipid bilayers such as those found in cellular membranes causes short-term depolarization of the membrane and formation of pores and other structural changes [17]. These so-called electropores allow the uptake of hydrophilic macromolecules such as plasmid DNA, siRNA, or proteins that are otherwise unable to diffuse passively through this highly regulated barrier. The use of high-voltage electrical pulses to permeabilize cell membranes was first described as a tool to deliver DNA into mammalian cells in 1982 (Wong and Neumann 1982 Neumann et al. 1982). In cuvette-based methods, cells are... 

Transfection efficacy of naked DNA can be increased by physical methods such as electroporation and sonication. Electroporation employs electric pulses to punch holes in the cell membrane, usually smaller than 10 nm but larger than oligonucleotides. With the use of electroporation, DNA was delivered into the cytosol of cells by diffusion. Since its introduction in 1982, in vivo transfection has been achieved in skeletal muscle, fiver, skin, tumors, testis, and the kidney. Tsujie et al. (2001) developed a method to target glomeruli using electroporation in vivo wherein injection of plasmid DNA via the renal artery was followed by application of electric fields. The kidney was electroporated by sandwiching the organ... 

Cell lysis under a high electric field is referred to as electroporation [6], Under these conditions, the cell membrane experiences dramatic changes in permeability to macromolecules. The main applications of the electroporation include the electrotransformation of cells and the electroporative gene transfer by the uptake of foreign DNA or RNA (in plants, animals, bacteria, and yeast). The electric field generates permeable microspores at the cell membrane, so that the nucleic acid can be introduced by electroosmosis or diffusion. 

Nearly any type of cell (prokaryotic or eukaryotic) can be transformed by the technique of electroporation. Protoplasts are first prepared by enzymatic or chemical disruption of the host-cell membrane polysaccharides. Next, the recombinant vector is introduced to the protoplast suspension residing in a very low ionic strength buffer (or distilled water). This DNA-protoplast suspension is then subjected to one or several 250-V pulses delivered from a cathode and anode placed directly into the solution. This applied voltage gradient will cause a certain population of the cells (—1010 per... 

Figure 6.35. Electroporation. Foreign DNA can be introduced into plant cells by electroporation, the application of intense electric fields to make their plasma membranes transiently permeable.

Extensive research on the attempts to overcome the bilayer membrane barrier has resulted in the emergence of several different methodologies for partial and temporary cell membrane permeabilization. The prominent among them are chemical permea-bilizers, liposomal interactions, ultrasonication, ionizing radiation, and electroporation. 

When cells are placed in external apphed electric fields, they experience an electric force. Electroporation involves the use of short, high voltage pulses to overcome barrier of the cell membrane. When a cell is submitted to an external electric field of high intensity and short duration (kV/cm, p,s), transient and dramatic increase in the permeability of the plasma membrane occurs beyond a point. This phenomenon is popularly called electroporation or electropermeabilization, which allows entry of otherwise impermeable exogenous molecules into the cell interior. This phenomenon has been an active area of research in biology and bioelectrochemistry for more than three decades [3,4] and has found many apphcations in cell biology. 

Composition of electroporation buffer is an important factor affecting electroporation yields. Ionic strength of cell suspension medium needs control, which determines resistance of the cell suspension and resultant RC time constant of the field pulse. Medium supplemented with Ca and Mg in mM concentration range is found to promote efficiency of transformation and cell viability. Erythrocytes electroporated in isotonic buffer in the presence of EDTA or membrane specific drugs showed significant modification in hemolysis response to electroporation [33,34]. Use of square wave pulse removes the medium conductivity mediated effects on cell/tissue electroporation outcome. Generally, cells are pulsed in suspensions of sucrose, mannitol, or sorbitol. Electroporation as well as incubation of pulsed cells can be carried out in medium containing usual cell culture recipes. 

Theoretical cell membrane before and after electroporation... 

Kinosita, K. et al.. Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope, Biophys. J., 53, 1015, 1988. 

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