Pulse electrofusion

The purpose of this paper is - besides a description of these new methods - to present a survey of main results in pulse electroporation (PEP), pulse electrofusion (PEF), and electrostimulation (PEMIC) illustrating the future possibilities and trends, too. 

Another cell operation is to use a pulsed electric field to conduct electroporation or electrofusion. For instance, electrofusion of liposomes (5 pm dia.) and RBC has been conducted on a microchip (see Figure 8.40) [903]. 

Although the mechanisms of electroporation, electrofusion, and electroinsertion are not known, biophysical data suggest that the primary field pulse effect is interfacial polarization by ion accumulation at the membrane surfaces. The resulting transmembrane electric field causes rearrangements of the lipids such that pores are formed1718. Electropores anneal slowly (over a period of minutes) when the pulse is switched off. 

When a DC pulse is applied to a couple of fluid-phase vesicles, which are in contact and oriented in the direction of the field, electrofusion can be observed. Vesicle orientation (and even alignment into pearl chains) can be achieved by application of an AC field to a vesicle suspension. This phenomenon is also observed with cells [164, 165] and is due to dielectric screening of the field. When the suspension is dilute, two vesicles can be brought together via the AC field and aligned. A subsequent application of a DC pulse to such a vesicle couple can lead to fusion. The necessary condition is that poration is induced in the contact area between the two vesicles. The possible steps of the electrofusion of two membranes are schematically illustrated in Figure 7.8a. In Sections 7.5.2.1 and 7.5.2.2, consideration will be given to the fusion of vesicles with different membrane composition or different composition of the enclosed solutions. 

Use of a high field to activate a membrane enzyme was first reported by Witt et al. (25) in 1976. They used dc pulses of approximately 1 kV/cm and of 1-ms duration to induce ATP synthesis by the chloroplast ATPase. Following this initial work, there have been many reports on 1-kV/ cm dc field-induced ATP synthesis in different ATP synthetic systems (see the literature cited in references 13 and 14). The main conclusion from these studies is that an applied field-induced transmembrane potential can facilitate ATP release from the enzyme whether a PEF can affect enzyme turnover is not clear. Because 1-kV/ cm dc fields also cause severe Joule heating of a sample suspension, thermal effects cannot be easily avoided except when very short electric pulses (microseconds) are used. Thus, the method has limited utility for electroactivation experiments. The PEF method is, however, quite popular for the study of electroporation and electrofusion of cell membranes (see the chapter by J. Weaver in this volume), electroinsertion of membrane proteins (26), and electrotransfection of cells (27). 

Electrofusion is the connection of two separate cell membranes into one by a similar pulse. It is believed that the process is based on the same field-induced restructuring of the bilayer lipid membranes, a process that may be reversible or irreversible. 

Urano, N., Sahara, H., Koshino, S. (1993). Conversion of a non-flocculant brewer s yeast to flocculant ones by electrofusion 1. identification and characterization of the fusants by pulsed field gel-electrophoresis. Journal of Biotechnology, 28, 237-247. 

The conductivity of sea water ranges between 1 and 9 S/m Therefore, only a small number of medical, pharmacological, chemical and environmental applications could be developed (mostly brief field-pulse techniques, such as electrofusion or electropermeabilisation [4] Because, this situation was unchanged over more than twenty years, the questions arises, in what respect there is physical limitation ... 

Figure 16.2 A series of phase contrast micrographs depicting the time sequence of electrofusion of giant liposomes of mixed PC PS (1 1). The recording times of the micrographs with respect to the 1.7kVcm , 250 ps pulse application are, from top to bottom, - 1 s. Os, Is, 5s, 30s and after the 150Vcm , 50kHz AC field is turned off, (bar= 10 pm).

Giant liposomes with 5 mol % of (PEG-DSPE) were used to test whether the fusion pulse width thresholds were shifted by the surface shielding polymers. Figure 16.6 shows the fusion threshold of liposomes with added PEG-DSPE. The surface shielding offered by the PEG moiety increases the energy barrier for electrofusion. As a result, the pulse widths required to fuse these surface-modified giant liposomes... 

Figure 26.6 Electroporation of GUV made from C14 (a) and (b) formation of a large pore with pulse durations of 10 ms and 1 ms, respectively (c) and (d) electroadhesion of two vesicles (e) and (f) electrofusion in which diameter increases from 54 to 58 pm (g) and (h) electroexpulsion of an internal vesicle. The scale bar represents 10 pm. In these pictures, the anode is placed at the upper side of the field of view with the cathode at the lower part, and the electric field between the electrodes is 0.5 kV cm . ...

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