Techniques of signal generation required for electropermeabilization. Survey of electropermeabilization devices.

Electropermeabilization is a phenomenon that transiently increases permeability of the cell plasma membrane. In the state of high permeability, the plasma membrane allows ions, small and large molecules to be introduced into the cytoplasm, although the cell plasma membrane represents a considerable barrier for them in its normal state. Besides introduction of various substances to cell cytoplasm, permeabilized cell membrane allows cell fusion or insertion of proteins to the cell membrane. Efficiency of all these applications strongly depends on parameters of electric pulses that are delivered to the treated object using specially developed electrodes and electronic devices--electroporators. In this paper we present and compare most commonly used techniques of signal generation required for electropermeabilization. In addition, we present an overview of commercially available electroporators and electroporation systems that were described in accessible literature.

Quantitative model of small molecules uptake after in vitro cell electropermeabilization.

Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution.

Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis.

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.