Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery

Mazères, Serge; Šel, Davorka; Golzio, Muriel; Pucihar, Gorazd; Tamzali, Y.; Miklavčič, Damijan

doi:10.1016/j.jconrel.2008.11.003

Cited by 56 publications

(30 citation statements)

References 41 publications

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“…To avoid an electric drift of the cells during pulsation, adherent cells were grown on a glass coverslip chamber for fluorescence microscopy observations (Lab-Tek™ II system, Nalge Nunc International). The EP chamber was designed using two stainless steel parallel rods (1 mm diameter, 10 mm length, 5 mm interelectrode distance) (45). The electrodes were connected to the pulse voltage generator and a uniform electric field was generated.…”

Section: Methodsmentioning

confidence: 99%

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Paganin-Gioanni

Bellard

Escoffre

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

116

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The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.

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Section: Methodsmentioning

confidence: 99%

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Paganin-Gioanni

Bellard

Escoffre

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

116

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“…The field distribution between the electrodes was homogenous in the central region between the electrodes, where the calculated field was equal to the applied voltage-toelectrode-distance ratio [43]. Different electrodes, with smaller interelectrode distance, had to be used for pulses of 150 ns duration, because of the high intensity of the field, required to electroporate the cells with such pulses.…”

Section: Electroporationmentioning

confidence: 99%

Equivalent Pulse Parameters for Electroporation

Pucihar

Krmelj

Reberšek

et al. 2011

IEEE Trans. Biomed. Eng.

188

109

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Abstract-Electroporation-based applications require the use of specific pulse parameters for a successful outcome. When recommended values of pulse parameters cannot be set, similar outcomes can be obtained by using equivalent pulse parameters. We determined the relations between the amplitude and duration/number of pulses resulting in the same fraction of electroporated cells. Pulse duration was varied from 150 ns to 100 ms, and the number of pulses from 1 to 128. Fura 2-AM was used to determine electroporation of cells to Ca 2+ . With longer pulses or higher number of pulses, lower amplitudes are needed for the same fraction of electroporated cells. The expression derived from the model of electroporation could describe the measured data on the whole interval of pulse durations. In a narrower range (0.1-100 ms), less complex, logarithmic or power functions could be used instead. The relation between amplitude and number of pulses could best be described with a power function or an exponential function. We show that relatively simple two-parameter power or logarithmic functions are useful when equivalent pulse parameters for electroporation are sought. Such mathematical relations between pulse parameters can be important in planning of electroporationbased treatments, such as electrochemotherapy and nonthermal irreversible electroporation.

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“…Pulse parameters (voltage and duration) were monitored through an oscilloscope (Enertec, France). The electropulsation was applied by using two stainless steel parallel rods (diameter 1 mm, length 10 mm, inter-electrode distance 4 mm) in contact with the bottom of the labtek or Petri dish (Figure 1) [18]. To follow the modifications induced by electric field pulses, we used the following permeabilization protocol: 0.7 kV.cm -1 , 5 ms, 1 Hz, x10 followed 2 min later by a single 5 Kv or 9 kV.cm -1 , 5 µs pulse, or we applied the single 5 kV or 9 kV.cm -1 , 5 µs pulse alone.…”

Section: Cell Culture and Electropermeabilizationmentioning

confidence: 99%

Double pulse approach of electropulsation: a fluorescence analysis of the nucleus perturbation at the single cell level

Bellard

Teissié

2009

IEEE Trans. Dielect. Electr. Insul.

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Plasmid Gene transfer and expression can be obtained by the application of electric pulses to a mixture of cells and plasmids (electrogenetherapy, EGT). Electropulsation is rather well characterized at the plasma membrane level. But, the transfer to and across the nuclear envelope remains a problem. Biological approaches showed that EGT was more effective during mitosis. Recently the group of Schoenbach showed that nanosecond ultra high field pulses may affect cytoplasmic organelles including the nucleus. The need for high field was linked on one hand on the time scale and on the other on the size of the target. Therefore we made an approach of the alteration of the nucleus induced by a microsecond high electric pulse (μs HV, up to 9 kV/cm, 5 µs). This perturbation was operated alone or a few seconds after EGT pulses (10x, 0.7 kV/cm, 5 ms) needed to introduce the plasmid in the cytoplasm. Structural alterations of the nucleus organization were investigated. This was obtained by a digitized fluorescence approach at the single cell level, using Hoechst dye as a probe with a high affinity to nucleic acids. The first train of pulses (EGT) induced a huge and rapid (<2min) swelling of cells and of their nucleus associated with a decrease of the mean fluorescence of the nucleus. Mean fluorescence level and volume changes were maintained along the next 10 minutes. The application of a μs HV pulse affects the cell volume and transiently the nucleus volume without any effects on the mean fluorescence level in the nucleus.

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Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery

Cited by 56 publications

References 41 publications

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Equivalent Pulse Parameters for Electroporation

Double pulse approach of electropulsation: a fluorescence analysis of the nucleus perturbation at the single cell level

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