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

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.