ELECTROPERMEABILIZATION BY UNIPOLAR AND SYMMETRICAL BIPOLAR RECTANGULAR ELECTRIC PULSES

T. Kotnik¹, L. M. Mir², D. Miklavčič^1
1Faculty of Electrical Engineering, University of Ljubljana,
Tržaška 25, SI-1000 Ljubljana, Slovenia
²UMR 8532 CNRS, Institute Gustave-Roussy,
39 rue C. Desmoulins, F-94805 Villejuif, France

Abstract: Coupling a high-frequency amplifier developed by our group to a programmable function generator, we have compared the efficiency of cell electroperme­abilization in vitro by unipolar and symmetrical bipolar rectangular electric pulses. With symmetrical bipolar pulses, the pulse amplitude required for permeabilization of 50% of the cells was approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the pulse amplitudes causing death of 50% of the cells. Bipolar pulses also led to more than 20% increase in the average uptake of lucifer yellow per cell. In addition, we have measured the concentrations of electrolytically released iron from stainless steel electrodes, and of aluminum from aluminum cuvettes. Such electrolytic contamination can be detrimental to the suspended cells, and also causes erosion of the electrodes. As our measurements show, the contamination is over tenfold lower with symmetrical bipolar pulses as compared to unipolar pulses of the same amplitude and total duration. A theoretical explanation can be provided for both the increased efficiency of permeabilization and the reduced electrolytic contami­nation.

INTRODUCTION

Electropermeabilization (electroporation) is an effective method of internalization of various molecules into biological cells, with an increasing number of applications in oncology, genetics, immunology, and cell biology. The efficiency of electropermeabilization in vitro depends on various physical and chemical parameters, such as the composition of the membrane and osmotic pressure, but above all on the parameters of electric pulses. Several studies have addressed the roles of the amplitude, number and duration of unipolar rectangular pulses. A systematic comparison of the efficiency of unipolar and bipolar rectangular pulses has been, to date, a subject of a single study, in which the authors showed that DNA transfection in vitro was significantly more efficient with a bipolar wave than with a unipolar wave [1]. While in this study, the authors applied continuous square waves, electropermeabilization is usually performed using sequences (trains) of rectangular pulses, with typical durations from hundreds of microseconds to tens of milliseconds, and sepa­rated by intervals from several milliseconds to several seconds. An increased efficiency of separate bipolar rectan­gular pulses in comparison to unipolar pulses of same amplitude and duration thus remained to be demonstrated.

METHODS

DC3F cells, a line of spontaneously transformed Chinese hamster fibroblasts, were grown in monolayers at 37°C and 5% CO₂. The culture medium was Eagle minimum essential medium (EMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 125 µg/ml streptomycin.

After trypsination, cells were centrifuged for 5 min at 1000 rpm and resuspended at 2x10⁷ cells/ml in Spinner minimum essential medium (SMEM, a calcium-depleted modification of EMEM). Electric pulses were generated by Tektronix AFG 310 programmable function generator and amplified to the required voltages (from -280V to +280 V) with a bipolar amplifier built in the Laboratory of Biocybernetics at the Faculty of Electrical Engineering of the University of Ljubljana. The pulses were delivered either to flat stainless steel electrodes, or to an aluminum cuvette. In both cases, the electrodes were 2 mm apart, and a 50-µl droplet of the cell suspension was placed between them.

The percentages of surviving and electropermeabilized cells were determined by their cloning efficiency after pulsation in pure SMEM and in SMEM containing 5 nM bleomycin [2], respectively. Uptake of exogenous molecules was determined by the cell fluorescence after pulsation in SMEM containing 1 mM lucifer yellow. Concentrations of Al³⁺ and Fe²⁺/Fe³⁺ were determined on Hewlett-Packard ICP-MS 4500 Series spectrometer, and on Beckman Coulter CX7 clinical system for chemical analysis, respectively.

RESULTS

Efficiency of electropermeabilization

We compared the efficiency of three trains of rectangular pulses: (i) a train of eight 1-ms unipolar pulses (8 × 1000µs), (ii) a train of eight 1-ms symmetrical bipolar pulses (8 × 500+500µs), and (iii) a train of four 2-ms symmetrical bipolar pulses (4 × 1000+1000µs). In each train, the pulses were delivered in 1-s intervals. For the unipolar train (i), the number and duration of pulses parameters were chosen by optimization, and the parameters of the bipolar trains (ii) and (iii) were then set to give each of these trains the same total duration of the pulses as for the train (i). Also, the train (ii) delivered the same number of pulses as (i), while in the train (iii) the duration of single polarity was the same as in (i).

Fig. 1 shows the percentages of electropermeabilized and surviving cells, as well as the fluorescence of internalized lucifer yellow as functions of the ratio between the applied voltage and the distance between the electrodes for these pulse trains. With bipolar pulses, the voltage-to-distance ratio required for the permeabilization of 50% of the cells was approximately 20% lower than with unipolar pulses, while no statistically significant difference was detected between the voltage-to-distance ratios causing the death of 50% of the cells. With bipolar pulses, the peak uptake of lucifer yellow was also more than 20% higher.

Electrolytic contamination

Fig. 2 shows concentrations of Al³⁺ ions released from aluminum cuvettes by application of the trains (i) and train (ii). At the same voltage-to-distance ratio, release of Al³⁺ was more than one order of magnitude lower with bipolar pulses than with unipolar ones. Similar results were obtained for release of Fe²⁺/Fe³⁺ from stainless steel electrodes.

Figure 1. Permeabilization (top), survival (middle), and uptake of lucifer yellow (bottom) with unipolar and bipolar pulses. P_50% and D_50% are voltage-to-distance ratios required for per­meabilization and death of 50% of the cells, respectively.

Figure 2. Release of Al³⁺ with unipolar and bipolar pulses.

DISCUSSION

Our results show that with bipolar pulses, efficiency of electropermeabilization is higher and electrolytic conta­mination lower than with unipolar pulses. A theoretical explanation can be provided for each of these two effects.

Increased efficiency of electropermeabilization. Bipolar pulses counterbalance the asymmetry of permeabilized areas at cell poles introduced by the resting transmembrane voltage, and they also increase the odds of permeabilization of cells with a nonspherical shape or a nonhomogeneous membrane. This model is presented in detail in Ref. [3].

Reduced electrolytic contamination. Symmetrical bipolar pulses are charge-balanced. In addition, in our setup the change of polarity within the bipolar pulse was practically instantaneous (< 2 µs). Further results and sources related to electrolytic contamination are given in Ref. [4].

Acknowledgments: This work was supported in part by the CNRS, the Institute Gustave-Roussy, the University of Paris XI, and the Ministry of Education, Science and Sport of the Republic of Slovenia. The bipolar amplifier was developed within the Cliniporator project (QLK3-1999-00484) under the framework of the 5th PCRD of the European Commission.

REFERENCES

[1]  E. Tekle, R.D. Astumian, P.B. Chock. “Electroporation by using bipolar oscillating electric field: An improved method for DNA transfection of NIH 3T3 cells,” Proc. Natl. Acad. Sci. USA, vol. 88, pp. 4230-4234, 1991.

[2] T. Kotnik, A. Maček-Lebar, D. Miklavčič, L.M. Mir. “Evaluation of cell membrane electropermeabilization by means of a nonpermeant cytotoxic agent,” Biotechniques, vol. 28, pp. 921-926, 2000.

[3] T. Kotnik, L.M. Mir, K. Flisar, M. Puc, D. Miklavčič. “Cell membrane electropermeabilization by symmetrical bi­polar rectangular pulses. Part I. Increased efficiency of per­meabilization,” Bioelectrochemistry, vol. 54, pp. 83-90, 2001.

[4] T. Kotnik, D. Miklavčič, L.M. Mir. “Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination,” Bioelec­trochemistry, vol. 54, pp. 91-95, 2001.