Elsevier

Bioelectrochemistry

Volume 141, October 2021, 107876
Bioelectrochemistry

Interference targeting of bipolar nanosecond electric pulses for spatially focused electroporation, electrostimulation, and tissue ablation

https://doi.org/10.1016/j.bioelechem.2021.107876Get rights and content

Highlights

  • Nanosecond stimuli can be structured to achieve maximum effect remotely.

  • Targeting is accomplished by cancellation of bipolar cancellation (CANCAN effect)

  • Interference targeting outweighs weakening of the electric field with distance.

  • Engaging the CANCAN effect improves the uniformity of ablation.

Abstract

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a “CANCAN effect”). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.

Introduction

Electric stimuli of nanosecond durations are increasingly explored as a novel tool for neuromodulation [1], [2], [3], [4], [5], activation of neuroendocrine and immune functions [6], [7], [8], [9], [10], [11], [12], [13], tumor and tissue ablation [9], [11], [12], [13], [14], [15], [16], and for biophysical analyses of cell membrane properties, permeabilization, and repair [17], [18], [19], [20], [21], [22]. Known primary effects of nsEP are stimulation by membrane depolarization and nanoelectroporation [4], [23], [24], [25], which can elicit or suppress action potentials [1], [26], [27], activate or inhibit voltage-gated channels [28], [29], [30], [31], and initiate the second messenger Ca2+ and PIP2 signaling even without the involvement of cell membrane receptors [7], [27], [32], [33], [34], [35]. Intense nsEP treatments cause cytoskeleton rearrangements, osmotic stress, cell swelling and blebbing, leading to the apoptotic or necrotic cell death [25], [34], [36], [37], [38], [39].

A recently discovered unique feature of nsEP is that the electric field reversal weakens their effects [40], [41], [42], [43], [44]. A bipolar nsEP can be less effective than a single phase of the same pulse applied as a unipolar pulse. Since a bipolar pulse is essentially a succession of two unipolar pulses, it means that the effect of one unipolar pulse can be canceled by a second unipolar pulse of the opposite polarity. Indeed, two unipolar pulses of the opposite polarity cause smaller effects even when separated by a short interval. This phenomenon was named “bipolar cancellation” and was consistently observed by independent groups [6], [45], [46], [47], [48] in different cell types (CHO, U937, GH3, cardiomyocytes, neurons, nerve fibers, chromaffin cells); in individual cells, cell suspensions, and 3D cultures; and for different endpoints (Ca2+ mobilization, excitation, membrane permeability and electrical conductivity, cell survival, phosphatidylserine externalization). However, the dependence of bipolar cancellation on nsEP parameters, such as phase duration and the ratio of phase amplitudes, differed profoundly between the studies, likely indicating diverse mechanisms involved in the bipolar cancellation phenomenon. For certain endpoints, such as the peripheral nerve excitation [44] and Ca2+ influx into chromaffin cells elicited by ultra-short 2-ns stimuli [47], bipolar cancellation was consistent with the assisted membrane discharge mechanism. For most other targets and endpoints, experimental findings could not be explained by the assisted discharge [16], [42] and the underlying mechanisms have not been identified. While the bipolar cancellation is observed almost universally with nsEP stimulation (including short microsecond pulses for some endpoints), the experimental data are indicative of several diverse biophysical mechanisms responsible for this effect.

The interest in the bipolar cancellation is not limited to mechanistic basic science research. We have recently presented a new concept of focusing stimulation and electroporation by interference targeting of bipolar nsEP [49]. The superposition of two properly shaped and synchronized bipolar nsEP can produce a unipolar pulse remotely from electrodes (Fig. 1A). In this example, two pairs of stimulating electrodes are arranged in a quadrupole and energized from two independent nsEP generators, one delivering a triphasic pulse to aa’ electrodes and the other one delivering a biphasic pulse to bb’ electrodes. The phase amplitude decreases from the first phase to the last phase. The bi- and triphasic pulses are synchronized in such a way that their superposition in cc’ area yields a unipolar pulse. While the electric field is unavoidably the strongest near the electrodes, it is inefficient at stimulation because of the bipolar pulse shape. The transition from bipolar to unipolar pulse shape, from the electrodes to the center of the quadrupole, cancels the bipolar cancellation (the CANCAN effect) and restores the biological efficiency of nsEP despite the weakening of the electric field with distance.

The fundamental novelty of the CANCAN targeting paradigm is that it modulates nsEP efficiency by the gradually changing pulse shape in the region encompassed by stimulating electrodes. The CANCAN paradigm enhances stimulation and electroporation and can be employed to achieve the maximum effect at a distance from electrodes, complementing other novel approaches for non-invasive electrostimulation [50], [51]. Likewise, employing CANCAN may assist tumor and tissue ablation by overcoming the inherent weakening of the electric field away from needle electrodes. At present, ablations by pulsed electric fields are restricted to small inter-electrode distances or require multiple electrode installations to achieve adequate coverage. While CANCAN stimulation does not change the electric field distribution, it alters the field’s ablation efficiency. One may expect a more uniform ablation within an area enclosed by the quadrupole due to the attenuation of the ablation near the electrodes (where the electric field is strong, but pulses are bipolar) and the enhancement of ablation towards the center of the array (where the electric field weakens, but pulses become unipolar).

Our previous study [49] introduced the CANCAN concept and tested it in a linear electrode array. This simplest electrode configuration (a “1D system”) was useful to prove the CANCAN principle, but it is inherently inefficient for practical applications. A more complex quadrupole configuration was proposed but testing it was beyond our equipment capabilities. We have since upgraded the nsEP exposure setup to reduce the cross-talk of two nsEP generators, increased the voltage output, and enabled the independent tuning of individual phases. We have also added an option for the rotation of the electric field in 90° steps, to refine the CANCAN effect and improve its uniformity. Finally, we improved the detection of nsEP effects in cell monolayers, by using a higher NA objective to automatically image multiple adjacent small areas and “stitch” them into one high-resolution image. This is the first study which accomplished CANCAN stimulation in a quadrupole array and demonstrated it feasibility for practical applications such as tumor and tissue ablation.

Section snippets

Cell lines and media

CHO-K1 (Chinese hamster ovary) and HEK 293 (human epithelial kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were propagated in Ham's F12K (CHO) or DMEM (HEK) medium (Mediatech Cellgro, Herdon, VA) supplemented with 10% fetal bovine serum (FBS). Bovine pulmonary arterial endothelial (BPAE) cells were kindly provided by Dr. J. Catravas (Center for Bioelectrics, ODU). BPAE cells were propagated in a low-glucose DMEM medium (GIBCO, Gaithersburg,

Bipolar cancellation and interference targeting of electroporation by nsEP in a cell monolayer

Fig. 2 compares the location and the extent of electroporation in BPAE cell monolayers after exposure to unipolar nsEP (panels A and C), bipolar nsEP (B and D), and their combinations (E-H). The graphs are the mean fluorescence values measured along two diagonals between the electrodes (see Fig. 1C), with zero in the geometrical center of the electrode array and the positive direction towards aa’ electrodes. The insets are the representative images of YP fluorescence in the monolayer with

Discussion

We consistently observed a robust enhancement of electroporation remotely from stimulating electrodes by engaging the CANCAN effect. Compared to our previous study with a linear electrode array, the CANCAN effect in a quadrupole was incomparably stronger, and the difference from control stimulation could often be discerned “by eye” even without quantitative measurements. The CANCAN effect was universally observed across three different cell lines and using diverse nsEP treatment parameters. We

Conclusions

Interference targeting of bipolar nanosecond electric pulses is a unique stimulation and ablation approach that overcomes the inevitable reduction of the electric field with distance. Its fundamental novelty is the modulation of biological efficiency by a gradual waveshape change in the space between nsEP-delivering electrodes. We have consistently and quantitatively achieved CANCAN-mediated enhancement of nsEP effects at a distance in monolayers of different cell lines, thus laying the

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [A.G.P. has stock options in Pulse Bioscience and authored patents on nsEP applications. Other authors declared no conflicts of interest.]

Acknowledgements

The study was supported by AFOSR MURI grant FA9550-15-1-0517 (to AGP). The authors also thank Dr. C. Zemlin (Washington University in St. Louis) for helpful discussions on the dosimetry and electric field simulations.

Author contributions

A.G.P. conceived the study; I.S. performed experiments; S.X. built nsEP generators; E.G. performed electric field simulations; I.S. and A.G.P. analyzed and interpreted the data; A.G.P. wrote the manuscript, with contributions and editing by all authors.

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      This dependence was observed for adherent (CHO) and suspension-based (U937) cell lines, as well as for primary ventricular cardiomyocytes. The dependence of cancellation on the electric field strength was qualitatively similar in three adherent cell lines (CHO, BPAE, and HEK)[36]. Cancellation was relatively weak or absent at the lowest electric field strengths.

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