# Threshold Interphase Delay for Bipolar Pulses to Prevent Cancellation Phenomenon during Electrochemotherapy

## TL;DR

This study investigates how adjusting the delay between pulses in electrochemotherapy can reduce the cancelation effect and improve treatment efficiency.

## Contribution

The study identifies threshold interphase delays for bipolar pulses to minimize the cancelation phenomenon during electrochemotherapy.

## Key findings

- Bipolar cancellation can be minimized with appropriate interphase delays, achieving efficiency similar to monophasic pulses.
- Bipolar cancellation significantly affects membrane permeabilization but has minimal impact on electrochemotherapy outcomes.
- Reduced interphase delays (0 ms and 0.1 ms) were tested in vitro with cisplatin and 10 Hz protocols.

## Abstract

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells’ plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.