(I-329) Optimization of Bipolar Microsecond Electric Pulses for Improved Electrochemotherapy

Introduction::

Electrochemotherapy (ECT) electrical pulses create transient pores on cell membranes, through a process known as reversible electroporation (RE), which improves transmembrane transport of chemotherapeutic agents. This enables the use of normally membrane impermeable agents and lower doses of membrane permeable agents, both of which have the potential to reduce systemic side effects associated with chemotherapy.  ECT has been previously demonstrated as a successful treatment for several solid tumor types; however, the induction of intense muscle stimulation by the electrical pulses limits the size of tumors amenable to the procedure. The use of bipolar pulses (bpRE) which are 50x shorter in duration than traditional ECT pulses have recently been proposed as a technique for eliminating muscle stimulation and increasing the overall volume of tissue affected with a single treatment. This study aimed to optimize bpRE for ECT.

Materials and Methods:: Cells were embedded in 3D collagen tissue mimics which enable cells to achieve their native morphology. A coaxial ring-and-pin electrode (Fig. 1) was used to deliver bpRE treatments with varied waveforms, doses, and delivery rates (Table 1) while the tissue mimics were maintained at 37°C treatment to replicate physiological conditions. Propidium iodide (PI), a cytotoxic fluorescent membrane impermeable molecule, was used as a surrogate for chemotherapeutic agents. Active temperature control was used during bpRE treatments to ensure temperature did not exceed 42°C, eliminating thermal damage as a confounding factor.  The mimics were imaged at 0 and 24 hours to assess chemotherapeutic uptake and viability.

A combination of COMSOL models and a MATLAB algorithm were then used determine the electric field intensities associated with ECT and irreversible electroporation (IRE) (Fig. 2) with optimal parameter sets being those which maximized the difference between these thresholds.

Results, Conclusions, and Discussions:: Of the treatment parameters tested, the 2-1-2 waveform demonstrated the highest average ratio of ECT to IRE (Fig. 3) indicating that this waveform maximally enhances transmembrane transport of the chemotherapeutic agent. We observed that the electric field thresholds for these phenomena varied based on cell type indicating a degree of cell-type selectivity. ANOVA analysis (Table 2) confirmed that all factors and their combined interaction have a statistically significant effect (p < 0.01) on both ECT and IRE thresholds with the resulting regression models fitting the experimental data with values of R2=0.970 and R2=0.971, respectively. Dose had the greatest effect on thresholds (Fig. 4). It was also found that the slower delivery rate resulted in lower electric field thresholds for both phenomena, but also lower ratios between these two values.

The use of high-throughput in vitro models coupled with computational modeling enables a broad range of parameters for electroporation to be examined. In this study, optimal waveform, dose, and delivery rates were found, and their importance in determining ECT outcomes demonstrated. Dose had the most noticeable effect on treatment outcomes. Surprisingly, increased energy delivery rates resulted in improved ECT outcomes. We hypothesize that this is due to increased pore formation kinetics over slower delivery rates and this warrants further study.

bpRE eliminates muscle stimulation associated with traditional ECT protocols. This enables the use of higher voltages to treat larger volumes and the use of a single applicator & grounding pad approach. Combined, these may enable a rapid and simplified approach to administering ECT to unresectable cholangiocarcinoma. Future work will focus on the optimization of these protocols in small and large animal models.

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