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Purpose <p>To determine if GET via charge-balanced patterned bipolar microsecond pulses could be improved, novel bursts of patterned bipolar microsecond pulsed electric fields were investigated in a cuvette and 3D tissue model to evaluate gene electrotransfer (GET) in vitro.</p> Methods <p>Various charge-balancing strategies were implemented to create bipolar microsecond waveforms. A cuvette model was used to identify optimal electric field intensities and plasmid concentrations for each protocol before cotransfection of two plasmids was evaluated in a cuvette and a 3D tissue model for a subset of best-performing protocols.</p> Results <p>The 2-1-1 unbalanced, 1-1-0.5 unbalanced, and 2-1-1 burst-balanced protocols were the top bipolar microsecond protocols tested on HEK 293 cells and achieved GET efficiencies comparable to the top-performing 8x100μs conventional GET protocol. Of the patterned bipolar pulses, the highest performing was 2-1-1 Unbalanced at 1000&#xa0;V/cm with a dose of 5&#xa0;ms, a delivery rate of 200&#xa0;μs/s, and a plasmid concentration of 1250&#xa0;µg/mL. C28 chondrocytes were also tested via the cuvette model with 2-1-1 burst-balanced exceeding even the top-performing 8x100μs conventional protocol in GET efficiency.</p> Conclusion <p>Patterned bipolar microsecond GET tested in this study has similar transfection capabilities as conventional GET settings while maintaining viability and requiring lower plasmid concentration for similar results. The novel patterned waveforms tested potentially enhance electrophoretic effects, reducing the need for high plasmid concentrations in vivo. These waveforms were developed based on &lt; 2&#xa0;μs bipolar pulses (H-FIRE and INSPIRE) which have been shown to reduce muscle stimulations in vivo.</p>

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Altering Charge-Balance via Patterned Bipolar Pulses for Successful Gene Electrotransfer

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月8日

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  • Alexia K. Cash,
  • Robert H. Williamson,
  • Driss Elhanafi,
  • Michael B. Sano

摘要

Purpose

To determine if GET via charge-balanced patterned bipolar microsecond pulses could be improved, novel bursts of patterned bipolar microsecond pulsed electric fields were investigated in a cuvette and 3D tissue model to evaluate gene electrotransfer (GET) in vitro.

Methods

Various charge-balancing strategies were implemented to create bipolar microsecond waveforms. A cuvette model was used to identify optimal electric field intensities and plasmid concentrations for each protocol before cotransfection of two plasmids was evaluated in a cuvette and a 3D tissue model for a subset of best-performing protocols.

Results

The 2-1-1 unbalanced, 1-1-0.5 unbalanced, and 2-1-1 burst-balanced protocols were the top bipolar microsecond protocols tested on HEK 293 cells and achieved GET efficiencies comparable to the top-performing 8x100μs conventional GET protocol. Of the patterned bipolar pulses, the highest performing was 2-1-1 Unbalanced at 1000 V/cm with a dose of 5 ms, a delivery rate of 200 μs/s, and a plasmid concentration of 1250 µg/mL. C28 chondrocytes were also tested via the cuvette model with 2-1-1 burst-balanced exceeding even the top-performing 8x100μs conventional protocol in GET efficiency.

Conclusion

Patterned bipolar microsecond GET tested in this study has similar transfection capabilities as conventional GET settings while maintaining viability and requiring lower plasmid concentration for similar results. The novel patterned waveforms tested potentially enhance electrophoretic effects, reducing the need for high plasmid concentrations in vivo. These waveforms were developed based on < 2 μs bipolar pulses (H-FIRE and INSPIRE) which have been shown to reduce muscle stimulations in vivo.