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Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro
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Altering Charge-Balance via Patterned Bipolar Pulses for Successful Gene Electrotransfer.

Alexia K Cash1, Robert H Williamson2, Driss Elhanafi2

  • 1North Carolina State University, Raleigh, NC, USA. akcash@ncsu.edu.

Annals of Biomedical Engineering
|April 8, 2026
PubMed
Summary

Novel patterned bipolar electric fields improve gene electrotransfer (GET) efficiency, matching conventional methods with lower plasmid needs. These microsecond pulses enhance electrophoretic effects for potential in vivo applications.

Keywords:
Bipolar pulsesCharge-balanced waveformsDNA deliveryElectroporationGene electrotransfer

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Bioengineering

Background:

  • Gene electrotransfer (GET) is crucial for gene delivery.
  • Conventional GET methods often require high plasmid concentrations.
  • Improving GET efficiency and reducing plasmid requirements are key research goals.

Purpose of the Study:

  • To investigate novel patterned bipolar microsecond pulsed electric fields for enhanced gene electrotransfer (GET).
  • To evaluate the efficacy of these novel waveforms in cuvette and 3D tissue models.
  • To compare the performance of patterned bipolar pulses against conventional GET protocols.

Main Methods:

  • Developed and implemented various charge-balancing strategies for bipolar microsecond waveforms.
  • Optimized electric field intensities and plasmid concentrations in a cuvette model.
  • Evaluated cotransfection of two plasmids in cuvette and 3D tissue models using best-performing protocols.

Main Results:

  • Patterned bipolar protocols (2-1-1 unbalanced, 1-1-0.5 unbalanced, 2-1-1 burst-balanced) achieved GET efficiencies comparable to conventional 8x100μs GET.
  • The 2-1-1 Unbalanced protocol showed optimal performance at 1000 V/cm with 5 ms dose and 1250 µg/mL plasmid concentration.
  • The 2-1-1 burst-balanced protocol in C28 chondrocytes exceeded conventional GET efficiency.

Conclusions:

  • Patterned bipolar microsecond GET offers comparable transfection to conventional methods with improved viability and lower plasmid concentration.
  • Novel patterned waveforms may enhance electrophoretic effects, reducing in vivo plasmid requirements.
  • These waveforms, inspired by H-FIRE and INSPIRE, show potential for reduced muscle stimulation.