Mechanistic Analysis of Electroporation-Induced Cellular Uptake of Macromolecules

Abstract

Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.

Activation of caspases and apoptosis in response to low-voltage electric pulses

“…Consistent with previous studies (20,21), PI uptake increased linearly with increased pulse duration and pulse number (Fig. 1).…”

Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes

“…This may be because, as previously stated, for 800 V/cm and 1200 V/cm irreversible damage is produced in the cell membrane. The obtained values are in accordance with observations made previously by other authors who also measured durations of around one second for the total lifetime of the short-term membrane resealing mechanisms [21,[54][55][56][57].…”

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

“…The lifetime and the size of electrically induced pores that would allow pDNA to pass through are still under debate because it is currently infeasible to measure them directly. 17,34,44,45 Whereas controversies remain over the biophysical nature of electrotransfection, there has been increasing evidence from various studies implicating the involvement of biological processes in the uptake and intracellular transport of electrotransfected pDNA. 46,47 Specifically, electrotransfection may involve endocytic pathways, 48 which are essential for nutrient uptake and regulation of receptor-mediated cell signaling.…”