New Insights into the Mechanisms of Gene Electrotransfer – Experimental and Theoretical Analysis

Abstract

Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.

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

“…Several studies have indicated that particles of different sizes could enter cells via different mechanisms. 8,83,85,86 Vesicles in clathrin-and caveolin-mediated endocytosis are small in size (<100 nm), which can significantly limit their transport capacities for pDNA with a similar size. In contrast, macropinocytosis, which is known to be responsible for cellular uptake of viruses, 87,88 nanoparticles, 89 and naked pDNA, 90 can generate vesicles with sizes of up to 5 mm that could transport a significantly larger amount of pDNA per vesicle.…”

“…7 Various gene delivery methods have been developed to circumvent the barriers. 8 In general, viral-based methods are more efficient and capable of integrating certain desired genes into the host genome for sustained gene expression. However, the viral-based systems have a number of drawbacks, including immunogenicity, cytotoxicity, and high cost.…”

Quantification of cell membrane permeability induced by monopolar and high-frequency bipolar bursts of electrical pulses

“…When biological membranes are exposed to sufficiently intense pulsed electric fields (PEFs), their permeability increases, enhancing the molecular exchange between the cell and its environment. This phenomenon, known as electroporation or electropermabilization, enables processes like gene transfection [1,2] and chemotherapy [3,4] to be performed much more efficiently. Molecular dynamics simulations have suggested that the formation of pores in the lipid bilayer occurs when water molecules align at the water-bilayer interface and are driven through the hydrophobic bilayer core by local electric field gradients [5,6,7,8].…”

Irreversible electroporation ablation area enhanced by synergistic high- and low-voltage pulses

“…From the above equations, the total electroporated area of the cell membrane that exceeds the critical transmembrane voltage U c is given by the equation below [38]: where S is the total surface area of the cell membrane. Eq 5 shows the positive proportional relationship between the electroporated area of the membrane and the external electric field.…”

“…Arena et al demonstrated greatly improved outcomes in heterogeneous tissues using high-frequency bipolar pulses [37]. Richard Heller and Mojca Pavlin found that a combination of short high-voltage pulses and long low-voltage pulses could enhance the efficiency of gene electrotransfer [38,39]. In addition, IRE has been very successful in the treatment of tumors [40].…”