The discovery and utilization of cell membrane electroporation date from decades ago [1], and the research on the electroporation mechanism has increasingly attracted attention. Most of the current mechanistic studies are based on macroscopic and static models such as calculating the electric field distribution with the electric circuit model and the transmembrane voltage measured experimentally. However, as a non-equilibrium process on a molecular scale, the electroporation mechanism is determined by the movement of phospholipid molecules, water molecules and ions. Classical static and macro-models cannot describe unbalanced microscopic processes, such as the kinetics of pore formation, dynamics behavior of pores, as well as the transport of small ions across the pores in membrane. Therefore, in this paper, in order to explore the relationship between the movement of molecules and the reversible electroporation process, the molecular dynamics simulation software GROMACS was used to simulate the electroporation dynamics to reveal the basic mechanism on a molecular scale.

In the earliest experiments, it was found that the permeability of cell membranes could be enhanced by exposing cells to a suitable electric field [1]. The transient pores that formed in the membrane allowed the intracellular delivery of drugs, DNA, and other molecules. Chang et al. were the first to use cryo-electron microscopy to observe the formation of nanoscale (20–120 nm) pores on the surface of the cell membrane [2]. They showed that the electroporation process was inconsistent with electrical breakdown, because electrical breakdown involved chemical reactions.

The theory that first explained the electroporation process treated the phospholipid membrane as a flexible surface [3] that was squeezed under the influence of an electric field. If the electric field strength was sufficiently large to increase the compression force above a critical value, the phospholipid membrane could no longer withstand it and broke. This model did not take the molecular structure of the phospholipid membrane into account. New models proposed in recent years have added information about pore structure on the surface of phospholipid membranes [4, 5]. Some researchers considered electroporation as a phase transition since the structure of the phospholipid bilayer was metastable and the formation of pores could be considered the nucleation process of the other phase [6]. The electric field influences the nucleation rate and determines whether it will develop into a ‘crystallization’ process with irreversible electroporation. In the transient hydrophilic pore model, the phospholipid molecule itself was assumed to randomly generate some penetrating channels due to fluctuations in its thermal motion [4, 7]. When placed in a suitable electric field, the polar head of a phospholipid molecule is pushed toward the interior of a randomly generated tunnel, causing it to increase in length.

None of the above theoretical models took into account the effects of atomic motion. The method of molecular dynamics simulation is considered a powerful tool for elucidating the electroporation process. Molecular dynamics simulation has been used for decades as a research method, and has become a basic research tool in fields such as nanomaterials, bioengineering, and biochemistry. Tieleman et al. were the first to use all-atom molecular dynamics to simulate the electroporation process of phospholipid membranes [8]. They found that 3.04 ns after electric field application, the membrane underwent intense deformation and water molecules entered the membrane from both sides. When the water molecules from both sides met, pore channels were formed and the water molecules assumed a strong orientation within them. Tarek also used an all-atomic molecular dynamics simulation to study the electroporation process [9], and his simulations showed that under an electric field, water molecules on each side of the phospholipid membrane moved to the inside and when they met in the middle, hydrophilic pores were formed, and at the same time, the polar head of the phospholipid molecules tilted towards the hydrophilic pores. Bockmann et al. [10] reported that pore formation proceeded quickly within 0.5 ns once the process started and suggested a four-state pore formation model. Pore radius of 0.5 nm was then determined with 10 lipid headgroups tilting into the hydrophobic core forming a hydrophilic pore, which was in agreement with the experiment results. Polak et al. [11] found that electroporation threshold differed substantially, which depended not only on the “electrical” properties of the membrane, i.e., its dipole potential, but also on the properties of its component hydrophobic tails. Kotnik et al. [12] reported that without the strong electric field, the pore formation rate was generally too slow to be observable in such simulations, which typically covered a submicrosecond time span, but in sufficiently strong electric fields, the pore formation rate increases dramatically, and pore initiation was well discernible on a nanosecond time scale. Delemotte et al. [13] showed that at the molecular level, the external electric field and charge imbalance produced similar effects: provided the transmembrane voltage are higher than a certain threshold, hydrophilic pores stabilized by the membrane lipid headgroups form within the nanosecond time scale across the lipid core and both methods induced similar electric field distributions within the membrane core.

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