Scientists often use electroporation to introduce drugs, vaccines or genes into cells. An electric field creates tiny pores in the lipid membrane of the cells, making them permeable. However, as an experiment has now shown, electroporation is based on different mechanisms than previously thought. The results also refute the theoretical standard model for this process, which has been in use for around 50 years, according to which the energy barrier for pore formation decreases with the square of the field strength. Instead, the researchers demonstrated a linear relationship. The new findings could now help to improve the transport of active substances in cells.
Our cells are shielded from their environment by an ingenious patent of nature: their lipid double membrane is only five millionths of a millimeter thick, but keeps most molecules and pathogens out. Only special protein channels and receptors in the membrane allow passage, but these usually only allow very specific messenger substances and molecules to pass through. If scientists want to smuggle their own active ingredients into the cell as part of medical research, vaccination or gene therapy, they either have to couple them to transport molecules or viral gene shuttles that can pass through the membrane, or they have to force their way in - so to speak - but without damaging the cell.
pores in the cell barrier
In these cases, electroporation is usually used. Scientists use a weak electrical DC voltage to disrupt the organization of the membrane lipids. This creates tiny pores in the membrane for a short time, through which water and substances dissolved in it can penetrate from the environment into the cell, such as drugs or other active ingredients through to RNA or DNA. Targeting such defects in membranes is an important technique in medicine and biotechnology, but also in food processing. The process of electroporation has been established for a long time, but there is still a lack of basic knowledge to optimize this method for various purposes, for example to introduce genetic material for gene therapy. For this it is important to understand the mechanism of pore formation under electric fields.
A standard theoretical model of electroporation, developed as early as the 1970s, assumes that the electric field exerts pressure on the lipids, increasing the likelihood of pore formation. "Another key prediction of the model is that the energy barrier for pore formation is lowered in proportion to the square of the electric field strength," explain Eulalie Lafarge of the University of Strasburg and her colleagues. Whether this assumption is correct, however, has so far been difficult to test experimentally. On the one hand, this is due to the difficulty of recording the formation of the electropores directly and, on the other hand, to the need to carry out a large number of such experiments in order to arrive at statistically reliable statements. Because the electropores show a very diverse, little stereotypical behavior.
Linear instead of square
Lafarge's team has now developed a method to examine the formation of electropores in more detail and to explore their laws. To do this, it used a microchip with many openings over which artificial lipid double membranes are stretched. The researchers used the ion current – the flow of electrically charged particles through the membrane – to determine whether pores formed in this membrane and under what circumstances. These particles, for example in the form of dissolved salts, are practically unable to penetrate intact membranes, but are transported through with the electric field as soon as a pore opens. This ion transport can be measured as a tiny electrical current of a few billionths to millionths of an ampere using electrodes and highly sensitive amplifiers. With the newly developed chip, the so-called microelectrode cavity array (MECA), Lafarge and her colleagues were able to produce hundreds of membranes in a relatively short time and measure and quantify the pore formation as a function of the strength of the DC voltage field.
This showed that, contrary to what was assumed by the previous model, the energy barrier for pore formation does not decrease with the square of the field strength, but linearly with it. Doubling the field strength only lowers the energy barrier by half. "This contradicts the predictions of the Standard Model of electroporation," the team said. As they explain, this suggests that the pore formation is due to a different mechanism than previously thought. Accordingly, a reorientation of the water molecules in the electric field leads to the destabilization of the interface between the lipid layer and the water. This result was also confirmed for membranes whose lipids were oxidized to varying degrees. This is of interest because lipid oxidation is a natural process in regulating the function of cell membranes and plays a role both in the natural aging of the organism and possibly in diseases such as Parkinson's and Alzheimer's. "Especially in view of the medical importance, we want to continue to pursue the topic, including optical methods, and thus achieve a real understanding of this important phenomenon," explains co-author Jan Behrends from the University of Freiburg.
Source: Eulalie Lafarge (Université de Strasbourg) et al., Proceedings of the National Academy of Sciences, doi: 10.1073/pnas.2213112120