Strong electric fields can be used to create pores in biofilms. The method is known as electroporation. Inducing such membrane defects in a targeted manner is an important technique in medicine and biotechnology, but also in food processing. A Franco-German research group led by Dr. Carlos Marquez of the École Normale Supérieure in Lyon/France and Professor Jan Bernds of the Institute of Physiology of the University of Freiburg has collected data that casts fundamental doubt on what has been accepted. Contracts as a standard form for this mechanism. “This is a challenge to build theory and numerical simulations in this field,” says Marquis. The results are now published in Proceedings of the Academy of Sciences. It can help improve the transport of active substances into cells.
Therapeutic substances enter the cells through electrical pores
Direct current electric fields above a certain intensity disrupt the organization of lipids, which are lipid-like molecules that form the basic structure of biological membranes in a bilayer stacked together in a kind of liquid crystal. The resulting electropores, which are usually only stable for a very short time, allow water and solutes dissolved in the surrounding medium — such as drugs or other active substances, including RNA or DNA — into the cell.
Because the lipid bilayer is very thin, measuring only five millionths of a millimeter, it is not necessary to apply a very high voltage to generate very high field strength (volts per meter). Thus, even at a voltage of 0.1 V across the membrane, the field strength is 20 million volts per metre. In air, for example, a spark discharge actually occurs at three million volts per meter. However, the direct current voltage must be; Alternating current fields in the megahertz-gigahertz range such as those generated by cell phones do not cause pores. While the technique is well established, electroporation of cell membranes for various purposes, such as the introduction of genetic material for gene therapy, still needs to be improved. For this purpose, it is important to accurately understand the mechanism of pore formation under electric fields.
Standard model with little empirical validation
The standard theoretical model of electroporation from the 1970s posits that the electric field applies pressure to the fat, increasing the potential for pore formation. To date, however, there is little empirical validation of the model. This is due, firstly, to the difficulty of directly detecting the formation of electrical pores and secondly, to the necessity of performing a very large number of such experiments in order to reach statistically defensible conclusions. This is because, in contrast to the pores formed by proteins, electric pores show very diverse and less modular behavior.
A method capable of detecting pore formation with great accuracy and temporal resolution is ionic current electrometry. Ions are positively or negatively charged components of salts found in all biological fluids, and therefore both inside and outside the cell. They are practically unable to penetrate intact membranes, but once a pore is opened, they are transported through it in the electric field. This transport of charged particles can be measured with highly sensitive amplifiers as a small electric current ranging from a few billionths to a millionth of an ampere. For this purpose, synthetic lipid bilayers are created in thin layers of Teflon through small holes of about 0.1 millimeter in diameter and placed between two electrodes. This technique of membrane formation is extremely prone to failure—only one membrane is formed at a time, which breaks easily, especially during high-voltage tests.
A new way to make greasy layers
For their experiments, the research group used a microchip with many openings, through which more stable lipid layers could be created very quickly and repeatedly using simplified procedures. This so-called microelectrode cavity array (MECA) was developed by the Jan Behrends research group and produced and made commercially available by Freiburg start-up Ionera Technologies GmbH founded in 2014.
With the help of this device, it is now possible for PhD candidate Eulalie Lafarge from the Charles Sadron Institute at the University of Strasbourg and Dr. Ekaterina Zaitseva from the Freiburg research group to generate and measure hundreds of membranes in a relatively short time. and determine pore formation as a function of direct current field strength. The results show that, contrary to the prediction of the old standard model, the energy barrier for pore formation decreases not with the square of the field strength but is proportional to the field strength. In other words, doubling the field strength reduces the energy barrier by only half, not fourfold. This points to a fundamentally different mechanism: instability of the lipid-water interface due to the reorientation of water molecules in the electric field.
Oxidized membranes have also been studied
This result is also confirmed for membranes in which lipids are oxidized to varying degrees. This is interesting because lipid oxidation is a natural process in regulating cell membrane function and plays a role in the normal aging of the organism and possibly also in diseases such as Parkinson’s and Alzheimer’s. “Because of the medical importance of this topic in particular, we want to pursue it more, including also optical methods, in order to come to a real understanding of this important phenomenon,” says Bernds.
