Strong electric fields can be used to create pores in biofilms. The method is known as electroporation. Inducing such defects in membranes in a targeted manner is an important technique in medicine and biotechnology, but also in food treatment. A Franco-German research team led by Dr. Carlos Marques from the Ecole Normale Supérieure in Lyon/France and Professor Dr. Jan Behrends from the Institute of Physiology at the University of Freiburg has now collected data that casts fundamental doubt on what has been accepted for decades as the standard model of this mechanism. “This is a challenge for theory building and numerical simulations in this area,” says Marques. The results have now been published on Proceedings of the Academy of Sciences. They could help improve the transport of active substances into cells.
Therapeutic substances enter the cells through an electropore
Direct current electric fields above a certain intensity disrupt the organization of lipids, fat-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 in the surrounding medium — such as drugs or other active substances, including RNA or DNA — to enter a cell.
Since the lipid bilayer is very thin, measuring only five millionths of a millimeter, it is not necessary to apply very high voltages to create very high field strengths (volts per meter). Thus, even at a voltage of 0.1 volt across the membrane, the field strength is 20 million volts per meter. In air, for example, the spark discharge already occurs at three million volts per meter. However, it must be DC voltage. Alternating current fields in the megahertz-gigahertz range, such as those generated by cell phones, do not induce sources. While the technique is well established, there is still a need to optimize electroporation of cell membranes for various purposes, such as introducing genetic material for gene therapy. To this end, it is important to precisely understand the mechanism of pore formation under electric fields.
A typical model with little experimental verification
A standard theoretical model of electroporation from the 1970s postulates that the electric field exerts pressure on the lipids, thereby increasing the likelihood of pore formation. So far, however, there is little experimental verification of the model. This is due, firstly, to the difficulty of directly detecting the formation of electropores and, secondly, to the necessity of conducting a very large number of such experiments in order to arrive at statistically reliable conclusions. This is because, in contrast to pores formed by proteins, electropores exhibit a very different, less stereotypical behavior.
A method capable of detecting pore formation with high precision and high temporal resolution is electrical ionic current measurement. Ions are positively or negatively charged components of salts present in all biological fluids and therefore inside and outside the cell. They practically cannot penetrate intact membranes, but once a pore opens, they are transported through it into the electric field. This transport of charged particles can be measured with very sensitive amplifiers as a tiny electric current of a few billionths to millionths of an ampere. For this purpose, artificial lipid bilayers are created in thin layers of Teflon through tiny openings about 0.1 mm in diameter and placed between two electrodes. This film formation technique is very prone to failure — only one film is formed at a time, which breaks easily, especially during tests at higher voltages.
New method of creating lipid layers
For their experiments, the research team used a multi-aperture microchip, through which much more stable lipid layers can be created very quickly and repeatedly using simplified procedures. This so-called microelectrode cavity array (MECA) was developed by the research group of Jan Behrends and has been produced and commercialized by the Freiburg startup Ionera Technologies GmbH founded in 2014.
With the help of this device, it was now possible for PhD candidate Eulalie Lafarge from the Charles Sadron Institute of the University of Strasbourg and Dr. Ekaterina Zaitseva from the Freiburg research group to create hundreds of membranes in a relatively short time and measure and quantify pore formation as a function of DC field strength. The results showed 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 proportionally with the field strength. In other words, doubling the field strength only halves, not quadruples, the energy barrier. This suggests a fundamentally different mechanism: a destabilization of the lipid-water interface due to a reorientation of the water molecules in the electric field.
Oxidized films were also studied
This result was also confirmed for membranes whose lipids were oxidized to varying degrees. This is interesting because lipid oxidation is a natural process in the regulation of cell membrane function and plays a role in the natural aging of the body and possibly in diseases such as Parkinson’s and Alzheimer’s. “Particularly in view of the medical importance of this topic, we want to pursue this further, including optical methods, in order to reach a real understanding of this important phenomenon,” says Behrends.