| Habilitation Sławomir Kalinowski
Subjects of workMonothematic series of publications selected as the basis of habilitation is entitled "Electroporation and electrostriction of thin lipid membranes."Description of researchOriginal creative works at the basis of my habilitation concern the influence of an electrical potential on the bilayer lipidmembranes, separating two aqueous solutions. This system is used as a functional model of the cell membranes. Lipidbilayers are the basic structural element of cell membranes, allowing the cells to maintain life processes. They arestructures very delicate mechanically, but very beneficial from the thermodynamic point of view. They exhibit several very characteristic features, such as a fluid structure, very low permeability to small ions and polar molecules, fairlygood water permeability, and a very low electrical conductivity placing them among the best insulators. Another important feature is their very high resistance to electrical breakdowns. This phenomenon occurs in a manner much different from the typical dielectrics. In the lipid bilayer the pore is created and is filled with the electrolyte adjacent to the membrane. These pores are formed at very high electric field strengths in the range of 105‑106 V/cm. Such a largeelectric field is also connected with a significant electrostriction (electrocompression) effect, leading to changes inmembrane thickness of ten or more percent. The presence of polar groups on the membrane surface in a strong electric field gives rise to another phenomenon - changes in the spatial orientation of these groups. The mechanism of formationof pores in lipid membranes is not yet fully understood. This phenomenon, however, has already found many practical applications, including inserting into the cell molecules such as drugs, DNA, gene therapy, treatment of cancer.Physico-chemical phenomena observed in artificial lipid membranes have a direct influence on the phenomena in cell membranes.An analysis of literature data showed that the stability of cell membranes and their resistance to harsh environmental conditions is associated with the presence of sterol molecules in the membranes and transmembrane lipids. Cholesterol inserted into artificial membranes formed from phosphatidylcholine facilitates the spontaneous formation of lipid bilayers and increases their resistance to electrical breakdowns. Transmembrane lipids have a length equal to the thickness of the membrane, and there are hydrophilic groups at the ends. These molecules are called bolaamphiphilic. Such molecules have been found in organisms living in extreme conditions, such as the boiling water of geysers or acidic volcanic lakes. This information formed the basis of the idea of synthesis of molecules that are both bolaamphiphilic molecules and contain sterol moiety in their structure. Three types of dimers of cholesterol were synthesized in collaboration with a group of organic chemists [1]. These dimers differed in the length and type of link between cholesterol molecules. The membranes were formed by the Mueller-Rudin method from phosphatidylcholine with cholesterol dimer [3]. The study showed that the membranes were gaining some positive features - lipid bilayer formed quickly, the membranes were thinner (no solvent residue remained between monolayers), the bilayers occupied most of the orifice in which they had been created, and had a higher resistance. What is more, their electrocompressibility reduced, they became more resilient. These features encourage the use of sterol dimers to form membranes used for practical purposes, such as biosensors. When working within the framework of the doctoral thesis, I sought methods of measuring the capacitance of lipid membranes, enabling the observation of the membrane formation process and assessing their quality. One of the techniques that I wanted to use was chronopotentiometry. When testing equipment and recording the first curves, I noticed that after an initial period of growth in the potential, a sudden drop in the potential occurs after starting the recording and it oscillates irregularly with a value of about 100 mV. These oscillations were observed within a relatively narrow range of currents of 0.2-2 nA. A literature review has shown that it is connected with the formation of membrane pores filled with the electrolyte solution. After the calculations of the size of the channel, which could be formed in the membrane, it can be concluded that only a single pore is formed under these conditions. It corresponded with a size of stable pores, determined by other methods [2]. In order to perform more detailed studies of the pore conductance, I have supplemented the program for recording the chronopotentiometric curves with procedures for the analysis of the curve. This program calculated and drew the conductance of the generated pore as a time function. The calculations included the membrane resistance without the pore and changes in the membrane capacitance caused by the potential. In the process of electroporation, an important step is to close the pores and return the cells to their normal physiological state. In order to study the process of closing the pores, I have written another program for chronopotentiometry with programming current. This program enabled the registration of chronopotentiometric curves with a variable intensity of the current flowing through the electrode. This current can consist of any combination of constant current levels and/or linear waveforms. In addition, you could insert an electrode shortcircuit stage of the current electrodes or disconnect the electrodes anywhere in the current program. This allows for, among other things, to force membrane potential equal to zero during the recording. It simulated a loss of membrane potential in cells and the state, which might be followed by recovering of the continuous structure of the membrane. These programs were used in the studies described in several publications [4-10]. A current of a right intensity causes a single pore to be created and maintained for a time depending only on the lifetime of a membrane. It is possible thanks to a negative feedback present in the system galvanostat and membrane pore. The creation of a pore results in an increase in the conductivity of the membrane, which leads to a drop in the membrane potential, which in turn prevents a further extension of the pore. Fluctuations observed in this system are related to fluctuations in the size of the pore. An analysis of the frequency spectrum of those fluctuation has shown that the power spectrum density depends on several factors, such as the composition of the membrane and the electrolyte, or the current intensity. When the diameter of the pore is no bigger than 1 nm, the power spectrum density is a 1/f function, which is a pink noise [5,7]. The electroporation phenomenon is preceded by additional phenomena: not only the electrostriction, but also the conformation of the lipids. My earlier research conducted within the doctoral thesis related to the capacitance dependence on the membrane potential, led me to the conclusion that the polar groups of lipids change their spatial orientation, depending on the membrane potential. We later considered this effect using an improved Pink model, for liquid and gel phase of lipids [9]. In the physiological range of membrane potentials, below 70 mV, the polar function groups of lipids did not change its orientation, but at potentials close to breakdown potential, these changes are significant. In cellular membranes, apart from phospholipids, other molecules exhibiting amphiphilic properties can be fund, e.g. cholesterol as an important structural element in bilayer lipid membranes, and α-tocopherol responsible for protecting lipids from oxidation and influencing the physiochemical properties of the bilayer [4]. The research described in that paper hinted at destabilizing influence of α-tocopherol in larger amounts (several percent of the sum of all the lipids in a membrane). It caused the time of bilayer formation to increase, lowered the membrane breakdown voltage, changed the characteristics of pore conductance oscillation after its formation, but also facilitated the reconstruction of the continuous structure of a membrane. Some of the results published concerned the influence of cholesterol on the electroporation process and the process of reconstructing the continuous structure of the membrane [5,8]. Electroporation of planar lipid membranes in chronopotentiometric conditions simulates the reaction of living cells undergoing the process of electroporation through regular means, like subjecting cells suspended in an electrolyte solution to a strong voltage pulse [10]. As the result of electroporation, an uncontrolled flow of ions between the interior and exterior of a cell, which causes depolarization and exhaustion of the cell’s energy supplies trying to restore its typical membrane potential. The chronopotentiometric studies have shown that pores can only be closed, when the membrane potential assumes a low enough value. To a cell, it means exhausting its supplies of energy. A result of the experience gained when researching planar lipid membranes was a patent application and a patent [11] concerning the electroporation in flow conditions. It describes a method enabling an electroporation of each cell flowing through a hole made in the hydrophobic material. The electroporation is conducted in current-clamp conditions. The electrodes are placed on the opposite sides of the channel. The moment a cell enters the hole, both solutions are separated by the cell, the resistance increases, which increases the voltage between the electrodes, resulting in pores forming in the cell membrane. The cell flowing through the hole is additionally under the influence of a change in pressure that further facilitates the flow of liquid through the pores, which enables insertion into the cell of molecules capable of passing a hole of several nm in diameter. This method can be useful in microbiological studies or genetic engineering. The advantage of this method is the ability to control the size of the pore through appropriate intensity of the current flowing through the electrodes. By recording the voltage between the electrodes, you can observe the process of electroporation and its effectiveness. Lately, I have additionally focused my research on monolayer and bilayer membranes supported on solid electrodes, whose purpose is to be applied as electrochemical sensors and biosensors. The basis of the operation of those sensors is the electroporation phenomenon studied through registration of capacitance-potential characteristics of such membranes. The construction, the principle of operation, the method of capacitance measurement and the application have been described in patent applications [12-15]. List of monothematic publications underlying the habilitation
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