Sławomir Kalinowski

Last changes: June 3, 2014

Subjects of work

Monothematic series of publications selected as the basis of habilitation is entitled "Electroporation and electrostriction of thin lipid membranes."

Description of research

Original 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

  1. J. Morzycki, S. Kalinowski, Z. Lotowski, J. Rabiczko, Synthesis of dimeric steroids as components of lipid membranes, Tetrahedron, 53 (1997) 10579-10590 (IF 3,011, citations 13). The contribution to the work (15%) regards the idea of photosynthesis and the predictable influence of the steroid dimers on the formation process and durability of lipid bilayers.
  2. S. Kalinowski, G. Ibron, K. Bryl, Z. Figaszewski, Chronopotentiometric studies of electroporation of bilayer lipid membranes, Biochim. Biophys. Acta, 1369 (1998) 204-212 (IF 4,647, citations 21). The contribution to the work (70%) regards the idea of applying chronopotentiometry to the studies of the bilayer lipid membrane electroporation process, the design and construction of computer-controlled measurement apparatus, writing software, carrying out some of the experimental research, formulating equations, calculations and conclusions about a single pore being formed in a membrane, preparing the drawings and drafting works.
  3. S. Kalinowski, Z. Łotowski, J.W. Morzycki, Influence of bolaamphiphilic steroid dimer on formation and structure of bilayer lipid membranes, Cell. Mol. Biol. Lett., 5 (2000) 107-128 (IF 1,455, citations 8). The contribution to the work (80%) regards carrying out the studies of physiochemical process of phospholipid membrane formation with addition of steroid dimers, their electrostriction, the electroporation process and their durability, the analysis and interpretation of the results, preparing the publication and drafting works.
  4. S. Koronkiewicz, S. Kalinowski, K. Bryl, Changes of structural and dynamic properties of model lipid membranes induced by alfa-tocopherol: implication to the membrane stabilization under external electric field, Biochim. Biophys. Acta, 1510 (2001) 300-306 (IP 4,647, citations 15). The contribution to the work (50%) regards the idea of applying chronopotentiometry to the studies of the process of electroporation and recovering of continuous structure of the bilayer lipid membranes, apparatus construction and writing software enabling measurements in programmable chronopotentiometry conditions, carrying out some of the experimental research, discussing the results and a contribution to their interpretation.
  5. M. Kotulska, S. Koronkiewicz, S. Kalinowski, Cholesterol induced changes in the characteristic of the time series from planar lipid bilayer membrane during electroporation, Acta Phys. Polonica B, 33 (2002) 1115-1129 (IF 0,664, citations 10). The contribution to the work (25%) regards carrying out some of the experimental research, contribution the experimental part of the work, consulting during article preparation and a role in its correction.
  6. S. Koronkiewicz, S. Kalinowski, K. Bryl, Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes, Biochim. Biophys. Acta, 1561 (2002) 222-229 (IF 4,647, citations 24). The contribution to the work (50%) regards constructing the measurement apparatus and writing software for studying electroporation and recovering of the continuous structure of membranes under programmable chronopotentiometry conditions, carrying out some of the experimental research, discussing the results and a contribution to their interpretation.
  7. M. Kotulska, S. Koronkiewicz, S. Kalinowski, Self-similar processes and flicker noise from fluctuating nanopore in a lipid membrane, Phys. Rev. E, 69 (2004) 319-329 (IF 2,352, citations 14). The contribution to the work (20%) regards carrying out and describing some of the experimental research, consulting during article preparation and a role in its correction.
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  9. S. Koronkiewicz, S. Kalinowski, Influence of cholesterol on electroporation of bilayer lipid membrane: chronopotentiometric studies, Biochim. Biophys. Acta, 1661 (2004) 196-203 (IF 4,647, citations 22). The contribution to the work (50%) regards the idea of applying chronopotentiometry to the studies of the process of electroporation, creating the apparatus and software, writing the software for calculating conductance of the generated pore as a time function, carrying out some of the experimental research, discussing the results and a contribution to their interpretation.
  10. M. Kotulska, K. Kubica, S. Koronkiewicz, S. Kalinowski, Modeling the induction of lipid membrane electropermeabilization, Bioelectrochemistry, 70 (2007) 64-70 (IF 3,52, citations 18). The contribution to the work (15%) regards confirming the validity of the simulation research results compared to the earlier experimental research, consulting during article preparation and a role in its correction.
  11. S. Kalinowski, S. Koronkiewicz, M. Kotulska, K. Kubica, Simulation of electroporated cell by chronopotentiometry, Bioelectrochemistry, 70 (2007) 83-90 (IF 3,52, citations 3). The contribution to the work (60%) regards the idea of applying chropotentiometry to modeling electrical properties of the pore generated in a cellular membrane in vivo, constructing the apparatus, writing the software, carrying out some of the experimental research, discussing the results and a contribution to their interpretation,  preparing some of the drawings and drafting works.
  12. S. Kalinowski, System for electroporation of cells in flow conditions, Polish patent application no. P.382661, June 14, 2007, decision of the Patent Office of RP to grant a patent - November 2011. The patent regards the electroporation of cells consecutively flowing through a channel similar in size to the cells. The applied conditions enable a controlled and effective insertion the desired molecules into a cell’s interior. A system to be used, among others, in genetic engineering.
  13.  S. Kalinowski, 3-electrode system for measurement of the capacitance of membranes supported on electrode, Polish patent application no. P.392104, August 11, 2010. The purpose of the device is to measure the capacitance and record capacitance-potential characteristics for mono- and bimolecular membranes supported on solid electrodes. The system enables measurement of the capacitance for a matrix of working electrodes and is meant for application, among others, to taste and smell detectors.
  14. S. Kalinowski, System for taste and smell detection and method for taste and smell detection, Polish patent application no. P.393352, December 20, 2010. A taste and smell detector system based on a matrix of electrodes covered with thin mono- and bimolecular membranes, e.g. lipid membranes, applying the electrostriction phenomenon and a change in a membrane’s capacitance-potential characteristics under the influence of analyzed substances.
  15. S. Kalinowski, 3-electrode system for measurement the capacitance of membranes supported on electrode, Polish patent application no. P.394034, February 25, 2011. A system meant for measuring capacitance and recording the capacitance-potential characteristics for mono- and bimolecular membranes supported on solid electrodes in which the working electrode is connected with the mass of the system. The system can co-operate with a quartz crystal microbalance and enables a simultaneous recording of both the capacitance-potential and mass-potential characteristics. A system meant for studying phenomena occurring on the surface of membranes supported on electrodes and application in biosensors, e.g. immunosensors.
  16. S. Kalinowski, Electrochemical detector for determination of detergents in aqueous solutions, Polish patent application no. P.394436, April 4, 2011. An electrochemical detector applying the electrostriction phenomenon of membranes supported on solid electrodes, meant for application to determination of amphiphilic molecules.
In addition to the original creative research works that form the basis of habilitation, the issues relating to lipid membranes are contained in two monographic works: 
  1. S. Kalinowski, Electrochemistry of lipid membranes. From biomembranes to biosensors (in Polish), Wydawnictwo UWM, Olsztyn 2004, 284 pp. ISBN 83-7299-325-4. Monograph including 116 figures, 47 tables, 127 structural formulas of chemical compounds.
  2. S. Kalinowski, Electrochemical methods and their application, in Advances in Planar Lipid Bilayers and Liposomes, Vol. 2, Ed. A. Ottova, Elsevier Inc. 2005,  p. 1-47, ISBN 0-12-369453-1.