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The integral conductance of planar lipid bilayer membranes in the presence of two Poloxamers (Pluronics) L61 and F68 with the same lengths of hydrophobic poly(propylene oxide) blocks and the different lengths of hydrophilic poly(ethylene oxide) blocks increases with an increase in the concentration of both Pluronics; however, the shape of the conductance-concentration curves is super linear for L61 and sublinear for F68. In the presence of both Pluronics, rare discrete current jumps are observed against the background of continuous current. At high concentrations, the I-V curves of membranes with both L61 and F68 became nonlinear at sufficiently low voltages but differed significantly. At voltages greater than 50 mV, the conductance of membranes with L61 increased sharply and quantized jumps were observed toward higher conductance, which could be interpreted as the appearance of additional pores. On the contrary, the conductance of membranes with F68 decreased and quantized jumps to lower conductance were observed, which could be interpreted as blocking of already existing pores. We attributed the differences in the conductance-concentration and I-V curves of these two Pluronics to their different effects on the dynamics of membrane hydration and, accordingly, on the probability of formation of conducting pores., (© 2023. The Author(s), under exclusive licence to EDP Sciences, SIF and Springer-Verlag GmbH Germany, part of Springer Nature.)

The conductive lipid pores occurring in planar bilayer membranes are known to manifest themselves experimentally as current fluctuations. Reliable recording of such fluctuations during phase transitions, as well as in membranes with various additives (for example, SDS), allows one to determine the characteristics of hypothetical hydrophilic pores, namely, their number, sizes, lifetimes, and duration of time intervals between pores. Because, in contrast with electroporation, the emergence of pores in a membrane does not require high voltages, this process is called soft poration. Studying the characteristics of pores under soft poration allows us to estimate the parameters of the Smoluchowski equation and compare them with the corresponding parameters used to describe electroporation. In this work, the experimental characteristics of current fluctuations in the membrane with the addition of SDS to the bulk solution were used to estimate the parameters of the Smoluchowski equation: the pore edge tension, the energy of the hydrophobic pore/hydrophilic pore barrier, the coefficient of pore diffusion in the radius space, the initial distribution density of the number of pores, and the attempt rate density of the lipids in a membrane. The obtained estimates are close to the parameter values used in studies of electroporation.

We describe the dynamics of lipoic acid (LA) alone, incorporated in liposomes and as a part of nanoemulsions. Mass spectrometry shows that LA in water forms aggregates of two or three molecules in the form of a negatively charged ion and a neutral molecule. Phosphatidylcholine (PC)-based nanoforms of LA as liposomes and nanoemulsions with a particle size equal to 145 nm are characterized by a high degree of incorporation of LA into the nanoparticles and long-term stability during storage at room temperature. Dynamic light scattering (DLS) gives the polydispersity index of the nanoforms (> 0.3), characterizing the homogeneity of the obtained nanodispersions. We found that such emulsions can significantly (5 ×) increase the concentration of LA in the aqueous phase (5-7 mg/mL) when compared with an aqueous solution of LA (1 mg/mL) and by 40% when compared with PC liposomes (4 mg/mL). Moreover, the inclusion of LA in liposomes and nanoemulsions from PC did not change the neutral ζ-potential characteristic of PC nanoforms. CryoTEM established that the structural organization of the liposomes practically did not differ from nanoemulsions and both nanoforms contained both multilayer and single-layer vesicles. When studying the release kinetics of LA from phosphatidylcholine nanoforms, we found that at 22 h, 45-55% of LA was released from nanoparticles, but that at the initial stage of the process LA was slowly released from the nanoemulsions and rapidly from the liposomes. Conductance measurements indicate that LA delivered in all the three forms increase membrane permeability, though this result is most marked with the LA in PC liposomes.

Detergent effects on lipid bilayers of artificial and biological membranes at subsolubilizing concentrations are known to include the membrane permeabilization which manifests itself through both a flip-flop of detergent molecules from the outer monolayer to the inner monolayer and the membrane leakage of entrapped solutes. We have explored the current fluctuations occurring in planar BLM of asolectin in the presence of ionic detergent SDS at subsolubilizing concentration. Two groups of current fluctuations which the average duration differs by two orders of magnitude can be distinguished. We assume that these differences in the duration of current fluctuations are associated with a different number of SDS molecules in the walls of the putative toroidal hydrophilic pores. We associated short pulses with the formation of short-lived lipid hydrophilic pores. Impulses of greater duration (steps) were associated with the formation of hydrophilic pores, the walls of which contain detergent. Taking into account the characteristics of these pores, we estimated the pore energy, as well as the edge energy of these two kinds of pores. We believe that the flip-flop of SDS molecules in liposomes is provided by long-lived pores, and the contents of the liposome leakage occurs through all pores., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A comparative study of antioxidant (radical-interceptor) properties of tryptanthrin (quinazoline alkaloid shows a high anti-inflammatory activity and it is found in many types of different families of higher plants and microorganisms, including the human microbiome) in the systems of 2,2'-azo-bis(2-methylpropionamidin)dihydrochloride-luminol and hemoglobin-hydrogen peroxide-luminol has been conducted and the influence on the permeability of planar bilayer lipid membranes is evaluated. Trolox was used as a reference antioxidant, and ascorbic acid and dihydroquercetin were taken as standards. Tryptanthrin exhibits very weak antioxidant activity, being markedly inferior to the reference standard and antioxidants while testing antioxidant activity in both studied systems. By the efficacy of antioxidative action the substrates in the systems studied can be arranged in the following order: dihydroquercetin > trolox > ascorbic acid > tryptanthrin. Antioxidant potential of tryptanthrin is approximately 1000 and 3000 times lower than that of trolox and bioflavonoid dihydroquercetine, respectively. Tryptanthrin causes no significant changes in the permeability of planar bilayer membranes in a dose range of 0.5 to. 10 μg/ml. Our data show that tryptanthrin displays no significant radical-interceptor and membranotropic activities. It can be assumed that the observed high anti-inflammatory activity of tryptanthrin is not related to the neutralizing effect against reactive oxygen species and the influence on the permeability of cell membranes. The anticipated mechanisms of biological activity of tryptanthrin are discussed.

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The release of cytochrome c (cyt c) from mitochondria is responsible for initiation of cell apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanism remains unclear. Cyt c binds to and penetrates lipid bilayer membranes of specific phospholipid cardiolipin (CL) contained in mitochondria. We present here the experimental results of monitoring planar BLM (pBLM) from mixtures of azolectin and of CL (4/1 by moles) by triangle voltage pulses of 100 mV in amplitude and frequency of 2 Hz. The BLM were modified by a successive addition of cyt c and of H(2)O(2) in water solution. It is shown that the addition of cyt c alone leads to a stepwise increase in the ionic conductance of the pBLM, indicating the appearance of transmembrane pores. Pore lifetimes then reached several seconds at an average pore diameter of ~2 nm. Current-voltage characteristics were then linear and passed through the origin which is characteristic for broad, nonselective ion pores. Subsequent addition of H(2)O(2) caused a dramatic increase in transmembrane current at retention of average pore size constant. Observed increase in membrane current is due to growth of a number of pores in an open state. We suggest that hydrogen peroxide in the presence of cyt c promotes a peroxidation of membrane phospholipids to form lysolipids, the embedding of which stabilizes the edge of the pore and the surface of lipid bilayer.

The metabolic damages and inflammatory diseases are closely connected with high increase of the contents of the reactive oxygen species (ROS) and lipid peroxidation products in organism. In connection with this natural antioxidants are actively used for efficient fight with oxidative stress. In current work we have estimated an antioxidative potential of rosmarinic acid (RA), which is broadly wide-spread amongst plants and possesses the useful spectrum of biological activities. It has been shown that RA noticeably exceeded known antioxidants in test on antioxidant activities in Hb-H2O2-luminol and 2,2'-azo bis (2-methylpropionamidine) dihydrochloride-luminol systems, as well as on inhibition of linoleic acid peroxidation. On antioxidant activities the investigated substances can be arranged in the following order: RA > dihydroquercetin > trolox > ascorbic acid. RA molecules are localized mainly in the lipid bilayer of polar groups, but have no significant effect on its structural and functional properties: RA does not cause significant changes in permeability of planar bilayer membranes in a dose range of 0.5 to 10 microg/ml. Spontaneous incorporating of 1 mol% of RA into the lipid bilayer is sufficient for the complete prevention of lipid peroxidation. Thus, the antioxidant activity of RA is based on its ability to inhibit the initiation stage of free radical reactions of lipid peroxidation, largely caused by the formation of ROS. In this regard RA, as a powerful and effective inhibitor of ROS and lipid peroxidation, has great potential for wide use not only in medicine but also in the food industry to preserve the quality of perishable food products.

Rosmarinic acid is found in many species of different families of higher plants and its chemical structure is phenol propanoid with various biological activity. In this paper, we conducted a comparative study of antioxidant (radical-scavenging) properties of rosmarinic acid in systems of 2,2'-azo-bis(2-methylpropionamidin)dihydrochloride-luminol and hemoglobin-hydrogen peroxide-lu- minol, determined its protective potential in preventing peroxidation of linoleic acid, and evaluated the effect on the permeability of planar bilayer lipid membranes. Linoleic acid peroxidation was assessed by iron-thiocyanate method. In these studies, trolox was used as a reference antioxidant, and ascorbic acid, and dihydroquercetin were taken as standards. Rosmarinic acid is significantly superior to trolox, ascorbic acid and dihydroquercetin in the tests for antioxidant activity in the systems studied, as well as in inhibition of linoleic acid peroxidation. According to their activity the investigated substances can be arranged in the following order: rosmarinic acid > dihydroquercetin trolox > ascorbic acid. Rosmarinic acid does not cause significant changes in the permeability of planar bilayer membranes in a dose range of 0.5 to 10 mkg/mL. Antioxidant activity of rosmarinic acid is due to the neutralization of reactive oxygen species and/or luminol radicals generated in model systems. The observed features of the antioxidant and membrane activity of rosmarinic acid, which may underlie the previously mentioned pharmacological effects are discussed.

Interaction of cytochrome c with cardiolipin in the presence of hydrogen peroxide induces peroxidase activity in cytochrome c and the ability to oxidize membrane lipids. These cytochrome c properties play a substantial role in the cytochrome c-mediated apoptotic reactions. In the present study the electric properties (specific capacitance and integral conductance) of the cardiolipin-containing asolectin planar bilayer lipid membranes (pBLM) in the presence of cytochrome c and hydrogen peroxide were studied. Cytochrome c interaction with cardiolipin-containing pBLM in the presence of hydrogen peroxide resulted in the dramatic increase of the conductance, pore production, their growth up to 3.5 nm diameter and subsequent membrane destruction. In the absence of hydrogen peroxide cytochrome c demonstrated almost no effect on the membrane capacitance and conductance. The data obtained prove the pivotal role of cytochrome c and membrane lipids in the permeabilization of pBLM. Correlation of apoptotic reactions and cytochrome c-mediated membrane permeability is discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.

The electrical capacity of planar bilayer lipid membranes (BLM) from natural hydrogenated egg lecithin (HEL) in n-decane at a temperature of phase transition was measured. The temperature of phase transition was determined calorimetrically to be 51 degrees C. The data obtained revealed a phase separation of HEL in BLM into two fractions, one freezing at 42-44 degrees C and one that is converted to a liquid-crystal state at 51-59 degrees C. It was assumed that the first fraction is rich in dipalmitoyl lecithin, and the second fraction is rich in distearoyl lecithin. Freezing and the transition to the liquid-crystal state were accompanied by an increase and decrease in membrane thickness, respectively, in part due to a displacement of the solvent from the torus to the planar part of the bilayer. The displacement of the solvent is explained by changes in the disjoining pressure in BLM, which arises across the lipid bilayer due to van der Waals forces of attraction between water layers on both sides of the BLM.

Polymyxin B in micromolar concentrations induces current fluctuations in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid identified as ion channels. The appearance of ion channels correlates with phase separation of the lipid in the presence of peptide polycations detected by differential scanning calorimetry. Ca2+ also induces the formation of ion channels in liquid crystalline bilayer lipid membranes from dipalmitoylphosphatidic acid followed by the phase transition of the phospholipid. The capacitive current, which indicates the possibility of structural transformations of bilayer-non-bilayer type (hexagonal phase II), precedes the formation of Ca(2+)-induced channels in bilayer lipid membranes from dipalmitoylphosphatidic acid.

The effect of the synthetic polycation VMII-DEMA (a potential antiheparin agent) on the ion transport through erythrocyte membranes was studied using ion-selective electrodes. We found that the passive ionic permeability of erythrocyte membranes in the presence of VMII-DEMA noticeably increased after the pretreatment of erythrocytes with long-chain fatty acids. When used separately, neither polycation nor fatty acids changed the membrane permeability at the same concentrations. The efficiency of the combined action of the polycation and fatty acids correlated with the length of the fatty acid (C9-C18) and the number of double bonds in it. The fatty acids C12:0, C13:0 and C18:2 were the most efficient. We suggest that in pathologies accompanied with deteriorations in lipid metabolism, polycations used for the correction of blood coagulation may damage the erythrocyte membrane.

It is shown that synthetic polycation quarternary ammonium salt polymetacryloil lupinine (poly-MACL) effects on passive ion permeability of erythrocytes membranes. The amplitude of Cl-/OH- exchange decreased in the buffer with SDS-polycation complex and simultaneously with pH elevation K+ exflux increased. On the contrary, in presence of heparin co-effect of poly-MACL and SDS vanished. Thus, synthetic polycation in the complex with long-chain anion (SDS) injure the membranes and effect depends on lipophilic-hydrophilic balance of polycation-SDS complex.

Mechanisms of potentiation of ionophoric effect of polymyxin M by free fatty acids was studied using measurement of electrical properties of bilayer lipid membranes. Among used unsaturated fatty acids the palmitic acid appeared to be the most effective. The obtained dependence of ionophoric effect of membrane antibiotic on the chain length of the fatty acid may be explained by the existence of the complex of special size appropriate to the membrane thickness. We suppose that free fatty acid and polymyxin M form the ion-conducting structure in lipid bilayer.

The aim of the work was to characterize the role of negatively charged lipids (cardiolipin and lauric acid) in the increase of ionic permeability of the membrane under the action of polymyxin B. Cardiolipin or lauric acid were added into membrane-forming solution in concentration close to that in the membrane of bacterial cell. It resulted in modification of membranes by these lipids. The obtained membranes had a higher ionic permeability in the presence of polymyxin B. However the comparison of different effects can lead to the conclusion that free fatty acid are responsible for the major part of the effect. It is concluded that the character of damage of the bilayer by polymyxin is diverse: polymyxin B forms ionic canals with lauric acid, whereas there is unspecific destruction of BLM by the polymyxin interacting with cardiolipin.

Polycationic peptide antibiotics, polymixins B and M, are studied for their effect on oxygen uptake by rat liver mitochondria. They both inhibit the process at state 3 and 3p according to Chance. Low concentrations of polymixin M do not affect oxygen uptake by mitochondria at state 4, but its high concentrations inhibit it. Concentration dependence of polymixin B effect on mitochondria at state 4 is of a bell-like character. Competitive interrelations are found between polymixins M and B in their effect on mitochondria at state 4. Chromatography reveals differences in peptide mobility in a thin layer. It is supposed that polymixin inhibition of oxygen uptake by mitochondria is associated with a disordering effect of the polymixins on the membrane structure during peptide binding to the lipid phase of the membranes. Low concentrations of polymixin B are likely to promote an increase in membrane permeability of mitochondria at state 4.

Effect of polymyxin B on the movement of K+ and H+ in polymyxin-sensitive cells of E. coli under different metabolic states has been studied. It was shown that polymyxin B induced the efflux of K+, decreased the efflux of H+ and inhibited the consumption of oxygen in bacterial cells. The effect of antibiotic on ion movement was independent of respiratory conditions. It was suggested that polymyxin B increased ion permeability and destroyed lipid-protein interactions of the respiratory chain simultaneously.

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.

Behavior of hydrophobic probes of 5-doxylstearol acid and pyrene in phosphatidylcholine liposomes modified by sterols isolated from Far East holothurian tissues was studied. The introduction of delta 7-sterol xylosides and delta 5-sterol sulfates into liposomes was shown to result in an increase of the membranes microviscosity. When the liposome suspension was added with holotoxin A1 a supplementary increase of microviscosity was observed, which was obviously a consequence of glycosides +non built into the membranes.

The membrane activity of 6 triterpenic glycosides was studied with respect to ionic permeability of the liposomes and flat bilayer lipid membranes. It was shown that the rate of the glycoside effect depended on both the sterol level in the membrane and the sterol type (cholesterol, ergosterol). The activity of cauloside C, a plant glycoside markedly depended on pH of the medium. The carbohydrate chain played an important role in the membrane activity of the glycosides. Correlation in the effect of triterpenic glycosides on the model lipid and cell membranes was noted.

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.

the mechanism of the effect of the peptide antibiotic polymixin B on respiration of rat liver mitochondria was studied. It was shown that polymixin B at concentrations of (1,6--2,0) . 10(-3) M inhibits mitochondrial respiration in state 3 and 3u irrespective of the oxidation substrate used and activates oxygen consumption in state 4 at lower concentrations. It is assumed that the antibiotic affects mitochondrial respiration by changing the ionic permeability of the membranes.

Conductivity of lecithin bilayers containing cholesterol and its esters was studied. It was shown that cholesterol causes a considerable decrease of conductivity, the original conductivity of bilayers being (5.8 +/- 0.9) . 10(-8) Ohm-1 cm-2. Maximal effect was observed when concentration of cholesterol was about 30 mol%. Further increase of cholesterol content does not change the conductivity of bilayers. It was found that cholesterol esters in contrast to cholesterol cause the increase of ion permeability of bilayers. The same effect was also observed at high membrane cholesterol content. The obtained results were discussed in relation to cell membrane destructions at atherosclerosis.