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Among potential macromolecule-based pharmaceuticals, polycations seem particularly interesting due to their proven antimicrobial properties and use as vectors in gene therapy. This makes an understanding of the mechanisms of these molecules' interaction with living structures important, so the goal of this paper was to propose and carry out experiments that will allow us to characterize these phenomena. Of particular importance is the question of toxicity of such structures to mammalian cells and, in the work presented here, two lines, normal fibroblasts 3T3-L1 and A549 lung cancer, were used to determine this. In this work, three well-defined cationic derivatives of barley-derived betaglucans obtained in a reaction with glycidyltrimethylammonium chloride (BBGGTMAC) with different degrees of cationization (50, 70, and 100% per one glucose unit) and electrostatic charge were studied. The studies address interactions of these polymers with proteins (bovine serum proteins and BSA), nucleic acids (DNA), glycosaminoglycans (heparin), and biological membranes. The results described in this study make it possible to indicate that toxicity is most strongly influenced by interactions with biological membranes and is closely related to the electrostatic charge of the macromolecule. The presentation of this observation was the goal of this publication. This paper also shows, using fluorescently labeled variants of polymers, the penetration and impact on cell structure (only for the polymer with the highest substitution binding to cell membranes is observed) by using confocal and SEM (for the polymer with the highest degree of substitution, and the appearance of additional structures on the surface of the cell membrane is observed). The labeled polymers are also tools used together with dynamic light scattering and calorimetric titration to study their interaction with other biopolymers. As for the interactions with biological membranes, lipid Langmuir monolayers as model membrane systems were used.

The Langmuir monolayer technique is one of the methods used to build models of cellular membranes and enables to investigate the interactions of membrane components with other biomolecules. This method has been applied to study the effect of edelfosine - a synthetic alkyl-lysophospholipid analog - on model lipid membranes in order to get insight into its mode of action and selectivity. Edelfosine is mainly known for its anticancer properties, although it is also applied in the treatment of other diseases, like autoimmune, anti-HIV and antiparasitic. In this review we focus on its antitumor activity (although some other aspects of its therapeutic effects are also indicated) and summarize the results obtained so far with use of the monolayer technique. The application of this method evidenced for a key role of cholesterol and membrane rafts in the mechanism of anticancer activity of edelfosine. As regards the selectivity of this drug, the obtained results proved that the difference in fluidity of tumor versus normal cell membrane is important but probably not the only factor determining an easier incorporation of edelfosine into cancer cells. Further studies show that edelfosine is of strong affinity to gangliosides, which may be considered as molecules targeting edelfosine into cancer cell membrane.

Edelfosine is a synthetic antitumor lipid of high selectivity. Its activity on membrane level inspired the investigations on edelfosine-lipid interactions to verify, which of the membrane components may be responsible for the selectivity of this drug. Because of overexpression of gangliosides in tumor progression and the ability of edelfosine to insert into membrane rafts, we have chosen two sphingolipids, i.e. sphingomyelin and ganglioside to investigate in mixtures with edelfosine. It was found that edelfosine shows strong affinity to ganglioside in contrast to sphingomyelin. Differences in the interactions of edelfosine with sphingolipids were analyzed from the point of view of the structure and shape of the interacting molecules. The comparison of the results with those previously reported for edelfosine mixed with other membrane components, allowed us to suggest that gangliosides may be considered as target molecules attracting edelfosine to tumor cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

One of the characteristic features of tumor membranes is altered concentration of gangliosides level and their over-expression in tumor progression. This fact should be considered when the mechanism of activity and selectivity of edelfosine (ED)--a membrane-active anticancer drug--is investigated. Strong affinity of this drug to ganglioside GM1, found in binary mixed monolayers, encouraged us for a deeper investigations on more complex model systems. In this work we have studied the influence of edelfosine on the interactions between molecules in model sphingomyelin/cholesterol monolayer, mimicking a tumor membrane, containing ganglioside in increasing proportion that is characteristic of cancer progression. It was found that edelfosine in very low concentration (1%) practically does not influence on model membrane system, independently of the proportion of ganglioside. On the other hand, at the increased ED concentration (10%), the interactions between the lipids in the mixed system become progressively stronger upon the increase of ganglioside concentration as compared to those in the model tumor membrane. The results of our investigations show that the affinity of edelfosine towards tumor cells may be correlated with over-expression of gangliosides in cancer cells., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (beta-sitosterol and stigmasterol) and saturated-monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than beta-sitosterol., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Sterols are essential constituents of membranes, both in the plant world and in human organisms. Therefore, their activity on model lipid systems has systematically been studied. Despite intensive investigations, differences in the effect induced by beta-sitosterol (beta-sito) and stigmasterol (stigma) (two major phytosterols) are very controversial and still under debate. To compare the influence of these compounds on model membranes, we have performed grazing incidence X-ray diffraction (GIXD) experiments on phytosterol/sphingomyelin (Sph) monolayers. The analysis of the X-ray scattering and the resulting in-plane parameters provided information on the lateral organization of pure lipid films and the mixed systems. The obtained results prove a nonideal mixing between the investigated lipids in the monolayers and the existence of strong interactions between phytosterols and Sph. Both the plant sterols incorporated into sphingolipid film condense the monolayer and order Sph chains. The results of GIXD experiments, compared with those obtained previously from Langmuir monolayer studies allowed us to observe the comparable influence of beta-sito and stigma on model membrane organization.

This work is aimed at comparing the effect of edelfosine (Ed) on normal and tumor cell membrane imitated by the Langmuir monolayers prepared from cholesterol and phosphatidylcholines. To keep the differences in the fluidity of these membranes, model membrane of normal cell has been composed from saturated phospholipid (DPPC) and contained a higher proportion of cholesterol (cholesterol:DPPC=0.67) than tumor cell model membranes prepared from unsaturated phosphatidylcholine (cholesterol:POPC=0.25). The results proved that the incorporation of edelfosine modifies the organization and interactions between molecules in both model systems. The interactions in cholesterol/DPPC/edelfosine monolayers are stronger than in cholesterol/DPPC film. On the other hand, the interactions in cholesterol/POPC/edelfosine system are weaker than in cholesterol/POPC monolayer, thus the incorporation of edelfosine is, from thermodynamical point of view, unfavorable for binary cholesterol/POPC monolayer. Edelfosine has been found to increase the fluidity of model membranes, however, at a lower concentration (up to 5% of edelfosine in model system) this compound affects only the condensation of tumor cell model membrane, while practically does not modify the organization of normal model system. It has been suggested that edelfosine inserts into tumor cellular membranes more easily than into normal cell membrane and cholesterol/edelfosine interactions determine the interactions between molecules in the investigated mixed monolayers.

In this work, the results of comparative studies on the effect of lysophospholipid analogue, edelfosine (ED), and its mixtures with the polyene antibiotic, amphotericin B (AmB), on model erythrocyte and parasite membranes are presented. Both compounds are known for their antileishmanian activity; however, the application of AmB is limited by its toxicity, while the treatment with alkyl-lysophospholipids (edelfosine) is expensive in addition to its lower therapeutic activity as compared to the polyene. An additional problem is the emergence of resistance to both groups of drugs. The foregoing facts inspired the investigations toward combined amphotericin B/alkyl-lysophospholipid therapy. In this aspect, the effect of edelfosine on model erythrocyte versus parasite membrane has been verified by the incorporation of the drug into binary sterol/phospholipid monolayer of the proportion corresponding to the respective cellular membrane. Afterward, amphotericin B/edelfosine mixtures of various proportions (1:9; 1:1, and 9:1) have been added into sterol/phospholipid model membranes in the concentration of 1, 5, and 10%. The interactions between amphotericin B and edelfosine in mixed monolayers have also been investigated. The obtained results indicate that differences in the organization of erythrocyte versus parasite membranes are of great importance for selectivity of edelfosine toward the parasite membrane. The mixtures of drugs practically do not modify the properties of model erythrocyte membrane; however, their incorporation in the parasite membrane is thermodynamically unfavorable and causes membrane destabilization. The strongest differences in the influence of drug mixtures on erythrocyte versus parasite model membrane occur for 1% content of 1:9 and 1:1 AmB/edelfosine mixture. Moreover, when edelfosine is combined with amphotericin B, a decrease in edelfosine concentration needed to induce a similar effect on parasite membranes as edelfosine alone does is observed. As a consequence of strong interactions between edelfosine and amphotericin B, the decrease of the activity of their mixtures on model membranes was found.

Fatty acids molecules have strong influence on membrane properties. In this work the interactions between fatty acids and phosphatidylcholines were studied in mixed binary and ternary Langmuir monolayers. The compounds investigated, both fatty acids and phospholipids were of the same hydrocarbon chain length and differed in their saturation degree (stearic acid (C18:0)), oleic acid (C18:1)), linoleic acid (C18:2) and distearoylphosphatidylcholine-DSPC and dioleoylphosphatidylcholine-DOPC). It was found that the investigated fatty acids interact more strongly with saturated phospholipid (DSPC) and more strongly affect the molecular organization of DSPC films as compared to DOPC monolayer. The saturated fatty acid (stearic acid) makes the phospholipid monolayers more rigid, while unsaturated fatty acids decrease the condensation of phospholipid films. The differences in the effect of the respective fatty acids on phosphatidylcholines monolayers were thoroughly analyzed from the point of view of geometry of the lipids molecules, which is determined by the saturation of the hydrocarbon chain.

In this work thermodynamic analysis of the interactions between lipids in ternary sphingomyelin/DPPC/sterol Langmuir films were performed to compare the effect of cholesterol, beta-sitosterol and stigmasterol on a model membrane. The condensing effect of the respective sterols and the interactions between molecules in ternary mixtures were analyzed on the basis of the excess area per molecule and the excess free energy of mixing values. The stability of the mixed monolayers was verified with the free energy of mixing values. The conclusions on the ordering effect of sterols were drawn from the analysis of the compression modulus values. It was found that the stoichiometry of the mixed films of the highest thermodynamic stability and of the strongest interactions is the same for all the sterols investigated. The results obtained prove that the mammalian sterol induces the strongest contraction of the area and reveals the strongest stabilizing and ordering effect among the investigated sterol. Stigmasterol was found to condense a model membrane in a weaker extent as compared to beta-sitosterol, however, the differences in ordering properties of both phytosterols are less pronounced. The magnitude of the influence of the investigated sterols on a model membrane was thoroughly discussed from the point of view of the structure of their side chain, which determines the geometry of a sterol molecule.

In this work, the Langmuir monolayer technique was applied to study the interactions between sphingomyelin and various sterols differing in the structure of the side chain (cholesterol, beta-sitosterol, stigmasterol). The mean area per molecule and the excess free energy of mixing values were analyzed in the context of sterol-induced condensing effect and interactions between molecules in the mixed monolayers. Moreover, the compression modulus values were calculated and widely discussed from the point of view of the ordering effect of sterols. It was found that all of the sterols investigated form the most stable monolayers with sphingomyelin at 2:1 sphingomyelin:sterol proportion and the strongest interactions exist between molecules in cholesterol-containing films. Moreover, cholesterol provokes the strongest area condensation and reveals the highest ordering properties, while plant sterols were found to differ only slightly with regards to their ordering properties. Additionally, the ordering effect of the sterols on dipalmitoylphosphatidylcholine (DPPC) films was analyzed and compared to that on sphingomyelin films.

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.

Owing to its distinct chemico-biological properties, chitosan, a cationic biopolymer, offers a great potential in multifarious bioapplications. One such application is as a dietary antilipidemic supplement to be used to reduce obesity/overweight and to lower cholesterol. The lipid-binding efficiency of chitosan, however, remains debatable. Accordingly, in this study we investigated the interactions of chitosan with selected lipids, cholesterol and fatty acids, the latter including saturated (stearic acid) and unsaturated (oleic, linoleic, alpha-linolenic) acids. The experiments were performed with the Langmuir monolayer technique, in which surface pressure-area isotherms were recorded for the lipid monolayers spread on the acetate buffer pH 4.0 subphase in the absence and presence of chitosan. We found that the presence of chitosan in the subphase strongly influenced the shape and location of the isotherms, proving that there existed attractions between chitosan and lipid molecules. The attractions were revealed by changes of the molecular organization of the monolayers. The common feature of these changes was that all the monolayers studied underwent expansion, in each case reaching saturation with increasing chitosan concentration. In agreement with the lipid molecular structures, the highest expansions were observed for the most unsaturated fatty acids, linoleic and alpha-linolenic, the lowest for stearic acid, with oleic acid and cholesterol being the intermediate cases. By contrast, the main distinguishing feature of these changes was that, although none of the monolayers studied changed its state when completely saturated with chitosan, compared to the parent ones the compactness of the monolayers was modified. The solid monolayers of stearic acid and cholesterol were loosened, whereas those of all the unsaturated acids, liquid in nature, were tightened. On the basis of these results we tentatively propose a mechanism of the chitosan action that includes both electrostatic and hydrophobic lipid-chitosan interactions as well as hydrogen bonding between them.

The aim of this work was to get insight into cholesterol distribution between two leaflets of a phospholipids bilayer. In this order, the thermodynamic analysis of the interactions between membrane lipids in binary (cholesterol/phospholipid) and ternary (phospholipid/ phospholipid/cholesterol) mixed Langmuir monolayers has been performed. For our investigation, phosphatidylcholine and phosphatidylethanolamine, which are the main types of phospholipids determining the distribution of cholesterol in membrane leaflets, were chosen and mixed in proportions corresponding to their molar ratios in the inner and outer layers of the natural human erythrocyte membrane. Into these mixed systems, various amount of cholesterol were incorporated. It has been found that despite strong differences in the phospholipid composition of both investigated ternary mixed systems, the influence of cholesterol is very similar, which indicates that cholesterol is symmetrically distributed between the inner and outer leaflets of the human erythrocytes membrane.

The paper presents a thorough characteristics of Langmuir monolayers formed at the air/water interface by a polyene macrolide antibiotic-nystatin. The investigations are based on the analysis of pi/A isotherms recorded for monolayers formed by this antibiotic at different experimental conditions. A significant part of this work is devoted to the stability and relaxation phenomena. It has been found that nystatin forms at the air/water interface monolayers of the LE state. A plateau region, observed during the course of the isotherm compression, is suggested to be due to the orientational change of nystatin molecules from horizontal to vertical position. Quantitative analysis of the desorption of the monolayer material into bulk water indicates that the solubility of nystatin monolayers increases with surface pressure. At low surface pressures, the desorption of nystatin from a monolayer is controlled both by dissolution and by diffusion. However, at the plateau and in the post-plateau region, the desorption does not achieve a steady state and the monolayer is less stable than in the pre-plateau region. However, the presence of membrane lipids, even at a low mole fraction, considerably increases the stability of nystatin monolayers. This enables the application of the Langmuir monolayer technique to study nystatin in mixture with cellular membrane components, aiming at verifying its mode of action and the mechanism of toxicity.

Nystatin (NYS), a polyene antifungal antibiotic, has been investigated in Langmuir monolayers alone and in mixtures with mammalian and fungi membrane sterols (cholesterol and ergosterol, respectively) as well as with a model phospholipid (DPPC). The interactions between film molecules have been examined both in a qualitative and quantitative way with the excess area per molecule (AExc), excess free energy of mixing (DeltaGExc) and the interaction parameter (alpha). The obtained results have been compared with those previously reported for another polyene antimycotic: amphotericin B (AmB) mixed with lipids. Higher affinity of NYS has been observed for ergosterol vs. cholesterol, however, the strongest attractions were found for its mixtures with DPPC. The obtained results have been verified with biological studies reported previously for both antibiotics (NYS and AmB). A thorough analysis of the Langmuir experiment results performed for both polyenes enabled us to conclude that the presence of DPPC can be considered as a key factor affecting their antifungal activity as well as their toxicity towards host cells.

Langmuir monolayers of cholesterol/ergosterol and dialkyldimethylammonium bromides (DXDABs) differing in alkyl chain length-14 (DTDAB), 16 (DHDAB), and 18 (DODAB)-spread at the air/water interface are examined. All the systems investigated are found to be nonideal and miscible. Negative values of the total free energy of mixing, proving film stability in the whole range of compositions and surface pressures, are observed for all the studied mixtures except for DTDAB/cholesterol. The strength of interactions, quantified with DeltaG(Exc) values, was found to be of the same order for mixtures of cholesterol/ergosterol and DHDAB or DODAB. Differences occurring for the mixtures of DTDAB with sterols indicate the affinity of DTDAB to ergosterol in contrast to cholesterol.

The interactions between tri-n-octylphosphine oxide (TOPO) and a series of dialkyldimethylammonium bromides (DXDAB) in mixed Langmuir monolayers have been investigated. The mean area per molecule in mixed monolayers (A12) and the excess area (AExc), exhibited deviations from ideal behavior for all the investigated systems. The molecular interactions have been quantified by the values of the excess free energy of mixing (DeltaGExc). The monolayers stability has been examined with the total free energy of mixing (DeltaGM) values. The obtained results indicate that the length of the hydrocarbon chain in dialkyldimethylammonium salts affects the molecular packing and determines the strength of interactions with TOPO molecules.

Mixtures of cholesterol and synthetic phospholipids, differing in saturation of phosphatidylcholine (PC) acyl chains, such as distearoyl phosphatidylcholine (DSPC), stearoyl-oleoyl phosphatidylcholine (SOPC) and dioleoyl phosphatidylcholine (DOPC) have been studied as floating Langmuir monolayers at the air/water interface. In order to examine the influence of a polar group, distearoyl phosphatidylethanolamine (DSPE) was chosen. The films were spread at room temperature on aqueous subphases and characterized by the surface pressure-area (pi-A) isotherms and compression modulus (C(s)(-1)) values. The interactions were examined by analyzing the mean molecular areas and quantified by the excess free energy of mixing values. The obtained results indicate that the affinity of cholesterol to saturated/unsaturated phosphatidylcholines does not differ significantly, and revealed strong influence of the kind of a polar group on the cholesterol-phospholipid interactions. On the other hand, the apolar group structure was found to modify the stoichiometry of sterol-PC complexes.