Paweł Wydro

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films.

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.

Variations in the Condensing Effect of Cholesterol on Saturated versus Unsaturated Phosphatidylcholines at Low and High Sterol Concentration

In this work, we have investigated the condensing and ordering effect induced by cholesterol on phosphatidylcholines (PCs). To perform the studies systematically, for the experiments we have selected phospholipids differing only in the number of cis monounsaturated chains (1,2-distearoyl-sn-glycero-3-phosphocholine--DSPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine--SOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine--DOPC) or in the length (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine--POPC vs SOPC) of sn-1 acyl chain. Because the cholesterol concentration in mammalian membranes can be as high as 70 mol % of total lipids, the investigations were performed in a wide range of the sterol content. The results of the Langmuir monolayer experiments evidence that the relation between the structure of hydrophobic part of PC and the magnitude of the effects induced by cholesterol found at lower sterol content is different from that observed at higher sterol concentration. At a lower concentration of sterol (up to 30%), the condensing effect of cholesterol is stronger on saturated DSPC than on PCs containing monounsaturated chain(s), which is consistent with the conclusions drawn by other authors. However, at higher sterol content (≥50%), saturated DSPC is less susceptible to the influence of sterol than the investigated unsaturated PCs. To explain these irregularities, we have considered the strength of van der Waals interactions as well as the influence of sterol on the tilt of polar heads of PCs. It was also found that in the whole range of sterol concentration the ordering effect is stronger on saturated DSPC as compared to unsaturated phospholipids. However, at lower sterol content (up to 30%) the ordering effect induced on unsaturated PCs is rather weak, and the ordering does not change drastically in comparison with pure PCs film.

Thermodynamic description of the interactions between lipids in ternary Langmuir monolayers: the study of cholesterol distribution in membranes.

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.

Sphingomyelin/phosphatidylcholine/cholesterol monolayers--analysis of the interactions in model membranes and Brewster Angle Microscopy experiments.

In this work the properties of two ternary sphingomyelin/phosphatidylcholine/cholesterol monolayers imitating erythrocyte membrane were studied at various content of sterol. Phosphatidylcholines chosen for experiments differ in the length of sn-1 saturated chain in the molecule (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine-SOPC vs. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - POPC). Langmuir monolayer experiments combined with Brewster Angle Microscopy prove that for both investigated systems the most favorable effect of cholesterol appears at 30% of sterol in the film. However, the interactions between molecules at 50% of sterol are only slightly weaker as compared to those for 1:1:1 films. It was also found that only at higher sterol concentration appear differences in the ordering effect of cholesterol on the systems containing PC molecules of various length of sn-1 saturated chain. Although the differences in the properties of POPC versus SOPC-containing monolayers were found, similarities in the morphology of the respective systems and stoichiometry of thermodynamically the most favorable mixture allow one to conclude that both SM/POPC/Chol as well as SM/SOPC/Chol monolayer can be used to mimic raft systems.

Cholesterol and phytosterols effect on sphingomyelin/phosphatidylcholine model membranes—Thermodynamic analysis of the interactions in ternary monolayers

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.

Edelfosine disturbs the sphingomyelin–cholesterol model membrane system in a cholesterol-dependent way – The Langmuir monolayer study

Synthetic alkyl-lysophospholipids, represented by edelfosine (ED), reveal strong anticancer activity and therefore are promising drugs used in anticancer therapy. Primary target for edelfosine is cellular membrane, which is in contrast to traditional cytostatics affecting DNA. The mechanism of antitumor activity of edelfosine was hypothesized to be related to its accumulation in membrane rafts. Inspired by these findings, we have performed the Langmuir monolayer studies on the influence of edelfosine on systems composed of sphingomyelin (SM) and cholesterol (Chol), being the principal components of membrane rafts. Sphingomyelin-cholesterol proportion in monolayers was varied to reflect the composition of solely membrane rafts (SM/Chol=2:1) and contain excess of cholesterol (SM/Chol=1:1 and 1:2). Into these systems, edelfosine was added in various concentrations. The analysis of surface pressure-area isotherms, complemented with films visualization with Brewster angle microscopy (BAM) allowed us to compare the effect of edelfosine on condensation and ordering of SM/Chol monolayers. The results evidenced that the influence of ED on the interactions in model membranes and its fluidizing effect is highly cholesterol-dependent. The strongest decrease of monolayer ordering was observed for model raft system, while the excess of cholesterol present in the remaining mixtures was found to weaken the fluidizing effect of the drug.

Interactions of serum with polyelectrolyte-stabilized liposomes: Cryo-TEM studies

Liposomes are used for in vitro or in vivo vectorization of drugs, proteins, or nucleic acids. However, the main problem with the application of liposomes for this purpose is their low stability in contact with blood serum. In this article, interactions between the whole serum and anionic liposomes, both bare and covered with strong polycations, were studied. The polycations of different chemical structures were prepared by the modification of poly(allylamine hydrochloride) (PAH). Dynamic light scattering (DLS), zeta potential and transmission cryo-electron microscopy (cryo-TEM) measurements showed that the adsorption of the polycations on the anionic liposomes induced a reversible aggregation of vesicles. The stable isolated polyelectrolyte-covered vesicles were obtained after the addition of sufficient amounts of the polycations. The effect of full serum on the morphology and stability of the polycation-coated liposomes was studied using cryo-TEM and a fluorescence method. The cryo-TEM analysis revealed that the introduction of serum caused the osmotic-driven destabilization of the bare liposomes or formation of twinned vesicles. Due to these processes the liposomes lost most of their content immediately after serum addition. The polycation-covered liposomes showed improved stability in the presence of serum. Partial deflation of the vesicles was observed, however, the loss of the content was significantly limited. The effect of the polymer structure, especially the position of the charged groups with respect to the main polymer backbone, on the stabilization of the polycation-covered liposomes in the presence of serum was discussed.

Lupane-type pentacyclic triterpenes in Langmuir monolayers: a synchrotron radiation scattering study.

Lupane-type pentacyclic triterpenes (lupeol, betulin, and betulinic acid) are natural products isolated from various plant sources. The terpenes exhibit a vast spectrum of biological activity and are applied in therapies for different diseases, among which the anticancer, anti-HIV, antihypercholesteremic, and antiinflammatory are the most promising. These chemicals possess amphiphilic structure and were proved to interact strongly with biomembranes, which can be the key stage in their mechanism of action. In our studies, we applied Langmuir monolayers as versatile models of biomembranes. It turned out that the three investigated terpenes are capable of stable monolayer formation; however, these monolayers differ profoundly regarding their physicochemical characteristics. In our research, we applied the Langmuir technique (surface pressure-mean molecular area (π-A) isotherm registration) coupled with Brewster angle microscopy (BAM), but the main focus was on the synchrotron radiation scattering method, grazing incidence X-ray diffraction (GIXD), which provides information on the amphiphilic molecule ordering in the angström scale. It was proved that all the investigated terpenes form crystalline phases in their monolayers. In the case of lupeol, only the closely packed upright phase was observed, whereas for betulin and betulinic acid, the phase situation was more complex. Betulinic acid molecules can be organized in an upright phase, which is crystalline, and in a tilted phase, which is amorphous. The betulin film is a conglomerate of an upright crystalline monolayer phase, tilted amorphous monolayer phase, and a crystalline tilted bilayer. In our paper, we discuss the factors leading to the formation of the observed phases and the implications of our results to the therapeutic applications of the native lupane-type triterpenes.

Investigation of the interactions of lupane type pentacyclic triterpenes with outer leaflet membrane phospholipids--Langmuir monolayer and synchrotron X-ray scattering study.

Lupane type pentacyclic triterpenes (LTs) are pharmacologically active natural products isolated from different plants. They have broad spectrum of therapeutic action ranging from anticancer via anti-HIV, antibiotic to anti-inflammatory and anti-protozoal activity. Many scientific papers underline that the key stage in the LT mechanism of action is their incorporation into cellular membrane and the interaction with the structural lipids. In our research we apply Langmuir monolayers as a versatile platform for the investigation of these phenomena, since till now important aspects concerning this issue are incomprehensible. We focus our attention on the interactions of lupeol and betulinic acid with choline-headgroup structural lipids: a representative of saturated glycerophosphatidylcholines (DPPCs), and octadecyl-sphingomyelin--a representative of membrane sphingolipids. Application of complementary physicochemical techniques such as the Langmuir technique, Brewster angle microscopy, and grazing incidence X-ray diffraction supported by thermodynamic analysis enabled us to investigate the intermolecular interactions in such binary model systems. Our results corroborate that LT is miscible with the outer leaflet membrane phospholipids, both DPPC and SM in the whole range of mole ratios. Moreover, the introduction of LT into the phospholipid film, even in small proportion, leads to the loss of periodical ordering of the phospholipid molecules and the disappearance of the diffraction signal as observed by GIXD. Our results also proved that LT does not form any surface complexes of fixed stoichiometry resembling the well characterized lipid rafts.

Spontaneous formation of densely stacked multilamellar vesicles in dioctadecyldimethylammonium bromide/oleosiloxane mixtures.

A double-tailed surfactant, for example, dioctadecyldimethylammonium bromide (DODAB), tends to form bilayer fragments rather than vesicles in aqueous solution upon sonication. This Letter reports on the morphological transformations of the membrane fragments leading to multilamellar vesicle formation. The surfactant membranes were swollen with tetra(11-methoxy-11-oxoundecyl)tetramethylcyclotetrasiloxane (3) (a cyclosiloxane with covalently attached fatty-acid esters) up to 50 mol % based on the surfactant content. That compound was synthesized by hydrosilylation of 1,3,5,7-tetramethylcyclotetrasiloxane with four molecules of methyl undec-10-enoate. Using cryo-transmission electron microscopy (cryo-TEM), it has been shown that densely stacked multilamellar vesicles form spontaneously in the sonicated DODAB dispersion containing 50 mol % of the compound 3 after adjusting pH to the value of 9.5. This occurs as a result of the hydrolysis of ester bonds of the compound 3 inside the membrane with formation of tetra(11-carboxyundecyl)tetramethylcyclotetrasiloxane (4). The carboxylic groups in compound 4 are ionized under experimental conditions. That results in the appearance of the negative charge at the surface which neutralizes the positively charged molecules of DODAB, thus reducing repulsive interactions between the membrane fragments. Additionally, as was shown using Langmuir monolayer measurements, the presence of compound 4 expands the DODAB film and decreases its bending rigidity. That makes the bending process considerably easier and promotes formation of multilamellar vesicles.