Michał Flasiński

X-ray grazing incidence diffraction and Langmuir monolayer studies of the interaction of β-cyclodextrin with model lipid membranes

The interactions of beta-CD with one component monolayers of cholesterol (chol), 1-stearoyl-sn-glycero-3-phosphocholine (lyso-PC), 1,2-dipalmitpyl-sn-phosphocholine (DPPC), sphingomyelin (SM) and the SM/chol and DPPC/chol mixtures have been investigated by the Langmuir monolayer technique and the synchrotron grazing incidence X-ray diffraction (GIXD). The investigated lipid monolayers have been studied with and without the 10(-3) M solution of beta-CD in the aqueous subphase. The surface pressure-area (pi-A) isotherms and the relaxation of the monolayers (surface pressure-time curves) were monitored. Our experiments reveal that there is not impact of beta-CD on the packing properties of the DPPC monolayers, while the presence of beta-CD in subphase changes the in-plane organization of SM molecules. Monolayers composed of pure chol molecules have been rapidly affected by the presence of the beta-CD in the subphase. Our data show that beta-CD can complex and desorb one-chain phospholipid (lyso-PC) but this process is relatively slow and, as indicated by the GIXD data, beta-CD molecules are present at the air/water interface. Subtraction of cholesterol by the beta-CD from mixed binary systems containing SM/chol (70/30, 50/50 and 30/70 mol ratio) and DPPC/chol (70/30 and 50/50 mol ratio) has also been investigated. Our experiments proved that cholesterol can be removed from the mixed monolayers only when it is unbound. The beta-CD was not capable to distract the monolayers of the SM/chol, forming a stable complex of the 2:1 stoichiometry (as observed in the model lipid raft). Interestingly, at the surface pressure of 30 mN/m also at the molar proportion of 50/50 no cholesterol removal was observed. This was interpreted by relatively strong SM/chol interactions and the tight packing of the mixed monolayer. For model membranes, in which cholesterol was in large excess (SM/chol, 30/70) the beta-CD extraction of cholesterol was observed, and the membrane composition evolves towards the lipid proportion corresponding to the stable complex stoichiometry (SM/chol 2:1).

Grazing Incidence Diffraction and X-ray Reflectivity Studies of the Interactions of Inorganic Mercury Salts with Membrane Lipids in Langmuir Monolayers at the Air/Water Interface

The interactions of mercury ions with the membrane phospholipids are considered to be of great importance regarding the toxicity of this metal in living organisms. To obtain deeper insight into this problem, we performed systematic studies applying the Langmuir technique complemented with synchrotron X-ray scattering methods (grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XR)). We focused our attention on the interactions of inorganic mercury salts dissolved in the aqueous subphase with lipid monolayers, formed by selected membrane phospholipids, namely, dipalmitoylphosphatidylglicerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), 1-octadecyl 2-sn-phosphatidylcholine (lyso-PC), and sphingomyelin (SM). Two different inorganic mercury salts, one of a hydracid, HgCl(2), and the other of an oxacid, Hg(NO(3))(2), have been investigated. Our results proved that the elastic properties of phospholipid monolayers are a key factor regarding the interactions with mercury ions. Significant differences in mercury ions complexation are observed with double-chain phospholipids (such as DPPG and DPPC) forming fluid layers of low compressibility and phospholipids forming more compressible films (like SM and lyso-PC). Namely, important changes in the monolayer characteristic were observed only for the latter kind of lipids. This is an important finding taking into account the accumulation of mercury in the central nervous system and its neurotoxic effects. SM is one of the most abundant lipids in neurons shells and therefore can be considered as a target lipid complexing mercury ions.

Effects of β-Cyclodextrin on the Structure of Sphingomyelin/Cholesterol Model Membranes

The interaction of beta-cyclodextrin (beta-CD) with mixed bilayers composed of sphingomylein and cholesterol (Chol) above and below the accepted stable complexation ratio (67:33) was investigated. Membranes with the same (symmetric) and different (asymmetric) compositions in their inner and outer leaflets were deposited at surface pressures of 20, 30, and 40 mN/m at the solid-liquid interface. Using neutron reflectometry, membranes of various global molar ratios (defined as the sum of the molar ratios of the inner and outer leaflets), were characterized before and after beta-CD was added to the subphase. The structure of bilayers with global molar ratios at or above the stable complexation ratio was unchanged by beta-CD, indicating that beta-CD is unable to remove sphingomyelin or complexed Chol. However, beta-CD removed all uncomplexed Chol from bilayers composed of global molar ratios below the stable complexation ratio. The removal of Chol by beta-CD was independent of the initial structure of the membranes as deposited, suggesting that asymmetric membranes homogenize by the exchange of molecules between leaflets. The interaction of beta-CD with the aforementioned membranes was independent of the deposition surface pressure except for a symmetric 50:50 membrane deposited at 40 mN/m. The scattering from 50:50 bilayers with higher packing densities (deposited at 40 mN/m) was unaffected by beta-CD, suggesting that the removal of Chol can depend on both the composition and packing density of the membrane.

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.

Interactions of pentacyclic triterpene acids with cardiolipins and related phosphatidylglycerols in model systems.

Pentacyclic triterpene acids (PTAs): betulinic (BAc), oleanolic (Ola) and ursolic (Urs) are potent pharmaceuticals applied in the therapy of cancer and bacterial infections. The mechanism of PTA action is multifactor, but the important step is their interaction with the lipids of mitochondrial and bacterial membranes. In our studies we applied the Langmuir monolayer technique to investigate the interactions between PTAs and cardiolipins (CLs) and phosphatidylglycerols (PGs). We applied two different mammalian mitochondrial CLs and one species extracted from the membrane of Escherichia coli. For comparison we performed the same experiments on the systems containing PTAs and 3 PGs strictly correlated structurally to the applied CLs. Our studies proved that PTAs can disturb the organization of CL-rich domains and affect the bacterial membrane fluidity by the interactions with phosphatidylglycerols, so anionic phospholipids are the targets of their membrane action. The thermodynamic interpretation of the results indicated that Urs has the highest membrane disorganizing potential among the 3 studied PTAs. The studies performed on model systems proved also that BAc can discriminate over structurally similar animal cardiolipin species, interacts specifically with BHCL - the main mammalian CL and can disturb its organization in the membrane. In contrast, Ola and Urs are much active as far as the interaction with bacterial CLs and PGs is concerned.

Does cholesterol preferentially pack in lipid domains with saturated sphingomyelin over phosphatidylcholine? A comprehensive monolayer study combined with grazing incidence X-ray diffraction and Brewster angle microscopy experiments

In this work, the Langmuir monolayers were used as a model for the analysis of the influence of cholesterol on 1,2-distearoyl-sn-glycero-3-phosphocholine - DSPC and stearoyl sphingomyelin - SSM, as well as their equimolar mixture. The aim of these studies was to compare the affinity of cholesterol to sphingomyelin and phosphatidylcholine and discuss the effectiveness of cholesterol packing with these phospholipids. The experiments involved the registration of the surface pressure-area isotherms combined with the application of Brewster angle microscopy (BAM) images and grazing incidence X-ray diffraction methods. We have performed a thorough analysis of the properties of both one-component DSPC and SSM films as well as their 1:1 mixture. Next, the effect of cholesterol on these systems was verified based on the results for 2:1 SSM/Chol, 2:1 DSPC/Chol, and 1:1:1 DSPC/SSM/Chol mixtures. It was found that both phospholipids form highly condensed monolayers, however, they differ in the orientation of acyl chains, namely the acyl chains are more tilted in DSPC film as compared to SSM monolayer as well as DSPC/SSM mixture. Furthermore, the area contraction provoked by the addition of cholesterol was found to be more pronounced for DSPC monolayer than in DSPC/SSM and SSM films. However, all the collected results allow one to postulate that the ability of cholesterol to form ordered domains with DSPC and SSM is similar and is predominantly driven by hydrophobic forces between molecules. The differences in the area condensation induced by cholesterol on the studied phospholipids films results from differences in molecular organization of pure phospholipids films rather than specific cholesterol-phospholipid interactions.

Comparative Studies on the Influence of β-Sitosterol and Stigmasterol on Model Sphingomyelin Membranes: A Grazing-Incidence X-ray Diffraction Study

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.

Natural vs synthetic auxin: studies on the interactions between plant hormones and biological membrane lipids.

Analysis of the interactions between two representatives of plant hormones: synthetic (1-naphthaleneacetic acid, NAA) as well as natural (indole-3-acetic acid, IAA) and phospholipids occurring in biological membrane of both plant and animal cells was the subject of present studies. The aim of undertaken experiments was to elucidate the problem of direct influence of these plant growth regulators on phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in monolayers at the air/water solution interface. The studied phospholipids differ not only as regards the structure of polar head-groups but also in the length of hydrophobic chains as well as their saturation degree. These differences result also in the main properties and functions of these phospholipids in biomembranes. The analysis of the results was based on the characteristics of the surface pressure (π)--area (A) isotherms registered for monolayers spread on the subphase containing plant hormone and as a reference on the surface of pure water. Moreover, as a complementary technique, Brewster angle microscopy was applied for the direct visualization of the investigated surface films. The obtained results revealed that auxins effectively influence phospholipids monolayers, regardless of the lipid structure, at the concentration of 10(-4)M. It was found that for this concentration, the influence of auxins was visibly larger in the case of PCs as compared to PEs. On the other hand, in the case of auxins solution of ≤ 10(-5)M, the observed trend was opposite. Generally, our studies showed that the natural plant hormone (IAA) interacts with the investigated lipid monolayers stronger than its synthetic derivative (NAA). The reason of these differences connects with the steric properties of both auxins; namely, the naphthalene ring of NAA molecule occupies larger space than the indole system of IAA. Therefore molecules of the latter compound penetrate easier into the region of phospholipids׳ polar head-groups. Moreover, the NH group of the indole moiety is capable of hydrogen bond formation with the acceptor groups in the polar fragment of lipid molecules. We proved also that among the investigated phospholipids, the highest susceptibility toward auxin influence show these lipids, for which during compression, surface film increases the degree of condensation.

Langmuir monolayer studies of the interaction of monoamphiphilic pentacyclic triterpenes with anionic mitochondrial and bacterial membrane phospholipids — Searching for the most active terpene

The interactions of three representative monoamphiphilic pentacyclic triterpenes (PTs) with cardiolipins (CL) and phosphatidylglycerols (PG) extracted from mitochondrial and bacterial membranes were comparatively characterized in binary Langmuir monolayers. The studied terpenes: lupeol, α- and β-amyrin are isomeric compounds known from their broad biological activity. Anticancer and antimicrobial activity of PTs is often correlated with their propensity of being incorporated into mitochondrial and bacterial membranes and their specific interactions with cardiolipins. In our studies on 18 model systems surface pressure (π)-mean molecular area (A) isotherms were registered at five different component proportions in each system. Thermodynamic analysis complemented by in situ Brewster angle microscopy visualization of the investigated mixed films enabled the thorough characterization of the studied systems. It turned out that the investigated terpenes interact more favorably with PG molecules as compared to CLs. For most of the system containing CLs the values of ΔG(exc) were positive which was interpreted as the ability of the terpenes to disintegrate the membranes rich in CLs. Our results confirmed also that in the light of thermodynamic criterion α-amyrin exhibited the highest potential to disintegrate the CL containing domains in mitochondrial and bacterial membranes. The probable origin of the observed specific interactions between α-amyrin and investigated phospholipids could be explained based on the phenomenon of chiral discrimination. The obtained results were also widely discussed in reference to the biological activity of the studied compounds.