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Colloids and Surfaces B: Biointerfaces 111 (2013) 43– 51

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb

nteractions between single-chained ether phospholipids andphingomyelin in mixed monolayers at the air/waternterface—Grazing incidence X-ray diffraction and Brewster angle

icroscopy studies

ichał Flasinski ∗, Katarzyna Hac-Wydro, Paweł Wydro, Marcin Broniatowski,atrycja Dynarowicz-Łatka

aculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

a r t i c l e i n f o

rticle history:eceived 25 January 2013eceived in revised form 12 May 2013ccepted 15 May 2013vailable online 23 May 2013

eywords:latelet activating factordelfosinephingomyelinixed Langmuir monolyersrazing incidence X-ray diffraction

a b s t r a c t

Single-chained ether phospholipids comprise a class of both natural (PAF, lyso-PAF) and synthetic (edelfo-sine, ED) compounds possessing confirmed extensive biological activities. Among them ED is knownto exhibit antineoplastic properties, while PAF and its lyso-precursor are lipids implicated e.g. in thefunctioning of organism immune system. In our study the interactions of ED, PAF and lyso-PAF with sphin-gomyelin (SM) being one of the main lipid found in a high concentration in membrane microdomains, likelipid rafts, were investigated in mixed monolayers at the air/water interface. The traditional Langmuirmethodology was complemented with modern physicochemical techniques: Grazing incidence X-raydiffraction and Brewster angle microscopy. The investigated compounds, i.e.: platelet activating factor(PAF), (lyso-PAF) and edelfosine were selected because of their highly different physiological propertiesdespite very similar chemical structure and evidenced membrane activity. The obtained results demon-strate that all the investigated three single-chained phospholipids cause strong modification of the model

rewster angle microscopy membrane properties in a concentration dependent manner. It has been proved that there are significantdifferences regarding the influence of the single-chained lipids on model SM membrane – in the region oflow concentration, edelfosine was found to be the most effective among all the investigated compounds.The collected data shed new light onto the membrane behavior of the investigated herein biochemicallyactive compounds, which can be of help in understanding their different biological activity and designingof new, more biocompatible drugs.

. Introduction

Ether lipids are vitally important class of molecules, which inuman tissues may comprise a large fraction of total choline andthanolamine-derived lipids [1,2]. For example, in the brain etheripids account for 30–50% of total PE lipids, while in the cardiac

uscle they cover ∼16% of PC species. These compounds partic-pate in essential intercellular processes, like signal transduction,ransport of ions, membrane metabolites and membrane fusion.hey also serve as the main building blocks of bilayers and regulateroteins’ activities, which – in consequence – leads to the mod-

lation of biomembrane properties. Moreover, these disturbances

n the level of particular ether lipids in mammalian body corre-ate with serious health disorders, like diabetes, cardiac diseases

∗ Corresponding author. Tel.: +48 12 663 20 82; fax: +48 12 634 05 15.E-mail address: flasinsk@chemia.uj.edu.pl (M. Flasinski).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.05.024

© 2013 Elsevier B.V. All rights reserved.

or cancer [1,3]. One of the well known molecules in this class oflipids is a single-chained ether phospholipid – Platelet ActivatingFactor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) [4].Interestingly, the chemical motif found in PAF molecule serves asan unique building element for a variety of synthetic compoundsdisplaying widely documented anti-tumor activity [5,6]. One of therepresentatives among this group is edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, abbr. ED), the molecularstructure of which is presented in Supplementary Materials SchemeS-1, together with structures of PAF and its deacetylated derivative;lyso-PAF.

Edelfosine has been proved to exhibit antineoplastic activity,similarly to other synthetic antitumor ether lipids. This propertyis related to their selective penetration and subsequent accumula-

tion in cancer cells’ membranes, which is in contrast to the mode ofaction of traditional cytostatics, which interact directly with DNA[5,6]. The analysis of literature data concerning various aspectsof biological activity of ED, PAF as well as the abovementioned

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4 M. Flasinski et al. / Colloids and Su

eacetylated PAF precursor (lyso-PAF) provide interesting conclu-ion that even subtle differences in the chemical structure of theseompounds strongly affect their biological properties. For example,delfosine in contrast to PAF, is found to be ineffective in plateletggregation [6,7], but on the other hand PAF does not cause apo-tosis in tumor cell lines sensitive to edelfosine [5]. As regards

yso-PAF, it is usually described as completely biologically inactivegent as compared to PAF [8–10], however, it has been found thatyso-PAF – in contrast to PAF – inhibits activation of neutrophilsnd aggregation of platelets [11].

It is worth to mention that in literature there are plenty oftudies connecting PAF bioactivity with direct interaction of thisolecule with specific G protein-coupled receptor (PAFR) local-

zed in cellular membrane, especially within domains enriched inaturated phosphoglycerides and sphingomyelin, like lipid rafts oraveolae [12]. On the other hand, it is also well known that PAFay act in cells throughout the receptor independent mechanism

13,14]; therefore biological membranes may be treated as one ofhe potential site of action of the investigated compounds.

All these three single-chained ether lipids are surface activeue to their amphiphilic structure and therefore are capable of

ncorporating into cellular membranes and, in consequence, modifyheir properties [15–18]. However, the investigations concerninghe influence of these three compounds on membranes have nevereen performed systematically. Therefore, it is impossible to drawny coherent conclusions regarding the relationship between thetructure of these molecules and their effect on biomembranes.or that reason, comparative studies performed in this context onodel systems are highly required and may be helpful in under-

tanding the observed differences in biological activity of theseompounds.

In this work we have undertaken Langmuir monolayers experi-ents complemented with Brewster angle microscopy and GIXD

echnique on the effect of ED, PAF and lyso-PAF (each having18 chains within the molecule) on sphingomyelin (SM) film.e have chosen this lipid because sphingomyelin, together with

hospatidylcholines (PCs), is a major phospholipid building thekeleton of the outer animal membrane layer and, additionally, it is

key component of lipid rafts [19]. In comparison to PCs having inheir molecules glycerol backbone, sphingomyelin is a derivativef amino alcohol: sphingosine; therefore possesses free hydroxylroup in the molecule. This difference causes that in the case ofM there is a higher probability of hydrogen bond formation witholecules incorporated into the membrane. Moreover, there are

lso results of studies suggesting that this specific structure of SMolecule affects the interactions with other lipids [20].Since in mammalian membranes sphingomyelin molecules pos-

essing fully saturated C18 and C16 chains prevail on the speciesaving unsaturated bond within the chains [19], for our investiga-ions we have selected synthetic C18 SM. Thermodynamic studiesn the interactions between the foregoing lipids in monolayers, thenalysis of morphology of the studied films as well as their molecu-ar organization performed on the basis of the collected monolayerata, BAM images and GIXD results allow us to explore the influ-nce of subtle differences in the ether lipids structure on their effectn the organization of model membrane.

. Experimental

.1. Materials and methods

The investigated compounds: C18PAF (1-O-octadecyl--acetyl-sn-glycero-3-phosphocholine) and C18lyso-PAF1-O-octadecyl-sn-glycero-3-phosphocholine) of high purity>99%) were purchased from Bachem AG Switzerland, whereas

B: Biointerfaces 111 (2013) 43– 51

edelfosine, ED (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), of purity ≥99.1%, was provided by BiaffinGmbH & Co. KG, Germany. Synthetic sphingomyelin (N-stearoyl-D-erythro-sphingosylphosphorylcholine), of purity ≥99%, waspurchased from Avanti Polar Lipids Inc., USA. Spreading solutionsof the lipids were prepared in chloroform/methanol 9/1 (v/v)mixture. Chloroform and methanol of spectroscopic purity (99.9%)were provided by Sigma–Aldrich. In all experiments ultrapurewater of the resistivity ≥18.2 M� cm from MilliQ system wasapplied as a subphase. Mixed solutions of the desired compositionswere prepared from the respective stock solutions and depositedonto aqueous subphase with the Hamilton microsyringe precise to1 �L.

�–A isotherms were recorded with the NIMA (Coventry, U.K.)Langmuir trough of total area of 300 cm2 equipped in a single mov-able barrier placed on anti-vibration table. The surface pressure wasmeasured with the accuracy of 0.1 mN/m using Wilhelmy balancewith a surface pressure sensor made of filter paper (ashless What-man). In each experiment, the monolayer was left to equilibratefor at least 5 min before the monolayer compression was initiatedwith the barrier speed of 20 cm2/min. Each isotherm was repeatedat least three times to ensure reproducibility of the curves to±2 A2. The circulating water system was used to control subphasetemperature (20 ◦C) during the experiments. For in situ visualiza-tion of the investigated monolayers Brewster angle microscope(ultraBAM, Accurion GmbH, Göttingen, Germany) with the lateralresolution of 2 �m was applied. Grazing incidence X-ray diffrac-tion (GIXD) experiments were performed at the surface pressure of30 mN/m with the liquid surface diffractometer at the BW1 (undu-lator) beamline in HASYLAB, DESY synchrotron center (Hamburg,Germany). The construction of the liquid surface diffractometerand the detailed theoretical background of grazing incidence X-raydiffraction technique were described in an exhaustive manner byus [21,22] and by other authors [23,24].

3. Results and discussion

In the first step of the studies the properties of ether lipids/SMmonolayers were analyzed based on the surface pressure–areaisotherms (Fig. 1) and the parameters calculated from these curves,namely the compressibility modulus (inset of Fig. 1) and theexcess free energy of mixing (Fig. 2). The �–A isotherms for onecomponent monolayers of the investigated ether lipids were pre-sented and discussed in details previously [25,26]. In short, theselipids form liquid-like monolayers and the curves for PAF andED are very similar in their course and position. In the isothermfor lyso-PAF monolayer a kink reflecting the formation of highlyordered domains was detected at � ≈ 32 mN/m [25]. On the otherhand, the isotherm obtained for the monolayer for synthetic C18:0sphingomyelin indicates much higher condensation of this film ascompared to ether lipids. Moreover, at � = 5 mN/m a plateau regionindicating a first order phase transition between liquid expandedand condensed state is clearly visible, which is in agreement withthe data published previously by Vaknin et al. [27].

Let us now proceed to the results obtained for the mixed films.As it was found the addition of even small amount of the respec-tive ether lipid into SM monolayer causes the shift of the isothermstoward larger areas and significant decrease of the compressionmodulus values. This suggests a lowering of the condensation ofthe monolayers in the presence of ether lipid molecules. Deeperanalysis of the results obtained for these systems evidences further

similarities in the characteristic of the isotherms for PAF/SM andED/SM mixtures. Namely, at 10% of PAF or ED in the system theisotherms do not differ drastically in their shape from the curvetaken for SM film. However, at Xether lipid = 0.3 two distinct collapses

M. Flasinski et al. / Colloids and Surfaces

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ig. 1. The �–A isotherms registered for monolayers of PAF/SM (a); lyso-PAF/SM (b)nd ED/SM (c) together with the compression modulus vs � plots.

an be distinguished in the curves. The first one occurs at surface

ressure of ca. 44 mN/m, whereas the second one at � = 67 mN/m.his may imply that phase separation occurs in the system, that ishe component collapsing at a lower collapse pressure (ether lipid)an be expelled from the mixed film when compressed to the higher

Fig. 2. The excess free energy of mixing (�GExc) calculated for the mixe

B: Biointerfaces 111 (2013) 43– 51 45

values of surface pressure, forming 3D phase. With the increase ofether lipid content in the monolayer, two collapses are no longervisible and the isotherms become similar in shape to those for purePAF or ED film. The results obtained for lyso-PAF/SM mixtures areslightly different from those for the remaining systems. Namely, theisotherm for monolayer of Xlyso-PAF = 0.1 differs significantly fromthe curve obtained for SM film, which is noticeable especially in theregion of � < 40 mN/m. These differences concern mainly the areaat which surface pressure starts to increase, namely in the case ofthe mixed film it is larger of ca. 10 A2/molecule. Moreover, it canbe noticed that the degree of curve inclination decreases reflect-ing more expanded character of the monolayer. The mentionedchanges are due to drastically different nature of lyso-PAF film, inwhich molecules are poorly organized at the interface, predomi-nantly as a result of the bulky head-group and on the other hand,single hydrophobic fragment. For a larger amount of lyso-PAF inthe binary film, the registered isotherms overlapped in their ini-tial course. Moreover, contrary to the monolayer of PAF(or ED)/SM0.3/0.7, in the curve of Xlyso-PAF = 0.3 there is only one collapse,which indicates that in this binary monolayer phase separation isnot privileged.

As it can be observed in Fig. 2 for all the mixed films stud-ied herein the values of the excess free energy of mixing (�GExc)are positive in the whole range of the surface pressures. Gener-ally, the positive values of this thermodynamic parameter indicatethat the miscibility of the respective components is thermodynami-cally unfavorable and the interactions between molecules are morerepulsive or at least less attractive in comparison to those existingin the respective one component films. Moreover, the positive val-ues of �GExc are often characteristic for the monolayers in whichthe phase separation takes place.

In order to visualize the structure of the investigated monolayersBAM images were recorded upon compression of the investigatedfilms.

As regards SM film at � = 0 mN/m, the structures characteristicfor the coexistence of a liquid expanded and gaseous phase canbe seen at the interface (Fig. 3). At � = 5 mN/m an irregular brightstructures of a condensed phase contrasting with a dark regionof an expanded (less dense) phase are visible. Such a texture ischaracteristic for the first order phase transition occurring in themonolayer of SM [28,29] as well as in the films of other structurallysimilar phospholipids bearing in their molecular structure cholinehead group [30,31]. Upon increasing of �, the condensed domainsgrow, whereas the distances between them diminish. Finally, at

� > 10 mN/m, the separated condensed domains merge and conse-quently monolayer becomes homogenous.

On the other hand, BAM images for the ether lipids films arehomogenous practically in the whole range of surface pressures

d monolayers of SM and respective single-chained phospholipid.

46 M. Flasinski et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 43– 51

Fig. 3. BAM images registered for the monolayer of SM as well as mixed monolayers composed of SM and a single-chained phospholipid at different stages of compression.In the case of ED/SM 0.1/0.9 monolayer (images not shown) the surface morphology was homogenous during entire compression.

rfaces B: Biointerfaces 111 (2013) 43– 51 47

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Fig. 4. Diffracted intensity plotted as a function of Qxy (Bragg peaks) registeredfor monolayer of sphingomyelin at � = 30 mN/m. The experimental points were fit-

M. Flasinski et al. / Colloids and Su

data not shown). For the monolayers of PAF and lyso-PAF, smallright structures of a dense phase are visible only at surface pres-ure >32 mN/m [21].

In Fig. 3 representatives BAM photos for the investigated etheripid/SM mixtures are presented. For the systems containing higherroportion of ether lipids (≥50%), surface textures are homogenousractically in the whole range of surface pressures.

In the case of PAF/SM 0.1/0.9 monolayer at � = 0.5 mN/m twohases of a different density coexist, namely bright regions of

high condensation and dark areas characteristic for expandedhase. At the region of phase transition (5 mN/m), small domainsf a snowflake shape appear. These structures are similar to thoseound for SM film and prove that liquid expanded – condensedhase transition occurs also in the mixed monolayer. The thor-ugh analysis of BAM images may lead to the conclusion that theseharacteristic condensed domains have slightly different (morelongated) shape as compared to pure SM film. This differenceay originate from the alteration of the electrostatic interactions

n the head-group region, since the organization of the polar frag-ents of PAF and SM molecules are very different. With further

ompression, monolayer texture becomes more uniform and theeparate domains are almost indistinguishable. At � = 30 mN/mlm attains homogeneity and the image remains unchanged untilollapse (data not shown). Very similar images were obtained foryso-PAF/SM 0.1/0.9 mixture. On the other hand, ED/SM 0.1/0.9 film

as found to be homogenous during entire compression (imagesot shown).

At 30% of ether lipid in the mixture, PAF/SM monolayer isomogenous at � ≈ 40 mN/m. At ca. 5 mN/m; characteristic con-ensed structures are formed, however, they are significantlymaller as compared to those in PAF/SM 0.1/0.9 film. On the otherand, the domains observed at � ∼ 10 mN/m reach comparable sizend shape.

The main conclusion resulting from the analysis of the BAMhotos affirms that the addition of the single-chained ether phos-holipid to the monolayer of SM reduces film condensation andonsequently leads to the reorganization of the double-chainedolecules at the interface. The latter statement will be discussed

omprehensively based on the analysis of the data obtained fromynchrotron radiation scattering experiments.

An interesting texture was found e.g. for PAF/SM 0.3/0.7 mono-ayer compressed to � = 30 mN/m, where blurred structures of aondensed phase coexist with a brighter small spots of anotherense phase. This is an important finding since monolayer at

≈ 30 mN/m displays properties that can be well correlated to therganization and structure of biomembranes [32]. The obtainedata indicate that in monolayer compressed to the relatively highurface pressure (30 mN/m) a phase separation occurs. This phe-omenon will be discussed together with the results obtained withhe application of GIXD technique. At this point of discussion itan be concluded that this centrally localized brighter spots seenn the characteristically shaped domains originate from the phaseartition in the condensed domains during introduction of theingle-chained compound into SM monolayer.

In the case of lyso-PAF/SM 0.3/0.7 film BAM pictures evidence strong decrease of SM monolayer condensation, namely smallright domains appear up to � = 10 mN/m, while with furtherompression the monolayer becomes homogenous until the col-apse. In the case of ED/SM 0.3/0.7 the sequence of the monolayertructures during increasing of the surface pressure is similar tohat found for the film of PAF/SM 0.3/0.7. Prior to the monolayerompression, foam-like structures of the expanded phase coex-

sting with the gaseous phase appear. Then initially small circular� = 5 mN/m) and subsequently larger star-shaped domains of aondensed phase (� = 10 mN/m) are formed. At � = 30 mN/m brightoalescent domains coexisting with small bright spots are visible

ted with the Lorentzian function (solid lines). The plot contains both the total andcomponent Bragg peaks.

at the interface. Such structures indicate that in the investigatedmonolayer two types of condensed domains are present.

Based on the results obtained with the application of BAMmicroscopy, one can find that all three investigated ether phos-pholipids are able to modify the surface texture characteristic formonolayer of sphingomyelin, however, their influence dependsstrongly on their content in binary mixtures. Generally, it canbe seen that for monolayers composed of ether phospholipid/SM0.3/0.7, the most pronounced effect was found when lyso-PAF wasadded. It was found that the condensed domains were significantlysmaller as compared to those detected for one component SM filmand they were visible in the narrow range of surface pressures. Thiseffect can be attributed to the difference in the polar head regionsobserved in the investigated ether lipids. First of all, in contrastto PAF and ED, only lyso-PAF possesses in its molecular structurefree hydroxyl group prone to hydrogen bond formation, both withthe neighboring head-groups as well as with water molecules. Sec-ondly, our previous studies proved that at the comparable surfacepressure lyso-PAF head-group reveals a lower degree of hydrationin comparison to PAF.

Comparing the results for PAF/SM and ED/SM (0.3/0.7) at theregion of a higher surface pressure (30 mN/m) it was found thatthe introduction of these ether phospholipids into monolayer ofSM causes the formation of the additional condensed phase atthe surface. This finding is of great importance from the point ofview of biological relevance of PAF and ED and keeping in minda well known correlation between the properties of monolayerscompressed to high surface pressure and bilayers [32].

The application of BAM enabled us to observe micrometerdomains in the investigated mixed monolayers nevertheless theinsight into Angström-scale molecular organization with this tech-nique is not possible. Therefore, in order to obtain high resolutioninformation regarding molecular organization of the investi-gated mixed films we applied grazing incidence X-ray diffraction.Although GIXD experiments were carried out for all of the investi-gated mixed films in a broad range of concentrations, the diffractionpattern was obtained only for binary monolayers of Xether lipid = 0.1and 0.3. For the larger amount of single-chained lipid the surfacefilms lost in-plane periodicity and thus did not scatter X-ray radia-tion.

In Fig. 4 the diffracted intensity was presented as a function ofthe in-plane scattering vector component Qxy. The detailed analysis

of the 2D diffraction pattern registered for the monolayer of SMpoints out that there are three diffraction signals. Among them, thepeak of the highest Qxy is localized very close to the horizon (Fig.

48 M. Flasinski et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 43– 51

Table 1Structural parameters calculated for the investigated monolayers on the basis of GIXD data.

Composition ofmonolayer

Bragg peak Qxy [A−1] Bragg rod Qz [A−1] Latticeparameters [A,A; deg] (±0.03;±0.1)

Ac [A2] (±0.2) Lxy [A] (±2) Tilt, � [deg] (±0.2) Lz [A] (±0.8)

SM 〈−1,1〉1.491 ± 0.001〈1,0〉1.469 ± 0.001〈0,1〉1.440 ± 0.002

〈−1,1〉0.12 ± 0.01〈1,0〉0.28 ± 0.03〈0,1〉0.43 ± 0.03

a = 4.86b = 4.96� = 118.3

21.2 192 17.0 21.5

PAF/SM 0.1/0.9 〈−1,1〉1.488 ± 0.001〈1,0〉1.467 ± 0.001〈0,1〉1.442 ± 0.002

〈−1,1〉0.13 ± 0.02〈1,0〉0.26 ± 0.04〈0,1〉0.30 ± 0.05

a = 4.88b = 4.96� = 118.2

21.3 160 12.8 14.6

PAF/SM 0.3/0.7 〈−1,1〉,〈−1,1〉′1.493 ± 0.001〈1,0〉1.466 ± 0.002〈0,2〉′1.353 ± 0.002

〈−1,1〉,〈−1,1〉′0.11 ± 0.03〈1,0〉0.24 ± 0.03〈0,2〉′0.18 ± 0.06

a′ = 4.77b′ = 9.28� = 90

22.1 189 7.6 13.3

Lyso-PAF/SM 0.1/0.9 〈−1,1〉1.489 ± 0.001〈1,0〉1.464 ± 0.002〈0,1〉1.435 ± 0.001

〈−1,1〉0.08 ± 0.02〈1,0〉0.24 ± 0.03〈0,1〉0.30 ± 0.07

a = 4.86b = 4.96� = 114.9

25.0 198 12.7 11.0

Lyso-PAF/SM 0.3/0.7 〈−1,1〉,〈−1,1〉′1.482 ± 0.001〈1,0〉1.459 ± 0.002〈0,2〉′1.369 ± 0.002

〈−1,1〉,〈−1,1〉′0.16 ± 0.03〈1,0〉0.26 ± 0.04〈0,2〉′0.19 ± 0.08

a′ = 4.77b′ = 9.17� = 90

21.9 92 8.1 10.9

ED/SM 0.1/0.9 〈−1,1〉,〈−1,1〉′1.490 ± 0.001〈1,0〉1.458 ± 0.002〈0,1〉1.428 ± 0.002〈0,2〉′1.348 ± 0.003

〈−1,1〉,〈−1,1〉′0.08 ± 0.02〈1,0〉0.31 ± 0.02〈0,1〉0.44 ± 0.06〈0,2〉′0.20 ± 0.08

a = 5.08b = 4.98�=113.9a′ = 4.77b′ = 9.32� = 90

23.122.2 171 17.58.5 13.3

ED/SM 0.3/0.7 〈−1,1〉,〈−1,1〉′1.492 ± 0.001〈1,0〉1.462 ± 0.002〈0,2〉′1.361 ± 0.002

〈−1,1〉,〈−1,1〉′0.06 ± 0.02〈1,0〉0.29 ± 0.04〈0,2〉′0.23 ± 0.05

a′ = 4.78b′ = 9.23� = 90

22.0 169 9.5 12.6

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, b, � – lattice parameters; Ac – area per hydrocarbon chain; Lxy – coherence scattehe errors for the GIXD parameters correspond to the maximum values estimated f

-1). The other two signals emerge at considerably higher Qz: 0.28nd 0.43 A−1 respectively. Such a distribution of the intensity in theiffraction pattern allows one to conclude that SM molecules arerganized in the 2D oblique unit cell with their acyl chains tiltedrom the surface normal toward the intermediate direction [33].he main structural parameters calculated on the basis of the GIXDesults are gathered in Table 1.

In Fig. 5 GIXD results obtained for binary monolayer composedf PAF and SM are presented. At first glance the results for PAF/SM.1/0.9 monolayer are comparable to those for pure SM film. Simi-

arly, three distinct Bragg peaks were acquired. The distribution ofhe signals in the diffraction pattern meets the requirement con-erning the location of the intensity maxima characteristic for theblique unit cell, i.e. Q 〈0,1〉

z ≈ Q 〈−1,1〉z + Q 〈1,0〉

z (Table 1). The calcu-ated surface parameters are similar to those found in the case ofure SM film, however some important differences can be easilyoticed. Namely, both the range of crystallinity (Lxy) as well as the

ength of the part of molecule that coherently scattered X-ray radia-ion (Lz) are smaller in the case of the binary mixture in comparisonith one component sphingomyelin film. Furthermore, the value

f molecular tilt decreases despite the fact that the incorporatedompound (PAF) is known to be poorly organized at the air/waternterface. Interestingly, similar effect was also observed in the casef PAF/DPPC mixed films [21].

The obtained results indicate that the addition of a small amountf single-chained phospholipid (XPAF = 0.1) into SM monolayer doesot lead to the change of 2D lattice type. On the other hand, theifferences in the calculated values of the unit cell parameters pointut that PAF molecules participate in the formation of periodicallyrdered phase.

For PAF/SM 0.3/0.7 monolayer, the situation is far more complex

in the GIXD diffractogram, a relatively broad intense peak appearst Qxy = 1.353 A−1 with the maximum at Qz = 0.18 A−1. The numberf the acquired signals as well as the distribution of their intensi-ies enables us to distinguish two independent periodically ordered

ength in the xy plain; Lz – coherence scattering length in z direction; � – tilt angle.he fitting procedure and calculated with the exact differential method.

phases: one with the intermediate molecular tilt direction (obliquephase) and the second of a rectangular centered symmetry withNNN tilt azimuth. The former one was assigned on the basis of thefollowing assumptions: first of all the recorded signals are localizedbeyond the horizon, i.e. at Qz > 0 as well as the two nondegeneratepeaks denoted as 〈−1,1〉 and 〈1,0〉 appear in the diffractogram atvirtually identical Qxy as compared to the signals acquired for onecomponent SM film.

Secondly, the overall intensity of the diffraction signals mea-sured for mixed PAF/SM monolayers decreases drastically with theincreasing of PAF content in two component systems. Integrationof the summary Bragg peaks for SM, PAF/SM 0.1/0.9 and PAF/SM0.3/0.7 systems leads to the following values (normalized withrespect to SM signal): 1:0.46:0.38. For this reason the intensities ofthe distinct reflections for mixed monolayer of XPAF = 0.3 becomevery weak and hence the third signal characteristic for the obliqueunit cell (〈0,1〉) was not separated from the background.

Generally, the mentioned propensity reveals that the intro-duction of single-chained lipid into a well-ordered monolayer ofsphingomyelin causes diminishing of the crystalline 2D phase.Moreover, the diffraction signal disappears for the mixed film ofPAF/SM 0.5/0.5, which means that in the case of such a large pro-portion of PAF, monolayer loses its periodicity. On the other hand,for mixture of XPAF = 0.3 both GIXD results and BAM images revealedthe coexistence of the two distinct crystalline phases.

The third diffraction signal registered at Qxy = 1.353 A−1 origi-nates from the second crystalline phase which was not observed inthe case of monolayer of PAF/SM 0.1/0.9. Due to the fact that PAFmolecules in one component film are not organized periodically atthe interface, this phase consists of both PAF and SM molecules,however their exact proportion is not known. The mentioned sig-

nal can be connected with the intense peak observed at the highestQxy value, i.e. 1.493 A−1 (Qz > 0), meaning that the second crystallinephase can be described as a centered rectangular with NNN tilt. Thevalues of the unit cell parameters calculated on the basis of such

M. Flasinski et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 43– 51 49

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tion from the GIXD data and shed new light on the problem of aphase coexistence in monolayer, in Fig. 8 the intensity map for theinvestigated mixed system was presented.

ig. 5. Diffracted intensity plotted as a function of Qxy (Bragg peaks) registered for moints were fitted with the Lorentzian function (solid lines). The plot contains both

n assumption were presented in Table 1. The signals marked byrime (′) correspond to the second crystalline phase with a centeredectangular symmetry.

It can be seen that the length of a coherently scattering part of aolecule (Lz) in the case of the system composed of PAF/SM 0.3/0.7

s smaller as compared to the binary monolayer of XPAF = 0.1 as wells SM film. This result indicates that the domains scattering X-rayadiations are composed of both sphingomyelin and PAF molecules.he latter compound of which molecules were found to be deeplymmersed into water subphase [25] causes reduction of an average

onolayer thickness.The interesting finding concerns the molecular tilt observed

n the discussed mixed monolayer, that is it turned out that thencrease of PAF content in the monolayer results in a significantiminishing of �. This is an unexpected result, since one can supposehat the incorporation of a compound that is disordered, stronglyilted and deeply immersed in the subphase to the well ordered SMlm will cause disorganizing effect and increase of a molecular tilt.urprisingly, the experiments prove an opposite tendency. It is pos-ible that the incorporation of molecules being deeply immersednto the water subphase (PAF) to the relatively well organized

onolayer of SM causes that molecules of sphingomyelin changeheir organization at the interface in the way imposing diminutionf the tilt. This effect may be caused by strong influence of bulkyAF fragment localized in aqueous subphase on polar head-groupf SM molecule. The situation described above is quite similar tohe case of the influence of ganglioside GM1 on monolayer of DPPC34]. Similarly to PAF, molecule of GM1 possesses bulky head group,hich affects its properties at the air/water interface and is theain reason of a large molecular tilt demonstrated by molecules

n monolayer of GM1. Interestingly, the incorporation of the men-ioned compound into the DPPC film causes a kind of condensingffect connected with increasing of the molecular ordering. Thenteractions between head-groups of both monolayer componentsive rise to the decrease of molecular tilt in the condensed domains.

To sum up the GIXD results obtained for the mixed monolayerf PAF/SM, in Fig. 6 a representation of the diffracted intensity dis-ribution was presented in the form of a schematic intensity map(Qxy,Qz).

Comparing the distribution of a measured intensities presentedn Fig. 6 it can be seen that although the molecules of the monolayerf SM as well as PAF/SM 0.1/0.9 give rise to the similar reflections,onolayer composed of PAF/SM 0.3/0.7 provides completely

yers of PAF/SM 0.1/0.9 (a) and PAF/SM 0.3/0.7 (b) at � = 30 mN/m. The experimentaltal and component Bragg peaks.

different diffraction pattern. The concept of a two different phasespresented above finds its corroboration in results obtained duringBAM visualization of a monolayer composed of PAF/SM 0.3/0.7.In the images registered for this system at � = 30 mN/m, thecoexistence of a two condensed phases was proved.

GIXD results obtained for the binary monolayers containinglyso-PAF and SM (Supplementary Materials, Fig. S-2) resemblethose registered for PAF/SM films.

The Bragg peaks plotted in Fig. 7 concern the investigated sur-face films of ED/SM 0.1/0.9 and 0.3/0.7.

The results obtained for the mixed monolayers of ED/SM arevery different as compared to the data registered for the previ-ously discussed systems. In the case of the ED/SM 0.1/0.9 binaryfilm four relatively intense diffraction signals can be distinguishedin the 2D diffractogram. Three Bragg peaks occurred at the highestQxy resemble those recorded for the one component monolayer ofsphingomyelin. The distribution of the signals’ intensity in this caseis characteristic for the organization of film-forming molecules intooblique unit cell. On the other hand, the Bragg peak localized atQxy = 1.348 A−1 indicates that at the interface second periodicallyordered phase is present. In order to attain additional informa-

Fig. 6. Schematic representation of the intensity distribution in the 2D diffractionpattern: I(Qxy ,Qz) map for the monolayers of SM (dark cyan); PAF/SM 0.1/0.9 (orange)and PAF/SM 0.3/0.7 (dark red). (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of the article.)

50 M. Flasinski et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 43– 51

F ompo0 funct

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Er

ig. 7. Diffracted intensity plotted as a function of the in-plane scattering vector c.3/0.7 (b) at � = 30 mN/m. The experimental points were fitted with the Lorentzian

It can be seen that the diffracted intensity obtained for theinary monolayer of ED/SM 0.1/0.9 is distributed along the Scher-er arc spanning from the horizon to Qz ∼ 0.55 A−1. Starting fromhe Bragg peak localized at 1.490 A−1 a smooth decay of the mea-ured intensity can be noticed. This finding provides a new insightnto the orientation of molecules’ hydrocarbon chains, namely itan be inferred that there is no uniform direction of the molecu-ar tilt. On the other hand, the relatively weak signal at the lowestxy can be associated with the most intense peak, which enabless to describe the parameters of the second ordered phase. Due tohe nonzero position of the most intense Bragg rod the rectangu-ar centered unit cell with the NNN tilt direction can be assignedo the second condensed phase. Interestingly, a very similar phaseequence was presented by Krasteva et al. for mixed monolayerf two representatives of monostearoyl-rac-glycerols investigatedith GIXD [35]. It was found that both single-chained compoundshen mixed in certain proportion tend to phase separate forming

t interface two condensed phases of a different lattice symmetry:blique and rectangular.

In Fig. 7b GIXD results obtained for monolayer composed ofD/SM 0.3/0.7 are presented. Comprehensive analysis of this dataeveals that there are three distinct low order reflections just like

Fig. 8. GIXD intensity maps, I(Qxy ,Qz) for monolayer of ED/SM 0.1/0.9.

nent Qxy (Bragg peaks) registered for monolayers of ED/SM 0.1/0.9 (a) and ED/SMion (solid lines). The plot contains both the total and component Bragg peaks.

in the case of previously presented results for PAF/SM and lyso-PAF/SM systems of similar sphingomyelin content (XSM = 0.7). It canbe seen that edelfosine of molar fraction of 0.3 affects the charac-teristics of SM monolayer in similar way like other single-chainedphospholipids presented herein.

Taking altogether the results obtained with the application ofGIXD technique, it can be concluded that the addition of a single-chained phospholipid to the monolayer of sphingomyelin causessignificant alteration of the film characteristics. Generally, theobtained results prove phase separation of the components in theinvestigated binary films, which has been postulated above on thebasis of the thermodynamic considerations complemented withBAM visualization. In the case of PAF/SM and lyso-PAF/SM mono-layers, the phase separation was evident especially for the systemscontaining 30% of the respective ether lipid. These monolayers werecharacterized by two distinct condensed phases of a different latticesymmetry. Additionally, a large fraction of molecules in the surfacefilm does not participate in the formation of ordered domains whichwas confirmed in the GIXD experiments.

On the other hand, it was proved that the addition of edelfo-sine to monolayer of SM causes strong modification of molecularordering at interface, even if the mentioned phospholipid is addedin a small amount (XED = 0.1). In this case, a significant alterationof the surface film properties occurs, which is manifested in dis-ordering of the molecular tilt azimuth. In contrast to edelfosine,the addition of both PAF and lyso-PAF into SM monolayer does notlead to such changes in the film characteristics. These phospho-lipids added in a small amount form mixed, highly ordered domainswith sphingomyelin, affecting mainly the average thickness of themodel membrane. The obtained results demonstrate the privilegedinteractions between edelfosine and sphingomyelin, which is ofa great significance taking into account that one of a possible EDmechanism of action is connected with the incorporation of thisdrug into membrane domains enriched in saturated phospholipids(especially sphingomyelin), i.e. lipid rafts [36,37].

4. Conclusions

The obtained results have demonstrated that the interactions

between molecules in PAF/SM, lyso-PAF/SM and ED/SM mixturesare generally thermodynamically unfavorable, which in conse-quence leads to the phase separation in the systems. It was alsofound that incorporation of the single-chained phospholipids in

rfaces

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M film causes a decrease of the monolayer condensation, whichan be easily observed as a diminishing of the condensed struc-ures at certain surface pressure. Undoubtedly decisive in ouromparative studies was the application of GIXD technique. Theollected parameters have evidenced similarities in the organi-ation of PAF/SM and lyso-PAF/SM 0.1/0.9 films, namely in bothases the investigated highly ordered domains are the structuresormed from mixed monolayer and coexist at the interface withlm fraction of disordered phase. On the other hand, at 10% of ED

n SM monolayer, in the GIXD pattern a set of reflections charac-eristic for two distinct crystalline phases appears. Moreover, ED

odifies strongly the azimuth of molecular tilt in the mixed mono-ayer as a consequence of the alteration of molecules’ orientationn water surface. In the case of the binary films composed of single-hained phospholipid and SM, where XSM = 0.7, at the interface twoondensed ordered phases coexist, which confirms the hypothesisegarding phase separation.

Finally, it should be stressed that monolayers consisting morehan 30% of single-chained ether phospholipids become homoge-ous and completely lose its periodicity. Our results proved that the

nvestigated single-chained ether phospholipids cause significantodification of SM monolayer properties in a concentration-

ependent manner. It is worth to underline that edelfosine, being well known anticancer drug, was found to be the most effec-ive on SM monolayer in the smallest studied concentration. Theesults obtained in this work provide a new evidence on the prop-rties and activities of three single-chained ether phospholipids.lthough these compounds posses very similar chemical struc-

ures, their behavior in membrane mimicking environment wasound to be different, which is of utmost importance in the con-ext of their pharmaceutical application. These findings collectedor binary ether phospholipid/membrane lipid monolayers allows to suggest that differences in the interactions of PAF, lyso-PAFnd ED with membrane lipids may diversify their biological activ-ty. This issue unquestionably requires further analysis based on

ore complex artificial membrane systems.

cknowledgements

This project was financed by the National Science Center (No.EC-2011/01/B/ST4/00910). We gratefully acknowledge HASYLAB,ESY (Hamburg) for granting us beamtime at the BW1 beamlinend would like to express our gratitude to Dr Bernd Struth for hiselp at BW1.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.colsurfb.2013.05.024.

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B: Biointerfaces 111 (2013) 43– 51 51

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