Lyso-phosphatidylcholines in Langmuir monolayers – Influence of chain length on physicochemical characteristics of single-chained lipids

Journal of Colloid and Interface Science 418 (2014) 20–30

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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis

Lyso-phosphatidylcholines in Langmuir monolayers – Influence of chain length on physicochemical characteristics of single-chained lipids Michał Flasin´ski a,⇑, Paweł Wydro b, Marcin Broniatowski a a b

Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 3, 30-387 Kraków, Poland Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

a r t i c l e

i n f o

Article history: Received 6 October 2013 Accepted 1 December 2013 Available online 10 December 2013 Keywords: Lyso-lipids Lyso-phosphatidylcholines Phase transition X-ray reflectivity Grazing incidence X-ray diffraction Brewster angle microscopy

a b s t r a c t Single-chained phospholipids constitute a class of membrane components found in normal cells in relatively low concentration; however, these group of compounds are known owing to their broad physiological activities. Despite that the knowledge concerning fundamental physicochemical properties of lyso-lipids is very limited and in contrast to double-chained phospholipids there is an obvious deficiency of studies focused on correlation between their amphipathic character and film-forming properties with biological activities. In the present paper we have attempted to explain the main issues regarding the characteristics of lyso-PCs in monolayers at the air/aqueous interface. Our results show that all the investigated phospholipids differing in the length of hydrophobic chain: C18lyso-PC, C22lyso-PC and C24lyso-PC form stable Langmuir monolayers of a relatively low degree of condensation. It was found that during compression the investigated monolayers significantly change their organization at the interface which is strongly connected with temperature of the subphase. The application of X-ray reflectivity confirmed that the bulky choline head-groups in molecules of lyso-PCs are strongly penetrated by water molecules, while the hydrophobic chains are significantly tilted from the surface normal. The obtained results show that the phase transitions observed in the course of the registered isotherms result from decrease in immersion of molecules in the subphase as well as from the decrease in hydrating water molecules. On the basis of GIXD experiments it turned out that in the monolayers of C22lyso-PC and C24lyso-PC at higher surface pressures (>20 mN/m) small fractions of periodically ordered phase appear at the interface. For the monolayer of C24lyso-PC in the periodically ordered fraction the untilted (U) to tilted (t) phase transition was found. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Physicochemical characteristics of biological membrane phospholipids for many years has drawn special attention of scientists and continuingly is a subject of extensive studies performed both on cell cultures in vitro as well as in a variety of artificial (model) systems. The driving force for such investigations is a need to understand the complicated processes that govern cells, in which the lipid membrane with its multiple functions and, in large extent still unrecognized properties, fulfill a significant role. Among suitable methods which are widely applied to study properties of membrane lipids, the Langmuir monolayer technique offers many incontestable advantages. This methodology enables the generations and study of monomolecular films of an amphiphile at the air/water interface, therefore can be adapted as a simple but well-designed model of a single bilayer leaflet [1,2]. In this technique both the experimental conditions as well as the composition ⇑ Corresponding author. E-mail address: fl[email protected] (M. Flasin´ski). 0021-9797/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcis.2013.12.003

and architecture of membrane-mimicking films can be easily adjusted. Among extensively studied membrane lipids special place belongs to phospholipids which comprise the main class of natural components in bilayers. These amphipathic compounds differ in their chemical structures both in terms of polar head-groups as well as hydrophobic chains [3]. Generally, the most typical representative of membrane phospholipid, also from the statistical point of view, is phosphatidylcholine bearing in molecular structure two acyl chains of 16 or 18 carbon atoms, among which one is fully saturated, while the other possess one double bond [4]. Of course, biological membranes vary significantly as regards lipids compositions which is caused, e.g. by different functions of particular organelles, cells and tissues [5,6]. It is also well known that phospholipids content in membrane may change in some inflammatory processes and diseases, like, e.g. diabetes [7] or cancer [8,9]. Despite their important structural function, double-chained phospholipids occurring in membranes in high percentage of total lipid composition serve also as a specific reservoir for compounds which are the products of enzymes action on these phospholipids. The important examples here are the processes catalyzed by

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phospholipase A2 (PLA2) – the enzyme which hydrolyzes the sn-2 ester linkage in molecule of diacyl phospholipids releasing two products: lyso-phospholipid and free fatty acid [10]. Lyso-phospholipids (lyso-PLs) are compounds found in cellular membrane in relatively low concentration, that is averagely of 0.5–6% of total lipid mass [11]; however, their content may increase in the case of some diseases like atherosclerosis, hyperlipidemia, arrhythmia or inflammatory processes [12]. The elevated concentrations of lyso-lipids in membranes cause significant alteration of their properties and structure. It was found that abnormally high concentration of lyso-compounds in lipid bilayers may lead to dysfunction of some membrane proteins, e.g. ion channels [13,14] and in extreme situation cause cell lysis [15]. On the other hand, there are also results of studies suggesting that concentration of lyso-PLs in cancer patients decrease [16]. In contrast to cell membranes, lyso-phosphatidylcholine is one of a major lipid component found in oxidized low density lipoproteins, oxLDL (40% of total lipid content) and in VLDL (very low density lipoprotein) [17]. Moreover, lyso-PC modulates the properties and functions of these lipoproteins, which is a reason why this molecule activity is frequently connected with cardiovascular diseases, especially atherosclerosis [10,18]. Lyso-phospholipids are implicated in a broad range of other important cellular processes including signal transduction, gene transcription, mitogenesis and vascular smooth muscle relaxation [19–21]. Moreover, lyso-PCs were found to be responsible for modulation of the intracellular calcium concentration [10,22] and are also recognized to be a chemotactic factor for monocytes [23]. Despite their broad functions, the mechanisms of activities displaying by lyso-phospholipids at the level of biological membrane still remain uncertain. It is also worth mentioning that lyso-lipids fulfill other very important role in membrane architecture, namely they are known to be inhibitors of biological processes connected with membrane fusion, like for example endo- and exocytosis, fertilization or some viral infections [11,24]. The main reason of specificity of lyso-PLs in this context is the characteristic chemical structure and in consequence the spatial arrangement of their molecules in natural bilayers. It is known that singlechained phospholipids possessing in their molecular structure bulky head-group and only one hydrophobic chain are often classified to the group of ‘inverted cone-shaped lipids’ or ‘micelle-forming lipids’ [25,26]. The direct reason of this fact is that the cross sectional area of the head-group is significantly larger than that of the single hydrocarbon tail. Such specific shape causes that molecules of lyso-PLs tend to spontaneously assemble into bent structures (e.g. micelles) rather than into flat layers. Lyso-PLs, especially these possessing large polar head-group (lyso-PCs rather than lyso-PEs and lyso-PA) have high positive spontaneous curvature in contrast to diacyl phospholipids of a negative curvature [27]. This means that incorporation of lysoPL molecules into membrane changes important mechanical properties of lipid bilayer, like tension or stress [28]. Of course, the effect of lyso-PLs on membrane curvature and in consequence fusion processes depends on their distribution between inner and outer leaflet [27]. For example, in membranes of human erythrocytes lyso-PC are present almost exclusively in the outer layer [29]. In contrast to their double-chained precursors, studies concerning physicochemical characteristic of lyso-phospholipids are rather rare. For example, in scientific literature there is evident lack of data as regards the fundamental properties of monomolecular films formed by lyso-lipids. To fulfill this gap we undertaken studies focused on the behavior of three representatives of lyso-phosphatidylcholines in monolayers at the air/water interface. For our

21

Scheme 1. Molecular structures of the investigated lyso-phospholipids: C18lyso-PC, C22lyso-PC and C24lyso-PC.

investigation we selected the following single-chained compounds: C18lyso-PC, C22lyso-PC and C24lyso-PC (Scheme 1). As it can be seen the molecules of the above presented lipids differ in the length of hydrophobic acyl chain, whereas the polar head-group is the same. It should be mentioned here that since C18lyso-PC is one of the dominating species of lyso-phosphatidylcholines found in cellular membranes, the other two derivatives having longer hydrophobic chains should rather be treated as traces in natural samples. On the other hand, because of high physiological activity of lyso-PLs, their exogenous administration could be considered in pharmacological treatment. The examples of single-chained compounds having long hydrophobic part (C22) can be found among molecules possessing promising pharmacological potential against cancer [30]. It is evident that increase in the hydrocarbon chain length affects significantly the main properties of these compounds, starting from their solubility, trough ability to modify membrane fluidity and permeability, and ending at molecular recognition by membrane receptors. In our studies we have applied the Langmuir monolayer technique to characterize interfacial properties of lyso-PCs differing in the length of hydrocarbon chains: C18lyso-PC, C22lyso-PC and C24lyso-PC. The undertaken experiments based on elementary monolayers characterization in terms of surface pressure (p) – molecular area (A) isotherms as well as stability tests. We have also applied Brewster angle microscopy (BAM) in order to directly visualize the morphology of the investigated films. The mentioned studies have been complemented with techniques based on synchrotron X-ray radiation scattering, that is X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). 2. Experimental 2.1. Materials The investigated single-chained phosphatidylcholines, namely C18lyso-PC (1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine), C22lyso-PC (1-behenoyl-2-hydroxy-sn-glycero-3-phosphocholine) and C24lyso-PC (1-lignoceroyl-2-hydroxy-sn-glycero-3-phosphocholine) of the highest purity available in stock (>99%) were purchased from Avanti Polar Lipids and used without further purification. Spreading solution of the lipids of the concentration close to 0.25 mg/ml were prepared in chloroform/methanol 9/1 (v/v) mixture. Chloroform of spectroscopic purity (99.9% stabilized by ethanol) as well as methanol (99.9% were provided by Sigma–Aldrich. In all experiments on Langmuir trough ultrapure water of the resistivity P18.2 MX cm from MilliQ system was applied as a subphase. 3. Methods 3.1. Langmuir experiments In routine experiments, p–A isotherms and stability measurements were recorded with the NIMA (Coventry, UK) Langmuir

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trough of total area of 300 cm2 equipped in single movable barrier placed on anti-vibration table. The surface pressure was measured with the accuracy of 0.1 mN/m using Wilhelmy balance equipped with a surface pressure sensor made of filter paper (ashless Whatman). The required amounts of lipid solutions were spread on pure water surface with Hamilton microsyringe precise to 2 lL. In each experiment, the monolayer was left to equilibrate for at least 5 min before the monolayer compression was initiated with the barrier speed of 20 cm2/min (12 Å2/moleculemin). The circulating water system was used to control subphase temperature. 3.2. BAM visualization The investigated monolayers were visualized with ultraBAM apparatus (Accurion GmbH, Göttingen, Germany). The light source was 50 mW laser emitting p-polarized light of a 658 nm wavelength direct to the air/water interface at the Brewster angle (53.2°). The lateral resolution of the BAM image was 2 lm. The microscope was installed over a KSV (Helsinki, Finland) double barriers Langmuir trough, model 2000 of total area of 700 cm2. The surface pressure was monitored during the experiment with the Wilhelmy plate made of filter paper (ashless Whatman). In all experiments at least 5 min were allowed for the spreading solvent to evaporate after which the symmetrical compression was initialized with the barriers speed of 20 cm2/min. The dimensions of the recorded images are 675  355 lm. 3.3. X-ray reflectivity experiments X-ray reflectivity (XR) measurements for the investigated single-chained phospholipids at different stages of monolayer compression were performed with the liquid surface diffractometer at the BW1 beamline in HASYLAB, DESY synchrotron center (Hamburg, Germany). The incidence X-ray wavelength of k = 1.303 Å was obtained by the reflection from a beryllium (2 0 0) monochromator crystal. In Langmuir monolayer experiment a single barrier trough of total area of 600 cm2 (R&K, Potsdam, Germany) placed in a gastight container mounted on the goniometer of the diffractometer was used. The surface pressure measurements were carried out with the Wilhelmy balance equipped with a filter paper stripe as a surface pressure sensor. In each experiment after spreading the lipid solution on water surface, container cover was sealed and the canister was flushed with helium in order to reduce the oxygen level. The purpose of this procedure is to minimize the beam damage during reflectivity scans and reduce the scattering background. After at least 40 min monolayer was compressed to the target surface pressure, which was then held constant during the entire experiment. Both the construction of BW1 beamline and the detailed theoretical background of X-ray reflectivity technique were described in an exhaustive manner in a several previous articles [31–35], thus we focus here only on the principal information. According to the definition, reflectivity is described as the intensity ratio of X-rays specularly scattered from an interface relative to the incident beam intensity. It is measured as a function of momentum transfer vector Qz in accordance with the equation:

Q z ¼ jkout  kin j ¼

4p sinðaÞ k

where k is the wavelength of incident X-ray beam and a is the angle equal to the both incident (ai) and reflected (af) angles. The experimental R(Qz) curve can be measured by a NaI scintillation detector moving on the af arc [31]. From the measurements of specular reflectivity the detail information concerning the laterally averaged electron density distribution (q) along the direction perpendicular to the monolayer plane

can be obtained. It is performed by modeling the deviation of measured X-ray reflectivity from the Fresnel’s law for the ideally sharp interfaces [34]. In analysis of our results we applied electron density model based on the construction of a stack of homogenous slabs (‘boxes’) with an assumption that each box possess constant electron density and thickness [31]. The boundaries between adjacent slabs were smeared out with Gaussian function of standard deviation r to account the roughness of interfaces caused by the thermally excited microscopic capillary waves and atomic interface roughness. Simulation of the reflectivity profile was carried out with Parratt32 software application by the Hahn-Meitner Institute, Berlin. This package is based on the Parratt’s recursive algorithm for stratified media using independent layers [36]. For our purpose we decided to apply the model with two independent boxes as an optimal assumption of monolayer formed by singlechained phospholipid. The first box contains the hydrophobic part of the molecule, that is the acyl chain, whereas the second one includes the polar head group and adjacent water molecules. In the routine procedure the parameters fitted were slabs thicknesses, electron densities as well as the interfacial roughness. The results of the best fitting models with the lowest v2 values were taken for the discussion in the next section. 3.4. Grazing incidence X-ray diffraction (GIXD) GIXD experiments were performed using the same apparatus, as described above in the case of XR. In GIXD experiment the quantitative information at the Å-scale can be obtained only if there is a periodicity within the monolayer. If this requirement is fulfilled, the incidence X-ray radiation is scattered and its intensity can be measured by position sensitive detector (PSD). Afterward, the intensity is customarily represented as a function of horizontal scattering vector (qxy) and the scattering vector component along the vertical z coordinate (qz) resulting in Bragg peak(s) and Bragg rod(s) respectively. The scattered intensity is measured by scanning over a range of horizontal scattering vectors Qxy, defined as:

Q xy 

  4p 2hxy sin k 2

ð1Þ

where 2hxy is the angle between incident and diffracted beam projected onto horizontal plane. Bragg peaks are resolved in the Qxy direction, by integrating the scattered intensity over the Qz. In contrast, the Bragg rod profiles are resolved in the Qz direction:

Qz ¼

2p sin af k

ð2Þ

where af is the exit angle of X-ray beam. Bragg rods are obtained by integrating the scattered intensity over Qxy corresponding to the Bragg peaks. On the other hand, lack of the signal in GIXD experiment is also valuable information, since it indicates that film-forming molecules are poorly organized in the monolayer plane and do not form two-dimensional ordered phase (2D crystals). The more detailed description of GIXD technique was featured in our previous articles [37–40] and in other papers [31,41]. 4. Results In the initial step of our investigation we performed the characteristics of monolayers formed by C18lyso-PC, C22lyso-PC and C24lyso-PC by means of surface pressure (p)–area (A) isotherms. The obtained results were presented in Fig. 1. As can be seen in Fig. 1 the compression curves recorded for monolayers of the investigated lyso-PLs differ significantly as

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Fig. 1. p–A isotherms registered for monolayers of the investigated lyso-phospholipids: C18 C22lyso-PC and C24lyso-PC together with the compression  lyso-PC,  modulus C 1 vs. surface pressure plots (inset). s

regards the location on the graph as well as the course and the shape. In the case of C18lyso-PC film, surface pressure starts to raise at relatively large mean molecular area of 125 Å2/molecule. In the registered curve of a mild inclination a slight change in the course can be noticed at p  33 mN/m, and then monolayer collapses at 41 mN/m. This characteristics reveals obviously in the calculated compression modulus dependency, defined as C 1 s ¼ Aðdp=dAÞ, vs. p, where at 33 mN/m a distinct minimum reflecting the mentioned kink in the isotherm can be found. The maximal values of the C 1 reach 61 mN/m which is in agreement with the data pubs lished previously for monolayers of single-chained phospholipids bearing 18 carbon atoms in their hydrophobic tail [37,42]. The abovementioned and additional parameters calculated on a basis of the presented in Fig. 1 plots and also obtained from static stability experiments (mean molecular area A–time dependency) were gathered in Table 1. Let us now proceed to the discussion of the p–A isotherm obtained for the monolayer of C22lyso-PC. As compared to C18lyso-PC film, the first striking difference concerns the course of the curve, in which two pseudo plateau regions can be noticed: first at surface pressure of ca. 12 mN/m and the second at p = 28 mN/m. Moreover, one can find that the compression curve for C22lyso-PC is shifted toward smaller molecular area in comparison with the isotherm recorded for C18lyso-PC. It is also worth mentioning that monolayer of C22 derivative posses more condensed character, especially above the surface pressure of the second kink in the curve. The analysis of the compression modulus leads to the conclusion that the degree of monolayer condensation increases notably during the compression with exceeding of the subsequent transition regions. The p–A isotherm registered for the third of the investigated compounds, that is C24lyso-PC reveals at 20 °C one phase transition at the surface pressure of 42 mN/m. Additionally, based on the calculated compression modulus curve it turns out that the monolayer of C24lyso-PC possess more condensed character as compared to the derivatives having shorter

hydrophobic chain, especially in the region below the kink. In this case the maximal value of C 1 exceeds markedly 100 mN/m, provs ing that monolayer is in the condensed (LC) state. It is worth mentioning that in view of the current X-ray diffraction data the mentioned monolayer state should be more precisely defined as a tilted condensed, since it is known that molecules in monolayer aligned parallel to each other are tilted from surface normal [43]. As far as the area at which surface pressure starts to increase is concerned it can be seen that the value of A0 is similar to that obtained for C22lyso-PC. Interestingly, one can find that the isotherms for monolayers of C22lyso-PC and C24lyso-PC cross each other at the surface pressure of 20 mN/m and moreover at the region of higher p (43–57 mN/m) they overlap. It should be also pointed out that monolayers of the investigated single-chained phospholipids reveal good stability when compressed to the surface pressure of 30 mN/m. In order to obtain additional information regarding morphology of monolayers at lm scale, the in situ visualization of surface structures during compression was carried out with application of Brewster angle microscopy (BAM). In Fig. 2. the selected images recorded for monolayers of C22lyso-PC and C24lyso-PC were gathered together with the compression curves registered at 20 °C. In the case of C18lyso-PC, monolayer was found to be homogenous in the whole range of surface pressures and temperatures between 10 and 35 °C (data not shown). In the case of monolayer of C22lyso-PC surface domains can be observed only for the pressures corresponding to the region of the isotherm between the kinks, while before the first plateau and after the second, monolayer is homogenous. The recorded structures presented in Fig. 2a possess irregular shape and relatively sharp edges. The observed moderate brightness distinguished them from the very bright domains characteristic for the monolayers in solid state as well as the structures found in the surface film of double-chained phosphatidylcholines compressed to the surface pressure corresponding to the LE ? LC phase transition [44]. This finding proves that monolayers of the investigated lyso-PLs have relatively low degree of condensation. Moreover, thorough analysis of the registered BAM photos suggests that the observed structures reveal differences in the grayscale caused by the optical anisotropy connected with the differences in the molecular tilt direction. On the other hand, BAM images registered for monolayer of C24lyso-PC reveal that bright domains are presented at the interface in considerably wider range of surface pressures as compared to C22lyso-PC. Small bright dots can be even found in the images registered for the monolayer at p  1 mN/m (data not shown). At surface pressure of ca. 2 mN/ m one can observe the coexistence of a circular and worm-like structures (Fig. 2b). In the case of higher p (20 mN/m) small, uniformly shaped and moderately bright domains cover nearly whole area of the image. The brightness of these textures resemble those observed for the monolayer of C22lyso-PC; however, they are markedly smaller. Above the transition region visible in the course of the isotherm at 42 mN/m image becomes uniformly gray; however, the closer to the collapse the more small light spots appear at the interface. This may indicate that at such small molecular area some fraction of molecules in the surface film form 3D (collapsed) phase.

Table 1 Parameters derived from p–A isotherms, compression modulus curves: C 1 s —p and stability tests: A vs. time.

C18lyso-PC C22lyso-PC C24lyso-PC

23

A0 (Å2/molec.)

Acoll (Å2/molec.)

pcoll (mN/m)

(mN/m) max C 1 s

Decrease in molec. area in stability test (1 h) at p = 30 mN/m (%)

125 87 81

33 24 23

41 57 59

61 126 153

16 19 27

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60

60

50

50

40

40

π [mN/m]

π [mN/m]

24

30

30

20

20

10

10

0

a 20

30

40

50

60

70

80

90

2

area [A /molecule]

0

b 30

40

50

60

70

80

90

2

area [A /molecule]

Fig. 2. p–A isotherms for the investigated monolayers of C22lyso-PC (a) and C24lyso-PC (b) registered at 20 °C together with the BAM images recorded at different stages of compression. White bar in the BAM photos corresponds to 100 lm.

In the next step of our study we investigated the influence of subphase temperature on the properties of monolayers formed by lyso-PCs. The aim of this experiment was to shed new light onto the phase behavior of the investigated compounds. The obtained results were collected in Fig. 3. In Fig. 3a p–A isotherms recorded for monolayer of C18lyso-PC at six different temperatures were presented. Analyzing the presented plots it can be seen that despite practically the same molecular areas in the case of these isotherms, their shape differ notably. The main discrepancy concerns the regions of a higher surface pressures, where changes in the course of the curves can be observed. One can find that the characteristic subtle kink in the isotherm disappears at higher temperature (>20 °C). Moreover, the surface pressure corresponding to the collapse of monolayer moves toward lower values with increasing of temperature. However, the mentioned influence of temperature on characteristics of monolayer formed by C18lyso-PC is evident, a far more interesting behavior was found for surface film of C22lyso-PC. In the case of the liquid expanded to condensed state transition, it can be seen that the surface pressure corresponding to the observed pseudo plateau increases with temperature. Although the mentioned values of transition pressures change significantly, i.e. from ca. 2 mN/m at 10 °C to nearly 29 mN/m at 35 °C, these alterations are not perfectly linear. Very similar irregularities were also reported by other authors investigated the influence of temperature on phase transition in monolayers of membrane phospholipids [45]. Interestingly, for the second transition, relation between surface pressure and temperature was found to be opposite as compared to 1st transition. It can be inferred that in the case of higher temperatures surface pressure corresponding to pseudo plateau region moves toward lower p. Furthermore, this characteristic kink in the curve can be observed only up to 20 °C. From the Fig. 3b it is clearly visible that the region of the isotherm between both transition points shrinks with increasing of temperature and that at 25 °C kinks in the curve melt into one at intermediate surface pressure. The trend regarding the influence of temperature on transition surface pressure described above for the monolayer of C22lyso-PC are very similar in the case of C24lyso-PC film, however it can be seen that the first phase transition in monolayer of the latter compound appears at higher temperature, that is P20 °C (Fig. 3c). This corresponds with the general finding that the longer is the hydrophobic part of surfactant molecule the higher is the temperature of

the phase transition [43]. On the other hand, the second phase transition in the isotherm of C24lyso-PC film can be seen in a broader range of temperatures, that is 10–30 °C. It is also worth to mention that from calculated C 1 s —p dependency it was inferred that the increase in the temperature results in mild diminishing of monolayer condensation. This finding was also proved on the basis of BAM images recorded at different temperature (data not shown). In order to visualize the changes in monolayer phases in the case of the investigated lyso-PCs in Fig. 4 phase diagrams (p–T dependencies) investigated lyso-PCs were shown. The experimental points shown in Fig. 4 were least square fitted in order to estimate the characteristic triple point (T0) temperatures for the investigated compounds. These values are equal 17.3 °C; 7.9 °C and 19.3 °C for monolayer of C18lyso-PC, C22lysoPC and C24lyso-PC respectively and indicate the temperature of a triple point characteristic for the coexistence of G/LE/LC phases. In contrast to the double chain phosphatidylcholines or sphingomyelins, [46,47] differing in the length of acyl chains, it can be seen that these temperatures differ markedly between the investigated even-chained lyso-PCs. T0 changes by ca. 6 °C per each CH2 group, whereas for fatty acids it was found that this difference is close to 10 °C. Moreover, one can find that in the case of lyso-PCs studied herein, the relationship between T0 and length of the hydrocarbon chain is perfectly linear (Fig. S1). In order to get a deeper insight into the properties of lyso-PLs molecules at the air/water interface, we performed X-ray reflectivity experiments (XR) for monolayers of the investigated phospholipids at different surface pressures. Because of a good stability of the studied films we were able to obtain good quality scans at different stages of the compression. The registered reflectivity curves were presented in Supplementary materials (Figs. S2–S4), whereas the main parameters calculated on the basis of this experiment were gathered in Table 2. All XR scans were performed for monolayers compressed on water subphase at 20 °C and to demonstrate the phase behavior of the investigated monolayers at this temperature we selected the values of surface pressure corresponding to all regions in the p–A isotherm differing as regards the degree of inclination. The parameters presented in Table 2 were acquired from fitting procedure with application of Parratt algorithm. The best quality results (the lowest values of v2 test) were obtained for the twobox model in which box 1 refers to the region of hydrophobic tail (smaller electron density), whereas box 2 (larger q) contains polar

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Fig. 4. Surface pressure p–temperature (T) phase diagrams for the investigated lyso-phospholipids: (a) C18lyso-PC (T0 = 17.3 °C); (b) C22lyso-PC (T0 = 7.9 °C, T⁄ = 25.7 °C); and (c) C24lyso-PC (T0 = 19.3 °C, T 1 ¼ 19:9  C, T 2 ¼ 34:6  C).

Fig. 3. p–A isotherms registered for monolayers of C18lyso-PC (a), C22lyso-PC (b) and C24lyso-PC (c) compressed at different temperature.

head-group and in many cases additionally hydrating water molecules. Both boxes were characterized by their thickness, electron density (q) and the number of electrons (calculated initially and obtained from the fitting procedure). In all cases electron density was calculated on the basis of molecular areas estimated from the p–A isotherms at given surface pressure. Additionally, in Table 2 the values of interfacial roughness were presented. Let us begin the analysis of the reflectivity results from the parameters acquired for monolayer of C18lyso-PC. The XR scans were taken for surface pressure of 20 and 36 mN/m, hence the organization of lipid molecules in surface films was monitored both below and above the kink point visible in the course of the isotherm. At the lower surface pressure it was found that the hydrophobic acyl chain is strongly tilted from the surface normal

and moreover, deeply immersed into aqueous subphase – six methylene groups are penetrated by water molecules. From that reasons the thickness of the first box was found to be only 7.00 Å. On the other hand, the dimension of the second slab is significantly larger and apart from lipid head group it contains nearly 5 hydrating water molecules (46 excess electrons). The situation changes significantly when the surface pressure rises (36 mN/m), namely it was found that both the thickness of the second box as well as the number of electrons in it increases which means that in this case the number of methylene groups immersed in water subphase reduces to one. Nonetheless, it should be stressed that the hydrophobic part of C18lyso-PC molecule is still strongly tilted toward water surface, since it is well known that for hydrocarbon chain in its all-trans conformation the length can be calculated
from the following formula: L ¼ n þ 98  1:265, where n is the number of CH2 groups and 9/8 is the correction for terminal methyl [48]. Thus, in the foregoing case for 16 carbon atoms in phospholipid chain calculated L = 20.40 Å, while the thickness of box 1 equals only 11.29 Å. Theoretically these values enable us to calculate the tilt angle; however, as we will shortly see analyzing GIXD results it cannot be done correctly. At this point it should be however reminded that XR technique provides laterally averaged information about monolayer, while to obtain a diffraction pattern periodical order of film forming molecules is required. For this

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Table 2 The parameters obtained from the fitting of X-ray reflectivity curves.

p (mN/m)

Tail (box 1) Thickness (Å)

Head (box 2) 2

q (Å )

r (Å)



Number of e

Thickness (Å)

q (Å2)

Box

Calc.

2.9 4.0

87 131

89 129

11.83 13.37

0.376 0.344

0.230 0.289 0.314

3.1 3.0 4.0

127 134 160

129 137 161

11.16 12.92 12.52

0.305 0.310

3.3 4.0

184 201

185 199

9.87 11.11

C18lyso-PC 20 36

7.00 11.29

0.233 0.310

C22lyso-PC 5 20 36

8.01 9.18 16.68

C24lyso-PC 20 43

14.92 22.64

r (Å)

Number of e Box

Calc.

3.6 2.6

237 (5H2O) 173 (2H2O)

191 159

0.317 0.408 0.414

3.0 3.3 4.1

244 (5H2O) 215 (3H2O) 159

191 183 159

0.408 0.428

3.0 3.9

163 (1H2O) 136

151 137

q – electron density. r – interface roughness.

reason in the case of monolayer containing both highly ordered condensed domains as well as a fluid-like disordered phase calculation of the average molecular tilt is pointless. Apart from the molecular tilt the another reason of the observed shortening of box 1 as compared to the theoretical chain length is presence of high concentration of gauche defects. This effect characteristic for monolayers containing molecules in fluid state reduces the number of methylene groups aligned along a straight line [49], therefore calculation of tilt angle leads in this case to serious errors [50]. It is also worth mentioning that during the monolayer compression one can observe that phospholipid polar head loses hydrating water, i.e. at p = 36 mN/m in second box only 2H2O molecules are accommodated. Moreover, the concentration of the gauche defects decreases with increasing of the surface pressure [51]. The more complicated but at the same time more interesting situation was found in the case of C22lyso-PC. Because for the monolayer compressed at 20 °C two phase transitions were found we performed XR experiments at three different surface pressures: 5 mN/m (before first kink); 20 mN/m (steep region between plateaus) and 36 mN/m (above second transition). The scan carried out at the lowest surface pressure revealed that the first box 8.01 Å thick contains hydrocarbon chain of 16 °C atoms, while in the box 2 the number of electrons equals 244 meaning that apart from six CH2 groups, phospholipid head-group 5 molecules of water are included (53 extra electrons). With the increase in the surface pressure to 20 mN/m, the length of the first box enlarges and the number of methylene groups becomes 17. On the same time, the head-group region loses 2 hydrating water molecules. This trend is visible with further compression and at p = 36 mN/m the thickness of box 1 equals 16.68 Å which corresponds to hydrocarbon chain of 20 carbon atoms. At this high surface pressure all hydrating water was found to be expelled from the second box. The transformation sequence described above for molecule of C22lyso-PC was schematically illustrated in Fig. 5. In the next step of our studies on interfacial properties of three single-chained phosphatidylcholines we applied Grazing Incidence X-ray Diffraction (GIXD) in order to obtain information regarding molecular organization in the monolayer plane. As can be seen in Fig. 6. a single Bragg signal was registered for monolayer of C22lyso-PC both at the surface pressure of 20 and 36 mN/m. Interestingly, the mentioned signal appears in the horizon (Qz = 0 Å1) with its maximum localized at Qxy = 1.456 and 1.454 Å1 respectively. Such a diffraction pattern with the single peak is characteristic for the monolayer in which molecules are oriented perpendicular to the air/water interface. At the first glance such a result seems unreasonable, since it was discussed above

Fig. 5. Schematic representation of C22lyso-PC molecule orientation at the air/ water interface at different stage of monolayer compression, from left: 5 mN/m; 20 mN/m and 36 mN/m.

that molecules are disordered and tilted rather than organized in the highly ordered hexagonal domains with their chains oriented upward. Hence, how to bring those results together? It should be underlined that all of the registered in this study diffraction signals are rather weak which means that only some small fraction of film forming molecules are involved in formation of the highly ordered hexagonal domains, while the remaining form disordered phase. Comparing the relative intensity of the Bragg peaks obtained for monolayer of C22lyso-PC at p = 20 and 36 mN/m it can be seen that in the latter case the signal is considerably more intense. This means that at the higher surface pressure the contribution of highly organized 2D crystalline phase in monolayer increases. The main structural parameters calculated on the basis of the recorded signals were presented in Table 3. Analyzing the structural parameters calculated for monolayer of C22lyso-PC it can be seen that their values are very similar

´ ski et al. / Journal of Colloid and Interface Science 418 (2014) 20–30 M. Flasin

27

Fig. 6. Bragg peaks registered for the monolayer of C22lyso-PC at surface pressure of 30 mN/m (a) and 36 mN/m (b). Solid lines were obtained by fitting of the experimental points with Lorentz function.

Table 3 Structural parameters calculated for investigated monolayers from GIXD data.

p (mN/m)

Bragg peak Qxy (Å1) ±0.003

Bragg rod Qz (Å1) ±0.02

Lattice parameters (Å, Å; °) ±0.03; ±0.01

Area (Å2) ±0.2

Lxy (Å) ±2

Tilt, s (°) ±0.2

C22lyso-PC

20 36

1.456 1.454

0 0

a = b = 4.98; c = 120 a = b = 4.99; c = 120

21.5 21.6

164 144

0 0

C24lyso-PC

20 43

1.432 1.503 1.436 1.377

0 0.16 0.30 0.50

a = b = 5.07; c = 120 a = 4.84; b = 5.05; c = 115.4

22.2 22.1

117 28

0 19.1

Monolayer

regardless of surface pressure at which monolayer was held during the experiment. It concerns also the obtained values of the unit cell area. It should be underlined here that both values, that is 21.5 and 21.6 Å2 calculated for monolayer at 20 and 36 mN/m respectively are typical for the phospholipids possessing in their molecules long saturated hydrocarbon chains [52,53]. In the discussed systems the correlation length; Lxy calculated from the Scherrer formula: 2p , where fwhmQ xy is the full width at half-maximum Lxy ¼ 0:9 fwhm Q xy

of Bragg peak, was found to be 164 and 144 Å respectively. These values are considerably smaller as compared to the monolayer formed by double-chained phospholipids having choline head-group or other polar fragments found naturally in membrane phospholipids. Namely, it was found that Lxy values are equal to 271 ± 6 Å for monolayer formed by molecules of DPPC [38], whereas 233 Å and 246 Å for monolayers of DPPE and DPPG respectively [40]. It can be therefore concluded that the highly ordered domains in monolayers of lyso-PCs are relatively small. The another important parameter that should be considered in the analysis of GIXD results is the coherence length in the direction perpendicular to the air/water interface: Lz. It allows to assume the thickness of the fraction in the surface film that coherently scatters X-ray radiation. For the investigated monolayer of C22lyso-PC Lz equals 9.7 and 9.9 Å at p = 20 and 36 mN/m

Lz (Å) ±0.8 9.7 9.9 12.3 14.0

respectively. Such values indicate that molecules forming highly ordered phase are strongly immersed into aqueous subphase, as it was postulated above on the basis of XR data. In Fig. 7. GIXD results registered for the monolayer of C24lyso-PC. For surface pressure of 20 mN/m in the diffraction pattern single narrow Bragg peak was registered, whereas at p = 43 mN/m the acquired signal is considerably broader and more intense. In the former case the signal is localized in the horizon which means that in the crystalline domains molecules are organized in the hexagonal unit cell. On the other hand, the detailed analysis of the diffractogram obtained for the monolayer compressed to 43 mN/m reveals that there are three distinct Bragg peaks localized beyond the horizon, that is at Qz > 0 Å1 (Fig. S5). Such a distribution of maxima denotes that molecules in this surface film are organized in the oblique unit cell. 5. Discussion The results presented above concern the physicochemical characteristics of three single-chained phosphatidylcholines in monolayers at the air/water interface. The aim of this study was to obtain the fundamental information about film-forming properties of the investigated lyso-lipids. Although this knowledge is of

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Fig. 7. Bragg peaks registered for the monolayer of C24lyso-PC at surface pressure of 20 mN/m (a) and 43 mN/m (b). Solid lines were obtained by fitting of the experimental points with Lorentz function.

utmost importance taking into account that these lipids exert their bioactivity at the level of biomembranes, in literature there is an evident lack of information concerning physicochemical properties of these phospholipids in surface films. Traditional characteristic based on isotherms measurement complemented with BAM visualization revealed that lyso-PCs form stable Langmuir monolayers which are characterized by relatively low degree of condensation. On the other hand, there are evident differences seen in the course of the compression curves registered for these three representatives. Namely, at 20 °C in the case of isotherm obtained for monolayer of C18 and C24 one, whereas for C22 film two kink points reflecting phase transitions can be observed. For that reason, the phase diagram for monolayer of C18lyso-PC is very simple – only one transition from LE to LC state can be observed, while in the case of derivatives possessing longer hydrophobic chains the situation is more complex (Fig. 4). For C22lyso-PC monolayer two condensed phases can be assigned: LC1 and LC2 which are observed in the diagram until the triple point denoted as a T⁄ = 25.7 °C. Further increase in temperature causes that only LE ? LC2 transition can be seen. As far as the transition between two condensed states is concerned it can be seen that the surface pressure decreases with temperature. Similar dependence was observed in the case of LC1/LC2 transition in fatty acids [54,55]. For the monolayer of C24lyso-PC the characteristic temperature point reflecting the coexistence of LE and two distinct condensed phases appear at T 2 ¼ 34:6  C. Interestingly, at temperature equals 19.9 °C (denoted as T 1 ) very close to the T0 point, in the phase diagram there is an evident change of the course of the line separating both condensed phases. Basing solely on the results obtained from the p–A isotherms registered at different temperatures it is not possible to find the origin of the second transition, therefore the interpretation of this phenomenon will be undertaken together with the discussion of the GIXD results. The another interesting conclusion comes from the analysis of the results obtained with application of XR technique. From the reflectivity experiments it could be inferred that the higher is the surface pressure, the longer is the box accommodating acyl chain and at the same time the number of methylene groups in it

increases (see Fig. 5). On the other hand, it can be seen that the thickness of the second slab changes only slightly, even though the mean molecular area decreases drastically during compression of the monolayer. As it was demonstrated, this effect is compensated by the diminishing of hydration shell and also by the conformational changes of the head-group. It is worth to mention here that at the surface pressure of 36 mN/m molecular area estimated from the p–A isotherm equals only 30.7 Å2 which means that molecules at the interface are packed closely. For comparison at the same surface pressure a molecule of palmitic acid occupies in monolayer the mean area of ca. 20 Å2/molecule, while doublechained phosphocholine: DPPC 44 Å2/molecule. Analyzing these data it can be concluded that in the case of lyso-PCs the main factor decisive of both orientation of molecules at the air/water interface as well as monolayer organization is a choline head-group. This relatively large polar part of molecule causes that monolayer of this lipid reveals expanded character up till rather high surface pressure. It is also likely that both the conformational changes in the head region as well as the alteration of hydrating water content are the main reasons of the observed transition between distinct monolayer states. On the other hand, the changes seen during compression in the hydrophobic part of C22lyso-PC molecule are at first glance rather minor, especially between 5 and 20 mN/m. Finally at the highest surface pressure investigated in the XR experiments (36 mN/m) hydrocarbon chain of C22lyso-PC emerges significantly from the water subphase and only one methylene group remains in the second box. In the case of monolayer formed by molecules of C24lyso-PC the situation is slightly different. First of all, at both of the investigated surface pressures (20 and 43 mN/m) nearly the entire hydrophobic part of molecule placed above the subphase. Namely, it was found that 23 and 24 carbon atoms of the chain accommodate in the first box at p = 20 and 43 mN/m respectively. Although the immersion of the chain virtually does not differ between those two situations, the thickness of box 1 increases significantly. It could be therefore concluded that in this case the phase transition is more likely to be connected with the alteration of molecular tilt and reduction of the gauche defects [51]. Secondly, the number of the hydrating water

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molecules is smaller as compared to the investigated lyso-PCs having shorter acyl chains. This findings stay in accordance with data obtained from initial experiments – at the surface pressure of 20 mN/m monolayer of C24lyso-PC possess considerably higher degree of condensation which was inferred from the calculated compression modulus. In the next step of our study we applied GIXD technique in order to achieve information about molecular organization in monolayer plane. Our intention was to find the answer for the question: Are molecules in the investigated monolayers ordered periodically? At the beginning it should be mentioned that in the case of monolayers revealing low condensation, that is being in the liquid expanded (LE), or more generally fluid state, diffraction signals are usually unable to obtain.  Indeed, for the monolayer of C18lyso-PC max C 1  60 mN=m compressed at 20 °C the 2D difs fraction pattern was not acquired regardless of the surface pressure applied in the experiment. The situation was different in the case of lyso-PCs possessing longer hydrophobic chains. GIXD results for C22lyso-PC revealed that regardless the fact that the experiments were carried out for the monolayer in LC1 and LC2 phase, the lattice parameters for 2D crystalline phases were nearly the same. From that reason in this case the origin of the second phase transition is not connected with the changes in the fraction of periodical phase. Alternatively, the reason can be found in the alterations observed with XR technique in the global monolayer organization. This refers to the changes in immersion of molecules into water subphase, their average tilt angle as well as the modifications in the head group region. In the case of C24lyso-PC the distribution of the peaks maxima confirmed that molecules are involved in formation of the oblique 2D crystal structure. The degeneracy of the signal in such a situation is lifted by the molecular tilt – for the mentioned case s = 19.1°. These three first order peaks registered for the abovementioned system prove that the azimuth of molecular tilt is intermediate between NN and NNN [33,34]. The observed effect is rather unexpected, since one may rather anticipated that the compression of the monolayer formed by molecules oriented upright does not lead to the deflection of chains from the surface normal. This finding can be explained based on the following arguments: first of all, the key is the large intensity of the registered signal as compared to the single peak of the hexagonal phase, i.e. the relative intensities were found to be 5.6:1. This means that in the case of C24lyso-PC monolayer at p = 43 mN/m, significantly larger fraction of phospholipid molecules are organized periodically participating in the overall intensity of the signal. From that reason the measured peak corresponds better to the global situation in the monolayer described previously based on XR results. Namely, it was proved that the dominating fraction of molecules is tilted from the surface normal, therefore when compressed to the high surface density form ordered periodical phase capable of X-ray radiation scattering. This is in contrast to the situation found at lower surface pressure, where only small fraction of molecules is organized periodically forming hexagonal phase. It is also worth mentioning that the alteration in symmetry of 2D domains and at the same time appearing of the molecular tilt takes place at the surface pressure above the phase transition. The similar situation was observed by the other researchers who described the untilting (U) to tilting phase (t) transition [56]. In our opinion the key parameter of this phenomenon in the investigated case is the strong discrepancy of the head to tail size ratio. For high surface pressure the interactions between bulky head-groups may impose the alteration of hydrocarbon chain orientation from untilted to tilted. Such argumentation can be supported by the results of theoretical calculation in which head/tail diameter was found to be the

29

crucial parameter responsible for U ? t and NNN ? NN transitions [57,58].

6. Conclusions The aim of this study was to perform a comprehensive physicochemical analysis of the film-forming properties of three single-chained lyso-phosphatidylcholines differing in the length of hydrophobic moiety: C18lyso-PC, C22lyso-PC and C24lyso-PC. We have undertaken this study since until now relatively little is known about the behavior of these compounds in monolayers formed at air/water interface [59]. On the other hand, the knowledge concerning this issue is of high importance bearing in mind that the representatives of this class of compounds fulfill variety of significant functions in living organisms [e.g. 10,15,19]. Moreover, a part of these activities can be realized due to the amphipathic properties of the discussed lyso-lipids. Our studies proved that the investigated single-chained phospholipids are capable of stable Langmuir monolayer formation and that the interfacial characteristics of these films is strongly modulated by temperature of the aqueous subphase. It was found that in the course of the registered p–A isotherms regions of the different degree of inclination exist meaning that during compression monolayers change their degree of condensation. This was additionally confirmed on the basis of BAM visualization. In the investigated range of temperature (10–35 °C) for the monolayer of C18lyso-PC – one, while for the monolayers of both C22lyso-PC and C24lyso-PC – two phase transitions can be observed. XR experiments confirmed that these transitions are connected with alterations of hydrophobic chain immersion into water subphase as well as with modification of the head-group conformation and hydration during compression. The latter besides alteration of the gauche defect concentration, was found to be the main factor determining the interfacial organization of lyso-PCs. Moreover, despite the fact that generally the investigated monolayers show low degree of condensation, in the case of two derivatives having the longest hydrophobic chains at surface pressures above 20 mN/m 2D crystalline domains were found. Apart from the monolayer of C24lyso-PC at 43 mN/m, in which periodically ordered molecules were found to be organized in oblique lattice, in the three other cases only one Bragg peak was acquired indicating unit cell of a hexagonal symmetry [43]. The results obtained from XR and GIXD measurements show that in monolayers of single-chained PCs small fractions of periodically ordered domains coexist with the disordered phase of a low condensation. Furthermore, the higher is the surface pressure, the larger is the fraction containing highly ordered domains and the better 2D crystalline phase reflects the real character of the entire surface film. The results obtained for the monolayer of C24lyso-PC indicate that similarly to the systems studied by other authors [56] in this case U ? t transition is possible, however to the best of our knowledge until now there was no information regarding such behavior in the case of membrane phospholipids. Our studies provide new facts concerning the interfacial properties of the selected single-chained phospholipids. The obtained results allow us to better understand the complexity of phospholipid molecular behavior in monomolecular layers at the air/water interface. We showed that the physicochemical properties of lyso-lipids differ significantly as compared to their double-chained analogs as well as other single-chained compounds possessing relatively small polar head-group. This knowledge is of great importance taking into account the important roles that these compounds fulfill in the functioning and architecture of biological membranes. Nevertheless, in order to obtain more detail characteristics of the behavior of these lyso-lipids in the presence of other membrane

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