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DPPC monolayers studied with Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy
Journal of Colloid and Interface Science 364 (2011) 133–139
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Properties of b-sitostanol/DPPC monolayers studied with Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy Katarzyna Ha˛c-Wydro a,⇑, Michał Flasin´ski a, Marcin Broniatowski a, Patrycja Dynarowicz-Ła˛tka a, Jarosław Majewski b a b
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland Lujan Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
a r t i c l e
i n f o
Article history: Received 23 June 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Langmuir monolayer Plant sterols Grazing Incidence X-ray Diffraction (GIXD) Brewster Angle Microscopy
a b s t r a c t Although the influence of structurally modified sterols on artificial membranes has been intensively investigated, studies on the properties of stanols, which are saturated analogs of sterols, are very rare. Therefore, we have performed Grazing Incidence X-ray Diffraction (GIXD) experiments aimed at studying in-plane organization of a plant stanol–b-sitostanol monolayer and its mixtures with 1,2-dipalmitoyl-snglycero-3-phosphocholine – DPPC at the air/water interface. The collected GIXD data, resulting in-plane parameters and BAM images provide information on molecular organization and in-plane ordering of the investigated films. It was found that the lateral organization of b-sitostanol/DPPC monolayers depends on their composition. The oblique structure of the in-plane lattice of tilted hydrophobic region of molecules, found for DPPC film, is maintained at 10 mol% of stanol in the system. However, at 30 and 90 mol% of stanol in the mixture, the arrangement of molecules is hexagonal and they are oriented perpendicularly to the interface. With the addition of stanol the extend of the in-plane order of the monolayers decreases. Moreover, in mixtures the ordered domains consist of both monolayer’s components. Additionally, b-sitostanol film is of similar in-plane organization as the corresponding sterol monolayer (b-sitosterol) and stanol induces condensing effect on DPPC. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Langmuir monolayers are widely used as models of biomembranes [see e.g. 1,2], which, in addition to the application in nanotechnology [see e.g. 3–6], represents the most important trends in the studies on these systems. The results of experiments on monomolecular films systematically provide valuable information on various issues related to the function and organization of membranes, properties of their components and mutual interactions or/and the effect of other molecules on membrane environment [7–10]. Although the scope of the monolayers studies on the properties of membranes is very broad, it seems that special attention is paid to the model systems containing cholesterol. It is not surprising since cholesterol is a crucial component of animal membranes that strongly determines molecular organization and mechanical properties of these structures. Cholesterol acts as condensing and ordering agent, which promotes the formation of liquid ordered phase with saturated lipids and in this way regulates membranes properties [11]. However, it should be pointed out that the comparative investigations on the influence of various sterols and sterols-derivatives on model membranes evidenced that also other ⇑ Corresponding author. Fax: +48 0 12 634 05 15. E-mail address: [email protected] (K. Ha˛c-Wydro). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.08.030
molecules reflect condensing, ordering and domains-promoting effect [12–16]. It was found that the foregoing properties of sterols depend on their molecular structure [12,17]. The basic structural conditions determining the ability of sterol to pack tightly with other lipids and influence the ordering of their hydrophobic chains as well as to form condensed domains are the presence of a tetracyclic ring, small polar group at position 3, a tail similar to that in cholesterol molecule and a small minimal area per molecule at the air/water interface [12,17] (Scheme 1). To the above mentioned group of membrane active sterols [17] belong also b-sitosterol and stigmasterol [14], which are phytocompounds naturally occurring in plant tissues, however, supplied with diet, are absorbed by human organism and, as it was evidenced, they can insert into human membranes [18 and references therein]. Diet is also the source of phytostanols, which differ from the molecule of the respective sterol only in the structure of ring B in the molecule i.e. stanol is a saturated sterol and its ring system is devoid of a double bond between carbons 5 and 6 [19] (Scheme 1). Stanols, similarly to sterols, may incorporate into human membranes and modify their organization [18 and references therein]. Since phytosterols/stanols are present in both plants and human organisms, the studies on the behavior of these molecules in model systems are required to verify the effect of these food-derived compounds on the properties of membranes. In addition to
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and P95% for stanol) purchased from Sigma. To prepare spreading solutions the lipids were dissolved in chloroform/methanol (4:1 v/ v) mixture (both chloroform and methanol were purchased from Aldrich, HPLC grade, P99.9%). From the respective stock solutions the mixed solutions were prepared and desirable volume of the latter was deposited onto the water subphase with the Hamilton micro syringe, precise to 1.0 lL. The measurements were performed at 20 °C ± 0.1 °C and the temperature was controlled thermostatically by a circulating water system. UltrapureMilli-Q water used as the subphase in the monolayer experiments at 20 °C ± 0.1 °C has surface tension of 72.6 mN/m and resistivity of 18 MX cm. 2.2. Methods
Scheme 1. The structure of b-sitosterol and b-sitostanol.
biological aspect of this kind of research, it is also of great interest to analyze the relationship between the structure of sterols and their properties. The investigations on the interactions of plant sterols with various membrane phospholipids as well as the analysis of their effect on multicomponent artificial membranes proved that phytosterols possess the condensing and ordering properties, however, they influence the packing properties of other lipids in a smaller extent as compared to cholesterol [14,20,21]. These differences are the consequence of the structure of aliphatic chain in animal versus plant sterols molecules. On the other hand, little is known on the effect of stanols on phospholipid membranes. In general, the studies on stanol molecules, especially compounds originating from plants, are very rare and the results presented in literature concern cholesterol derivative, namely cholestanol [13,22,23]. Therefore, Langmuir monolayers experiments to investigate the condensing and ordering effect of plant stanol (bsitostanol) and its miscibility and interactions with various phospholipids have been performed recently [24]. Moreover, based on these results it was concluded that the influence of b-sitostanol and corresponding sterol (i.e. b-sitosterol) on the respective phospholipids is similar [24]. Since the plant stanols incorporated into membranes can affect their properties, it is required to perform a comprehensive analysis of the properties of these biologically active molecules and to analyze the molecular arrangement of model systems composed of stanol and other membrane lipids. Therefore, we have studied the in-plane organization and morphology of b-sitostanol/DPPC monolayers by using Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy (BAM) techniques to obtain detailed characterization of the ordering, morphology and structure of the investigated films. The analysis of BAM images and GIXD data obtained for b-sitostanol and its mixture with phopholipid provide completely new information on molecular organization of stanol/DPPC monolayers. 2. Experimental 2.1. Materials The investigated compounds: phospholipid (1,2-dipalmitoylsn-glycero-3-phosphocholine – DPPC) and b-sitostanol (dihydrob-sitosterol) were synthetic products of high purity P99% for DPPC
Brewster Angle Microscopy experiments were performed with ultraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10 magnification objective, polarizer, analyzer and a CCD camera. The spatial resolution of the BAM was 2 lm. The experiments were carried out with KSV 2000 Langmuir trough (KSV Instruments Ltd., Helsinki, Finland) (total area = 870 cm2) equipped with two movable barriers and placed on antivibration table. The surface pressure was measured with the accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. After spreading, the monolayers were left for solvent evaporation for 20 min. and then the compression was initiated with the barrier speed of 5 cm2/min (2.5 Å2 molec1 min1). X-ray scattering experiments were performed at the BW1 (undulator) beamline at the HASYLAB synchrotron source (Hamburg, Germany) using a dedicated liquid surface diffractometer [25] with an incident X-ray wavelength k 1.3 Å. A Teflon thermostatted Langmuir trough (Riegler&Kirstein, Potsdam, Germany), equipped with a movable barrier for monolayer compression, was placed in a gastight container and mounted on the diffractometer. After spreading the solution onto the subphase, at least 40 min were allowed for the trough container to be flushed with helium to reduce the scattering background and to minimize beam damage during X-ray scans. Then, the monolayers were compressed to the surface pressure of 30 mN/m (the surface pressure at which the properties of monolayer can be compared with those of bilayers in the natural membrane [26]), at which the X-ray experiments were performed. As far as the GIXD experiments are concerned, the X-ray scattering theory and the liquid diffractometer used here have been described previously [27–29]. GIXD experiments were carried out to obtain lateral ordering information of the samples. The scattered intensity was measured by scanning over a range of horizontal scattering vectors Qxy; Q xy 4kp sinð2hxy =2Þ where 2hxy is the angle between the incident and diffracted beam projected on the liquid surface. The GIXD intensity resulting from a powder of 2D crystallites can be represented as Bragg peaks, resolved in the Qxy direction, by integrating the scattered intensity over the Qz direction, which is measured by the position-sensitive detector placed perpendicular to the air–water interface. Conversely, the Bragg rod profiles were resolved in the Qz direction (Q z ¼ 2kp sin af ; where af is the X-ray exit angle) and obtained by integrating the scattered intensity over Qxy corresponding to the Bragg peak. From the positions of the Bragg peaks the d-spacing, d = 2p/Qxy (where the Qxy is the position of the maximum of the Bragg peak along Qxy) and the unit cell parameters for the 2D lattice was obtained. From the line width of the peaks, using the Scherrer formula, it is possible to determine the 2D crystalline coherence length, Lxy, the average distance in the direction of the reciprocal lattice vector Qxy, over which the ordering extends. The analysis of the intensity distribution along Qz (Bragg rods) provides the information about the
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magnitude and the direction of the molecular tilt and the coherently scattering length of the molecule, Lz. The analysis procedure has been described in details elsewhere [29,30]. 3. Results and discussion The experiments were performed for pure DPPC and b-sitostanol monolayers and their mixtures of various molar compositions (Xb-sitostanol = 0.1; 0.3 and 0.9), (i.e. for films dominated by one of the lipids (10 and 90 mol% of stanol) and for the mixture, for which the strongest contraction of area per molecule was found in the previously published experimental results (30 mol% of stanol) [24]. For the clarity of presentation in Fig. 1 the surface pressure–area isotherms (A) and compressional modulus ðC 1 S Þ values vs. surface pressure plots (B) for the investigated films are shown. The BAM images recorded at different stages of the compression of the monolayers formed by the investigated lipids (DPPC and bsitostanol) and their mixtures of various compositions are presented in Fig. 2. As it is seen for pure DPPC monolayer, at the surface pressure p = 0 mN/m and large areas, in the images 2D foam reflecting the coexistence of gas and liquid expanded (LE) phase can be observed. With the compression of the monolayer, the gaseous phase vanishes and monolayer becomes homogenous. Further decrease of the area per molecule leads to the formation of liquid condensed (LC) domains. The region of LE/LC phase coexistence corresponds to the well known plateau in the surface pressure–area curve for DPPC film (see Fig. 1A). The foregoing LE/LC phase transition manifests also as a minimum in the compressional modulus versus surface pressure plot (see Fig. 1B). The size of LC domains increases
Fig. 1. The surface pressure–area isotherms (A) and compressional modulus ðC 1 S Þ values vs. surface pressure plots (B) for the investigated monolayers.
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with the compression and finally they fuse together, forming uniform LC phase at p > 12 mN/m. It is worth noting that the condensed domains exhibit regions of distinct reflectivities. Moreover, these domains show inversion of the contrast in the reflected light intensity with rotation of the BAM analyzer. This proves its optical anisotropy induced by different tilt-azimuthal orientations of the phospholipid molecules, which has been also found by other authors [31–33]. As it was found previously [24], the addition of 10 mol% of stanol into DPPC film practically does not change the shape of the isotherm, however, causes its shift to lower areas per molecule and slightly increases the surface pressure at the region corresponding to LE–LC phase coexistence (Fig. 1A). Also, BAM images recorded for this system are very similar to those obtained for pure DPPC film. However, the LC domains appearing in the region of plateau are smaller as compared to those detected for pure DPPC monolayer. Further addition of stanol (Xb-sitostanol = 0.3) into DPPC film strongly changes the isotherm as it is steeper and shifted to lower mean molecular areas (Fig. 1A). The BAM images recorded for this system indicate that the condensed phase appears at a lower surface pressure than for DPPC film and the LE–LC phase transition is not visible. For b-sitostanol monolayer the course of the recorded p-A isotherm (Fig. 1A) (i.e. the steep shape of the curve and small area per lipid molecule) and high values of the compressional modulus (Fig. 1B) indicate that this compound, similarly to b-sitosterol, forms tightly packed and ordered films. High condensation of stanol monolayers is reflected also in BAM images. As it can be observed in presented pictures, at large areas per molecule 2D foam corresponding to gas – condensed phases coexistence appears. At smaller areas, but still at the surface pressure p = 0 mN/m, a dark region of the gaseous phase coexists with the bright condensed phase. Further compression of the film induces the formation of one condensed phase and the images are homogenous. At very high surface pressures (p > 36 mN/m) small bright spots appear, which correspond to 3D nuclei of collapsed phase. For the mixture containing 10 mol% of DPPC (Xb-sitostanol = 0.9) the BAM images indicate on similar morphology of the monolayers as it was found for pure stanol film. GIXD data collected for DPPC monolayer at surface pressure p = 30 mN/m are presented in Fig. 3A and B. Fig. 3A shows the diffracted intensity as a function of the inplane scattering vector component Qxy (Bragg diffraction peaks) for different values of out-of-plane scattering vector component Qz, whereas Fig. 3B depicts the diffracted intensity (Bragg rods) along the direction normal to the interface, integrated over small Qxy intervals. For DPPC monolayer three different Bragg rods and therefore three Bragg peaks can be distinguished, which corresponds to an oblique structure of the in-plane lattice of tilted alkyl chains. This is in agreement with the results reported also by other authors [34,35]. The parameters obtained from the fitting the X-ray intensity distribution along Bragg peaks and Bragg rods are listed in Table 1. For pure b-sitostanol monolayer the Bragg diffraction peak and Bragg rod profiles are presented in Fig. 4. The values of structural parameters obtained from Bragg profiles are compiled in Table 1. In this case only one Bragg peak and Bragg rod with the maximum at Qz 0 can be observed. This indicates that, at the investigated surface pressure, the molecules pack in the 2D hexagonal lattice and they are oriented perpendicularly to the air/water interface. For both investigated lipids (DPPC and b-sitostanol) the values of the area per lipid molecule derived from GIXD experiments agree well with those obtained from the surface pressure–area measurements. Namely, at the surface pressure of 30 mN/m for stanol monolayer: 38.3 ± 0.1 Å2 from GIXD vs. 38.4 ± 0.2 Å2 from the isotherm, and for DPPC: 44.6 ± 0.1 Å2 (for DPPC the area per
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Fig. 2. BAM images taken for the investigated monolayers at different stages of compression.
molecule = 2 Auc due to the presence of two acyl chains in the molecule) from GIXD vs. 44.4 ± 0.2 Å2 from the isotherm. This indicates that in both cases practically the whole monolayer material forms the condensed domains.
In the case of b-sitostanol/DPPC mixture containing 10% of stanol, diffracted intensity as a function of the in-plane scattering vector component Qxy integrated over all Qz values, shows one broad Bragg peak. However, the analysis of the in-plane diffraction scans
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Fig. 3. Background-subtracted GIXD diffraction data (points) and fit (solid lines) for DPPC monolayers compressed to 30 mN/m. (A) Bragg peak profiles I(Qxy) integrated over the Qxy regions indicated on the graph. The Bragg peaks were fitted using Lorenzian function. (B) Corresponding Bragg rod profiles I(Qz) – for clarity of presentation the data have been offset vertically. The Bragg rods were integrated over the Qxy regions indicated on the graph. The distribution of the scattered intensity along the Bragg rods was fitted using Gaussian function.
for different Qz intervals (Fig. 5A) as well as Bragg rods (Fig. 5B), clearly indicates that, similarly to DPPC film, three low order diffraction peaks, indicating an oblique lattice with tilted hydrophobic parts of molecules, can be distinguished. However, the positions of these peaks lead to slightly smaller lattice constants and the tilt of the hydrophobic parts is reduced from s = 29° for DPPC monolayer to s = 23° for the mixed film (Table 1). Moreover, significant broadening of the Bragg peaks suggests that the extend of in-plane order is much smaller as compared to one-component phospholipid film, especially for the (1, 1) reflection. Further addition of stanol into DPPC monolayer (30% of b-sitostanol in the mixture) causes significant changes in X-ray scattering
Fig. 4. Background-subtracted GIXD diffraction data (points) and fit (solid lines) for b-sitostanol monolayers compressed to the surface pressure of 30 mN/m. (A) Bragg peak profile I(Qxy) integrated over the relevant Qz region; (B) corresponding Bragg rod profile I(Qz). The Bragg peaks and Bragg rods were fitted using Lorentzian and Gaussian function, respectively.
data and derived parameters as compared to DPPC (see Table 1). For this system only one Bragg peak (Fig. 5C) as well as one Bragg rod with maximum at Qz 0 (Fig. 5D) were obtained. This suggests hexagonal packing of molecules in the monolayer and their orientation perpendicular to the interface. Since the Bragg peak is located between those for DPPC and stanol films, it can be concluded that the scattering domains are not formed by individual lipids but they consist of an ordered array of both monolayer’s components. Moreover, considering Lxy value it can be stated that extend of in-plane order is smaller than in pure monolayers of the respective components and than in DPPC/stanol film of Xb-sitostanol = 0.1. For the system of high stanol content (Xb-sitostanol = 0.9) also only one diffraction peak and one Bragg rod with maximum at Qz 0 have been found (Fig. 5E and F respectively). The peak is significantly broader meaning that the ordered regions are smaller than
Table 1 In-plane structural parameters obtained from GIXD experiments for DPPC, b-sitostanol and their mixed monolayers at the surface pressure p = 30 mN/m: d – distance between scattering planes, a and b – the lengths of the unit cell vectors, c – the angle between the vectors a and b, s – tilt angle, Lxy – in-plane coherence length, Auc – area of the 2D unit cell, Lz – the length of the coherently scattering molecular moiety. The errors for the respective parameters represent maximum values estimated from the fitting procedure and calculated with the exact differential method.
DPPC
Xb-sitostanol = 0.1
Xb-sitostanol = 0.3 Xb-sitostanol = 0.9 b-sitostanol *
d* (Å) (±0.006)
a, b* (Å) (±0.006)
c (°)
s (°)
(±0.3)
(±1)
d{1,1} = 4.251 d{0,1} = 4.408 d{1,0} = 4.520 d{1,1} = 4.266 d{0,1} = 4.354 d{1,0} = 4.444 4.416 5.681 5.761
4.934 5.059
116.7
29
4.927 5.028
117.9
23
5.099 6.560 6.652
120 120 120
0 0 0
For hexagonal unit cell d{1,1} = d{0,1} = d{1,0}, a = b, and L{1,1} = L{0,1} = L{1,0}.
Lxy (Å) (±1)
Lz (Å) (±0.5)
Auc (Å2) (±0.1)
L{1,1} = 209 L{0,1} = 72 L{1,0} = 60 L{1,1} = 69 L{0,1} = 68 L{1,0} = 58 24 39 133
18.1
22.3
17.2
21.9
15.1 15.1 16.6
22. 5 37.3 38.3
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Fig. 5. Bragg peak I(Qxy) profile(s) (left panel) and corresponding Bragg rod profile(s) I(Qz). (right panel) for b-sitostanol/DPPC monolayers of various composition compressed to the surface pressure of 30 mN/m. (A, B) 10% of stanol (the Qxy and Qz integration regions are indicated on the graph); (C and D) 30% of stanol; (E and F) 90% of stanol, The solid lines are the curves fitted to background-subtracted GIXD diffraction data (points). For clarity of presentation the data have been offset vertically (A and B). For (C and D) and (E and F) the X-ray intensity integrations were performed over the relevant Qxy and Qz regions. The Bragg peaks and Bragg rods were fitted using Lorentzian and Gaussian function, respectively.
in one component stanol monolayer. Similarly to the mixture containing 30 mol% of stanol the domains are consisted of both monolayer components and the molecules are oriented perpendicularly to the interface. The lengths, Lz, of the coherently scattering molecular moieties, calculated from the Bragg rod intensity distributions, show a decreasing trend with the amount of stanol molecules in the system (see Table 1). For pure DPPC monolayer the Lz is equal to 18.1 Å, which is in agreement with the previously reported data [36]. Upon addition of stanol to DPPC monolayer the Lz deceases from 17.2 Å to 15.1 Å and 15.1 Å for the Xb-sitostanol = 0.1, 0.3 and 0.9, respectively. This is an indication that in all mixtures the ordered scattering domains are form by both DPPC and stanol molecules. Stanol molecules are shorter than the length of the all-trans DPPC acyl tails. Therefore, the addition of stanol molecules into the DPPC matrix decreases the amount of van der Waals contact between its tails and increases the number of gauche defects along the molecular backbones. This, in turn, decreases the average length of the coherently scattering molecular length in the ordered domains. It is worth to notice that the calculated Lz of pure stanol monolayer is 16.6 Å, which is bigger than the Lz obtained for
Xb-sitostanol = 0.3 and 0.9. This can indicate that not full length of the stanol molecule form the coherently scattering moieties in the mixtures with DPPC. It is also worthy comparing the mean molecular areas obtained for the mixed monolayers from the recorded isotherms [24] (Fig. 1A) with those calculated based on the area of unit cell (Auc) derived from GIXD experiments. Both oblique and hexagonal unit cells found for stanol/DPPC mixtures of Xb-sitostanol = 0.1, 0.3 and 0.9, respectively, contain one scattering moiety. However, in contrast to DPPC monolayer, in the case of mixtures the mean molecular area cannot be calculated by simple multiplying of the area of unit cell by two. This results from the fact that one DPPC molecule possesses two scattering parts (acyl chains) whereas stanol contains only one scattering moiety. Thus, in the case of b-sitostanol/DPPC mixtures the mean molecular area should be calculated as follows: Amix = Auc (2 XDPPC + Xstanol). The values of the mean molecular area calculated from the foregoing equation (41.6, 38.3, and 41.0 Å2/molecule for Xb-sitostanol = 0.1, 0.3 and 0.9, respectively) agree with those estimated from the isotherms (42.1, 38.5 and 39.0 Å2/molecule for Xb-sitostanol = 0.1, 0.3 and 0.9, respectively) at the same surface pressure p = 30 mN/m. This indicates that
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whole monolayer material is highly condensed and that the composition of scattering domains is the same as the composition of the monolayer. Since the values of the mean area per molecule are smaller as compared to those resulting from additivity rule (i.e. linear combination of the areas of the single components with the coefficients of their molar fractions) [24] thus the condensing effect of stanol on DPPC is reflected also in GIXD data. Similar results were found previously for sphingomylin/b-sitosterol mixtures as well as for sphingomylin/stigmasterol, for which the variations in the position of Bragg peaks positions were detected [37]. This clearly shows that b-sitostanol as well as b-sitosterol and stigmasterol do not form complexes of fixed stoichiometry with the investigated lipids. These findings are consistent with the results published for cholesterol/DPPC mixtures [38], for which it was concluded that the composition of the ordered domains changes with the proportion of lipids in the mixed film. The performed experiments allow us also to compare the properties of two phytocompounds namely b-sitostanol versus b-sitosterol, differing only in the structure of tetracyclic ring skeleton. b-sitostanol is a saturated analog of b-sitosterol, i.e. stanol molecule lacks the double bond between carbons 5 and 6 of the B ring (see Scheme 1). As it was evidenced in the characteristics of the isotherms [20,24], both these plant compounds form tightly packed and ordered monolayers. Moreover, the GIXD data collected in this work for b-sitostanol are similar to those obtained previously for b-sitosterol [37] at the same surface pressure values (p = 30 mN/m). Thus, it can be concluded that the difference in the structure of both compounds seems to be not so important for the in-plane organization within the monolayer. 4. Conclusions The presented results provide new and valuable information on the lateral organization of the investigated plant stanol/DPPC monolayers. The analysis of BAM images for stanol/DPPC mixtures shows that stanol influences morphology and molecular packing of DPPC monolayer: in the system containing 30 mol% of the plant stanol the condensed phase appears at lower surface pressure as compared to DPPC film and the LE–LC phase transition, clearly observed for pure DPPC film, vanishes. When the system is composed of 90 mol% of stanol, BAM images show similar texture as those taken for pure stanol film. However, at the surface pressure, at which the GIXD experiments were performed, no differences in BAM images for the investigated mixtures were found – for all the systems the monolayers are highly condensed and homogenous. On the other hand, GIXD data obtained at the surface pressure of 30 mN/m indicated that stanol/DPPC monolayers differ in their lateral organization for different compositions. The results obtained for the mixed monolayer containing 10 mol% of stanol evidence an oblique in-plane lattice of tilted hydrophobic region of molecules, similarly to pure DPPC film. However, the molecular tilt found for the mixture is lowered as compared to DPPC monolayer and the 2D scattering domains are smaller. With further addition of stanol into DPPC monolayer (i.e. 30 and 90 mol% of stanol in the mixture), the packing of molecules changes from oblique to hexagonal and the molecules are oriented perpendicularly to the interface. The ordered 2D domains are even smaller than those at 10 mol% content of stanol in the monolayer. For mixtures (Xb-sitostanol = 0.1, 0.3 and 0.9) the ordered domains are composed of both monolayers components. Summarizing the obtained results, it can be concluded that the composition of scattering structures formed in b-sitostanol/DPPC mixture depends on the composition of the monolayer. Moreover, in-plane organization within b-sitostanol film is similar to that
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found for b-sitosterol monolayer, despite of differences in the structure of both these compounds. Acknowledgments The Authors are grateful to DESY-HASYLAB, Hamburg (Germany) for granting synchrotron beam time for the realization of the project. The research was carried out with the equipment (ultraBAM) purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08). Lujan Neutron Scattering Center at LANSCE is funded by the DOE Office of Basic Energy Sciences and Los Alamos National Laboratory under DOE Contract DE-AC52-06NA25396.C. References [1] C. Peetla, A. Stine, H. Labhasetwar, Mol. Pharm. 6 (2009) 1264. [2] M.G. Sarpietro, M.Ch. Giuffrida, S. Ottimo, D. Micieli, F. Castelli, J. Nat. Prod. 74 (2011) 790. [3] C.-W. Wang, H.-G. Liu, X.-T. Bai, Q. Xue, X. Chen, Y. Lee, J. Hao, J. Jiang, Cryst. Growth Des. 8 (2008) 2660. [4] F.-W. Hsiao, Y.L. Lee, C.-H. Chang, Colloids Surf., B 73 (2009) 110. [5] J.J. Park, W.R. Lee, S.S. Bae, Y.J. Kim, K.H. Yoo, J. Cheon, S. Kim, J. Phys. Chem. B 109 (2005) 13119. [6] A. Heyman, I. Medalsy, O. Dgany, D. Porath, G. Markovich, O. Shoseyov, Langmuir 25 (2009) 5226. [7] S. Yokoyama, Y. Ohta, H. Sakai, M. Abe, Colloids Surf., B 34 (2004) 65. [8] S. Steinkopf, A. Simeunovic´, H.J. Bustad, T.H. Ngo, H. Sveaass, A.U. Gjerde, H. Holmsen, Biophys. Chem. 152 (2010) 65. [9] P. Wydro, S. Knapczyk, M. Łapczyn´ska, Langmuir 27 (2011) 5433. [10] S. Belem-Goncalves, G. Matar, P. Tsan, D. Lafont, P. Boullanger, V.M. Salim, T.L.M. Alves, J.-M. Lancelin, F. Besson, Colloids Surf., B 75 (2010) 466. [11] H. Ohvo-Rekila, B. Ramstedt, P. Leppimaki, J.P. Slotte, Prog. Lipid Res. 41 (2002) 66. [12] M.E. Beattie, S.L. Veatch, B.L. Stottrup, S.L. Keller, Biophys. J. 89 (2005) 1760. [13] X. Xu, E. London, Biochemistry 39 (2000) 843. [14] X. Xu, R. Bittman, G. Duportail, D. Heissler, C. Vilcheze, E. London, J. Biol. Chem. 276 (2001) 33540. [15] J. Aittoniemi, T. Róg, P. Niemelä, M. Pasenkiewicz-Gierula, M. Karttunen, I. Vattulainen, J. Phys. Chem. B 110 (2006) 25562. [16] T. Róg, L.M. Stimson, M. Pasenkiewicz-Gierula, I. Vattulainen, M.J. Karttunen, J. Phys. Chem. B 112 (2008) 1946. [17] Y. Barenholz, Prog. Lipid Res. 41 (2002) 1. [18] A. de Jong, J. Plat, R.P. Mensink, J. Nutr. Biochem. 14 (2003) 362. [19] S. Rozner, N. Garti, Colloids Surf. A 282–283 (2006) 435. [20] K. Ha˛c-Wydro, P. Dynarowicz-Ła˛tka, J. Phys. Chem. B 112 (2008) 11324. [21] K.K. Halling, J.P. Slotte, Biochim. Biophys. Acta 1664 (2004) 161. [22] K. Borrenpohl Lintker, P. Kpere-Daibo, S.J. Fliesler, A. Barnoski Serfis, Chem. Phys. Lipids 161 (2009) 22. [23] T.A. Daly, M. Wang, S.L. Regen, Langmuir 27 (2011) 2159. [24] K. Ha˛c-Wydro, A. Zaja˛c, P. Dynarowicz-Ła˛tka, J. Colloid Interface Sci. 360 (2011) 681. [25] J. Majewski, R. Popovitz-Biro, W.G. Bouwman, K. Kjaer, J. Als-Nielsen, M. Lahav, L. Leizerowitz, Chem. Eur. J. 1 (1995) 304. [26] D. Marsh, Biochim. Biophys. Acta 1286 (1996) 183. [27] T.R. Jensen, K. Kjaer, in: D. Mobius, R. Miller (Eds.), Novel Methods to Study Interfacial Monolayers, Elsevier, Amsterdam, The Netherlands, 2001, p. 205. [28] J. Als-Nielsen, K. Kjaer, in: T. Riste, D. Sherrington (Eds.), Proceedings of the NATO AdVanced Study Institute, Phase Transitions in Soft Condensed Matter, Plenum Press, New York, 1989, p. 113. [29] J. Als-Nielsen, D. Jacquemain, K. Kjaer, F. Leveiller, M. Lahav, L. Leiserowitz, Phys. Rep. 246 (1994) 252. [30] M. Tanaka, M.F. Schneider, G. Brezesinski, Chem. Phys. Chem. 4 (2003) 1316. [31] G. Weidemann, D. Vollhardt, Colloids Surf. 100 (1995) 187. [32] J. Miñones Jr., J.M. Rodriguez Patino, O. Conde, C. Carrera, R. Seoane, Colloids Surf., A 203 (2002) 273. [33] J. Miñones Jr., S. Pais, J. Miñones, O. Conde, P. Dynarowicz-Ła˛tka, Biophys. Chem. 140 (2009) 69. [34] G. Brezesinski, A. Dietrich, B. Struth, C. Bohm, W.G. Bouwman, K. Kjaer, H. Mohwald, Chem. Phys. Lipids 76 (1995) 145. [35] K. Wagner, G. Brezesinski, Chem. Phys. Lipids 145 (2007) 119. [36] M. Flasin´ski, M. Broniatowski, J. Majewski, P. Dynarowicz-Ła˛tka, J. Colloid Interface Sci. 348 (2010) 511. [37] K. Ha˛c-Wydro, M. Flasin´ski, M. Broniatowski, P. Dynarowicz-Ła˛tka, J. Majewski, J. Phys. Chem. B 114 (2010) 6866. [38] A. Ivankin, I. Kuzmenko, D. Gidalevitz, Phys. Rev. Lett. 104 (2010) 108101.
Contents lists available at SciVerse ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Properties of b-sitostanol/DPPC monolayers studied with Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy Katarzyna Ha˛c-Wydro a,⇑, Michał Flasin´ski a, Marcin Broniatowski a, Patrycja Dynarowicz-Ła˛tka a, Jarosław Majewski b a b
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland Lujan Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
a r t i c l e
i n f o
Article history: Received 23 June 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Langmuir monolayer Plant sterols Grazing Incidence X-ray Diffraction (GIXD) Brewster Angle Microscopy
a b s t r a c t Although the influence of structurally modified sterols on artificial membranes has been intensively investigated, studies on the properties of stanols, which are saturated analogs of sterols, are very rare. Therefore, we have performed Grazing Incidence X-ray Diffraction (GIXD) experiments aimed at studying in-plane organization of a plant stanol–b-sitostanol monolayer and its mixtures with 1,2-dipalmitoyl-snglycero-3-phosphocholine – DPPC at the air/water interface. The collected GIXD data, resulting in-plane parameters and BAM images provide information on molecular organization and in-plane ordering of the investigated films. It was found that the lateral organization of b-sitostanol/DPPC monolayers depends on their composition. The oblique structure of the in-plane lattice of tilted hydrophobic region of molecules, found for DPPC film, is maintained at 10 mol% of stanol in the system. However, at 30 and 90 mol% of stanol in the mixture, the arrangement of molecules is hexagonal and they are oriented perpendicularly to the interface. With the addition of stanol the extend of the in-plane order of the monolayers decreases. Moreover, in mixtures the ordered domains consist of both monolayer’s components. Additionally, b-sitostanol film is of similar in-plane organization as the corresponding sterol monolayer (b-sitosterol) and stanol induces condensing effect on DPPC. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Langmuir monolayers are widely used as models of biomembranes [see e.g. 1,2], which, in addition to the application in nanotechnology [see e.g. 3–6], represents the most important trends in the studies on these systems. The results of experiments on monomolecular films systematically provide valuable information on various issues related to the function and organization of membranes, properties of their components and mutual interactions or/and the effect of other molecules on membrane environment [7–10]. Although the scope of the monolayers studies on the properties of membranes is very broad, it seems that special attention is paid to the model systems containing cholesterol. It is not surprising since cholesterol is a crucial component of animal membranes that strongly determines molecular organization and mechanical properties of these structures. Cholesterol acts as condensing and ordering agent, which promotes the formation of liquid ordered phase with saturated lipids and in this way regulates membranes properties [11]. However, it should be pointed out that the comparative investigations on the influence of various sterols and sterols-derivatives on model membranes evidenced that also other ⇑ Corresponding author. Fax: +48 0 12 634 05 15. E-mail address: [email protected] (K. Ha˛c-Wydro). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.08.030
molecules reflect condensing, ordering and domains-promoting effect [12–16]. It was found that the foregoing properties of sterols depend on their molecular structure [12,17]. The basic structural conditions determining the ability of sterol to pack tightly with other lipids and influence the ordering of their hydrophobic chains as well as to form condensed domains are the presence of a tetracyclic ring, small polar group at position 3, a tail similar to that in cholesterol molecule and a small minimal area per molecule at the air/water interface [12,17] (Scheme 1). To the above mentioned group of membrane active sterols [17] belong also b-sitosterol and stigmasterol [14], which are phytocompounds naturally occurring in plant tissues, however, supplied with diet, are absorbed by human organism and, as it was evidenced, they can insert into human membranes [18 and references therein]. Diet is also the source of phytostanols, which differ from the molecule of the respective sterol only in the structure of ring B in the molecule i.e. stanol is a saturated sterol and its ring system is devoid of a double bond between carbons 5 and 6 [19] (Scheme 1). Stanols, similarly to sterols, may incorporate into human membranes and modify their organization [18 and references therein]. Since phytosterols/stanols are present in both plants and human organisms, the studies on the behavior of these molecules in model systems are required to verify the effect of these food-derived compounds on the properties of membranes. In addition to
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and P95% for stanol) purchased from Sigma. To prepare spreading solutions the lipids were dissolved in chloroform/methanol (4:1 v/ v) mixture (both chloroform and methanol were purchased from Aldrich, HPLC grade, P99.9%). From the respective stock solutions the mixed solutions were prepared and desirable volume of the latter was deposited onto the water subphase with the Hamilton micro syringe, precise to 1.0 lL. The measurements were performed at 20 °C ± 0.1 °C and the temperature was controlled thermostatically by a circulating water system. UltrapureMilli-Q water used as the subphase in the monolayer experiments at 20 °C ± 0.1 °C has surface tension of 72.6 mN/m and resistivity of 18 MX cm. 2.2. Methods
Scheme 1. The structure of b-sitosterol and b-sitostanol.
biological aspect of this kind of research, it is also of great interest to analyze the relationship between the structure of sterols and their properties. The investigations on the interactions of plant sterols with various membrane phospholipids as well as the analysis of their effect on multicomponent artificial membranes proved that phytosterols possess the condensing and ordering properties, however, they influence the packing properties of other lipids in a smaller extent as compared to cholesterol [14,20,21]. These differences are the consequence of the structure of aliphatic chain in animal versus plant sterols molecules. On the other hand, little is known on the effect of stanols on phospholipid membranes. In general, the studies on stanol molecules, especially compounds originating from plants, are very rare and the results presented in literature concern cholesterol derivative, namely cholestanol [13,22,23]. Therefore, Langmuir monolayers experiments to investigate the condensing and ordering effect of plant stanol (bsitostanol) and its miscibility and interactions with various phospholipids have been performed recently [24]. Moreover, based on these results it was concluded that the influence of b-sitostanol and corresponding sterol (i.e. b-sitosterol) on the respective phospholipids is similar [24]. Since the plant stanols incorporated into membranes can affect their properties, it is required to perform a comprehensive analysis of the properties of these biologically active molecules and to analyze the molecular arrangement of model systems composed of stanol and other membrane lipids. Therefore, we have studied the in-plane organization and morphology of b-sitostanol/DPPC monolayers by using Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy (BAM) techniques to obtain detailed characterization of the ordering, morphology and structure of the investigated films. The analysis of BAM images and GIXD data obtained for b-sitostanol and its mixture with phopholipid provide completely new information on molecular organization of stanol/DPPC monolayers. 2. Experimental 2.1. Materials The investigated compounds: phospholipid (1,2-dipalmitoylsn-glycero-3-phosphocholine – DPPC) and b-sitostanol (dihydrob-sitosterol) were synthetic products of high purity P99% for DPPC
Brewster Angle Microscopy experiments were performed with ultraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10 magnification objective, polarizer, analyzer and a CCD camera. The spatial resolution of the BAM was 2 lm. The experiments were carried out with KSV 2000 Langmuir trough (KSV Instruments Ltd., Helsinki, Finland) (total area = 870 cm2) equipped with two movable barriers and placed on antivibration table. The surface pressure was measured with the accuracy of ±0.1 mN/m using a Wilhelmy plate made of filter paper (ashless Whatman Chr1) connected to an electrobalance. After spreading, the monolayers were left for solvent evaporation for 20 min. and then the compression was initiated with the barrier speed of 5 cm2/min (2.5 Å2 molec1 min1). X-ray scattering experiments were performed at the BW1 (undulator) beamline at the HASYLAB synchrotron source (Hamburg, Germany) using a dedicated liquid surface diffractometer [25] with an incident X-ray wavelength k 1.3 Å. A Teflon thermostatted Langmuir trough (Riegler&Kirstein, Potsdam, Germany), equipped with a movable barrier for monolayer compression, was placed in a gastight container and mounted on the diffractometer. After spreading the solution onto the subphase, at least 40 min were allowed for the trough container to be flushed with helium to reduce the scattering background and to minimize beam damage during X-ray scans. Then, the monolayers were compressed to the surface pressure of 30 mN/m (the surface pressure at which the properties of monolayer can be compared with those of bilayers in the natural membrane [26]), at which the X-ray experiments were performed. As far as the GIXD experiments are concerned, the X-ray scattering theory and the liquid diffractometer used here have been described previously [27–29]. GIXD experiments were carried out to obtain lateral ordering information of the samples. The scattered intensity was measured by scanning over a range of horizontal scattering vectors Qxy; Q xy 4kp sinð2hxy =2Þ where 2hxy is the angle between the incident and diffracted beam projected on the liquid surface. The GIXD intensity resulting from a powder of 2D crystallites can be represented as Bragg peaks, resolved in the Qxy direction, by integrating the scattered intensity over the Qz direction, which is measured by the position-sensitive detector placed perpendicular to the air–water interface. Conversely, the Bragg rod profiles were resolved in the Qz direction (Q z ¼ 2kp sin af ; where af is the X-ray exit angle) and obtained by integrating the scattered intensity over Qxy corresponding to the Bragg peak. From the positions of the Bragg peaks the d-spacing, d = 2p/Qxy (where the Qxy is the position of the maximum of the Bragg peak along Qxy) and the unit cell parameters for the 2D lattice was obtained. From the line width of the peaks, using the Scherrer formula, it is possible to determine the 2D crystalline coherence length, Lxy, the average distance in the direction of the reciprocal lattice vector Qxy, over which the ordering extends. The analysis of the intensity distribution along Qz (Bragg rods) provides the information about the
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magnitude and the direction of the molecular tilt and the coherently scattering length of the molecule, Lz. The analysis procedure has been described in details elsewhere [29,30]. 3. Results and discussion The experiments were performed for pure DPPC and b-sitostanol monolayers and their mixtures of various molar compositions (Xb-sitostanol = 0.1; 0.3 and 0.9), (i.e. for films dominated by one of the lipids (10 and 90 mol% of stanol) and for the mixture, for which the strongest contraction of area per molecule was found in the previously published experimental results (30 mol% of stanol) [24]. For the clarity of presentation in Fig. 1 the surface pressure–area isotherms (A) and compressional modulus ðC 1 S Þ values vs. surface pressure plots (B) for the investigated films are shown. The BAM images recorded at different stages of the compression of the monolayers formed by the investigated lipids (DPPC and bsitostanol) and their mixtures of various compositions are presented in Fig. 2. As it is seen for pure DPPC monolayer, at the surface pressure p = 0 mN/m and large areas, in the images 2D foam reflecting the coexistence of gas and liquid expanded (LE) phase can be observed. With the compression of the monolayer, the gaseous phase vanishes and monolayer becomes homogenous. Further decrease of the area per molecule leads to the formation of liquid condensed (LC) domains. The region of LE/LC phase coexistence corresponds to the well known plateau in the surface pressure–area curve for DPPC film (see Fig. 1A). The foregoing LE/LC phase transition manifests also as a minimum in the compressional modulus versus surface pressure plot (see Fig. 1B). The size of LC domains increases
Fig. 1. The surface pressure–area isotherms (A) and compressional modulus ðC 1 S Þ values vs. surface pressure plots (B) for the investigated monolayers.
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with the compression and finally they fuse together, forming uniform LC phase at p > 12 mN/m. It is worth noting that the condensed domains exhibit regions of distinct reflectivities. Moreover, these domains show inversion of the contrast in the reflected light intensity with rotation of the BAM analyzer. This proves its optical anisotropy induced by different tilt-azimuthal orientations of the phospholipid molecules, which has been also found by other authors [31–33]. As it was found previously [24], the addition of 10 mol% of stanol into DPPC film practically does not change the shape of the isotherm, however, causes its shift to lower areas per molecule and slightly increases the surface pressure at the region corresponding to LE–LC phase coexistence (Fig. 1A). Also, BAM images recorded for this system are very similar to those obtained for pure DPPC film. However, the LC domains appearing in the region of plateau are smaller as compared to those detected for pure DPPC monolayer. Further addition of stanol (Xb-sitostanol = 0.3) into DPPC film strongly changes the isotherm as it is steeper and shifted to lower mean molecular areas (Fig. 1A). The BAM images recorded for this system indicate that the condensed phase appears at a lower surface pressure than for DPPC film and the LE–LC phase transition is not visible. For b-sitostanol monolayer the course of the recorded p-A isotherm (Fig. 1A) (i.e. the steep shape of the curve and small area per lipid molecule) and high values of the compressional modulus (Fig. 1B) indicate that this compound, similarly to b-sitosterol, forms tightly packed and ordered films. High condensation of stanol monolayers is reflected also in BAM images. As it can be observed in presented pictures, at large areas per molecule 2D foam corresponding to gas – condensed phases coexistence appears. At smaller areas, but still at the surface pressure p = 0 mN/m, a dark region of the gaseous phase coexists with the bright condensed phase. Further compression of the film induces the formation of one condensed phase and the images are homogenous. At very high surface pressures (p > 36 mN/m) small bright spots appear, which correspond to 3D nuclei of collapsed phase. For the mixture containing 10 mol% of DPPC (Xb-sitostanol = 0.9) the BAM images indicate on similar morphology of the monolayers as it was found for pure stanol film. GIXD data collected for DPPC monolayer at surface pressure p = 30 mN/m are presented in Fig. 3A and B. Fig. 3A shows the diffracted intensity as a function of the inplane scattering vector component Qxy (Bragg diffraction peaks) for different values of out-of-plane scattering vector component Qz, whereas Fig. 3B depicts the diffracted intensity (Bragg rods) along the direction normal to the interface, integrated over small Qxy intervals. For DPPC monolayer three different Bragg rods and therefore three Bragg peaks can be distinguished, which corresponds to an oblique structure of the in-plane lattice of tilted alkyl chains. This is in agreement with the results reported also by other authors [34,35]. The parameters obtained from the fitting the X-ray intensity distribution along Bragg peaks and Bragg rods are listed in Table 1. For pure b-sitostanol monolayer the Bragg diffraction peak and Bragg rod profiles are presented in Fig. 4. The values of structural parameters obtained from Bragg profiles are compiled in Table 1. In this case only one Bragg peak and Bragg rod with the maximum at Qz 0 can be observed. This indicates that, at the investigated surface pressure, the molecules pack in the 2D hexagonal lattice and they are oriented perpendicularly to the air/water interface. For both investigated lipids (DPPC and b-sitostanol) the values of the area per lipid molecule derived from GIXD experiments agree well with those obtained from the surface pressure–area measurements. Namely, at the surface pressure of 30 mN/m for stanol monolayer: 38.3 ± 0.1 Å2 from GIXD vs. 38.4 ± 0.2 Å2 from the isotherm, and for DPPC: 44.6 ± 0.1 Å2 (for DPPC the area per
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Fig. 2. BAM images taken for the investigated monolayers at different stages of compression.
molecule = 2 Auc due to the presence of two acyl chains in the molecule) from GIXD vs. 44.4 ± 0.2 Å2 from the isotherm. This indicates that in both cases practically the whole monolayer material forms the condensed domains.
In the case of b-sitostanol/DPPC mixture containing 10% of stanol, diffracted intensity as a function of the in-plane scattering vector component Qxy integrated over all Qz values, shows one broad Bragg peak. However, the analysis of the in-plane diffraction scans
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Fig. 3. Background-subtracted GIXD diffraction data (points) and fit (solid lines) for DPPC monolayers compressed to 30 mN/m. (A) Bragg peak profiles I(Qxy) integrated over the Qxy regions indicated on the graph. The Bragg peaks were fitted using Lorenzian function. (B) Corresponding Bragg rod profiles I(Qz) – for clarity of presentation the data have been offset vertically. The Bragg rods were integrated over the Qxy regions indicated on the graph. The distribution of the scattered intensity along the Bragg rods was fitted using Gaussian function.
for different Qz intervals (Fig. 5A) as well as Bragg rods (Fig. 5B), clearly indicates that, similarly to DPPC film, three low order diffraction peaks, indicating an oblique lattice with tilted hydrophobic parts of molecules, can be distinguished. However, the positions of these peaks lead to slightly smaller lattice constants and the tilt of the hydrophobic parts is reduced from s = 29° for DPPC monolayer to s = 23° for the mixed film (Table 1). Moreover, significant broadening of the Bragg peaks suggests that the extend of in-plane order is much smaller as compared to one-component phospholipid film, especially for the (1, 1) reflection. Further addition of stanol into DPPC monolayer (30% of b-sitostanol in the mixture) causes significant changes in X-ray scattering
Fig. 4. Background-subtracted GIXD diffraction data (points) and fit (solid lines) for b-sitostanol monolayers compressed to the surface pressure of 30 mN/m. (A) Bragg peak profile I(Qxy) integrated over the relevant Qz region; (B) corresponding Bragg rod profile I(Qz). The Bragg peaks and Bragg rods were fitted using Lorentzian and Gaussian function, respectively.
data and derived parameters as compared to DPPC (see Table 1). For this system only one Bragg peak (Fig. 5C) as well as one Bragg rod with maximum at Qz 0 (Fig. 5D) were obtained. This suggests hexagonal packing of molecules in the monolayer and their orientation perpendicular to the interface. Since the Bragg peak is located between those for DPPC and stanol films, it can be concluded that the scattering domains are not formed by individual lipids but they consist of an ordered array of both monolayer’s components. Moreover, considering Lxy value it can be stated that extend of in-plane order is smaller than in pure monolayers of the respective components and than in DPPC/stanol film of Xb-sitostanol = 0.1. For the system of high stanol content (Xb-sitostanol = 0.9) also only one diffraction peak and one Bragg rod with maximum at Qz 0 have been found (Fig. 5E and F respectively). The peak is significantly broader meaning that the ordered regions are smaller than
Table 1 In-plane structural parameters obtained from GIXD experiments for DPPC, b-sitostanol and their mixed monolayers at the surface pressure p = 30 mN/m: d – distance between scattering planes, a and b – the lengths of the unit cell vectors, c – the angle between the vectors a and b, s – tilt angle, Lxy – in-plane coherence length, Auc – area of the 2D unit cell, Lz – the length of the coherently scattering molecular moiety. The errors for the respective parameters represent maximum values estimated from the fitting procedure and calculated with the exact differential method.
DPPC
Xb-sitostanol = 0.1
Xb-sitostanol = 0.3 Xb-sitostanol = 0.9 b-sitostanol *
d* (Å) (±0.006)
a, b* (Å) (±0.006)
c (°)
s (°)
(±0.3)
(±1)
d{1,1} = 4.251 d{0,1} = 4.408 d{1,0} = 4.520 d{1,1} = 4.266 d{0,1} = 4.354 d{1,0} = 4.444 4.416 5.681 5.761
4.934 5.059
116.7
29
4.927 5.028
117.9
23
5.099 6.560 6.652
120 120 120
0 0 0
For hexagonal unit cell d{1,1} = d{0,1} = d{1,0}, a = b, and L{1,1} = L{0,1} = L{1,0}.
Lxy (Å) (±1)
Lz (Å) (±0.5)
Auc (Å2) (±0.1)
L{1,1} = 209 L{0,1} = 72 L{1,0} = 60 L{1,1} = 69 L{0,1} = 68 L{1,0} = 58 24 39 133
18.1
22.3
17.2
21.9
15.1 15.1 16.6
22. 5 37.3 38.3
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Fig. 5. Bragg peak I(Qxy) profile(s) (left panel) and corresponding Bragg rod profile(s) I(Qz). (right panel) for b-sitostanol/DPPC monolayers of various composition compressed to the surface pressure of 30 mN/m. (A, B) 10% of stanol (the Qxy and Qz integration regions are indicated on the graph); (C and D) 30% of stanol; (E and F) 90% of stanol, The solid lines are the curves fitted to background-subtracted GIXD diffraction data (points). For clarity of presentation the data have been offset vertically (A and B). For (C and D) and (E and F) the X-ray intensity integrations were performed over the relevant Qxy and Qz regions. The Bragg peaks and Bragg rods were fitted using Lorentzian and Gaussian function, respectively.
in one component stanol monolayer. Similarly to the mixture containing 30 mol% of stanol the domains are consisted of both monolayer components and the molecules are oriented perpendicularly to the interface. The lengths, Lz, of the coherently scattering molecular moieties, calculated from the Bragg rod intensity distributions, show a decreasing trend with the amount of stanol molecules in the system (see Table 1). For pure DPPC monolayer the Lz is equal to 18.1 Å, which is in agreement with the previously reported data [36]. Upon addition of stanol to DPPC monolayer the Lz deceases from 17.2 Å to 15.1 Å and 15.1 Å for the Xb-sitostanol = 0.1, 0.3 and 0.9, respectively. This is an indication that in all mixtures the ordered scattering domains are form by both DPPC and stanol molecules. Stanol molecules are shorter than the length of the all-trans DPPC acyl tails. Therefore, the addition of stanol molecules into the DPPC matrix decreases the amount of van der Waals contact between its tails and increases the number of gauche defects along the molecular backbones. This, in turn, decreases the average length of the coherently scattering molecular length in the ordered domains. It is worth to notice that the calculated Lz of pure stanol monolayer is 16.6 Å, which is bigger than the Lz obtained for
Xb-sitostanol = 0.3 and 0.9. This can indicate that not full length of the stanol molecule form the coherently scattering moieties in the mixtures with DPPC. It is also worthy comparing the mean molecular areas obtained for the mixed monolayers from the recorded isotherms [24] (Fig. 1A) with those calculated based on the area of unit cell (Auc) derived from GIXD experiments. Both oblique and hexagonal unit cells found for stanol/DPPC mixtures of Xb-sitostanol = 0.1, 0.3 and 0.9, respectively, contain one scattering moiety. However, in contrast to DPPC monolayer, in the case of mixtures the mean molecular area cannot be calculated by simple multiplying of the area of unit cell by two. This results from the fact that one DPPC molecule possesses two scattering parts (acyl chains) whereas stanol contains only one scattering moiety. Thus, in the case of b-sitostanol/DPPC mixtures the mean molecular area should be calculated as follows: Amix = Auc (2 XDPPC + Xstanol). The values of the mean molecular area calculated from the foregoing equation (41.6, 38.3, and 41.0 Å2/molecule for Xb-sitostanol = 0.1, 0.3 and 0.9, respectively) agree with those estimated from the isotherms (42.1, 38.5 and 39.0 Å2/molecule for Xb-sitostanol = 0.1, 0.3 and 0.9, respectively) at the same surface pressure p = 30 mN/m. This indicates that
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whole monolayer material is highly condensed and that the composition of scattering domains is the same as the composition of the monolayer. Since the values of the mean area per molecule are smaller as compared to those resulting from additivity rule (i.e. linear combination of the areas of the single components with the coefficients of their molar fractions) [24] thus the condensing effect of stanol on DPPC is reflected also in GIXD data. Similar results were found previously for sphingomylin/b-sitosterol mixtures as well as for sphingomylin/stigmasterol, for which the variations in the position of Bragg peaks positions were detected [37]. This clearly shows that b-sitostanol as well as b-sitosterol and stigmasterol do not form complexes of fixed stoichiometry with the investigated lipids. These findings are consistent with the results published for cholesterol/DPPC mixtures [38], for which it was concluded that the composition of the ordered domains changes with the proportion of lipids in the mixed film. The performed experiments allow us also to compare the properties of two phytocompounds namely b-sitostanol versus b-sitosterol, differing only in the structure of tetracyclic ring skeleton. b-sitostanol is a saturated analog of b-sitosterol, i.e. stanol molecule lacks the double bond between carbons 5 and 6 of the B ring (see Scheme 1). As it was evidenced in the characteristics of the isotherms [20,24], both these plant compounds form tightly packed and ordered monolayers. Moreover, the GIXD data collected in this work for b-sitostanol are similar to those obtained previously for b-sitosterol [37] at the same surface pressure values (p = 30 mN/m). Thus, it can be concluded that the difference in the structure of both compounds seems to be not so important for the in-plane organization within the monolayer. 4. Conclusions The presented results provide new and valuable information on the lateral organization of the investigated plant stanol/DPPC monolayers. The analysis of BAM images for stanol/DPPC mixtures shows that stanol influences morphology and molecular packing of DPPC monolayer: in the system containing 30 mol% of the plant stanol the condensed phase appears at lower surface pressure as compared to DPPC film and the LE–LC phase transition, clearly observed for pure DPPC film, vanishes. When the system is composed of 90 mol% of stanol, BAM images show similar texture as those taken for pure stanol film. However, at the surface pressure, at which the GIXD experiments were performed, no differences in BAM images for the investigated mixtures were found – for all the systems the monolayers are highly condensed and homogenous. On the other hand, GIXD data obtained at the surface pressure of 30 mN/m indicated that stanol/DPPC monolayers differ in their lateral organization for different compositions. The results obtained for the mixed monolayer containing 10 mol% of stanol evidence an oblique in-plane lattice of tilted hydrophobic region of molecules, similarly to pure DPPC film. However, the molecular tilt found for the mixture is lowered as compared to DPPC monolayer and the 2D scattering domains are smaller. With further addition of stanol into DPPC monolayer (i.e. 30 and 90 mol% of stanol in the mixture), the packing of molecules changes from oblique to hexagonal and the molecules are oriented perpendicularly to the interface. The ordered 2D domains are even smaller than those at 10 mol% content of stanol in the monolayer. For mixtures (Xb-sitostanol = 0.1, 0.3 and 0.9) the ordered domains are composed of both monolayers components. Summarizing the obtained results, it can be concluded that the composition of scattering structures formed in b-sitostanol/DPPC mixture depends on the composition of the monolayer. Moreover, in-plane organization within b-sitostanol film is similar to that
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found for b-sitosterol monolayer, despite of differences in the structure of both these compounds. Acknowledgments The Authors are grateful to DESY-HASYLAB, Hamburg (Germany) for granting synchrotron beam time for the realization of the project. The research was carried out with the equipment (ultraBAM) purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08). Lujan Neutron Scattering Center at LANSCE is funded by the DOE Office of Basic Energy Sciences and Los Alamos National Laboratory under DOE Contract DE-AC52-06NA25396.C. References [1] C. Peetla, A. Stine, H. Labhasetwar, Mol. Pharm. 6 (2009) 1264. [2] M.G. Sarpietro, M.Ch. Giuffrida, S. Ottimo, D. Micieli, F. Castelli, J. Nat. Prod. 74 (2011) 790. [3] C.-W. Wang, H.-G. Liu, X.-T. Bai, Q. Xue, X. Chen, Y. Lee, J. Hao, J. Jiang, Cryst. Growth Des. 8 (2008) 2660. [4] F.-W. Hsiao, Y.L. Lee, C.-H. Chang, Colloids Surf., B 73 (2009) 110. [5] J.J. Park, W.R. Lee, S.S. Bae, Y.J. Kim, K.H. Yoo, J. Cheon, S. Kim, J. Phys. Chem. B 109 (2005) 13119. [6] A. Heyman, I. Medalsy, O. Dgany, D. Porath, G. Markovich, O. Shoseyov, Langmuir 25 (2009) 5226. [7] S. Yokoyama, Y. Ohta, H. Sakai, M. Abe, Colloids Surf., B 34 (2004) 65. [8] S. Steinkopf, A. Simeunovic´, H.J. Bustad, T.H. Ngo, H. Sveaass, A.U. Gjerde, H. Holmsen, Biophys. Chem. 152 (2010) 65. [9] P. Wydro, S. Knapczyk, M. Łapczyn´ska, Langmuir 27 (2011) 5433. [10] S. Belem-Goncalves, G. Matar, P. Tsan, D. Lafont, P. Boullanger, V.M. Salim, T.L.M. Alves, J.-M. Lancelin, F. Besson, Colloids Surf., B 75 (2010) 466. [11] H. Ohvo-Rekila, B. Ramstedt, P. Leppimaki, J.P. Slotte, Prog. Lipid Res. 41 (2002) 66. [12] M.E. Beattie, S.L. Veatch, B.L. Stottrup, S.L. Keller, Biophys. J. 89 (2005) 1760. [13] X. Xu, E. London, Biochemistry 39 (2000) 843. [14] X. Xu, R. Bittman, G. Duportail, D. Heissler, C. Vilcheze, E. London, J. Biol. Chem. 276 (2001) 33540. [15] J. Aittoniemi, T. Róg, P. Niemelä, M. Pasenkiewicz-Gierula, M. Karttunen, I. Vattulainen, J. Phys. Chem. B 110 (2006) 25562. [16] T. Róg, L.M. Stimson, M. Pasenkiewicz-Gierula, I. Vattulainen, M.J. Karttunen, J. Phys. Chem. B 112 (2008) 1946. [17] Y. Barenholz, Prog. Lipid Res. 41 (2002) 1. [18] A. de Jong, J. Plat, R.P. Mensink, J. Nutr. Biochem. 14 (2003) 362. [19] S. Rozner, N. Garti, Colloids Surf. A 282–283 (2006) 435. [20] K. Ha˛c-Wydro, P. Dynarowicz-Ła˛tka, J. Phys. Chem. B 112 (2008) 11324. [21] K.K. Halling, J.P. Slotte, Biochim. Biophys. Acta 1664 (2004) 161. [22] K. Borrenpohl Lintker, P. Kpere-Daibo, S.J. Fliesler, A. Barnoski Serfis, Chem. Phys. Lipids 161 (2009) 22. [23] T.A. Daly, M. Wang, S.L. Regen, Langmuir 27 (2011) 2159. [24] K. Ha˛c-Wydro, A. Zaja˛c, P. Dynarowicz-Ła˛tka, J. Colloid Interface Sci. 360 (2011) 681. [25] J. Majewski, R. Popovitz-Biro, W.G. Bouwman, K. Kjaer, J. Als-Nielsen, M. Lahav, L. Leizerowitz, Chem. Eur. J. 1 (1995) 304. [26] D. Marsh, Biochim. Biophys. Acta 1286 (1996) 183. [27] T.R. Jensen, K. Kjaer, in: D. Mobius, R. Miller (Eds.), Novel Methods to Study Interfacial Monolayers, Elsevier, Amsterdam, The Netherlands, 2001, p. 205. [28] J. Als-Nielsen, K. Kjaer, in: T. Riste, D. Sherrington (Eds.), Proceedings of the NATO AdVanced Study Institute, Phase Transitions in Soft Condensed Matter, Plenum Press, New York, 1989, p. 113. [29] J. Als-Nielsen, D. Jacquemain, K. Kjaer, F. Leveiller, M. Lahav, L. Leiserowitz, Phys. Rep. 246 (1994) 252. [30] M. Tanaka, M.F. Schneider, G. Brezesinski, Chem. Phys. Chem. 4 (2003) 1316. [31] G. Weidemann, D. Vollhardt, Colloids Surf. 100 (1995) 187. [32] J. Miñones Jr., J.M. Rodriguez Patino, O. Conde, C. Carrera, R. Seoane, Colloids Surf., A 203 (2002) 273. [33] J. Miñones Jr., S. Pais, J. Miñones, O. Conde, P. Dynarowicz-Ła˛tka, Biophys. Chem. 140 (2009) 69. [34] G. Brezesinski, A. Dietrich, B. Struth, C. Bohm, W.G. Bouwman, K. Kjaer, H. Mohwald, Chem. Phys. Lipids 76 (1995) 145. [35] K. Wagner, G. Brezesinski, Chem. Phys. Lipids 145 (2007) 119. [36] M. Flasin´ski, M. Broniatowski, J. Majewski, P. Dynarowicz-Ła˛tka, J. Colloid Interface Sci. 348 (2010) 511. [37] K. Ha˛c-Wydro, M. Flasin´ski, M. Broniatowski, P. Dynarowicz-Ła˛tka, J. Majewski, J. Phys. Chem. B 114 (2010) 6866. [38] A. Ivankin, I. Kuzmenko, D. Gidalevitz, Phys. Rev. Lett. 104 (2010) 108101.