Chemistry and Physics of Lipids 183 (2014) 60–67

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Surface Gibbs energy interaction of phospholipid/cholesterol monolayers deposited on mica with probe liquids Małgorzata Jurak * Department of Physical Chemistry – Interfacial Phenomena, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska Sq. 3, 20031 Lublin, Poland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 6 March 2014 Received in revised form 20 May 2014 Accepted 21 May 2014 Available online 2 June 2014

The mica supported binary monolayers containing phospholipids: 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-oleoyl-1-palmitoyl-snglycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG), and cholesterol (Chol), mixed at different molar fractions, were investigated by measurements of the contact angles of water, formamide and diiodomethane. This allowed calculation of apparent surface Gibbs energy (further in the paper termed as ‘surface free energy’) of the monolayers according to the theoretical approach developed by Chibowski (contact angle hysteresis model, CAH). Then, based on the surface free energy values, the molar interaction Gibbs energy of the lipid molecules with the given probe liquid was evaluated. These values correlate with the values of excess area, interpreted as an indicator of the condensing effect of cholesterol on phospholipid monolayers at the air–water interface. The results indicate that the thermodynamic parameters of interactions depend on the monolayer composition and the probe liquid used to their determination. Changes of the parameters are discussed in relation to the monolayer packing, ordering, tilting of the molecules, and properties of the probe liquids as well. ã 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cholesterol Phospholipids Excess area Surface Gibbs energy Molar interaction Gibbs energy

1. Introduction Interaction of biological membranes with the surrounding water molecules and interaction in the membrane between cholesterol and phospholipids having different acyl chains have been a topic of great interest (Svanberg et al., 2009; van Uitert et al., 2010). The seminal papers of Keough's group evidence that the nature, stoichiometry and magnitude of cholesterol–phospholipid interactions is very sensitive to the degree of unsaturation of the hydrocarbon chains (Davis and Keough, 1983; Jackman et al., 1999; Kariel et al., 1991; Keough et al., 1989). The thermotropic phase behavior of binary mixtures of cholesterol with phospholipids containing one or two unsaturated fatty acyl chains is quite different from that of binary mixtures of cholesterol with saturated phospholipids. Typical role of cholesterol in the membrane is to strengthen the interactions between individual phospholipid molecules forming the membrane which causes an increase of the layer stability and lowers its permeability for water and ions (Ohvo-Rekilä et al., 2002; Róg et al., 2009). Because of the condensing effect, the area per

* Tel.: +48 81 537 5547; fax: +48 81 533 3348. E-mail address: [email protected] (M. Jurak). http://dx.doi.org/10.1016/j.chemphyslip.2014.05.007 0009-3084/ ã 2014 Elsevier Ireland Ltd. All rights reserved.

molecule in the monolayer is much lower if compared to the system of ideal mixing, the presence of cholesterol induces thickening of the membrane (Bennett et al., 2009). The increase of the membrane thickness causes reorganization of its structure by microdomains formation, known as the lipid rafts (Pike, 2006, 2009). These domains being rich in cholesterol are thought to be involved in signal transduction, lipid trafficking, and protein function (Fantini et al., 2002). Therefore, understanding of the mechanisms of cholesterol-induced domain formation has motivated many researchers to investigate cholesterol–phospholipid interactions. Different models have been proposed to explain the non-ideal behavior of cholesterol–phospholipid mixtures, such as the superlattice model (Chong, 1994; Chong et al., 2009; Somerharju et al., 1999), the umbrella model (Ali et al., 2007; Huang and Feigenson, 1999; Parker et al., 2004), the condensedcomplexes model (McConnell and Radhakrishnan, 2003; Radhakrishnan and McConnell, 1999; Radhakrishnan et al., 2000; Ratajczak et al., 2009), the mesoscopic water–lipid–cholesterol model (de Meyer and Smit, 2009), and others (Ivankin et al., 2010; Subczynski et al., 2012). Studies indicate that cholesterol within the membranes exhibits the solubility limit. The researchers are usually focused on narrow range of cholesterol concentration (upto 50%) (Hung et al., 2007; Pan et al., 2008a). However, above the solubility

M. Jurak / Chemistry and Physics of Lipids 183 (2014) 60–67

threshold, which depends on the composition of membrane, monohydrate crystals (Huang and Feigenson, 1999) or cholesterol bilayer domains are formed (Jacob et al., 1999; Subczynski et al., 2012). The latter ones are commonly recognized as a symptom of pathology, but they can also play a positive physiological function. For example, it was found that the membranes in the human eye lens are overloaded with cholesterol which possibly maintains lens transparency protecting against cataract (Jacob et al., 1999; Mason et al., 2003). A hypothesis has been put forward that cholesterol induces smoothing of the membrane surface thus causing a decrease in the light-scattering, and keeping the lens transparency (Plesnar et al., 2012; Subczynski et al., 2012). Additionally, the high level of cholesterol is associated with gap junctions where cholesterol takes part in maintaining fiber-to-fiber stability during visual accommodation (Biswas et al., 2010). The composition of the eye-lens membrane changes significantly with aging, and an increase in total cholesterol/phospholipid (Chol/PL) molar ratio can be observed (Raguz et al., 2009). Other function of cholesterol includes a raise of the hydrophobic barrier for polar molecules and an increase of the rigidity barrier for nonpolar molecules (Subczynski et al., 2012). This is revealed in the membrane permeability for small polar and nonpolar molecules. This specific role of cholesterol can manifest through cholesterol–membrane interactions with surrounding environment. Therefore, it is believed that better understanding of these interactions can be achieved from wettability studies of model membranes as a function of their composition. In the previous paper some correlation between the condensing and ordering effects in the phospholipid monolayers induced by cholesterol and the apparent surface free energy of the lipid films were shown (Jurak, 2013). The condensing effect was analyzed qualitatively through mean area per molecule (A12), as well quantitatively considering the excess Gibbs energy of mixing (DGexc) values. Moreover, the ordering effect was also analyzed by the compression modulus values (Cs
1) as a function of composition. It was concluded that cholesterol causes an increase in packing and ordering of the phospholipid monolayers which reflects in a systematic decrease of the mean area per phospholipid molecule with increasing amount of cholesterol in the mixed film. In the presence of cholesterol the films of saturated phospholipids are found to be more condensed and chain-ordered than those comprised of unsaturated ones. This also reflected in the changes of surface free energy of the films which was evaluated from van Oss et al. (1986, 1988) approach. Moreover, to some extent the results are also in line with the condensedcomplex model. In this paper the magnitude of area-condensing effect, expressed by the excess area, is used as a rough indicator of relative strength of the interaction between cholesterol and phospholipids. This parameter is associated with the apparent surface free energy determined from the contact angles hysteresis (CAH) (Chibowski, 2002, 2005; Chibowski and Jurak, 2011) of three probe liquids (water, formamide, and diiodomethane). In this paper a new method of the molar interaction energy calculation is presented. The calculated values are consistent with those obtained by other researchers. Moreover, the changes of surface free energy and molar interaction Gibbs energy as a function of the monolayers composition are explained with the help of different models of the phospholipid/cholesterol interactions. To better understand physiological functions of cholesterol in the membrane which depend on its content, knowledge of the membrane interactions with water and other polar and non-polar liquids is needed, that is the membrane permeability for different molecules. This is possible to learn by determining the surface free energy and the molar interaction energy.

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2. Materials and methods 2.1. Materials 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl1-palmitoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3phospho-rac > -1-glycerol (DPPG) and cholesterol (Chol) were supplied by Sigma (purity 99%) and used as received. Chloroform (p.a.) and ethanol (96%, p.a.) were from POCH S.A., Poland, and were applied as solvents for the lipids. The solutions were prepared by dissolving about 1 mg of the investigated compound (DPPC, POPC, DOPC, and Chol) in 1 ml of chloroform or the mixture of chloroform/ ethanol 4:1 (v/v) (DPPG). Mixed solutions were obtained from the respective stock solutions. Ultrapure water from Milli-Q Plus system (resistivity 18.2 MVcm) was used both as a subphase for preparation of the Langmuir monolayers and as a probe liquid in contact angle measurements. Other probe liquids used were formamide (98%, Aldrich) and diiodomethane (99%, Aldrich). Freshly cleaved mica plates (Continental Trade, Poland) of 38 mm  26 mm  1 mm size were used as a solid support for the lipids. 2.2. Determination of p–A isotherms and excess areas The surface pressure–molecular area (p–A) isotherms were recorded with a computer-controlled KSV standard-trough (KSV Instruments Ltd., Finland) placed on an anti-vibration table in a plexi-glass box. Two movable barriers of the apparatus enabled symmetric film compression. Surface pressure was measured with the accuracy of 0.1 mN m
1 using a platinum Wilhelmy plate which was flamed before using. The purity of water subphase was checked during its surface compression in the absence of lipid. If upon compression the change of surface pressure of water was less than 0.3 mN m
1 its surface was recognized to be pure. Then, needed amount of the lipid solution was dropped onto the water subphase from a Hamilton microsyringe, and 10 min was allowed to evaporate the solvent completely, and afterwards the constant rate compression was applied with a barrier speed of 10 mm min
1. In all experiments, the temperature of the subphase was kept constant at 20  0.1  C by a circulating water system. All experiments were repeated at least three times. From the p–A isotherms, at the surface pressure of 35 mN m
1 mimicking the lateral pressure in biological membranes (Marsh, 1996), the excess area Aexc was calculated from equation (Dynarowicz-Ła?tka and Kita, 1999; Gaines, 1966): Aexc ¼ A1;2
ðx1 A1 þ x2 A2 Þ

(1)

where A1,2 is the mean molecular area in the mixed monolayer at 35 mN m
1, A1 and A2 are the molecular area in the pure monolayer of components 1 and 2 at the same surface pressure, x1 and x2 are mole fractions of the components in the mixed film. 2.3. Langmuir–Blodgett (LB) monolayer formation Before the monolayer deposition onto a solid support the clean mica plate was attached to the dipper of trough and immersed into the subphase. Then the monolayer was spread onto water, compressed to a final surface pressure of 35 mN m
1, and transferred by withdrawing the mica support from the subphase through the monolayer at a rate of 5 mm min
1. The surface pressure of lipid monolayer, the rate of deposition, and the temperature (20  0.1  C) were kept constant. The monolayer transfer efficiency during the withdrawal was 1.00  0.01. Thus obtained lipid films were dried in a vacuum apparatus overnight under the pressure of 117 mbar at room temperature. The structure

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of the transferred monolayers onto mica was confirmed by atomic force microscopy (AFM) images (see Supplementary data). 2.4. Contact angle measurements The advancing and receding contact angles of water, formamide, and diiodomethane were measured on the monolayers described above. The measurements were conducted by means of contact angle meter (GBX, France) equipped with a camera and an automatic drop deposition system. The contact angle was calculated by the software from the droplet shape. The readings were taken both on the left and right sides of the 2D droplet's profile for all three liquids. The droplets of 3 ml volume were used to measure the advancing contact angles, then after sucking 1 ml of the liquid from droplet into the syringe, the receding contact angles were determined. The measurements were conducted in a closed chamber under constant flow rate of nitrogen. The temperature of 20  C was kept by an external circulating water system. The contact angles were measured in three separate series and their reproducibility was within 2–4 . 2.5. Surface free energy calculation The model proposed by Chibowski, called the contact angle hysteresis (CAH) model, was applied to the surface free energy determination (Chibowski, 2002, 2005; Chibowski and Jurak, 2011). Using this approach, three measurable parameters: advancing (u a) and receding (ur) contact angles (hysteresis of contact angle), and surface tension (g L) of only one probe liquid are needed to calculate apparent solid surface free energy (g S):

gS ¼

g L ð1 þ cosua Þ2 ð2 þ cosur þ cosua Þ

(2)

where g L is the surface tension of the probe liquid, ua is the advancing contact angle, u r is the receding contact angle. The g S calculated from contact angle hysteresis of one liquid, i.e. water, formamide, or diiodomethane, is denoted by superscript W, F, DM, i.e. g SW, g SF, and g SDM, respectively. 2.6. Interaction Gibbs energy calculation Assuming that on mica surface, the mean area occupied by one molecule in the mixed monolayer transferred at 35 mN m
1 is preserved, the interaction energy, g mol =
(DGmol), of one mole of the film molecules with water, formamide or diiodomethane was calculated from the determined surface free energy. For example, taking the area occupied by one DPPC molecule at 35 mN m
1 (A = 0.418 nm2) (Jurak, 2013) and the surface free energy determined from the contact angle hysteresis of water (g SW = 27.1 mJ m
2), the energy of interaction of one mole of DPPC molecules with water (molar interaction Gibbs energy),
(DGmol), was calculated to be 6.83 kJ mol
1 DPPC. It should be stressed that the area per molecule in the deposited mixed film used for the calculations is an averaged one, i.e. it is the same for the molecules of the two components. Also, their uniform distribution in the monolayer is assumed. 3. Results and discussion 3.1. Monolayer condensation To analyze the magnitude of condensation and to confirm nonideal behavior of the mixtures induced by an addition of cholesterol to the phospholipid film, the values of excess area per molecule (Aexc) were calculated according to Eq. (1). These results are presented as a function of Chol concentration in Fig. 1.

Fig. 1. Excess area (Aexc) in the film versus cholesterol molar fraction (xChol) in mixed monolayers: DPPC/Chol, POPC/Chol, DOPC/Chol and DPPG/Chol at the film surface pressure of 35 mN m
1.

The excess area results from the kind of interactions in the mixed monolayers. For the binary films of zwitterionic phospholipids (DPPC, POPC and DOPC) and cholesterol the Aexc values are negative which allows one to conclude that lateral interactions between the molecules are more attractive as compared to those in one component monolayers. In contrary, the value of excess area in DPPG/Chol mixtures indicates for nearly ideal behavior of these systems, nevertheless, small attractive or repulsive interactions may appear. This is in agreement with the results reported by Borochov et al. (1995) who prove that the low miscibility of Chol in the gel states of saturated phosphatidylglycerol bilayers is due to a concomitant phase separation of the two lipid components. The occurring minima of Aexc indicate the largest condensation of the phospholipid monolayer induced by the cholesterol addition. Therefore, due to stronger attractive interactions between the molecules, thermodynamically favorable binary monolayers of a defined stoichiometry can be formed, i.e. 3:1 for DPPC/Chol, and 1:1 for POPC/Chol or DOPC/Chol. In the next section the monolayer condensing effect is discussed via interactions of the films with different liquids, which are expressed by values of the monolayers surface free energy. 3.2. Surface free energy Lipid films transferred onto a solid support with a help of Langmuir–Blodgett trough and then desiccated in vacuum are directly linked to the supporting surface and their structure is retained (Günster and Souda, 2006) due to strong interactions between the negatively charged mica surface and the lipid heads (Qi et al., 2009). Moreover, some studies indicate that lipid monolayers at the air–water interface and compositionally similar planar bilayers on mica in the pressure regime of 30 mN m
1 at nearly the same temperature possess equivalent structures, i.e. tilt of the lipid chains and the area per lipid molecule (Brewer et al., 2010; Pan et al., 2008b). This author hopes the same is true of the monolayer transferred onto mica. The surface free energies of the lipid monolayers were calculated from the contact angle hystereses of water, formamide and diiodomethane applying the CAH approach (Eq. (2)). The results are plotted in Fig. 2. The surface tensions (g L) are published elsewhere (Chibowski and Jurak, 2013; van Oss et al., 2001). Water and formamide are polar liquids while diiodomethane is apolar one. They have different size (volume) of their molecules, as well as surface tension and its components. Water has the smallest molecule and it possesses strong electron-donor

M. Jurak / Chemistry and Physics of Lipids 183 (2014) 60–67

Fig. 2. Surface free energy, g S, of the mixed monolayers: DPPC/Chol, POPC/Chol, DOPC/Chol and DPPG/Chol deposited on mica at 35 mN m
1, calculated from the water (W), formamide (F) and diiodomethane (DM) contact angle hystereses versus cholesterol molar fraction (xChol).

g L
and electron-acceptor g L+ interactions while formamide shows only strong g L
parameter (Chibowski and Jurak, 2013; van Oss et al., 2001). On the contrary, diiodomethane molecules practically interact by London dispersion forces only. These properties reflect in different strength of interactions with the monolayers expressed by the surface free energy values (Fig. 2). The results obtained for pure phospholipid monolayers (DPPC, DPPG, POPC and DOPC) (Fig. 2) show that the surface free energy of DPPC determined from the contact angle hysteresis of water (27.1 mJ m
2) and formamide (28.5 mJ m
2) is lower than that obtained from the contact angle hysteresis of diiodomethane (34.6 mJ m
2). These values suggest that both polar liquids interact with DPPC film principally by dispersion forces. Moreover, this indicate that the layer have a tight structure with the hydrophobic tails of molecules faced outward. The tight packing of lipids reduces water permeability (Lande et al., 1995). This correlates well with the decreased area per lipid molecule (Mathai et al., 2008). According to Róg et al. cholesterol favorably interacts with DPPC than water as indicated by deeper location of cholesterol molecules in the DPPC-Chol bilayer (Róg et al., 2009).

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On the other hand, if the phosphoglycerol group in DPPG is exposed outwards instead of the phosphocholine group, it significantly changes the monolayer interaction with water and higher interaction g SW = 41.5 mJ m
2 than with diiodomethane g SDM = 35.3 mJ m
2 occurs. However, the interaction with formamide is practically the same as it was with the pure DPPC layer (g SF = 26.9 mJ m
2). This is probably due to strong tendency of DPPG to form hydrogen bonds with water by the hydrogen acceptors (phosphate and carbonyl) and/or the hydrogen donor (OH of the glycerol) (Borochov et al., 1995). Similarly, the interactions of unsaturated POPC and DOPC with polar liquids are stronger than with apolar diiodomethane. They are also stronger than the interactions of these liquids with the saturated DPPC and DPPG. This suggests significant contribution of hydrogen bonds to the surface free energy of these monolayers. In all of the investigated systems, the addition of cholesterol affects interactions with water, formamide and diiodomethane, they generally increase with increasing cholesterol molar ratio. At low cholesterol concentration (xChol = 0.25) the surface free energy determined from the contact angle hysteresis of water increases in the following order DPPC < POPC < DOPC < DPPG and this tendency remains also at higher mole fraction of cholesterol, except for DPPC for which the energy drastically increases at xChol = 0.5. The results on lipid hydration obtained by Bach et al. (Bach and Miller, 2005; Bach and Wachtel, 2003) indicate that the number of water molecules bound or immobilized by the phospholipids increases steeply in the region of phase separation of cholesterol. The phase separation of cholesterol takes place above a defined molar ratio, e.g. for the DPPC/Chol mixture it is about 1:1 (Bach and Miller, 2005; Bach and Wachtel, 2003). It justifies the energy changes presented in Fig. 2. In the case of unsaturated phosphatidylcholines, the surface free energy of the mixed DOPC/Chol layers is comparable to, or higher than, that of POPC/Chol, irrespective of which the probe liquid is used (Fig. 2). The reason for this is that the presence of unsaturated double bond(s) weakens the close packing of the molecules, which occurs for saturated tails, resulting in weaker apolar interactions. Moreover, the incorporation of Chol into the membrane increases the distance between phospholipid headgroups. Therefore, the interactions between the polar groups are weaker, carbonyl and phosphate oxygen atoms are more accessible to water, and hydration of polar headgroups increases (Murzyn et al., 2001). Such monolayer possesses looser structure and packing which makes easier permeability for water and other small molecules. In consequence, a higher apparent surface free energy is determined. It is worth again stressing that among these mixed films that with DPPC/Chol at xChol = 0.25 water interacts principally by dispersion forces as the results in Fig. 2 show. This must be result of very closely packed monolayer, also confirmed by the minimum of the excess area appearing at the same composition (Fig. 1). It should be also mentioned that the molecule volume and size of the probe liquid may have an effect on the strength of solid/liquid interactions. The volume of water molecules is about 4.5 times smaller than that of the diiodomethane molecule and 2.2 times smaller than that of the formamide molecule (Chibowski and Jurak, 2013). Moreover, the area per molecule of water is 0.059 nm2 while that of diiodomethane is 0.215 nm2 thus being over 3.6 times larger (Chibowski and Jurak, 2011). Therefore, water molecules can penetrate more easily into cavities of the monolayer. Consequently, it may be concluded that the sizes of cavities in DPPC/Chol (xChol = 0.25) monolayers are smaller than the water molecule diameter and it cannot penetrate into the tightly packed film. It is found that at this component stoichiometry (xChol = 0.25) also interactions of polar formamide molecules are weaker than with pure DPPC (the minimum in the film surface free energy) (Fig. 2). Such behavior supports the hypothesis of condensed DPPC/Chol

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M. Jurak / Chemistry and Physics of Lipids 183 (2014) 60–67

complex formation of the 3:1 stoichiometry (McConnell and Radhakrishnan, 2003; Radhakrishnan and McConnell, 1999; Radhakrishnan et al., 2000; Ratajczak et al., 2009). The results in Fig. 2 show that the apparent surface free energy if determined from the CAH model depends to some extent on the kind of probe liquid applied. It is concluded that the strength of interactions originating from the film surface depends on the kind and strength of interactions coming from the probe liquid being in contact with the surface, as well the spacing between the solid and liquid interacting molecules is important. It means that thus calculated values of the surface free energy are apparent and depend on its roughness too. Moreover, it appears that the averaged values of the energy (g Stot) from the CAH approach (Fig. 3), which are an arithmetic mean of the values determined from the contact angles hystereses of water, formamide and diiodomethane (Eq. (2)), agree very well with the mean values from van Oss et al. (1986, 1988) approach, if the same triad of the probe liquids are used for advancing contact angle measurements (Jurak, 2013). Namely, the trend of surface free energy changes calculated from the two approaches in relation to the monolayer composition is practically the same. However, the surface free energy values determined from the CAH approach are slightly larger. The reason of it can be found in that the receding contact angles for the energy calculation are also used. Therefore, thus calculated the energy values seem to express the interfacial interaction occurring at closer distances. 3.3. Molar interaction Gibbs energy Assuming that the mean area occupied by one molecule in the mixed Langmuir monolayer at 35 mN m
1 is preserved in the film transferred on mica surface, the calculated surface free energy of deposited layers allows for calculation of the apparent interaction Gibbs energy of one mole of given film molecules with water, formamide or diiodomethane (see Experimental, p. 2.6). The obtained results of the molar interaction Gibbs energy,
DGmol, for all the investigated monolayers are plotted in Fig. 4. As can be seen the molar energy of interactions of the monolayers depends on the kind of liquid. For pure DPPC monolayer and DPPC/Chol at xChol = 0.25 with water it is lower than that calculated for diiodomethane. This suggests importance of the dispersion interaction contribution to the molar interaction energy in some of the monolayers while in others the hydrogen bonds interactions play a major role. Analyzing molar interaction energy of the mixed lipid/cholesterol monolayers with water, the pronounced energy

Fig. 3. Averaged surface free energy, g Stot, of the mixed monolayers: DPPC/Chol, POPC/Chol, DOPC/Chol and DPPG/Chol deposited on mica at 35 mN m
1, calculated from the CAH approach versus cholesterol molar fraction (xChol).

Fig. 4. Molar interaction energy, g mol =
(DGmol), of the mixed monolayers: DPPC/ Chol, POPC/Chol, DOPC/Chol and DPPG/Chol, deposited on mica at 35 mN m
1, with water (W), formamide (F) and diiodomethane (DM) versus cholesterol molar fraction (xChol).

minima, or inflections, appear at xChol = 0.5 in POPC or DOPC, and at xChol = 0.25 in DPPC or DPPG. These changes correspond very well with the changes of excess Gibbs energy of mixing, where at the corresponding monolayer compositions the minima are observed (Jurak, 2013). The excess Gibbs energy of mixing indicates the strength of the intermolecular interactions, and the strongest ones appear in DPPC/Chol system at xChol = 0.25. In the case of unsaturated phospholipids the energy minima appear at different composition, i.e. xChol = 0.5. It is quite logical that the stronger interactions between molecules lead to more condensed structure of the monolayer which becomes less permeable for water molecules. Then an increase of molar interaction energy (Fig. 4) and the topographical images of the monolayers studied (see Supplementary data) indicate for the phase separation at characteristic phospholipid/cholesterol ratio, i.e. 1:1 for DPPC/ Chol, 1:3 for POPC/Chol and DOPC/Chol, and 3:1 for DPPG/Chol. On the other hand, in spite of the phase separation in the lipid monolayers the apparent contact angles of macroscopic (i.e. millimeter-sized) liquid droplets were measured reproducibly. The

M. Jurak / Chemistry and Physics of Lipids 183 (2014) 60–67

obtained macroscopic advancing and receding contact angles result from an averaging over the spatially varying, microscopic contact angles (Decker et al., 1999). Therefore, it is believed that the determined values of the energy faithfully reflect real energetic state of the layers. In case of diiodomethane, its interactions are practically of dispersive nature only, while those with water or formamide are also of the electron-donor and electron-acceptor (hydrogen bonding) nature. In other words, in the case of polar probe liquids (water or formamide) the calculated values of molar interaction Gibbs energy, first of all express the polar interactions, hydrogen bonding, but if the molar energy is calculated from the apolar diiodomethane contact angles, its value characterizes the London dispersion interactions of the lipids molecules (Fig. 4). The dispersion interaction energy well correlates with the mean area of condensation presented in the previous paper (Jurak, 2013), i.e. the energy changes run in a similar way as the changes of average molecular area. Because of similarity in the changes of these two quantities one may conclude that the molar dispersion interaction energy can be also used as a parameter of relative strength of the interactions between cholesterol and phospholipids. 3.4. The calculated molar interaction Gibbs energy in the light of some literature interactions models For DPPC and water the molar interaction energy agrees well with some literature data. Taking the mean molecular area of DPPC at 35 mN m
1 as 41.8 Å2, the calculated –(DGmol) equals 6.83 kJ mol
1. This value is comparable to experimental value determined from solute transfer experiments where a measure of the hydrophobic interaction in the aqueous medium is the Gibbs energy of transfer of a hydrocarbon molecule from a hydrocarbon solvent to an aqueous medium (Reynolds et al., 1974), as well with the value of the standard free energy of transfer of a solute from water to a nonpolar solvent (Kyte, 2003). Thus determined values vary from 16 to 33 cal mol
1 Å
2 (Chothia, 1976; Eisenberg and McLachlan, 1986; Hermann, 1972; Reynolds et al., 1974), which recalculated for DPPC/water, taking 41.8 Å2 molecule
1, they range from 2.80 to 5.77 kJ mol
1, respectively. Also, using molecular dynamics simulations calculation of hydrophobic interactions in aggregates of small hydrophobic solutes in water gives 24 cal mol
1 Å
2, i. e. 4.20 kJ mol
1 (Raschke et al., 2001). Possibly, a bit larger value (6.83 kJ mol
1) calculated from the contact angle hysteresis is caused by hydrogen bonding of water with phospholipid polar groups. On the other hand, Lazaridis and Paulaitis expressed the standard free enthalpy of solvation of a simple solute (methane) in water as 2.0 kcal mol
1, i.e. 8.37 kJ mol
1 (Lazaridis and Paulaitis, 1992). Tanford (1979) derived the interfacial free energy of hydrocarbon/water interaction from the interfacial tension as 51 mJ m
2, which gives 12.8 kJ mol
1. In the light of the aforementioned literature data it may be concluded that the molar interaction energy values calculated via contact angles hysteresis (Eq. (2)) reflect real interfacial interactions, and somehow the distances between the interacting molecules of solid–liquid too. Moreover, the interactions between phospholipid/cholesterol layer and liquids can be interpreted with a help of different structural models. Subczynski et al. found that the saturating amounts of cholesterol, whose rigid steroid-ring is located between carbon atom C9 and C10 positions in the membranes, ensures the hydrophobicity increase of the bilayer (Subczynski, 2012). Therefore the activation energy for polar molecules needed to pass through the membrane greatly increases and the rigidity barrier for nonpolar molecules near the membrane surface increases as well (Subczynski, 2012). Hence, one may conclude that the lowest values of molar interaction energy of DPPC/Chol

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with water are due to the presence of densely packed binary monolayer. This conclusion is in agreement with the condensedcomplex model (McConnell and Radhakrishnan, 2003; Radhakrishnan and McConnell, 1999; Radhakrishnan et al., 2000; Ratajczak et al., 2009), according to which cholesterol can form selectively associates with saturated chains having lower affinity to unsaturated acyl chains. If an excess amount of cholesterol is present, it simply mixes with the complexes (or forms a separate phase), and then an increase in molar interaction energy is observed. The changes of molar interaction energy (Fig. 4) indicate favorable monolayer composition for the binary complex formation, which is 3:1 for DPPC/Chol and 1:1 for POPC/Chol (inflections on the plots), and suggest that in DOPC/Chol films no complexes are present. However, the behavior of cholesterol in the DOPC membranes can be explained by the umbrella model (Huang and Feigenson, 1999). For instance, the solubility limit of cholesterol in unsaturated DOPC bilayers, measured by light scattering and optical microscopy, was found to be 0.67  0.02 (Parker et al., 2004) below which cholesterol is evenly distributed in the lipid bilayer. Above this value, cholesterol monohydrate crystals are formed and the lipid packing in the bilayers becomes less ordered (Huang and Feigenson, 1999; Parker et al., 2004). Following this model, at xChol = 0.5 the cholesterol solubility limit in POPC and DOPC is not exceeded; the monolayers are condensed and ordered, which is proved by the minima in the excess area (Fig. 1). This is also reflected in the lowest value of the interaction energy appearing at this stoichiometry (Fig. 4). At xChol = 0.75 the cholesterol precipitation may take place. Therefore, the mixed monolayers become less condensed, and thus more disordered, which is evidenced by the excess area decrease (Fig. 1). This makes the monolayers easier permeable for water resulting in the interaction energy increase. Moreover, the model of Ivankin et al. suggests that the tilting of polar groups and hydrocarbon chains of the phospholipid molecules change depending on the cholesterol content in the membrane (Ivankin et al., 2010). At higher concentration of cholesterol (xChol = 0.7 and 0.85) packing density of the upper part of the acyl chains is reduced by their tilting. Hence, the layer becomes more permeable. On the other hand, the tilt angle of cholesterol ring with respect to the membrane normal is a good measure of the sterol ability to induce order in a membrane. The tilt angle was found to be larger in DOPC matrices (24 ) in comparison with DPPC (20 ). Hence, the unsaturated bilayers are less sensitive to sterol structure compared to the saturated ones (Róg et al., 2009). Thus, the membrane condensation and ordering induced by cholesterol are weaker in membranes consisting of unsaturated lipids than saturated ones (Róg et al., 2009). 4. Conclusions In this study the surface free energy of the phospholipid/ cholesterol monolayers and their molar interactions energy with water, formamide and diiodomethane were determined via advancing and receding contact angle measurements of three probe liquids and then applying the contact angles hysteresis (CAH) model. It was found that if the surface free energy value of the monolayer is considered for the particular probe liquid, together with the mean molecular area per molecule, thus calculated interactions in relation to the layer composition suggest formation of the tightly packed structure of the phospholipidcholesterol membrane or the phase separation. Moreover, the results suggest that the strength of the interactions originating from the solid surface depends on the kind and strength of interactions acting from the probe liquid whose droplet is in contact with the film surface, where also the separation distance between the interacting molecules is significant. It is believed that these results would be helpful for better understanding the

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