Surface Behavior of Oleoyl Palmitoyl Phosphatidyl Ethanolamine

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Langmuir 2004, 20, 11414-11421

Surface Behavior of Oleoyl Palmitoyl Phosphatidyl Ethanolamine (OPPE) and the Characteristics of Mixed OPPE-Miltefosine Monolayers I. Rey Go´mez-Serranillos,† J. Minõnes, Jr.,† P. Dynarowicz-Ła¸ tka,*,‡ J. Minõnes,† and O. Conde† Department of Physical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain, and Jagiellonian University, Faculty of Chemistry, Ingardena 3, Krako´ w 30-060, Poland Received February 26, 2004. In Final Form: September 21, 2004 Langmuir monolayers of oleoyl palmitoyl phosphatidyl ethanolamine (OPPE) were investigated at the air/water interface by means of surface pressure (π)-area (A) isotherms complemented with Brewster angle microscopy images upon film compression/expansion. The characteristic phase transition appearing in the course of π/A isotherms was attributed to the coexistence of two liquid-expanded phases of different molecular ordering. The interactions between OPPE and hexadecylphosphocholine (miltefosine) were studied at different subphase pHs (2, 6, and 10) at 20 °C and analyzed with mean molecular area (A12)-, excess area of mixing (Aexc)-, and excess free energy of mixing (∆Gexc)-composition plots. The obtained results indicate that at pH 10, where both OPPE and miltefosine polar groups are negatively charged, attractive interactions are observed (reflected by negative deviations from ideality), contrary to expectation. This peculiar behavior is explained as being due both to water molecules, which surround negatively charged polar groups and increase the distance between them, weakening in this way the electrostatic repulsion forces; and to positively charged counterions present in the diffuse double layer, neutralizing their charge. In this way, the van der Waals attraction forces between hydrocarbon tails of both molecules predominate and are responsible for the observed negative deviations from ideal behavior. Similar explanations are given for the observed negative deviations at pH 2 where both polar groups are positively charged. At pH 6, the observed negative deviations at low surface pressures and positive deviations at high pressures are interpreted as being due to a change in orientation of polar groups upon monolayer compression.

Introduction The investigations of the miscibility and interactions between cell membrane components and drugs in Langmuir monolayers, which can serve as one of the models of biomembranes in vitro, can be considered as one of the crucial factors leading to the understanding of the mechanism of action of many drugs. This paper is a subsequent work of our previous research on the film-forming properties and surface behavior of an anticancer drug, namely, hexadecylphosphocholine, known as miltefosine, by itself1 and in mixtures with cholesterol,2 aiming primarily at getting deeper insight into miltefosine’s mode of action, which has not been elucidated so far. A number of hypotheses have been brought forward,3 and each of them evidences a significant importance of the cellular membrane in the pharmacological action of miltefosine. Therefore, we have undertaken studies on the miscibility and interaction between this drug and the main lipids of cellular membranes. In general, three groups of lipids constitute a cellular membrane, i.e., phospholipids, sterols (mainly cholesterol), and glycosphingolipids.4 * Corresponding author. Address: Jagiellonian University, Faculty of Chemistry, Department of General Chemistry, Ingardena 3, 30-060 Krako´w, Poland. Tel: +48-12-6336377 ext 2236. Fax: +48-12-6340515. E-mail: [email protected]. † University of Santiago de Compostela. ‡ Jagiellonian University.

(1) Rey Go´mez-Serranillos, I.; Minõnes, J., Jr.; Dynarowicz-Ła¸ tka, P.; Iribarnegaray, E.; Casas, M. Phys. Chem. Chem. Phys. 2004, 6, 1580. (2) Rey Go´mez-Serranillos, I.; Minõnes, J., Jr.; Dynarowicz-Ła¸ tka, P.; Minõnes, J.; Iribarnegaray, E. Langmuir 2004, 20, 928. (3) Kosano, H.; Takatani, O. Cancer Res. 1988, 48, 6033.

Phospholipids form the framework of the cellular membrane. Different membranes contain various types of phospholipids. For example, mamalian membranes contain a high proportion of lecithins (diacyl glycerophosphocholines) and cephalins (diacyl glycerophosphoethanolamines), both saturated and unsaturated. The amount of unsaturated ones increases from the outside part of the cellular membrane toward its interior.5 Also, the distribution of phospholipids with different polar groups varies depending on the membrane side.6 For example, the membrane of human red cells contains mainly phosphatidylcholines in the external region of the membrane, while the majority of phospholipids with ethanolamine polar groups are situated in its inner part.7 Because of the asymmetrical composition of both sides of the lipid bilayer, for our studies with miltefosine, we have chosen different lipids that are representative for each side of the cellular membrane. Herein we show our studies of mixed monolayers of miltefosine and one of the phospholipids, namely, β-oleoyl-γ-palmitoyl, L-R-phosphatidylethanolamine, abbreviated as OPPE. The presence of an unsaturated hydrophobic tail and ethanolamine polar group makes this compound a typical phospholipid forming the inner part of the membrane. In a subsequent study,8 we will focus our attention on the other membrane component, that is, a ganglioside (GM1) which is situated (4) Biological Membranes: Physical Fact and Function; Chapman, D., Ed.; Academic Press: London, 1968; p 7. (5) Robinson, G. B. In Biological Membranes; Parson, D. S., Ed.; Academic Press: London, 1975; p 19. (6) Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. In Molecular Biology of the Cell; Garland: New York, 1983. (7) Guirr, M. I.; James, A. T. In Lipid Biochemistry, 3rd ed.; University Press: Cambridge, 1980.

10.1021/la040036v CCC: $27.50 © 2004 American Chemical Society Published on Web 11/19/2004

Surface Behavior of OPPE

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in the external region of the cellular membrane, while our former paper2 deals with the interaction between miltefosine and cholesterol, which is placed between both sides of the biomembrane. Experimental Section Both miltefosine and OPPE were supplied by Sigma (99%). The compounds were stored in a refrigerator without the access of light. Spreading solutions were prepared by weighing a proper amount (typically 2-3 mg) of the investigated compound (miltefosine/OPPE) on the analytical balance (accurate to 0.1 mg) and dissolving each of the compounds in a 4:1 mixture of chloroform/ absolute ethanol (Merck, p.a.) in a 10 mL flask. Mixed solutions were prepared from the respective stock solutions of both compounds. The number of molecules spread on a water subphase (3.2 × 1016 molecules) with a Microman Gilson microsyringe, precise to (0.2 µL, was kept constant in all experiments. Ultrapure water (produced by a Nanopure water purification system coupled to a Milli-Q water purification system, resistivity ) 18.2 MΩ cm) was used as a subphase. The subphase temperature was controlled to within 0.1 °C by a circulating water system from Haake. HCl or NaOH was added into water to adjust the pH value of the subphase. Experiments were carried out with a NIMA 601 (Coventry, U.K.) trough (total area ) 525 cm2), placed on an antivibration table. Surface pressure was measured with the accuracy of (0.1 mN/m using a Wilhelmy plate made from chromatography paper (Whatman Chr1) as the pressure sensor. After spreading, monolayers were left for 10 min for the solvent to evaporate, and afterward the compression was initiated with a barrier speed of 15.6 Å2/(molecule min). Brewster angle microscopy (BAM) images and ellipsometric measurements were performed with a BAM-ELLI 2000 (NFT, Germany) equipped with a 30 mW laser emitting p-polarized light at 690 nm wavelength which was reflected off the air/water interface at approximately 53.1° (Brewster angle). In measuring the relative reflectivity of the film, a camera calibration was necessary as described elsewhere.9 The reflectivity at each point of the BAM image depends on the local thickness and film optical properties and can be measured by determining the light intensity at the camera and analyzing the polarization state of the reflected light employing the method based on the Fresnel reflection equation.10 At the Brewster angle

I ) |Rp|2 ) Cd2

(1)

where I is the relative reflectivity, C is a constant, d is the film thickness, and Rp is the p-component of the light. The lateral resolution of the microscope was 2 µm, and the images were digitized in order to obtain high-quality BAM images.

Results Monolayers of Pure Components. Miltefosine was found to form stable liquid-expanded Langmuir monolayers,1 which are scarcely influenced by diverse experimental conditions, such as compression rate, number of spread molecules, subphase temperature (within the range of 10-25 °C), and subphase pH (within the range of 2-12). BAM images taken along the full monolayer compression at 20 °C on water (pH 6) are completely homogeneous (Figure 1, images A and B). Only at surface pressures close to the monolayer collapse (ca. 39 mN/m) do some small domains start to appear (Figure 1, image C) which increase in amount as the collapse proceeds. OPPE monolayers show a transition region, visualized as a pseudoplateau on the π-A isotherms (Figure 2), at surface pressures of around 35 mN/m (pH 2), 38.5 mN/m (pH 6), and 42 mN/m (pH 10). This first-order phase (8) Rey Go´mez-Serranillos, I.; Minõnes, J., Jr.; Dynarowicz-Ła¸ tka, P.; Iribarnegaray, E.; Casas, M. In preparation. (9) Rodriguez Patino, J.; Sanchez, C. C.; Rodriguez Ninõ, M. R. Langmuir 1999, 15, 2484. (10) Azzam, R. M. A.; Bashara, N. M. In Ellipsometry and Polarized Light, 1st ed.; North-Holland: Amsterdam, 1992.

Figure 1. Surface pressure (π)-area (A) isotherm of miltefosine spread on water, pH 6, at 20 °C, together with BAM images taken at the surface pressures indicated by arrows. Inset: compression modulus (Cs-1) vs surface pressure (π) curve.

Figure 2. Surface pressure (π)-area (A) isotherms of OPPE spread at 20 °C on aqueous subphases of pH 2, 6, and 10. Inset: Compression modulus (Cs-1)-surface pressure (π) dependencies.

transition appears in the compression modulus (or elasticity) (Cs-1) vs surface pressure (π) curves as a pronounced minimum (see the inset of Figure 2). Maximum Cs-1 values for OPPE monolayers spread on different subphase pHs range from 80 to 120 mN/m, which suggests their liquidcondensed (LC) character. The time evolution of relative reflectivity (I-t curves) and surface pressure (π-t curves) during a compressionexpansion cycle for OPPE monolayers spread on an aqueous subphase of pH 6, at 20 °C, is shown in Figure 3. Upon compression, in the liquid-expanded-liquidcondensed transition, the relative reflectivity is maintained constant at I ) 0 (arbitrary units) until the area of 135 Å2 (t ) 450 s) is reached. At this area the surface pressure is still zero; however, the relative reflectivity reaches the value of 1 × 10-6 (arbitrary units), and upon further compression, it keeps on increasing monotonically without any visible discontinuity until the collapse is reached at about 53 mN/m. In the collapse region, a number of noise peaks appear due to the formation of 3D structures. Upon film decompression, the relative reflectivity-time curve is practically symmetrical to that recorded on compression, similarly to the π-A isotherm (although in the latter there is a visible discontinuity at 35 mN/), proving the quasi-reversible behavior of the monolayer during the compression-decompression cycle. In another words, we may state that the monolayer adopts the same surface states upon monolayer compression and

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Figure 3. The relative reflectivity (I) vs time (t) dependence and surface pressure-area isotherm of an OPPE monolayer spread on water, pH 6, at 20 °C. Inset: Compression-decompression cycle.

Rey Go´ mez-Serranillos et al.

expansion, with identical phase transition in both cases, although the molecular area values show some hysteresis (see the inset of Figure 3). BAM images for an OPPE monolayer spread at pH 6 are shown in Figure 4. At zero surface pressure, the presence of a number of bright domains immersed in the expanded phase (gray zone) can be seen (image A). These domains exhibit optical anisotropy, clearly seen by rotating the analyzer to 60° (image B), which evidences a different orientation of the aliphatic tail groups. Because of a low viscosity of the expanded phase, the domains are continuously moving. At 15.5 mN/m, the domains increase in number and are immersed in water (black zones) (image C). As the transition region is reached (surface pressures between 36 and 39 mN/m), the domains still exhibit optical anisotropy (some domains are less bright than the others, image D taken at 36 mN/m), i.e., domains having the tilt azimuth of the molecules in the p-plane look bright,

Figure 4. BAM images of an OPPE monolayer spread on water, pH 6, at 20 °C at different stages of compression.


Figure 5. Surface pressure (π)-area (A) isotherms of OPPE, miltefosine, and their mixtures spread on an aqueous subphase of pH 2 at 20 °C. Inset: Compression modulus (Cs-1)-surface pressure (π) dependencies.

Figure 6. Surface pressure (π)-area (A) isotherms of OPPE, miltefosine, and their mixtures spread on an aqueous subphase of pH 6 at 20 °C. BAM images taken for XM ) 0.6 at surface pressures indicated by arrows. Inset: Compression modulus (Cs-1)-surface pressure (π) dependencies.

whereas those containing molecules with the tilt azimuth orthogonal to the p-plane look dark when the analyzer is in the p-plane. Such a contrast in domain brightness can also be observed at surface pressures above the transition (see image E, at π ) 39.6 mN/m). The collapse region is visualized as the presence of broad stripes of high intensity (image F). When the monolayer is decompressed until its initial area and zero surface pressure and then it is relaxed for 15 min, white domains of condensed phase can be seen (image G). They are responsible for the appearance of noise (visible in Figure 3) at the end of film decompression. Mixed Monolayers of Miltefosine and OPPE. Figures 5-7 show the π/A isotherms of miltefosine (M), OPPE (P), and their mixtures of miltefosine molar fraction XM ) 0.2, 0.4, 0.6, and 0.8, spread at 20 °C on aqueous subphases of pH 2 (Figure 5), pH 6 (Figure 6), and pH 10

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Figure 7. Surface pressure (π)-area (A) isotherms of OPPE, miltefosine, and their mixtures spread on an aqueous subphase of pH 10 at 20 °C. Inset: Compression modulus (Cs-1)-surface pressure (π) dependencies.

(Figure 7). As can be observed, the progressive addition of miltefosine into the OPPE film affects the transition surface pressure, elasticity (Cs-1), stability (quantified by the collapse pressure, πc), and the lift-off area (Ao) value of the phospholipid monolayer. Regarding the phase transition, the influence of miltefosine is twofold. At pH 2, the addition of miltefosine to OPPE (mixed films of XM ) 0.2 and 0.4) shifts the transition surface pressure toward higher values. However, further increase in miltefosine proportion causes the disappearance of the plateau transition. In general, the higher the pH value, the smaller the amount of miltefosine required for the transition disappearance. The compression modulus-surface pressure curves clearly indicate that the transition (sharp minimum) disappears at pH 2 for mixtures exceeding XM ) 0.4 and at pH 6 for mixtures of XM > 0.2. Interestingly, at pH 10 none of the mixed films exhibit the sharp minimum in the Cs-1-π curves (nor the plateau in the π-A isotherms). In the expanded region, all mixed films show homogeneous structure as exemplified for the mixture of XM ) 0.6 at pH 6 in Figure 6, image A. Upon compression, no changes in film structure are observed, which indicates a liquid character of the monolayers, until the collapse is reached (image B, Figure 6). The Cs-1-π plots also indicate the decrease in film elasticity upon incorporation of miltefosine into the OPPE monolayer. At all the investigated pH values, mixed films that exhibit the plateau transition are characterized by Cs-1 values of ca. 100-120 mN/m in the preplateau region, indicating their LC state. On the other hand, the maximum compression modulus values for mixtures without visible transition are below 100 mN/m, proving their liquidexpanded character. Another important parameter which is influenced by the presence of miltefosine in mixed monolayers is the film stability, quantified by the collapse surface pressure (πc) value: the higher the value of πc, the more stable the monolayer (see Table 1). The analysis of the values of the collapse pressure clearly indicates that for mixtures with plateau transition (XM ) 0.2-0.4 at pH 2 and XM ) 0.2 at pH 6) the stability is nearly the same as for pure OPPE,

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Figure 8. (A) Dependence of the mean molecular area (A12) on the molar fraction of miltefosine (XM) in mixed films with OPPE, spread on aqueous subphases of pH 2, 6, and 10 at 20 °C. (B) Plots of the excess area of mixing (Aexc) versus molar fraction of miltefosine (XM) in mixed films with OPPE, spread on aqueous subphases of pH 2, 6, and 10 at 20 °C. Table 1. Compression Modulus (Cs-1, mN/m) and Collapse Pressure (πc, mN/m) Values for Monolayers of OPPE, Miltefosine, and Their Mixtures at Different Subphase pHs at 20 °C pH 2 monolayer composition

Cs-1 1st maximum

Cs-1 2nd maximum

OPPE XM ) 0.2 XM ) 0.4 XM ) 0.6 XM ) 0.8 miltfosine

125 113 104 81 64 43

117 71 35

pH 6 πc

Cs-1 1st maximum

Cs-1 2nd maximum

53.4 52.8 54.0 48.0 50.4 36.5

85 80 86 57 56 47

75 62

since their collapse pressures nearly coincide. The stability of mixed monolayers with higher miltefosine proportion lies between those for pure components. At pH 10, none of the mixed films exhibit a phase transition. In this case, the mixtures of XM ) 0.2-0.6 are almost as stable as OPPE alone, and only the mixed monolayer of XM ) 0.8 shows the decreased stability. The extent of contraction/expansion of mixed monolayers in respect to pure components is a measure of interaction between film-forming components and can be clearly visualized on diagrams representing mean molecular area as a function of film composition (A12 ) f(XM)). Such dependencies have been plotted in Figure 8A for

pH 10 πc

Cs-1 1st maximum

Cs-1 2nd maximum

56.3 54.1 51.3 49.9 49.9 39.5

112 109 86 65 54 48

70

πc 49.4 51.6 51.0 50.4 44.5 37.5

different subphase pH values at constant surface pressures of 5, 20, and 30 mN/m. The dotted lines illustrate the additive relationship for the ideal system: A12 ) A1X1 + A2X2,11 wherein A12 is the mean molecular area of the mixed monolayer, X1 and X2 denote the mole fractions of components 1 and 2, and A1 and A2 stand for molecular areas of pure components at the same surface pressure as A12 was determined. Within the whole range of the investigated pHs, deviations from ideality can be observed both in low and high surface pressure regions. The kind of deviation, however, differs depending on the subphase pH. At pH 10, negative deviations occur within the whole range of mole fraction studied. On the other hand, at pH


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6 negative deviations appear only at low surface pressures, while at higher pressures the behavior is different. Namely, the mixed monolayer of XM ) 0.4 shows positive deviations while that of XM ) 0.8 exhibits negative deviations from ideality. Mixed monolayers containing 20 and 60 mol % of miltefosine show ideal behavior. At acidic pH, the film of XM ) 0.8 shows positive deviations, while the others either behave ideally or exhibit negative deviations. Regarding the magnitude of the discussed deviations, the strongest are observed at pH 10, especially for the mixture of XM ) 0.6. Comparing the A12-XM plots at the three different pH values studied, one may conclude that the interactions between miltefosine and OPPE, responsible for the observed deviations, diminish in the following order: pH 10 > pH 6 > pH 2. Independent of subphase pH, the extent of these interactions diminishes as the surface pressure increases. Quantitatively the interactions can be well defined by the excess functions, like the excess areas of mixing (Aexc) and excess free energy of mixing (∆Gexc). Values of Aexc can be calculated by comparing the area of the mixed monolayer (A12) with that for unmixed, one-component monolayers (A1 and A2), according to the equation Aexc ) A12 - (A1X1 + A2X2). If the mixture is ideal or its components are immiscible, Aexc is zero and the dependence Aexc ) f(Xi) is linear. Deviations from these conditions indicate miscibility and nonideality.12,13 Figure 8B shows the results of Aexc calculated for the investigated mixtures at different subphase pHs. On an acidic subphase, the mixed monolayer of equimolar composition shows the lowest negative values while that of XM ) 0.8 exhibits the highest positive deviations. As can be seen, the strongest deviations occur in the low surface pressure region. At pH 6, the excess area values are completely different as compared to those at pH 2, i.e., at low surface pressure the deviations are negative for all mixed monolayers. At higher pressures, the mixed films of low miltefosine contents exhibit positive deviations while those containing a high proportion of miltefosine show negative deviations. At pH 10, negative values of Aexc, observed within the whole composition range, prove the film condensation, or rather contraction, independent of surface pressure. In this case, the most negative Aexc values appear for XM ) 0.6. A more quantitative analysis of the intermolecular interactions is based on calculation of the excess free energy of mixing (∆Gexc), which can be determined from the following relation derived by Goodrich11 and Pagano and Gershfeld:14

∆Gexc ) N

∫0π(A12 - X1A1 - X2A2) dπ

(2)

Figure 9 shows ∆Gexc values at three different pH values, 20 °C, at surface pressures within the range of 5-30 mN/ m, as a function of miltefosine composition in mixed monolayers. For the acidic subphase, the most stable mixtures (showing the most negative ∆Gexc values) are those of XM between 0.4 and 0.5. On the contrary, the mixed monolayer of XM ) 0.8 is unstable, showing positive excess free energies, which are increasing with surface pressures. Different behavior is observed at pH 6 wherein the mixed monolayer of XM ) 0.8 is the most stable, contrary to pH 2. (11) Goodrich F. C. In Proceedings of the 2nd International Congress on Surface Activity; Butterworth: London, 1957; Vol. 1, p 85. (12) Costin, I. S.; Barnes, G. T. J. Colloid Interface Sci. 1975, 51, 106. (13) Bacon, K. F.; Barnes, G. T. J. Colloid Interface Sci. 1978, 67, 70. (14) Pagano, R. E.; Gershfeld, N. L. J. Phys. Chem. 1972, 76, 1238.

At pH 10, the values of ∆Gexc are negative, irrespective of a particular monolayer composition and surface pressure, which is consistent with thermodynamic conditions for miscibility and strong interactions in binary monolayers.11 The lowest ∆Gexc value, observed for XM ) 0.6, evidences the most thermodynamically stable mixture because of the strongest attractive interactions between both components. Discussion OPPE Monolayer. At areas larger than 90 Å2/molecule, where the surface pressure is nearly zero (Figure 2), BAM images show the presence of condensed bright domains immersed in gray zones (Figure 4A) which suggest that in this region there exists a phase coexistence between the liquid-expanded phase (gray zones) and the liquidcondensed phase (bright domains). This transition region is depicted in both surface pressure-area and relative reflectivity-time plots as a well-marked plateau (Figures 2 and 3) the length of which, however, is different in these two plots. Such a phenomenon, observed also for other phospholipids,15 can be attributed to a different sensitivity of both surface techniques, i.e., the results observed at the microscopic level (BAM) cannot necessarily be reflected at the macroscopic level (π-A isotherms). It may be assumed that the surface density of the OPPE monolayer in this transition region is not high enough to provoke the increase of surface pressure, although is quite sufficient to cause an abrupt change of film reflectivity. BAM images recorded at the beginning and at the end of the plateau at 35 mN/m (Figure 4, images D and E) are very similar; i.e., both contain condensed phase domains. Therefore it is logical to classify both regions separated by the plateau as liquid-condensed states: LC and LC′, in which molecular ordering is different. Similar elasticity values (Cs-1) before and after this transition (Figure 2, inset) also confirm that both phases are of the same nature. Along the transition, the condensed domains undergo aggregation, which can be attributed to a nucleation process that involves their fusion and increase in size.16 The orientation of OPPE molecules in both LC phases is not exactly vertical. As can be seen in Figure 4D,E, the brightness of domains differs; some of them are brighter than the others, which evidences a different orientation of molecules (a different azimuth tilt orientation of their tails). The coexistence of two LC surface states has been reported for fatty acids.17-19 Although in some cases the presence of two LC states was not detected in the course of the π/A isotherms, other techniques evidenced their existence.20 The limiting area of the OPPE monolayer spread on a subphase of pH 6, obtained by extrapolating the condensed region above the plateau transition to π ) 0, is 56 Å2. Interestingly, for the lecithin containing the same as OPPE apolar tail, i.e., OPPC (β oleoyl-γ palmitoyl, L-R phosphatidylcholine), the limiting area was found to be larger under the same experimental conditions (around 85 Å2/ molecule).21 This difference, however, cannot be simply (15) Minõnes, J., Jr.; Rodrı´guez Patino, J. M.; Conde, O.; Carrera, C.; Seoane, R. Colloids Surf., A 2002, 203, 273. (16) Saulnier, P.; Fousard, F.; Boury, F.; Proust, J. E. J. Colloid Interface Sci. 1999, 218, 40. (17) Durbin, M. K.; Malik, A.; Richter, A. G.; Ghaskadvi, R.; Gog, T.; Dutta, P. J. Chem. Phys. 1997, 106, 8216. (18) Overbeck, G. A.; Mo¨bius, D. J. Phys. Chem. 1993, 97, 7999. (19) Schwartz, D. K.; Knobler, C. M. J. Phys. Chem. 1993, 97, 8849. (20) Fischer, B.; Teer, E.; Knobler, C. M. J. Chem. Phys. 1995, 103, 2365. (21) Minõnes, J., Jr.; Dynarowicz-Ła¸ tka, P.; Conde, O.; Minõnes, J.; Iribarnegaray, E.; Casas, M. Colloids Surf., B 2003, 29, 205.

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Figure 9. The excess free energy of mixing (∆Gexc) as a function of mole fraction of miltefosine (XM) in mixed films with OPPE, spread on aqueous subphases of pH 2, 6, and 10 at 20 °C.

due to the larger size of the phosphatidylcholine polar group as compared to phosphatidylethanolamine, since (on the basis of crude calculations of their cross-section areas considering bond lengths and angles between them) their areas were found to be very similar. These calculations, however, do not take into account the hydration of both polar groups, which may contribute to their crosssectional area at the air/water interface. Indeed, the phosphatidylcholine group (PC) is found to be more solvated than phosphatidylethanolamine (PE),22 and this could explain the observed differences in limiting areas. Moreover, the larger size of the PC polar group could prevent the vertical tail arrangements of OPPC molecules, as compared to OPPE,23 which also contributes to the observed differences in limiting areas. At last, intermolecular interactions in OPPC monolayer between positively charged amino group and negative phosphate group of (22) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968, 163, 301. (23) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. Rev. Mod. Phys. 1999, 71, 779.

the neighboring molecules are not as strong as for OPPE,24 and this also makes a contribution to the observed difference in limiting areas in both cases. The behavior of OPPE monolayers spread on subphases of different pH (Figure 2) can be explained taking into account that the polar phosphatidylethanolamine group is zwitterionic at pH 7 ( 2.23 Therefore, at pH 2 OPPE molecules are ionized (positively charged), which provokes their repulsion and, consequently, gives rise to the expansion of the monolayer as compared to films spread on pH 6 or 10. At pH 10, the polar heads of OPPE molecules are also ionized (negatively charged), although in this case the repulsion forces between film molecules are not as strong as on an acidic subphase, since this pH value is close to the conditions at which OPPE is zwitterionic. Only at high surface pressures, when the monolayer molecules are closely packed, do small repulsion forces become apparent. Mixed Monolayers. The results obtained indicate that in the investigated system the strongest attractive in(24) Almog, R.; Berns, D. S. J. Colloid Interface Sci. 1981, 81, 332.


teractions between film components occur at pH 10. At this particular subphase condition, one may list the following observations: (i) The collapse pressure of mixed films is higher than for pure components, except for the mixture of XM ) 0.8, which collapses at intermediate surface pressure (Table 1). (ii) The characteristic LC-LC′ phase transition of OPPE does not appear in the course of any mixed isotherm (Figure 7). (iii) Mixed systems exhibit negative deviations from ideality within the whole composition range, in both high and low surface pressure regions (Figure 8A,B). (iv) The thermodynamic stability of the mixed monolayers, expressed by ∆Gexc values, is always higher than for the ideal system, independent of both film composition and surface pressure (Figure 9). All the above observations evidence the miscibility of film components as well as the existence of attractive intermolecular interactions (reflected by the negative deviations from ideal behavior). However, taking into account that both miltefosine and OPPE are negatively charged at pH 10, a repulsive interaction (positive deviations from ideality) should rather be expected, contrary to the experimental results. What kind of phenomena occurring at the interface could thus be responsible for such a peculiar behavior? First, because OPPE is isoelectric at pH 5-9,23 in a mixed monolayer the proportion of negatively charged molecules to those existing in the neutral form (zwitterions) is not very significant at pH 10. Second, the polar groups of both film components are anchored in the aqueous subphase and are capable of strong hydrogen bond formation with surrounding water molecules.25 Water molecules, separating polar groups, increase the distance between charged polar groups, and the repulsive interactions are weakened. The strength of electrostatic repulsions is additionally weakened by the presence of positively charged counterions, which exist in the diffuse layer beneath negatively charged polar groups and neutralize their charge. In consequence, the van der Waals attraction forces between hydrocarbon tails of miltefosine and OPPE predominate, giving rise to the observed negative deviations from ideality. (25) Shapovalov, V. L. Thin Solid Films 1998, 327, 599.

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A similar explanation can be put forward at pH 2, where polar groups of miltefosine and OPPE are both positively charged. However, it can be supposed that, at this subphase pH, the proportion of charged miltefosine molecules is rather small (since the phosphatidylcholine group becomes zwitterionic at pH 3),26-28 and therefore the repulsive forces between miltefosine and solvated ammonium groups of OPPE play an insignificant role, especially for mixed monolayers of small miltefosine proportion. This could explain the observed negative deviations from ideality, resulting from the attractions between hydrocarbon chains, which prevail over the repulsion between polar groups. Only for monolayers of high miltefosine proportion do the repulsion forces become more pronounced as the amount of charged miltefosine molecules becomes significant. This is illustrated by the positive deviations from ideality (Figure 8). At pH 6, the stability of mixed monolayers is higher as compared to the ideal behavior; however, at higher surface pressures for some mixed films, the existence of positive deviations can be noticed (Figure 9). This can be due to a change in orientation of polar groups upon compression; i.e., at low surface pressures the zwitterions of both components have coplanar (to the interface) orientation, responsible for the electrostatic intermolecular attraction (negative deviations). Upon compression, their orientation changes into vertical to the interface (at high surface pressures), causing the appearance of repulsive interactions between polar groups (positive deviations). Slightly less pronounced deviations from ideal behavior as observed herein for miltefosine-OPPE were recently reported by Rakotamanga et al.29 for mixed films of miltefosine-OPPC at pH 5.6. Acknowledgment. This financial support of Xunta de Galicia (Project PGIDT99PXI20302B) is gratefully acknowledged. LA040036V (26) Anderson, P. J.; Pethica, B. A. In Biochemical Problems of Lipids; Popjak and Le Breton: London, 1956; p 24. (27) Papahadjopoulos, D. Biochim. Biophys. Acta 1968, 163, 240. (28) Gong, K.; Feng, S. S.; Lin Go, M.; Hsing Soew, P. Colloids Surf., A 2002, 207, 113. (29) Rakotomanga, M.; Loiseau, P. M.; Saint-Pierre-Chazalet, M. Biochim. Biophys. Acta 2004, 1661, 212.

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