Mixed Monolayers of Amphotericin B−Dipalmitoyl Phosphatidyl

Langmuir 2002, 18, 2817-2827

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Mixed Monolayers of Amphotericin B-Dipalmitoyl Phosphatidyl Choline: Study of Complex Formation J. Min˜ones, Jr.,† J. Min˜ones,*,† O. Conde,† J. M. Rodriguez Patino,‡ and P. Dynarowicz-Latka§ University of Santiago de Compostela, Faculty of Pharmacy, Department of Physical Chemistry, Campus Sur, 15706 Santiago de Compostela, Spain, University of Seville, Faculty of Chemistry, Department of Chemical Engineering, c/Prof. Garcı´a Gonza´ lez, s/n. 41012 Seville, Spain, and Jagiellonian University, Faculty of Chemistry, Ingardena 3, 30-060 Kra´ kow, Poland Received August 31, 2001. In Final Form: January 2, 2002 Mixed Langmuir monolayers of amphotericin B (AmB) and dipalmitoyl phosphatidyl choline (DPPC) were investigated by recording surface pressure-area (π-A) isotherms in addition to Brewster angle microscopy. The analyses of π-A and compressional modulus curves indicate the existence of interactions in the AmB-DPPC system; the greatest were found to occur for a ca. 2:1 AmB/DPPC mixture. For this mixed monolayer (XAmB ) 0.66), the formation of a stable complex, composed of two horizontally oriented AmB molecules and one DPPC molecule in a vertical position, is suggested. For mixtures containing AmB in excess as compared to the stoichiometric mixture for the complex formation (XAmB > 0.66), the filmforming components are miscible and the mixed monolayer consists of AmB-DPPC complexes together with horizontally (at surface pressures below the first transition) or vertically (at pressures above this transition) oriented free AmB molecules. On the other hand, mixtures of AmB-DPPC complex and DPPC in excess (at XAmB < 0.66) were found to be miscible at surface pressures below the second transition. However, the system becomes immiscible at higher surface pressures.

Introduction Amphotericin B (AmB) has potent antimycotic properties1-3 as is demonstrated by the fact that after more than 30 years of use and although other antifungal agents such as miconazole and ketoconazole have been introduced into the pharmaceutical industry, it continues to be the drug of choice in the treatment of the majority of fungal infections. This may be due to the fact that there is practically no resistance to this antibiotic and, moreover, to the fact that the spectrum of the antifungal activity of AmB is very wide. The mechanism of AmB’s action is still not well-known. The most widely accepted view holds that the polyene antibiotics act at the fungi cellular membrane level, causing enhanced permeability and the loss of a number of vital elements from the cell.4,5 In this way, cellular metabolism is altered along with vital cellular functions, which provokes lysis and the ultimate death of the cells. The administration of amphotericin B induces an important loss of potassium ions6,7 with a subsequent loss (within 10 min) of magnesium ions. Likewise, the cellular transport of amino acids is significantly reduced and * To whom correspondence should be addressed. Prof. J. Min˜ones, Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, 15706 Santiago de Compostela, Spain. Fax: 34-981-594912. E-mail: [email protected]. † University of Santiago de Compostela. ‡ University of Seville. § Jagiellonian University. (1) Medoff, G.; Brajtburg, J.; Kobayashi, G. S. Annu. Rev. Pharmacol. Toxicol. 1983, 23, 30. (2) Sabra, R.; Branch, R. A. Drug Saf. 1990, 5, 2, 94. (3) Gallis, H. A.; Drew, R. H.; Pickard, W. W. Rev. Infect. Dis. 1990, 12, 308. (4) Busch, H.; Lane, M. In Chemotherapy. An introductory Text; Year Book Medical: Chicago, 1967. (5) Bolard, J. Biochim. Biophys. Acta 1986, 864, 257. (6) Kinsky, S. C. J. Bacteriol. 1961, 82, 889. (7) Zygmunt, W. A. Appl. Microbiol. 1966, 14, 953.

protein synthesis in the fungi is inhibited. The synthesis of RNA and the uptake of glucose are completely stopped within 20 min. Amphotericin B applied in higher doses also causes the loss of other essential elements from the cell, such as, for example, nucleic acids.8 In any event, the key role in all the observed effects on cellular functions seems to be played by the loss of potassium and magnesium ions. It is generally believed that the enhanced permeability of membranes caused by AmB is due to the formation of transmembrane ion-permeable pores or channels (Figure 1), in which the AmB molecules are in a quasi-parallel orientation, with the polar sides (hydroxyl groups) pointing toward the inside of the pore and their lipophilic parts interacting with the lipid environment.9-11 However, it has been reported that AmB’s activity is limited to membranes that contain sterols,12,13 such as those that can be found in fungi, protozoa, higher algae, and mammalian erythrocytes. Other organisms, like bacteria or viruses, which do not contain sterols, are insensitive to AmB.14 Furthermore, in vitro studies based on model lipid membranes have shown that AmB has a higher affinity for the ergosterol-rich membranes than for those containing cholesterol.15-19 Nonetheless, despite all the (8) Kinsky, S. C. Annu. Rev. Pharmacol. 1970, 10, 119. (9) Holtz, R.; Filkenstein, A. J. Gen. Physiol. 1970, 56, 125. (10) Ermishkin, L. N.; Kasumov, Kh. M.; Potzeluyev, V. M. Nature 1976, 262, 698. (11) Andreoli, T. E. Ann. N.Y. Acad. Sci. 1974, 235, 469. (12) De Kruijff, B.; Demel, R. A. Biochim. Biophys. Acta 1974, 339, 57. (13) Lampen, J. E.; Arrow, P. M.; Safferman, R. S. J. Bacteriol. 1960, 80, 200. (14) Weete, J. D. Photochemistry 1973, 12, 1843. (15) Kinsky, S. C.; Luse, S. A.; Van Deenen, L. L. Fed. Proc. 1966, 25, 1503. (16) Gruda, J.; Nadeau, P.; Brajtburg, J.; Medoff, G. Biochim. Biophys. Acta 1980, 602, 260. (17) Vertut-Croquin, A.; Bolard, J.; Chambert, M.; Gary-Bobo, C. M. Biochemistry 1983, 22, 2939.

10.1021/la011378t CCC: $22.00 © 2002 American Chemical Society Published on Web 02/09/2002

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Figure 1. Schematic representation of the mechanism of action of AmB.

aforementioned knowledge, the actual details of the molecular mechanism of the interaction of amphotericin B with biological membranes, as well as the formation of the membrane pore structures, are still not well understood, and in view of recent reports, it seems likely that the mechanism of AmB’s action is more complex than was previously believed. Interestingly enough, when studies are performed on model membrane systems, the interpretation does not become simpler as one might expect; rather, in fact, contradictory results are found in the literature on the subject. For example, it has been postulated that amphotericin B becomes active in cholesterol-containing membranes only in the self-associated form.20,21 Balakrishnan et al.22 have shown that AmB was capable of undergoing a concentration-dependent aggregation when incorporated into bilayer membrane systems without sterols and have proposed the existence of an organized multimolecular structure, in which AmB interacts with the acyl chains, forming a 1:1 complex. Other authors23,24 have suggested that the interactions between drug molecules (AmB/AmB), but not the AmB/sterol complex, are responsible for the antifungal activity of amphotericin B. This model has been supported by the results of Cohen25 and Lambing et al.,26 who proposed that AmB oligomers themselves may act as protochannels, independent of sterols. Fujii et al.27 also reported that the presence of sterols is not essential for the activity of AmB, since it can interact specifically with phospholipids and thus form pores in biological membranes. In this way, the sterols would only act indirectly, modifying the packing arrangement of phospholipids in the membrane and thereby making it more accessible for the antibiotic molecules.28-30 According to this mechanism, the interaction AmB(18) Readio, J. D.; Bittmann, R. Biochim. Biophys. Acta 1982, 685, 219. (19) Brutyan, R. A.; McPhie, P. J. Gen. Physiol. 1996, 107, 69. (20) Bolard, J.; Vertut-Croquin, A.; Cybulska, B.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1981, 647, 241. (21) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry 1991, 30, 5707. (22) Balakrishnan, R.; Easwaran, K. R. K. Biochemistry 1993, 32, 4139. (23) Chapados, C.; Barwicz, J.; Gruda, J. Biophys. Chem. 1994, 51, 71. (24) Tancrede, P.; Barwicz, J.; Jutras, S.; Gruda, J. Biochim. Biophys. Acta 1990, 1030, 289. (25) Cohen, B. E. Biochim. Biophys. Acta 1992, 1108, 49. (26) Lambing, H. E.; Wolf, B. D.; Hartsel, S. C. Biochim. Biophys. Acta 1993, 1152, 185. (27) Fujii, G.; Chang, J. E.; Coley, T.; Steere, B. Biochemistry 1997, 36, 4959. (28) Marty, A.; Finkelstein, A. J. Gen. Physiol. 1975, 65, 515. (29) Kleimberg, M. E.; Finkelstein, A. J. Membr. Biol. 1984, 80, 257. (30) Hsuchen, C. C.; Feingold, D. S. Biochem. Biophys. Res. Commun. 1973, 51, 972.

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membrane requires that the latter be found in a disordered state, which cannot always be achieved. More recent papers by Cotero et al.31 and Ruckwardt et al.32 also advance the idea of the indirect involvement of sterols in the channel formation and postulate that AmB can form channels in sterol-free membranes. However, the idea that AmB does not require sterols to form ion-selective channels has been criticized20 on the grounds that egg phospholipids, commonly used in these experiments, contain small quantities of sterols, which can associate with AmB and form channel structures. Given such contradictory results, it is obvious that clear evidence of the mechanism, which unambiguously explains the action of polyene antibiotics, does not yet exist. It is likely that AmB possesses several distinct mechanisms of channel activity, depending on the specific conditions. In this paper, we show the existence of significant interactions in monolayers between AmB and a synthetic model membrane phospholipid, namely, dipalmitoyl phosphatidyl choline (DPPC), since it is quite possible that these interactions compete with the interaction between the polyene antibiotic and sterols in the cellular membrane. Experimental Section Materials. L-R-Phosphatidylcholine dipalmitoyl (DPPC) (99%) was purchased from Sigma. Squibb (Bristol-Myers Lab) supplied amphotericin B, and its purity was specified as higher than 95%. The phospholipid was dissolved in a chloroform/ethanol mixture (4:1 v/v), and AmB was dissolved in a mixture of dimethylformamide and 1 M HCl (3:1 v/v). The spreading solutions of mixtures were prepared from the respective stock solutions of both components. The solutions were prepared every 2 days and stored at 4 °C in a desiccator saturated with the spreading solvent. The number of molecules spread on the subphase (4.25 × 1016), deposited by a Microman Gilson microsyringe (precise to (0.2 µL), was kept constant in all experiments. Ultrapure water, used as a subphase, was obtained from the Milli RO, Milli Q reverse osmosis system (Millipore Corp.), containing two carbon and two ion-exchange columns. Finally, water was purified through a 0.22 µm Zetapore filter. The resistivity of purified water was 18 MΩ cm. The absence of active surface contaminants in the water subphase was checked by surface tension measurements. No aqueous solution with a surface tension other than that accepted in the literature was used (72-73 mN/m at 20 °C). The subphase temperature was maintained constant at 20° C. Surface Film Balance. The Langmuir-Blodgett KSV-5000 (Finland) trough, equipped with two symmetrical compartments, each 71 × 12 cm2, was used to record the surface pressure/area (π-A) curves. The isotherms were obtained by simultaneous compression of two monolayers spread on the water subphase of each compartment. Compression was carried out with two barriers, moving with the same speed from the edge of each compartment toward its central part where the Wilhelmy plate, used as the surface pressure sensor, was placed. This procedure enabled us to register simultaneously, under identical experimental conditions, the compression isotherms corresponding to the individual monolayer spread in each compartment. Good reproducibility of the isotherms, recorded in two successive compression experiments, was the evidence for reliable results. Each π-A isotherm shown in this paper represents the average of four independent experiments. The monolayers were compressed with a barrier speed of 6.2 Å2/molecule min. Brewster Angle Microscopy. For microscopic observation of the monolayers’ topography, the Brewster angle microscope, BAM 2 (NFT, Go¨ttingen, Germany), equipped with a 30 mW laser emitting p-polarized light at a 690 nm wavelength, was used; light was reflected off the air/water interface at ap(31) Cotero, B. V.; Rebolledo-Antu´nez, S.; Ortega-Blake, I. Biochim. Biophys. Acta 1998, 1375, 43. (32) Ruckwardt, T.; Scott, A.; Scott, J.; Mikulecky, P.; Hartsel, S. C. Biochim. Biophys. Acta 1998, 1372, 283.

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Figure 2. Surface pressure-area (π-A) isotherms for mixed monolayers of AmB and DPPC of low AmB content (XAmB < 0.5) spread on water at pH 6 and at 20 °C. Inset: compressional modulus-surface pressure (Cs-1-π) curves. proximately 53.1° (Brewster angle). The lateral resolution of the microscope was 2 µm, and the images were digitized and processed in order to obtain high-quality images. The measurements were performed upon monolayer compression using different shutter speeds (within the range of 1/125-1/500 s). Camera calibration was necessary in order to determine the relationship between the intensity unit (i.e., “gray level”) (GL) and relative reflectivity (I) for different shutter speeds.33,34

Results Surface Pressure-Area (π-A) Isotherms and Compressibility of AmB-DPPC Mixed Monolayers. Figure 2 shows the π-A isotherms for both pure phospholipid and AmB-DPPC mixtures (for XAmB ranging from 0.1 to 0.5) spread on a pure water subphase (pH ) 6). The monolayer of DPPC exhibits, at a surface pressure of 4 mN/m, a typical LE-LC phase transition evidenced as a plateau in the π-A isotherm. This plateau gradually disappears when AmB is incorporated into the phospholipid monolayer (XAmB ) 0.1 and 0.3), and the transition shifts to higher surface pressures (5.7 and 9 mN/m, respectively). This can also be observed in the surface compressional modulus [Cs-1 ) -A(dπ/dA), where (dπ/ dA) is the slope of the π-A isotherm] versus surface pressure (Cs-1-π) curves, shown in the inset of Figure 2, wherein the first minimum (L) corresponds to the above(33) Rodriguez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R. Langmuir 1999, 15, 2484. (34) Rodriguez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R. Food Hydrocolloids 1999, 13, 401.

mentioned phase transition. For mixtures with increasing amounts of AmB, this minimum appears at respectively higher surface pressures, and the increasing values of Cs-1 indicate its progressive disappearance. As for curves 2-4, at surface pressures of about 16-17 mN/m, a second minimum (M) can also be distinguished. In the case of curve 2, the minimum is not very distinct and, in fact, it is reduced to a simple inflection. This minimum M, which corresponds to a second phase transition in the mixed monolayer, is not visualized in the π-A isotherm. However, the Cs-1-π plot demonstrates its existence. With respect to the mixed monolayer of XAmB ) 0.5, the pressure/ area isotherm shows an elongated pseudo-plateau region wherein the surface pressure is maintained nearly constant. The corresponding Cs-1-π plot (curve 4) exhibits a broad minimum M, thus demonstrating the existence of a phase transition. Figure 3 shows the results for mixed monolayers containing AmB in proportions exceeding 50% (in moles). The mixed film of XAmB ) 0.7 still exhibits the phase transition M at a surface pressure of 16.5 mN/m. The corresponding Cs-1-π curve also shows a characteristic broad region where the surface compressional modulus values remain practically constant. The mixture of XAmB ) 0.9 exhibits a transition at about 12 mN/m (a surface pressure slightly higher than that for the transition of pure AmB). A second transition, at higher surface pressures, can hardly be distinguished. However, in the corresponding Cs-1-π plot, in addition to the first minimum (A) representing the phase transition characteristic

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Figure 3. Surface pressure-area (π-A) isotherms for mixed monolayers of AmB and DPPC, containing a high proportion of AmB (XAmB > 0.5), spread on water at pH 6 and at 20 °C. Inset: compressional modulus-surface pressure (Cs-1-π) curves.

of AmB, a second minimum, M, less defined, is observed at a surface pressure of about 18 mN/m. Finally, the monolayer for pure AmB shows three distinct regions: the first that corresponds to the monolayer in its liquid expanded state; the second, of a liquid condensed nature, that is characterized by high Cs-1 values; and the third, that spans between both of the abovementioned regions and that is represented by a broad plateau of constant surface pressure at about 10 mN/m, which is due to the phase transition from the liquid expanded to the liquid condensed state. In summary, we can conclude that the addition of AmB into the DPPC monolayer causes film expansion and provokes the disappearance of the phase transition (L), which is characteristic of pure phospholipids. However, the mixed monolayers show a phase transition M at higher surface pressures than those corresponding to the phase transition of pure phospholipid (4 mN/m) or the antibiotic (10 mN/m). The surface pressure of this transition hardly varies as the composition of the mixed films changes as can be clearly seen in Table 1, which also compiles the results obtained for subphases at pH 3 and 10, whose isotherms are very similar to those recorded at pH 6 (results not shown). Brewster Angle Microscopy. Brewster angle microscopy allows us to directly visualize the phase transitions observed in both the π-A and Cs-1-π curves. Figure 4 shows relative reflectivity (I) as a function of the surface pressure for monolayers of AmB, DPPC, and their mixture of XAmB ) 0.5 spread on water (pH ) 6). For the

Table 1. Transition Surface Pressures for AmB-DPPC Mixed Monolayers Spread on Aqueous Subphases at Different pHs transition surface pressure (mN/m) molar fraction of AmB (XAmB) 0 (DPPC) 0.1 0.3 0.5 0.7 0.85 0.9 1.0 (AmB)

pH ) 3 L

M

pH ) 6 A

3 4.8 13.3 7.7 14.0 14.3 14.4 14.5 10.3 9.7

L

M

pH ) 10 A

L

M

A

3.9 5.7 17.1 9.0 16.2 16.0 16.5

5.1 5.6 15.0 7.2 15.1 7.6 16.2 16.2 15.9 11.2 18.0 11.9 15.6 11.1 9.9 9.9

phospholipid, the shutter speed was adjusted to 1/125 s, while for AmB the value of 1/500 s was applied since a longer exposure time causes camera saturation. For the mixed film, the value of 1/250 s was used. The results obtained for the pure AmB monolayer show that in the liquid expanded (LE) state, at surface pressures below 10 mN/m, the relative reflectivity is low (1.5 × 10-6) and increases upon film compression up to 2.5 × 10-6, at the beginning of the liquid expanded to liquid condensed (LC) transition. It is thus evident that the monolayer in the LE phase is thin. However, when the LE-LC transition is attained (i.e., above 10 mN/m), relative reflectivity increases rapidly. Finally, in the LC state its value reaches 1.1 × 10-5 and is maintained nearly constant throughout this region. As relative reflectivity (I) and

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Figure 4. Relative reflectivity-surface pressure (I-π) plot for the compression of AmB, DPPC, and the AmB-DPPC mixed monolayer of XAmB ) 0.5 spread on water at pH 6 and at 20 °C.

monolayer thickness (d) are related by the expression35 I ) Cd2 (wherein C is a constant), it is evident that a ca. 10-fold increase in relative reflectivity upon compression of the AmB monolayer from the expanded to the condensed phase corresponds to a 3-fold increase in its film thickness. The relative reflectivity curve for the DPPC monolayer shows only a small increase of I at 5 mN/m, which is related to the LE-LC phase transition. In this case, the absence of reflectivity peaks as well as the small values of relative reflectivity indicate that the domains of DPPC are significantly smaller than those of AmB, as is confirmed by images A and B in Figure 5. At surface pressures lower than 5 mN/m, the relative reflectivity plot for the mixed monolayer resembles that for pure AmB, except for the absence of reflectivity peaks characteristic of the latter. As the monolayer compression proceeds, a discontinuity appears in the I-π curve at a surface pressure of 16-18 mN/m. This break in the plot may be attributed to the phase transition M described above. At higher surface pressures (above ca. 25 mN/m), relative reflectivity is maintained practically constant and the corresponding BAM image is almost completely homogeneous (Figure 5C). For the mixed monolayer containing XAmB ) 0.7, an abrupt increase in relative reflectivity above π ∼ 16 mN/m was observed (Figure 6). However, the signals were more intense than those observed for the mixture of XAmB ) 0.5. Moreover, the I-π plot exhibits a number of reflectivity peaks, similar to those of the pure AmB monolayer, which indicates the presence of compact domains of AmB (Figure 5, image D) with optical anisotropy (Figure 5, image D′ taken with the analyzer rotated at 90°). (35) Azzam, R. M. A.; Bashara, N. M. In Ellipsometry and Polarized Light, 1st ed.; Noth Holland: Amsterdam, 1997.

The I-π curve for the mixed monolayer of XAmB ) 0.9 (Figure 7) is similar to that for pure AmB at π values lower than 10 mN/m. Both systems are characterized by the presence of a number of reflectivity peaks of intermediate intensity. This behavior is due to the fact that at the interface the AmB structure predominates over that of phospholipid as its proportion in the mixed monolayer also prevails. However, the presence of DPPC in the mixed film was observed at π higher than 10 mN/m. In fact, at this surface pressure the relative reflectivity for the pure AmB monolayer exhibits an abrupt increase (due to the LE-LC phase transition of the polyene), but for the mixed film the values of I begin to rise at higher surface pressures, at about 17 mN/m. This is in accordance with the results of the π-A isotherms shown in Figure 2, where the transition is attained at higher surface pressures for the mixed monolayer (16-17 mN/m) than for pure AmB (10 mN/m). In the post-transition region, both reflectivity curves (for AmB and the mixture) coincide and only a few reflectivity peaks can be registered, which is probably due to camera saturation despite the highest shutter speed applied here (1/500 s). The predominance of the AmB structure over that of DPPC when both compounds were spread as mixed films was confirmed by the BAM images. The presence of a number of rectangular domains, typical of AmB, can be seen at low surface pressures, below 10 mN/m (Figure 5E). Upon film compression up to 19 mN/m, these domains are packed together to form more compact structures. Above this pressure, the film structure becomes practically uniform, although large domains of AmB, responsible for the reflectivity peaks, are visible (Figure 5F). Mean Molecular Area of AmB-DPPC Mixed Monolayers. The plots of the mean molecular area (A)

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Figure 5. BAM images corresponding to (A) the AmB monolayer at π ) 1 mN/m; (B) the DPPC monolayer at π ) 15.7 mN/m; (C) the AmB-DPPC mixed monolayer at XAmB ) 0.5 at π ) 54 N/m; (D) the AmB-DPPC mixed monolayer at XAmB ) 0.7 and at π ) 4.4 mN/m; (D′) as above, with the analyzer rotated at 90°; (E) the AmB-DPPC mixed monolayer at XAmB ) 0.9 and at π ) 7.2 mN/m; (F) as in (E) at π ) 19 mN/m.

as a function of XAmB for mixed monolayers are shown in Figure 8. For the low (5 mN/m) and intermediate surface pressures (15 mN/m), the deviations from the ideality are

positive for all compositions, except that for the AmB-rich mixture of XAmB ) 0.9. The highest deviations occur for the mixed films of XAmB ) 0.5 and 0.7. For the high surface

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Figure 6. Relative reflectivity-surface pressure (I-π) curves corresponding to the compression of AmB, DPPC, and the AmBDPPC mixed monolayer at XAmB ) 0.7, spread on water at pH 6 and at 20 °C.

pressure region (30 mN/m), the experimental results of mean molecular area nearly coincide with theoretical values, calculated on the basis of the additivity relation. Discussion AmB-DPPC Complex Formation and Its Stoichiometry. AmB-DPPC mixtures show several common features. First, all of the mixed films exhibit a transition between LE-LC structures at surface pressures of about 16-18 mN/m, which is ascribed to the presence of AmB in the mixed monolayer since this component has a characteristic first-order phase transition, albeit at significantly lower surface pressures (10 mN/m). This phase transition has been attributed36,37 to the change in orientation of AmB molecules from the horizontal position (in the liquid expanded phase) to the vertical position when the molecules are in the liquid condensed state. The relative reflectivity values (Figure 4) confirm that AmB undergoes an orientational change as reflectivity increases from 1.5 × 10-6 to 1.1 × 10-5 (≈10 times) upon monolayer compression from the expanded to the condensed state, which corresponds to a 3-fold increase in the film thickness. This value closely approximates that calculated from the length of the AmB molecule in the vertical position (24 or 20 Å2 depending on whether the polar group is considered to be immersed in the subphase)38,39 and in the horizontal position (7 Å2). (36) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyrat, M.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1988, 944, 477. (37) Min˜ones, J., Jr.; Carrera, C.; Dynarowicz-Latka, P.; Min˜ones, J.; Conde, O.; Seoane, R.; Rodriguez Patino, J. M. Langmuir 2001, 17, 1477. (38) Wo´jtowicz, K.; Gruszecki, W. I.; Walicka, M.; Barwicz, J. Biochim. Biophys. Acta 1998, 1373, 220.

The phase transition for mixed monolayers can also be attributed to orientational changes. However, the reorientation of AmB in the mixtures is hindered by the presence of phospholipid molecules, due to the existence of interactions between both components. Therefore, the LE-LC transition occurs at higher surface pressures (∼17 mN/m) for mixed films, instead of 10 mN/m as for the pure AmB monolayer. In the absence of interactions between film-forming components (or when film-forming molecules are immiscible at the surface), the phase transition of AmB should not be altered by the presence of DPPC. That is, the transition should occur at the same surface pressure value, that is, at 10 mN/m for pure and mixed monolayers. Thus, the results prove the existence of interactions (and miscibility) between AmB and DPPC at the air/water interface: the higher the surface pressure of transition, the greater the interaction. Furthermore, the length and the flatness of the pseudo-plateau can also be useful in quantifying the extent of the interaction, reaching the maximum value at XAmB ) 0.7, that is, approximately when the film components are in the proportion of 2:1 (AmB/DDPC), which exactly corresponds to XAmB ) 0.66. This is an intermediate value between those of XAmB ) 0.5 and XAmB ) 0.7, for which the highest deviations from ideality were observed in the mean molecular area versus XAmB plots (Figure 8). In conclusion, the existence of the maximum interaction in the mixture at XAmB ) 0.66 allows us to conclude that under these conditions the film components form a stable complex, which is denoted as C(h-v), composed of two horizontally oriented AmB molecules and one DPPC molecule in the vertical position (Figure 9). In mixed (39) Van Hoogevest, P.; de Kruiff, B. Biochim. Biophys. Acta 1978, 511, 397.

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Figure 7. Relative reflectivity-surface pressure (I-π) curves for the compression of AmB, DPPC, and the AmB-DPPC mixed monolayer at XAmB ) 0.9, spread on water at pH 6 and at 20 °C.

Figure 9. Schematic representation of the complex C(h-v) structure. Figure 8. Mean molecular areas for mixed AmB-DPPC monolayers spread on water (pH 6), plotted as a function of the molar fraction of AmB in the mixture.

monolayers of different compositions, such a complex coexists with an excess of either phospholipid (XAmB < 0.66) or AmB (XAmB > 0.66) molecules. The formation of a complex with this stoichiometry has been previously reported,36,40-42 and the existence of molecules with (40) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Biochim. Biophys. Acta 1998, 1375, 73. (41) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Langmuir 1999, 15, 3570. (42) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Langmuir 1999, 15, 5567.

different orientations in miscible mixed films was suggested by Handa et al.43 for mixtures of horizontally oriented hydroxypalmitic acid and vertically oriented stearic (or palmitic) acid molecules. In addition, the presence of a complex at the interface with a specific stoichiometry was proposed by a number of authors44-48 to explain the nonideal behavior in mixed systems. (43) Handa, T.; Tomita, K.; Nakagaki, M. Colloid Polym. Sci. 1987, 265, 250. (44) Gershfeld, N. L. J. Biophys. 1978, 22, 469. (45) Dervichian, D. J. In Surface Phenomena in Chemistry and Biology; Danielli, J. F., Pankhurst, K. G. A., Riddiford, A. C., Eds.; Pergamon: New York, 1958; p 70. (46) Shah,D. O. J. Colloid Interface Sci. 1971, 37, 744. (47) Albrecht, O.; Gruler, H.; Sackmann, H. E. J. Colloid Interface Sci. 1981, 79, 319.

Mixed Monolayers of Amphotericin B-DPPC

Figure 10. Phase diagram for AmB-DPPC mixed monolayers. C(h-v) denotes the complex formed by two AmB horizontally oriented molecules and one DPPC molecule, vertically oriented. AmBh denotes a horizontally oriented AmB molecule; AmBv corresponds to an amphotericin B molecule oriented vertically. Phosp denotes phospholipid in the expanded (Phospexp) or condensed (Phospcond) state. L corresponds to the LE-LC phase transition for DPPC, A corresponds to the phase transition of AmB, and M corresponds to the phase transition of mixed monolayers. P1, P2, P3, P4, P5, and P6 denote homogeneous phases of the system.

Miscibility of the Complex with an Excess of Particular Components (AmB or DPPC). For mixed monolayers with a different composition to that for the stoichiometric complex formation (XAmB ) 0.66), there exists an excess of either DPPC (at XAmB < 0.66) or AmB (at XAmB > 0.66) molecules. Under these conditions, there are two possibilities for the association of the film-forming components at the interface: (i) the component in excess can form a homogeneous (miscible) system with the AmBDPPC complex or (ii) the component in excess may constitute a heterogeneous and immiscible monolayer. With the aid of the phase diagram and by application of the Crisp phase rule,49,50 -F ) C - P + 1 (where F is the number of degrees of freedom, C is the number of components, and P denotes the number of phases), the number of surface phases at the equilibrium and the miscibility or immiscibility of the film-forming components in the mixed system can be determined. (a) Mixtures Containing AmB in Excess (XAmB > 0.66). The surface pressure values corresponding to the first phase transition for mixtures containing AmB in excess, as compared to the stoichiometric mixture for the complex formation (XAmB > 0.66), are compiled in column A of Table 1 and are depicted in Figure 10 as line A. In this case, C ) 2 (AmB and DPPC) and F ) 1, so the transition pressure varies with the composition of the mixed system. Therefore, the phases in equilibrium are two: P1 and P2. At surface pressures below that for transition A (π < πA), the mixed monolayer contains only one surface phase P1, in which the complexes C(h-v) are miscible with excess AmB horizontally oriented molecules (AmB(h)) (Figure 11a). It is quite possible that there exists a kind of interaction between AmB-DPPC complexes and free AmB molecules, (48) Alsina, M. A.; Mestres, C.; Valencia, G.; Garcı´a Anto´n, J. M.; Reig, F. Colloids Surf. 1989, 40, 145. (49) Crisp, D. J. In Surface Chemistry (Supplement to Research); Butterworth: London, 1949; pp 17-35. (50) Gaines, G., Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Prigogine, I., Ed.; Interscience: New York, 1966; p 283.

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since difficulties in the reorientation of free polyene molecules, caused by the presence of the complex, make the transition appear at higher surface pressures than for pure AmB. At surface pressures above the transition (π > πA), the mixed monolayer exists in another surface phase (P2), quite distinct from the previous one, which is composed of C(h-v) complexes together with AmB molecules, vertically oriented (AmB(v)) in this case (Figure 11b). Upon compression, mixed monolayers exhibit a second phase transition, denoted as M, which may be attributed to the change of orientation of AmB molecules which are embedded in the C(h-v) complex. In this situation, F ) 0 (since the surface pressure corresponding to the phase transition M practically does not vary with the monolayer composition), and three phases coexist in equilibrium (P ) 3) (see line M, Figure 10). The first one, P2, described above, is composed of mixed C(h-v) complexes with the excess of vertically oriented AmB molecules (Figure 11b). The other two phases are composed of the components segregated from the complex, that is, by phospholipid molecules (Phosp) (phase P3), which are immiscible with AmB(v) molecules (phase P4), forming two separate phases (Figure 11c). Therefore, it is clear that the change in the orientation of AmB molecules included in the complex provokes the separation of the complex’s components and leads to the formation of an immiscible system of vertically oriented AmB and DPPC molecules. (b) Mixtures Containing DPPC in Excess (XAmB < 0.66). When the amount of AmB is lower than that corresponding to the stoichiometric complex formation (XAmB < 0.66), the excess of phospholipid molecules coexists with the AmB-DPPC complex. The surface phase rule can also be applied in this case to find out whether the film-forming components of such a system are miscible or immiscible. In fact, at the transition M, the number of degrees of freedom, F, is zero (invariant system) (Figure 10). Therefore, three phases exist in equilibrium (P ) 3) along this transition. To explain the origin of these phases, the following two hypotheses can be postulated: (1) at surface pressures below the transition M, π < πM, the system components (C(h-v) complex and DPPC molecules in excess) are immiscible, and they mix at higher surface pressures; (2) the reverse situation to that described above, that is, the system is miscible at π < πM and immiscible at π > πM. According to the phase rule, in each case there exist three phases in equilibrium during the transition. The first hypothesis should be ruled out. In fact, as can be seen in Figure 10, the LE-LC transition of the phospholipid monolayer (L) varies with the composition of the mixed system. So, there exist two phases in equilibrium, namely, P5, formed by the C(h-v) complex mixed with the phospholipid in its expanded state (Phospexp), and P6, which is composed of the same complex (C(h-v)) and the phospholipid molecules in the condensed state (Phospcond). The miscibility between DPPC and the complex explains the increase of the LE-LC transition pressure with the increasing proportion of the complex in the mixed system (Figure 10) and hinders the condensation due to interactions between DPPC and the complex. If this were not the case, the surface pressure at the LE-LC transition should be independent of the mixture composition. We may thus definitely state that the C(h-v) complex and DPPC are miscible at surface pressures below the transition, that is, at π < 17 mN/m. This interpretation is compatible with the second hypothesis. In fact, at surface pressures below the transition M, the system is composed of one phase, P6,

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Figure 11. Schematic representation of the structure of AmB and C(h-v) complexes in mixed monolayers at XAmB > 0.66: (a) below the phase transition A, (b) above the phase transition A (or below the phase transition M), and (c) above the phase transition M (segregation of the complex and formation of an immiscible system).

which contains a mixture of C(h-v) complexes and an excess of phospholipid molecules (Phospcond) (Figure 12a). However, at higher surface pressures, the system becomes immiscible and contains two separate phases, P3 and P4, which are formed by the segregated components of the complex (Figure 12b). Deviations from Ideal Behavior. The immiscibility of film-forming components at surface pressures higher than that of the transition M is demonstrated by the mean molecular area versus mixed film composition plots (Figure 8). Indeed, at π ) 30 mN/m, both the additivity of the molecular areas and the nearly constant values of the transition surface pressures (Table 1) indicate the immiscibility between AmB and DPPC under these conditions, although in this situation the values for the

molecular area of AmB are uncertain, due to the instability of the AmB monolayer.51-53 On the other hand, at surface pressures below that for the transition M, the existence of attractive interactions might be expected, due to the formation of the 2:1 (AmB/ DPPC) complex that could provoke the condensation of the film components at the surface and thus the occurrence of negative deviations from ideal behavior. However, the experimental results (Figure 8) show the existence of (51) Min˜ones, J., Jr.; Min˜ones, J.; Conde, O.; Seoane, R.; DynarowiczLatka, P. Langmuir 2000, 16, 5743. (52) Rey-Go´mez-Serranillos, I.; Min˜ones, J., Jr.; Seoane, R.; Conde, O.; Casas, M. Prog. Colloid Polym. Sci. 1999, 112, 29. (53) Rey-Go´mez-Serranillos, I.; Dynarowicz-Latka, P.; Min˜ones, J., Jr.; Seoane, R. J. Colloid Interface Sci. 2001, 234, 351.

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Figure 12. Schematic representation of the structure of DPPC and C(h-v) complexes in mixed monolayers at Iamb < 0.66: (a) below the phase transition M and (b) above the phase transition M (segregation of the complex).

positive deviations. Similar results have been obtained by Saint-Pierre-Chazalet et al.36 for the AmB-cholesterol system, where the positive deviations from ideal behavior were attributed to the interactions between both components, which prevented the AmB molecules’ desorption from the surface. In a like fashion, it is possible to explain the present results since our previous studies51-53 unambiguously confirmed the occurrence of the significant loss of AmB molecules at the interface. The presence of DPPC molecules can substantially reduce the AmB desorption by interacting with the polyene molecules. This

phenomenon provokes the increase of the mean molecular area of the film, in comparison to that which it would occupy in an ideal (or immiscible) situation. As such, the stronger the interaction, the higher the deviation, as is confirmed in Figure 8 where the highest deviations occur for the mixed films of XAmB ) 0.5 and 0.7. Acknowledgment. The Xunta de Galicia (Project PGIDT99PXI20302B) supported this work. LA011378T