Interactions between Amphotericin B and Sterols in Monolayers

Langmuir 1999, 15, 5567-5573

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Interactions between Amphotericin B and Sterols in Monolayers. Mixed Films of Amphotericin B-Cholesterol R. Seoane, J. Min˜ones,* O. Conde, E. Iribarnegaray, and M. Casas Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain Received May 13, 1998. In Final Form: April 26, 1999 π-A isotherms of mixed monolayers composed of cholesterol and amphotericin B (AmB) spread on aqueous buffers of various pH and temperatures show the existence of interactions between the two components, which are more pronounced when the mole fraction of AmB is 0.7. As a consequence of the interactions, the excess areas and excess free energies of mixing are negative at low surface pressures and positive at high surface pressures. These results suggest that negative deviations of the additivity rule are due to the formation of a hydrogen-bonded AmB-cholesterol complex in which AmB molecules are oriented horizontally at the interface and cholesterol molecules lie vertically. The positive excess areas of mixing at high surface pressures could be due to AmB being less desorbed in the substrate by compositiondependent van der Waals interactions between the apolar moieties of the components, both oriented in this situation in a vertical position at the A/W interface.

Introduction The amphiphilic polyene antibiotic amphotericin B (AmB) is currently the drug of choice for treatment of severe fungal infections,1 including candidiasis,2 histoplasmosis,3 and cryptococcosis.4 Its antifungal activity, like that of other polyene antibiotics, is widely believed to be largely or wholly due to its inducing cell lysis by creating, in the fungal cell membrane, pores through which small intracellular molecules and ions are lost.5,6 De Kruij and Demel7 have suggested that the pores are constituted by AmB-sterol complexes composed of alternated antibiotic and sterol molecules whose polar groups face toward the inside of the pore, where they form hydrogen bonds with water molecules, and whose lipophilic groups contact the aliphatic chains of membrane phospholipids. According to Filkenstein et al.8,9 the function of sterols may be to order the packing of the AmB molecules. Other authors10 proposed that the action of sterols is by altering membrane phospholipid packing and thus faciliting the penetration of polyene molecules. The above theories have been questioned on the grounds that the activity of AmB depends on cell growth phase and other factors as well as on the dose of AmB and the way in which it is administered.11 It has also been reported that in some cases AmB has fungicide action even at concentrations that are too low to alter membrane permeability,12 which suggests that it may act partly or * To whom the correspondence should be addressed. Fax: +3481-594912. E-mail: [email protected]. (1) Kobayashi, G. S.; Medoff, G. Annu. Rev. Microbiol. 1977, 31, 291. (2) Hospenthal, D.; Gretzinger, K.; Rogers, A. L. J. Med. Microbiol. 1978, 30, 193. (3) Lo´pez-Berenstein, G.; McQueen, T.; Mehta, K. Cancer Drug Delivery 1985, 2, 183. (4) Dromer, F.; Barbet, J.; Bolard, J.; Charreire, J.; Yeni, P. Antimicrob. Agents Chemother. 1990, 34, 2055. (5) Bolard, J. Biochim. Biophys. Acta 1986, 864, 257. (6) Holz, R.; Finkelstein, A. J. Gen. Physiol. 1970, 56, 125. (7) De Kruij, B.; Demel, R. A. Biochim. Biophys. Acta 1974, 339, 57. (8) Marty, A.; Finkelstein, A. J. Gen. Physiol. 1975, 65, 515. (9) Kleinberg, M. E.; Finkelstein, A. J. Membr. Biol. 1984, 80, 257. (10) Hsuchen, C. C.; Feingold, D. S. Biochem. Biophys. Res. Commun. 1973, 51, 972. (11) Hartsel, S. C.; Benz, S. K.; Petterson, R. P.; White, B. S. Biochemistry 1991, 30, 77.

wholly via some other mechanism. However, in the case of filipin, Demel et al.13 have demostrated that these molecules interact specifically with steroles forming complexes of defined stoichiometry, but in the case of AmB or nystatin evidence of such complex is not clear. Ockman14 concluded, on the basis of a study of polarized absorption spectra, that cholesterol increases the ability of AmB to penetrate in lecithin monolayers, probably because it increases the degree of order among the lipid chains, and Saint-Pierre-Chazalet et al.,15 studying monolayer compression isotherms, showed that AmB-sterol complexes dissociated upon addition of a phospholipid which competed with AmB for the sterol. To obtain more information about the stoichiometry of AmB-sterol complexes, the conditions under which they exist, and the selectivity of the action of AmB, we have studied mixed monolayers of AmB and either cholesterol or ergosterol. We report here the results obtained with cholesterol. Materials and Methods AmB was provided in 95.6 purity by Bristol-Myers Squibb as a yellow powder that was not further purified. Cholesterol (Chol) (99%) was supplied by Fluka AG (Switzerland). Each component was dissolved in a 3:1 v/v mixture of dimethylformamide and 1 M hydrochloric acid (both Merck pa products), and these solutions were used to make up AmB/Chol mixtures with the desired mole ratios, which were stored in the dark in a refrigerator. Dimethylformamide was employed because AmB is insoluble in the more usual solvents (including hexane, chloroform, petroleum ether, and mixtures of these solvents with ethanol or methanol), and AmB/Chol mixtures can only be prepared if both AmB and Chol are each initially dissolved in the solvent that dissolves both components. (12) Brajtburg, J.; Powderly, W. G.; Kobayashi, G. S.; Medoff, G. Antimicrob. Agents Chemother. 1990, 34, 183. (13) Demel, D. A.; Van Deenen, L. L. M.; Kinsky, S. J. Biol. Chem. 1985, 240, 2749. (14) Ockman, N. Biochim. Biophys. Acta 1974, 345, 263. (15) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyrat, M.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1988, 944, 477.

10.1021/la980571t CCC: $18.00 © 1999 American Chemical Society Published on Web 06/25/1999

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Seoane et al. Table 1. Values of the Transition Surface Pressures, Collapse Pressures, and Compressional Modulus for Mixed Monolayers of AmB and Chol mole fraction of AmB 0.1 0.3 0.5 0.7 0.9

Figure 1. π-A curves of monolayers with various proportions of AmB/Chol spread on water at 20 °C. XAmB ) mole fraction of amphotericin B.

The AmB/Chol mixtures were spread on aqueous substrates (reagent grade water of resistivity 18 MΩ cm obtained using a Milli-RO Milli-Q system from Millipore Corp. and brought to the desired pH by addition of HCl or NaOH) in the Teflon trough of an FW-1 surface balance (Lauda, Germany) with an available spreading area of 562 cm2. Temperature was controlled by circulating water from a Grant LC10 thermostat through the trough base. Compression isotherms were recorded at a compression rate of 43 Å2/(molecule‚min). Previously, the isotherms obtained over the range 5.7-43 Å2/(molecule‚min) were found to be similar irrespectively of the compression rate used. Except where otherwise stated, the total number of AmB and Chol molecules that was deposited in each experiment was 2.3 × 1016; this allowed direct comparison of π-A curves obtained in different experiments with different mixtures. Results Results Obtained at 20 °C. Figure 1 shows the compression isotherms obtained at 20 °C for AmB, Chol, and AmB/Chol mixtures. Pure Chol (curve 1) formed a typical condensed monolayer with a compressibility of 2.88 × 10-3 m/mN (compressional modulus Cs-1 ) 347.2 mN/ m), a collapse pressure of 45 mN/m, and a limiting area (A0,min) of 38 Å2 (estimated by extrapolation of the steep, high-pressure, linear part of the π-A curve to zero surface pressure). Other authors have reported similar values of the collapse pressure16,17 and A0,min.18,19 As the measured equilibrium spreading pressure was 38 mN/m, according with previously reported values of 3620 and 39.9 mN/m,21 (16) Ries, H. E., Jr.; Swift, H. J. Colloid Interface Sci. 1978, 64, 111. (17) Ga´lvez Ruiz, M. J.; Cabrerizo Vilchez, M. A. Colloid Polymer Sci. 1991, 269, 77.

compressional modulus (mN/m) 5 25 45 130.0 95.0 52.0 37.7 42.5

120.0 80.0 87.5 34.0 45.8

189.0 112.5 108.0 100.0 135.0

transition pressure (mN/m)

collapse pressure (mN/m)

10.0 9.0, 32,5 8.0, 25.0 21.0 10.0

48.8 54.3 56.3 60.0 63.8

the monolayer at the collapse region is not in thermodynamic equilibrium. Monolayers of AmB (curve 7) exhibited two regions of different compressibilities separated by a transition phase in which the surface presure is around 10 mN/m rising gently during the compression from 130 to 70 Å2/molec probably due to the high compression rate used. The surface compressional moduli of the liquid-expanded and liquid-condensed states were 50.0 and 104.2 mN/m, respectively, and the extrapolated area at zero pressure corresponding to the condensed region was about 25 Å2, whereas the molecular area at the collapse pressure, 65 mN/m, was about 12 Å2, which clearly shows that AmB molecules are desorbed during compression. The compression isotherms of all the mixed monolayers except the 70:30 AmB/Chol film (curve 5) show plateau regions with a soft change of surface pressure beginning at surface pressures of 8-10 mN/m. As AmB concentration decreases, the area range covered by the plateau region also falls and the slope of the π-A curve in this region increases. The films with AmB mole fractions XAmB of 0.3 and 0.5 (curves 3 and 4) also exhibit a shoulder (indicated by arrows in Figure 1) at 25 and 32 mN/m, respectively. The general effect of increasing Chol concentration is to condense the expanded phase and expand the condensed phase of the AmB film. A similar effect was observed by Candenhead and Phillips.22 The π-A curve of the 70:30 AmB/Chol film (curve 5) shows no plateau region at about 10 mN/m. Instead, the transition from the surface-expanded to the surfacecondensed phase occurs at about 21 mN/m. In all regions, this film is significantly more compressible than any other (Table 1). For no monolayer other than the pure Chol film was it possible to determine the collapse pressures under the conditions used to record the compression isotherms, because the barrier reached the end of its stroke before film collapse. The collapse pressures of mixed films, recorded in supplementary experiments carried out using larger numbers of film molecules, increased with the proportion of AmB in the mixture (Table 1). To reveal more clearly the interaction between the two components of the mixtures, the mean area per molecule was plotted as a function of the mole fraction of AmB at five surface pressures, 5, 8, 20, 25, and 30 mN/m (Figure 2). The dashed lines are the mean molecular areas calculated by assuming the additivity rule. The results show that for surface pressures below the plateau in the π-A isotherms (5 and 8 mN/m) negative deviations from (18) Motomura, K.; Terazono, T.; Matuo, H.; Matuura, R. J. Colloid Interface Sci. 1976, 57, 52. (19) Mu¨ller-Landau, F.; Cadenhead, D. A. Chem. Phys. Lipids 1979, 25, 299. (20) Phillips, M. C.; Hauser, H. J. Colloid Interface Sci. 1974, 49, 31. (21) Tajima, K.; Gershfeld, N. L. Biophys. J. 1978, 22, 489. (22) Candenhead, D. A.; Phillips, M. C. Adv. Chem. Ser. 1968, No. 8, 131.

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of Chol monolayers were unaffected.24 The mixed films were rather more compressible than at neutral pH, to the point that the plateau regions in the π-A curves of monolayers with AmB contents of 30, 50, or 70% disappeared or became very much less clearly defined. Excess Free Energies of Mixing. The excess free energy of mixing was calculated from the experimental π-A curves of the mixed and pure films, following Goodrich25 and Pagano and Gershfeld,26 as

∆Gex ) N

Figure 2. Mean molecular areas, at various surface pressures, of mixed monolayers of AmB/Chol spread on water at 20 °C. XAmB ) mole fraction of amphotericin B.

ideal behavior were observed with a maximum for the mixture of XAmB ) 0.7. At surface pressures above that corresponding to the phase transition (20, 25, and 30 mN/m), deviations from the additivity rule were positive (Figure 2) and specially marked for the mixtures of XAmB ) 0.5-0.7. As the surface pressure increases, the positive deviations were less pronounced. Influence of Temperature. Compression isotherms recorded for the mixed films at intervals of 5 °C over the range 5-30 °C were in all cases morphologically similar to those of Figure 1, but temperature significantly affected mean molecular area and, as in the case of pure AmB,23 the surface pressure of the transition region fell with increasing temperature (Table 2). Below the surface pressure of the π-A plateau region, the excess areas of mixing (Aex ) A - XAmBAAmB - XCholAChol) were increasingly negative as temperature rose to 10-15 °C and thereafter became less negative as temperature continued to rise (Figure 3, lower panel). At higher surface pressures there was a general trend for the positive Aex values to decrease with increasing temperature (Figure 3, upper panel). At all surface pressures, the absolute values of Aex were generally greatest at or near a mole fraction XAMB of 0.7 (Figure 4). Influence of pH. Compression isotherms recorded at pH 3 and 10 were very similar (Figure 5 shows those obtained at pH 3), as were the corresponding graphs of excess areas of mixing against composition (Figure 5, inset), which at surface pressures above that of the transition region always peaked at XAmB ) 0.7. As reported previously,23 the molecular areas of AmB monolayers were smaller at pH 3 and 10 than at neutral pH, whereas those (23) Seoane, J. R.; Vila Romeu, N.; Min˜ones Trillo, J.; Conde O.; Dynarowicz, P.; Casas, M. Prog. Colloid Polym Sci. 1997, 105, 173.

∫0π(A1,2 - X1A1 - X2A2) dπ

where N is Avogadro’s number, A1, A2, and A1,2 are the molecular areas of the pure components and of the mixture, and X1 and X2 are the mole fractions of the components in the mixture. For the upper limit of integration values of 5, 8, 25, and 30 mN/m were used. Figure 6 shows the composition dependence of the excess free energy of mixing at 25 °C and pH 7. At low surface pressure, the stabilization of mixed films of all compositions was reflected by their negative ∆Gex values. At surface pressures higher than that of the plateau region, excess free energy of mixing was negative for low AmB content but positive for XAmB g 0.3 (increasingly so as surface pressure increased), peaking at XAmB ) 0.7. Plotting ∆Gex against temperature failed to afford straight lines, not allowing calculation of ∆Sex as per other authors.27,28 Discussion Low Surface Pressure Region. Chol molecules are believed to be always oriented vertically, anchored in the interface by their sole hydrophilic group and giving rise, in the absence of other molecules, to densely packed films stiffened by strong intermolecular hydrophobic interactions.29 Since several studies have reported that the components of mixed films are mutually miscible only when their molecules have the same orientation relative to the interface,30-32 it might therefore be thought that at low surface pressure one of the following two possibilities must hold: AmB and Chol are surface immiscible or they are miscible and the AmB molecules in their mixed films are held vertical by hydrophobic interaction with Chol molecules. Furthermore, since most of the π-A curves of the mixed AmB/Chol monolayers in this study showed plateau regions at surface pressures of about 10 mN/m similar to that appearing in the π-A curve of the pure AmB monolayers, which is thought to reflect the transition from a state in which the AmB molecules lie horizontally in the interface to a state in which they are oriented vertically,15,23 it might be reasoned that at low surface pressure the AmB molecules of the mixed films must be horizontal and the two components of the films immiscible. However, the finding that the surface pressure at which the AmB molecules appear to switch from horizontal to (24) Min˜ones, J.; Garcı´a Ferna´ndez, S.; Sanz Pedrero, P. An. Fis. Quim. 1967, 63(B), 833. (25) Goodrich, F. C. Proc. 2nd Int. Congr. Surf. Act. 1957, 1, 85. (26) Pagano, R. E.; Gershfeld, N. L. J. Colloid Interface Sci. 1972, 41, 311. (27) Bacon, K. J.; Barnes, G. T. J. Colloid Interface Sci. 1978, 67, 70. (28) Min˜ones, J. Yebra-Pimentel, E.; Conde, O.; Iribarnegaray, E.; Casas, M. Langmuir 1994, 10, 1888. (29) Yamauchi, H.; Takao, Y.; Abe, M.; Ogino, K. Langmuir 1993, 9, 300. (30) Gabrielli, G.; Puggelli, M.; Baglioni, P. J. Colloid Interface Sci. 1982, 86, 485. (31) Gabrielli, G.; Madii, A. J. Colloid Interface Sci. 1978, 64, 19. (32) Gabrielli, G.; Baglioni, P.; Madii, A. J. Colloid Interface Sci. 1981, 79, 268.

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Table 2. Surface Pressure Values (mN/m) Corresponding to Transition Regions at Different Temperatures for Mixed Monolayers of AmB and Chol 5 °C XAmB 0.1 0.3 0.5 0.7 0.9 1

first plateau 12.5 11.3 12.2 12.0 12.5

10 °C

second plateau

first plateau

37.0 27.5

12.0 10.7 9.8 11.2 12.3

15 °C

second plateau 36.5 25.0 25.0

first plateau 10.5 9.5 8.5

20 °C

second plateau 32.5 25.0 21.5

11.5 11.0

first plateau 10.0 9.0 8.0 10.0 9.3

25 °C

second plateau 32.5 25.0 21.0

first plateau 10.7 7.0 5.8 8.0 8.5

30 °C

second plateau

first plateau

20.0 25.0

10.5 7.2 6.0 9.3 8.2 8.3

second plateau

20.0

Figure 4. Excess areas of mixing, at temperatures of 5, 10, 15, 20, 25, and 30 °C, of mixed monolayers of AmB/Chol spread on water at various surface pressures. XAmB ) mole fraction of amphotericin B.

Figure 3. Excess areas of mixing, at surface pressures of 8 mN/m (lower panel) or 20 mN/m (upper panel), of monolayers with various proportions of AmB/Chol spread on water at various temperatures. XAmB ) mole fraction of amphotericin B.

vertical orientation depends on the composition of the monolayer (Figure 1 and Table 1) implies, according to Crisp’s phase rule,33 that the two components must in fact be mutually miscible in all proportions. This is not the first report of miscible mixed films in which the molecules of the two components have different orientations. Handa et al.,34 Nakagahi et al.,35 and (33) Crisp, D. J. In Surface Chemistry Suppl. Research; Butterworths: London, 1949; pp 17-23

Candenhead et al.36 concluded that horizontally oriented 16-(9-anthroyloxy)palmitic acid (16AP) and vertically oriented compounds form mixed monolayers with nonideal behavior, which were interpreted in terms of the surface regular solution theory. It seems likely that in the present case the miscibility of the differently oriented AmB and Chol molecules must be favored by the formation of polyene-sterol complexes of the kind postulated by Herve´ et al.,37 in which a water molecule bridges between the sterol 3β-OH group and the carboxyl and protonated amino groups of an AmB molecule (34) Handa, T.; Tomita, K.; Nakagaki, M. Colloid Polymer Sci. 1987, 265, 250. (35) Nakagaki, M.; Tomita, K.; Handa, T. Biochemistry 1985, 24, 4619. (36) Candenhead, D. A.; Kellner, B. M. J.; Jacobson, K.; Papahadjopoulus, D. Biochemistry 1997, 16, 5386. (37) Herve´, M.; Debouzy, J. C.; Borowski, E.; Cybulska, B.; GaryBobo, C. M. Biochim. Biophys. Acta 1989, 980, 261.

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Figure 7. Schematic hypothetical hydrogen-bonded complex formed by amphotericin B, cholesterol, and a water molecule at low surface pressures.

Figure 5. π-A curves of monolayers with various proportions of AmB/Chol spread on aqueous substrate of pH 3 at 20 °C. XAmB ) mole fraction of amphotericin B. Inset: excess areas of mixing, at 20 °C, of mixed monolayers of AmB/Chol spread on aqueous substrates of pH 3 (upper panel) or pH 10 (lower panel) at various surface pressures.

Figure 6. Excess free energies of mixing, at various surface pressures, of mixed monolayers of AmB/Chol spread on water at 25 °C. XAmB ) mole fraction of amphotericin B.

(Figure 7), direct polyene-sterol hydrogen bonds being prevented by steric hindrance. The formation of such complexes would be in keeping with the observation of negative low-pressure excess free energies of mixing at all film compositions (Figure 6). The particular stability of the 70:30 AmB/Chol monolayer, shown by the absence of an transition region in π-A curve (Figure 1) and by having the most negative excess areas of mixing (Figures 3 and 4), suggests that this composition must be that of a particularly favorable packing of the AmB-Chol com-

plexes, as has been proposed by other authors to explain similar behavior in other films.38-42 High Surface Pressures Region. The presence of two distinct plateau regions in the π-A curves of 30:70 and 50:50 AmB/C monolayers (Figure 1 and Table 1) suggests that in these films the AmB-H2O-Chol complexes are not disrupted when the AmB molecule first becomes vertical but only at a higher surface pressure. When the complexes do break up, the water molecule no longer acts as a spacer and the higher-pressure inflection region reflects further compression favored by attractive van der Waals forces between the hydrophobic parts of the AmB and Chol molecules. Compression isotherms with multiple inflection points have likewise been observed in the case of other mixed monolayers in which, at low surface pressure, one component is horizontal and the other vertical, specifically in the 16AP-palmitic acid system investigated by Handa et al.34 and in the valinomycin/ dipalmitoylphosphatidic acid system studied by Zaitsev et al.,43 in which the dipalmitoylphosphatidic acid molecules are vertically oriented at the interface and the valinomycin molecules horizontally. The finding that the excess areas of mixing at moderately high surface pressure are positive (instead of negative, as normally corresponds to the existence of an attractive interaction) may be attributed to the AmB molecules being retained in the interface by hydrophobic interactions with Chol molecules, whereas in pure AmB monolayers there is a strong tendency for AmB molecules to be desorbed from the interface at high surface pressure before the collapse of the monolayer occurs. The fact that maximum interaction between the AmB and Chol molecules, as reflected by excess area of mixing, occurred in (38) Dervichian, D. G. In Surface phenomena in chemistry and biology; Danielli, J. F., Pankhurst, K. G. A., Riddiford, A. C., Eds.; Pergamon: New York, 1958; p 70. (39) Shah, D. J. Colloid Interface Sci. 1971, 37, 744; 1981, 79, 319. (40) Albrecht, O.; Gruler, H.; Sackmann, E. J. Colloid Interface Sci. 1981, 79, 319. (41) Gershfeld, N. L. Biophys. J. 1978, 22, 469. (42) Alsina, M. A.; Mestres, C.; Valencia, G.; Garcı´a Anto´n, J. M.; Reig, F. Colloid Surf. 1989, 40, 145. (43) Zaitsev, S. Y.; Zubov, V. P.; Mo¨bius, D. Colloid Surf. 1995, 94, 75.

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Figure 9. Collapse pressures πc of monolayers with various proportions of amphotericin B and cholesterol spread on water at 20 °C, as observed experimentally and calculated as per Joos.41 XAmB ) mole fraction of amphotericin B. Table 3. Values Corresponding to Joss and Goodrich Interaction Parameters with Values of Interaction Energy for Mixed Monolayers of AmB and Chol mole Joos interaction Goodrich interaction fraction interaction energy ∆E ∆Gexc interaction energy of AmB param (ξ) (cal/mol) (J/mol) param (R) (∆h) (J/mol) 0.1 0.3 0.5 0.7 0.9

Figure 8. Schematic hypothetical arrangement of amphotericin B and cholesterol molecules at the air/water interface at high surface pressure.

monolayers with XAmB between 0.5 and 0.7 (depending on temperature and pressure; see Figure 4) supports SaintPierre-Chazalet et al.’s15 suggestion that at these surface pressures a 2:1 AmB-Chol complex is formed in which each Chol molecule is sandwiched by a pair of AmB molecules whose hydrophilic moieties face those of a neighboring complex and whose hydrophobic moieties interact with the Chol (Figure 8). The shift of the excess area of mixing peak toward lower AmB content as surface pressure rose (Figure 4) may have been due to desorption of AmB leading to a mixture of 2:1 and 1:1 AmB-Chol complexes with a growing proportion of the latter. Collapse Region. The existence of strong attractive interactions between the components of the mixed AmB/ Chol monolayers at high surface pressure is apparently corroborated by their measured collapse pressures (Table 1) being significantly greater than those calculated under the assumption of no molecular interaction as per Joos44 (Figure 9) and by their negative values of the Joos interaction parameter ξ and interaction energy ∆E (Table 3). However, the fact that ∆E becomes less negative as AmB content increases (whereas the π-A curves and the data for excess area of mixing and excess free energy of mixing suggest increasing molecular interaction as xAmB increases from 0 to 0.5-0.7) suggests that the assumption of hexagonal packing on which Joos’ method is based45,46 (44) Joos, P. Bull. Soc. Chim. Belg. 1969, 78, 207.

-20.7 -4.78 -2.0 -1.5 -1.5

-2021.7 -465.6 -195.7 -153.2 -141.9

-577 -878 -715 -665 -652

-2.68 -1.75 -1.20 -1.32 -3.02

-1068.5 -696.8 -476.7 -527.8 -1207.4

is not fulfilled by the AmB/Chol monolayers, the very different mean molecular areas of the components of which (35 Å2 for Chol and 135 Å2 for AmB) probably lead to the packing arrangement depending on the composition of the monolayer.47 Similarly, the negative values of the interaction parameter R and the interaction energy ∆h calculated for a surface pressure of 5 mN/m from the excess free energies of mixing using the equations46

R ) (1/RT)∆Gex/(XAmBXChol2 + XCholXAmB2) and

∆h ) RTR/(zL) are at odds with the other measures of intermolecular interaction when a value of 6 is used for the coordination number zL and, hence, show that hexagonal packing cannot be assumed for these monolayers. Influence of Temperature. Raising the temperature decreases the surface pressures of both plateau regions exhibited by the π-A curves of the mixed AmB/Chol monolayers (Table 2). In the case of the low-pressure plateau, this may be attributed to a reduction in the degree of hydrogen bonding between the AmB hydroxyl groups and water, which allows AmB molecules to become vertical at a lower surface pressure than at low temperature. (45) Joos, P.; Ruyssen, R.; Min˜ones, J.; Garcı´a Ferna´ndez, S.; Sanz Pedrero, P. J. Chim. Phys. 1969, 66, 1665. (46) Mestres, C.; Alsina, M. A.; Espina, M.; Rodrı´guez, L.; Reig, F. Langmuir 1992, 8, 1388. (47) Kasselouri, A.; Coleman, A. W.; Baszkin, A. J. Colloid Interface Sci. 1996, 180, 384.

Amphotericin B and Sterols in Monolayers

Similarly, the lowering of the surface pressure of the highpressure inflection kink may be attributed to higher temperatures facilitating the disruption of hydrogenbonded AmB-H2O-Chol complexes and so allowing the transition to a more condensed state at lower surface pressure than at lower temperatures. Also, the fall in excess areas of mixing observed at high surface pressure as temperature rises (Figures 3 and 4) is doubtless due to thermal weakening of the van der Waals interactions between AmB and Chol molecules in the condensed state, which must facilitate desorption of the AmB molecules; and the reduction of negative excess areas of mixing observed at low surface pressure when temperature rises above 15 °C (Figures 3 and 4) may be attributed to increased thermal motion following the cleavage of AmBChol complexes. The increase in negative excess areas of mixing observed at low surface pressure when temperature rises from 5 to 15 °C (Figures 3 and 4) may possibly be due to the increased mobility of water molecules favoring their appropriate orientation for formation of AmB-H2O-Chol complexes (Figure 7), 15 °C being the point at which increased complex formation begins to be dominated by increased thermal disruption. Influence of pH. The fact that the mixed monolayers were more compressible at pH 3 and 10 than at neutral pH is attributable to the AmB amino group being protonated and its carboxyl group undissociated at pH 3, giving it a net positive charge, while at pH 10 it has a net negative charge because its carboxyl group is dissociated and its amino group unprotonated. These net charges facilitate the solubility of AmB in acid and alkaline substrates,23 and the fact that in both cases either the amino or the carboxyl group is uncharged means that only one of the two AmB-H2O hydrogen bonds shown in Figure 7 can be formed, which must make the AmBH2O-Chol complexes less rigid and the monolayer more compressible. Furthermore, repulsion between the charged AmB molecules must facilitate their adoption of a vertical orientation, which explains why the low-pressure plateau region of the π-A curves disappears or is less well defined than at neutral pH.

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General Conclusions The shape of the π-A curves of mixed monolayers of cholesterol-AmB indicates the presence of strong interactions between the components when XAmB equals to 0.7 and much weaker ones for mixtures with XAmB being 0.3 and 0.5. These interactions are responsible for the existence of negative values of the area and free energy of mixing at low surface pressures and positive values of the above-mentioned functions at higher pressures. The change of temperature and pH of the subphase has some influence on the area excess values, however, practically, the change does not affect the position at which minima or maxima on the Aex - XAmB dependences are observed. It is suggested that at low surface pressures, below those corresponding to the phase transition of AmB, the polyene-sterol complex is formed in which cholesterol, with one water molecule forming a “bridge” to 3β-OH group, orientates vertically to the interface, while the carboxylic and protonated amino groups of AmB are oriented horizontally. At higher surface pressures, above the transition, the reorientation of AmB molecules to the vertical position permit the establishment of the attractive van der Waals forces between apolar parts of the components, preventing the desorption of AmB molecules from the interface which explains the existence of positive values of the excess functions. The existence of strong attractive interactions between the components of the mixed AmB/Chol monolayers is proved by their measured collapse pressures being significantly greater than those calculated from the Joos equation under the assumption of ideal behavior of the components. Acknowledgment. This work was supported by the Counsellery for Education of the Xunta de Galicia (Spain) under Project XUGA 20313B96. LA980571T