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Langmuir 1999, 15, 3570-3573
Interaction between Amphotericin B and Sterols in Monolayers. Mixed Films of Ergosterol-Amphotericin B R. Seoane, J. Min˜ones,* O. Conde, M. Casas, and E. Iribarnegaray Departamento de Quı´mica Fı´sica, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, 15706 Santiago de Compostela, Spain Received May 7, 1998. In Final Form: November 25, 1998
Analysis of the compression isotherms of ergosterol/amphotericin B (AmB) mixed monolayers spread on aqueous substrates shows the existence of interactions between the two components at AmB mole fractions between 0.1 and 0.7. At low surface pressure AmB molecules appear to lie horizontally in the A/W interface and ergosterol molecules to stand vertically, while at higher surface pressures the molecules of both components are vertically oriented at the interface.
Introduction
Material and Methods
Amphotericin B (AmB) is the antibiotic of choice for fungal infections,1,2 probably because of its acting on fungal cell membranes to form pores or channels through which small intracellular molecules and ions are lost, causing cell lysis and death.3-5 It has been suggested that the pores are constituted by AmB-sterol complexes composed of alternating antibiotic and sterol molecules,6 and that the sterols either order the packing of the AmB molecules7,8 or alter membrane phospholipid packing and thus facilitate the penetration of the polyene (AmB).9 It has also been suggested that the selective targeting of fungi by AmB rather than bacteria or host cells is due to its having greater binding affinity for ergosterol, the predominant sterol in fungal cell membranes, than for cholesterol, the predominant sterol in mammalian cell membranes.10-12 Bolard et al.13 have further suggested that whereas the conventional model of the AmB-sterol complex may be a valid explanation of pore formation in ergosterol-rich membranes, AmB must be in a self-associated form in order to create pores in cholesterol-rich membranes. To throw light on 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 AmB/ergosterol monolayers.
Bristol-Myers Squibb provided AmB as a yellow powder that was not further purified. Fluka A.G. (Germany) supplied ergosterol with a purity of 99%. Each component was dissolved in a 3:1 v/v mixture of dimethylformamide and 1 M hydrochloric acid (both Merck p.a. products), and these solutions were used to make up AmB/ergosterol 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/ergosterol mixtures can only be prepared if both AmB and ergosterol are each initially dissolved in the solvent that dissolves both components. The AmB/ergosterol mixtures were spread on aqueous substrates (reagent grade water of resistivity 18 MΩ. cm obtained using a Milli-RO Milli-Q system from Millipore Corporation and brought to the desired pH by the 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. The temperature was controlled by circulating water from a Grant LC10 thermostat through the trough base. Compression isotherms were recorded at a compression rate of 99.0 cm2/min after preliminary experiments had shown no significant differences among isotherms recorded at rates of 13.2-99.0 cm2/min. Except where otherwise stated, the total number of AmB and ergosterol molecules that were deposited in each experiment was 2.3 × 1016; this allowed a direct comparison of π-A curves obtained in different experiments with different mixtures.
* To whom correspondence should be addressed. Fax: +34-81594912. E-mail:
[email protected].
Results
(1) Hospental, D.; Gretzinger, K.; Rogers, A. L. J. Med. Microbiol. 1978, 30, 193. (2) Stam, A. M.; Diaso, R. B.; Dismukes, W. E.; Shadomy, G. A.; Cluod, C. A.; Bowles, G.; Hand Karam, A. Am. J. Med. 1987, 83, 236. (3) Hamilton-Miller, J. M. T. Adv. Appl. Microbiol. 1974, 17, 109. (4) Bolard, J. Biochim. Biophys. Acta 1986, 864, 257. (5) Cohen, B. E. Biochim. Biophys. Acta 1986, 857, 117. (6) De Kruij, B.; Demel, R. A. Biochim. Biophys. Acta 1974, 339, 57. (7) Marty, A.; Finkelstein, A. J. Gen. Physiol. 1975, 65, 515. (8) Kleinberg, M. E.; Finkelstein, A. J. Membr. Biol. 1984, 80, 257. (9) Hsuchen, C. C.; Feingold, D. S. Biochem. Biophys. Res. Commun. 1973, 51, 972. (10) Vertut-Croquin, A.; Bolard, J.; Gary-Bobo, C. M. Biochem. Biophys. Res. Commun. 1984, 125, 360. (11) Kother-Brajtburg, J.; Price, H. D.; Medoff, G.; Schelessinger, D.; Kobayashi, G. S. Antimicrob. Agents Chemother. 1974, 5, 377. (12) Norman, A. W.; Spielvogel, A. M.; Wong, R. G. Adv. Lipids Res. 1976, 14, 127. (13) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry 1991, 30, 5707. (14) Gaines, G. L., Jr. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishing: New York, 1966; p 260.
Like those of other sterols, ergosterol monolayers spread on water at 20 °C were highly condensed, with a surface compression modulus, Cs-1 ) -A (∂π/∂A)T, of 275 mN/m (Figure 1, curve 1) and a limiting molecular area, A0, of 33 Å2, estimated by extrapolation of the steep linear part of the π-A curve (the difference between this value of A0 and the value reported by Gaines,14 37.5 Å2, is probably due to the different purities of the ergosterol samples used). The films collapsed at a surface pressure of 51.2 mN/m and a molecular area of 26.6 Å2. The π-A curves of 1:9 AmB/ergosterol monolayers were similar to those of pure ergosterol except for the presence of a weak shoulder that began at a surface pressure of 22.5 mN/m and separated two regions of different compressibility (see Table 1 and curve 2 of Figure 1). Similar transition regions were observed at surface pressures of
10.1021/la980541q CCC: $18.00 © 1999 American Chemical Society Published on Web 04/15/1999
Mixed Films of Ergosterol-Amphotericin B
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Figure 2. Mean molecular areas, at various surface pressures, of monolayers with various proportions of amphotericin B and ergosterol spread on water at 20 °C. xAmB ) mole fraction of amphotericin B. Figure 1. π-A curves of mixed monolayers of amphotericin B and ergosterol spread on water at 20 °C. xAmB ) mole fraction of amphotericin B. Table 1. Compression Moduli Cs-1 at Surface Pressures of 5, 25, and 50 mN/m, Transition Surface Pressures and Collapse Surface Pressures of Mixed Monolayers of Ergosterol and Amphotericin B Spread on Water at 20 °C mole fraction of AmB 0.1 0.3 0.5 0.7 0.9
compressional modulus (mN/m) 5 25 50 72.0 50.0 82.0 35.0 27.5
110.0 87.5 84.0 60.0 73.0
280.0 113.1 96.0 122.0 155.0
transition pressure (mN/m)
collapse pressure (mN/m)
22.5 25.0 27.5 10.0 and 30.0 8.6
53.7 60.6 62.2 63.1 64.0
25-27 mN/m in the π-A curves of 3:7 and 1:1 AmB/ ergosterol monolayers (Figure 1, curves 3 and 4). The π-A curve of 7:3 AmB/ergosterol monolayers (Figure 1, curve 5) differed from those with lower AmB content in exhibiting two transition regions: one similar to those of the lowAmB films at a surface pressure of about 30 mN/m, and the other at about 10 mN/m, a value similar to the transition pressure of the pure AmB monolayer (Figure 1, curve 7). Finally, the π-A curve of 9:1 AmB/ergosterol monolayers (Figure 1, curve 6) was similar to that of pure AmB, with a single plateau region at low surface pressure. Plotting mean molecular area against the mole fraction of AmB shows negative deviations from ideal behavior below the transition pressure of the AmB film and positive deviations above this surface pressure (Figure 2). At 5 mN/m the partial molecular area of AmB is 71 Å2 in monolayers with AmB contents of 10-30% and 124 Å2 in 30-70% AmB monolayers (cf. 146 Å2 for pure AmB). Thus, the condensing effect of ergosterol on AmB molecules rises from 22 to 75 Å2/molecule as the proportion of sterol increases. The positive deviations from ideal behavior
observed at surface pressures of 20-30 mN/m peak at an AmB mole fraction between 0.5 and 0.6. The negative excess molecular areas observed at low surface pressures are practically independent of temperature over the range 5-20 °C and decrease as the temperature rises further (Figure 3); at all temperatures, the values for monolayers with AmB mole fractions of 0.3, 0.5, and 0.7 are very similar. The positive excess areas observed at high surface pressure exhibit similar temperature dependence and at all temperatures fall with increasing surface pressure. To investigate the orientation of AmB molecules in mixed films at low surface pressure, equimolar amounts of the two components were spread separately (AmB first) and the resulting mixed film was left for a short or a longer while (10 or 120 min) before compression isotherms were recorded. Whereas the π-A curve of the monolayer obtained by spreading a 1:1 AmB/ergosterol mixture has a single transition region at about 27.5 mN/m (Figure 4 curve 1), that of the monolayer formed by the separately spread components left for a short time, 10 min (Figure 4, curve 2), has two transition regions, the lower of which corresponds to the same surface pressure (about 10 mN/ m) as that of the assumed transition from horizontal to vertical molecular orientation in pure AmB monolayers, while that of the film formed by the separately spread components left for a longer time, 2 h (Figure 4, curve 3), has a single plateau region around 17.5 mN/m. Discussion According to Saint-Pierre-Chazalet et al.,15 the negative deviations of AmB-sterol monolayers from ideal behavior that they have observed at surface pressures below the (15) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyrat, M.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1988, 105, 173.
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Figure 4. π-A curves of 1:1 AmB/ergosterol monolayers spread on water at 20 °C. (1) After the two components were mixed as a bulk spreading solution, waiting 10 min before compression isotherms were recorded; (2) when the two components were spread separately and left only for 10 min before compression isotherms were recorded; (3) when the two components were spread separately and left for 2 h before compression isotherms were recorded.
Figure 3. Excess mean molecular areas, at temperatures of 5, 10, 15, 20, 25, and 30 °C, of mixed monolayers of amphotericin B and cholesterol spread on water at various surface pressures. xAmB ) mole fraction of amphotericin B.
transition pressure of the pure AmB monolayer are due to the AmB molecules being held perpendicular to the interface by attractive AmB-sterol hydrophobic interactions, whereas in pure AmB monolayers they are thought to be horizontal, the transition reflected in the π-A curves consisting precisely in their becoming vertical.15,16 The composition dependence of π-A curve morphology, collapse pressure and transition pressure observed in the present study (Table 1) is certainly evidence that ergosterol and AmB are mutually miscible and interact quite strongly at all surface pressures.17 However, the finding that when ergosterol and AmB are spread separately on the substrate there is a gradual rise in the surface pressure of the lowpressure π-A inflection region as the two components diffuse into each other (Figure 4) suggests that in all cases this region reflects the transition of AmB molecules from horizontal to vertical orientation, and that the rise in surface pressure is due to an increasing, presumably nonhydrophobic, attractive interaction between AmB and (16) Seoane, J. R.; Vila Romeu, N.; Min˜ones, J.; Conde, O.; Dynarowicz, P.; Casas, M. Prog. Colloid Polym. Sci. 1997, 105, 173. (17) Crisp, D. J. Surface Chemistry, (Suppl. Research); Butterworths: London, 1949; pp 17-23. (18) Handa, T.; Makagaki, M. Colloid Polym. Sci. 1979, 257, 374. (19) Zaitsev, S. Y.; Zukov, V. P.; Mo¨bius, D. Colloids Surf. 1995, 94, 75. (20) Do¨rfler, H. D.; Kru¨ger, H. Colloid Polym. Sci. 1991, 269, 1026. (21) Do¨rfler, H. D.; Koth, C. Colloid Polym. Sci. 1992, 270, 384. (22) Do¨rfler, H. D.; Koth, C.; Rettig, W. J. Colloid Interface Sci. 1996, 180, 478.
ergosterol molecules (minimal when the monolayer components are spread separately and left only for a short time (10 min) before film compression (Figure 4, curve 2), greater when the components are spread separately but left for a longer time (2 h) allowing mutual diffusion (Figure 4, curve 3), and greatest when the component mixture is prepared before the film is spread (Figure 4, curve 1)). The slow, spontaneous mixing of initially unmixed monolayer components has been observed by several authors.18-22 Attractive interaction between AmB and ergosterol molecules would also account for the transition region of curve 1 in Figure 4 occurring at smaller mean molecular areas than the low-pressure transition region of curve 2. It is hoped that further research using other techniques such as BAM or fluorescence microscopy may confirm these findings. Attractive interaction at low surface pressure between the vertical sterol molecule and the horizontal AmB molecule must be nonhydrophobic. It seems likely that it consists of hydrogen bonds creating AmB-H2O-ergosterol complexes The positive excess areas of AmB/ergosterol monolayers at surface pressures above the transition pressure may be attributed to desorption of AmB molecules being hindered by their attractive interaction with sterol molecules, which at these surface pressures, at which both the ergosterol and the AmB molecules are vertical, includes the contribution of hydrophobic interactions between the two. The influence of temperature on excess areas may be attributed to the thermal disruption of AmB-sterol interactions. (23) Herve´, M.; Debouzy, J. C.; Borowski, E.; Cybulska, B.; GaryBobo, C. M. Biochim. Biophys. Acta 1989, 980, 261.
Mixed Films of Ergosterol-Amphotericin B
The above results show that AmB-ergosterol interaction is greater, and is manifest over a greater range of monolayer compositions (0.1 e xAmB e 0.7), than AmBcholesterol interaction (unpublished results), and hence support the hypothesis12,13,23 that there is greater affinity for ergosterol than for cholesterol that makes the antibiotic activity of AmB selective for fungi, which have cell
Langmuir, Vol. 15, No. 10, 1999 3573
membranes with higher ergosterol content than those of bacterial or animal cells. Acknowledgment. This work was supported by the Counsellery for Education of the Xunta de Galicia (Spain) under Project XUGA 20313B96. LA980541Q
