Orientational Changes of Amphotericin B in Langmuir Monolayers

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Orientational Changes of Amphotericin B in Langmuir Monolayers Observed by Brewster Angle Microscopy J. Min˜ones, Jr.,† C. Carrera,‡ P. Dynarowicz-Ła¸ tka,§ J. Min˜ones,*,† O. Conde,† R. Seoane,† and J. M. Rodrı´guez Patino‡ Faculty of Pharmacy, Department of Physical Chemistry, University of Santiago de Compostela, Campus Sur, 15 706 Santiago de Compostela, Spain, Faculty of Chemistry, Department of Chemical Engineering, University of Sevilla, c/Prof. Garcı´a Gonza´ llez, s/nu´ m. 41012 Seville, Spain, and Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krako´ w, Poland Received June 15, 2000. In Final Form: October 23, 2000 The morphology of amphotericin B monolayers spread at the air/water interface was investigated at the microscopic level with a Brewster angle microscope. The images were recorded simultaneously with the surface pressure (π)/area (A) isotherms. The transformation from the domain-like structure to homogeneous solid phase was observed upon monolayer compression. Quantitative analysis was made from the measurements of relative reflectivity during monolayer compression/decompression. A 3-fold increase in relative monolayer thickness throughout the compression from the expanded to a condensed state is observed and related to orientational changes of the monolayer molecules from a horizontal to a vertical position.

Introduction Surface pressure (π)-area (A) isotherms, frequently associated with electric surface potential (∆V) measurements, are primary and basic methods for researching a Langmuir monolayer formed at the air/water interface.1 Although these traditional techniques have been known and applied since the beginning of this century, they are still widely used. However, research based solely on the analysis of π(∆V)/A isotherms is certainly not sufficient for a full characterization of a monolayer since it does not provide any direct information regarding the morphology and orientation order of molecules in a monolayer. The application of other complementary techniques in conjunction with traditional methods is usually a must. In the past 2 decades a number of sophisticated, surfacesensitive physicochemical techniques, which include spectroscopic and microscopic techniques, have been developed for probing “in situ” the organization of molecules on the free water surface.2,3 Recently one of the microscopical techniques, namely, Brewster angle microscopy (BAM),4 has made rapid progress to visually probe the 2D organization of monolayer materials, including the size and shape of domains and heterogeneity in Langmuir films.5,6 Unlike fluorescence microscopy, it does not require the addition of the fluorescent probes, and this decreases the risk of experimental artifacts. The principle of BAM measurements has been described in detail elsewhere.4-6 Usually, there exists a good correlation * To whom correspondence may be addressed. Fax: 00 34 981 594912. E-mail: [email protected]. † University of Santiago de Compostela. ‡ University of Sevilla. § Jagiellonian University. (1) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience: New York, 1966. (2) Knobler, C. M. Adv. Chem. Phys. 1990, 77, 397. (3) Dynarowicz-Ła¸ tka, P.; Dhanabalan, A.; Oliveira, O. N., Jr. Adv. Colloid Interface Sci., in press. (4) He´non, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936. (5) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (6) Vollhardt, D. Adv. Colloid Interface Sci. 1996, 64, 143 and references therein.

between observations from surface pressure and surface potential isotherms and BAM. Since direct observation of monolayer structures with BAM has been found to be helpful in identifying the phenomena occurring at the transition regions which appear in the course of the π/A isotherms,7 we have applied this method to get further insight into the plateau which can be seen when amphotericin B (AmB) (a polyene antifungal antibiotic8) monolayer is being compressed at the air/water interface. The pioneering work on the Langmuir monolayers of AmB dates back to 1988 when Saint-Pierre-Chazalet et al.9 reported the π/A characteristic of pure AmB as well as in its mixtures with sterols. It has been suggested there9 that the plateau region in the π/A isotherms is due to progressive changes in orientation of AmB molecules, from a lying to an erect position.9 Recently we have been systematically investigating the properties of amphotericin B both alone10 and in mixtures with membrane sterols.11-13 Successive compressionexpansion experiments10 have shown that the monolayer in the preplateau region is stable. This enabled us to support the interpretation of the plateau region suggested by Saint-Pierre-Chazalet.9 It was confirmed that in the expanded state, amphotericin molecules, oriented horizontally to the interface, are strongly anchored to the free water surface by hydrogen bonds. However, in the condensed state (postplateau region), AmB molecules are assumed to be vertically oriented and their linkage with the interface is considerably reduced, which in turn leads (7) Dynarowicz-Ła¸ tka, P.; Dhanabalan, A.; Cavalli, A.; Oliveira, O. N., Jr. J. Phys. Chem. B 2000, 104, 1701. (8) Hospental, D.; Gretzinger, K.; Rogers, A. L. J. Med. Microbiol. 1978, 30, 193. (9) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyrat, M.; Gary-Babo, C. M. Biochim. Biophys. Acta 1988, 944, 477. (10) Seoane, J. R.; Vila Romeu, N.; Min˜ones, J.; Conde, O.; Dynarowicz, P.; and Casas, M. Prog. Colloid Polym. Sci. 1997, 105, 1173. (11) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Biochim. Biophys. ActasBiomembr. 1998, 1375, 73. (12) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Langmuir 1999, 15, 3570. (13) Seoane, R.; Min˜ones, J.; Conde, O.; Iribarnegaray, E.; Casas, M. Langmuir 1999, 15, 5567.

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Figure 1. Surface pressure-area isotherm of amphotericin B spread on a water subphase (pH ∼6) at 20 °C, speed of compression 30 mm/min. The designated points correspond to the BAM images of Figure 2: (A) [, number of molecules spread, 4.25 × 1016; (B) 9, number of molecules spread, 1.4 × 1016.

to a marked hysteresis of the π/A isotherms when the monolayer is compressed to high surface pressure, above the plateau.10 This hypothesis regarding orientational changes was based solely on the results of surface pressure measurements. To provide further evidence for this, and rule out other potential phenomena for the appearance of the plateau,7 we analyze herein the morphological features in AmB monolayers with BAM. The monolayer as observed with BAM is described, and the images recorded upon compression are correlated with the respective π/A isotherm. The quantitative image analysis is also made with the measurements of the relative reflectivity. Experimental Section The AmB was supplied by Bristol-Myers-Squibb (95.6% purity) and used without further purification. Spreading solutions were prepared by dissolving the AmB in a 3:1 (v/v) mixture of dimethylformamide and 1 M hydrochloric acid (Merck, p.a.) and depositing onto the surface of ultrapure water, obtained from a Milli-RO, Milli-Q reverse osmosis system (Millipore) (18 MΩ cm-1 resistivity, pH 6) in the area of 562 cm2 of the Langmuir film balance from LAUDA (Germany). To investigate monolayers in the expanded state, aliquots of 61.81 µL (1.4 × 1016 molecules) were spread on the water surface. However, to be able to observe the monolayer characteristics at low areas per molecule, more monolayer material (4.25 × 1016 molecules) was deposited. After spreading, the solutions were left for 10 min in order to ensure complete evaporation of the solvent before compression was initiated, with the speed of 33 mm/min (49.5 cm2/min). The surface pressure of the floating monolayer was measured to an accuracy of 0.1 mN m-1 All the measurements were performed at room temperature (20 °C). Brewster Angle Microscope (BAM). A commercial Brewster angle microscope (BAM 2 plus, NFT, Go¨ttingen, Germany) was used to image monolayer structures and determine the relative film thickness. The principles of BAM have been described in detail.4-5,14

The p-polarized light from a 690-nm 30-mW laser was reflected off the air-water interface with an angle of incidence close to the Brewster angle (53.1°). The lateral resolution was 2 µm, and the BAM images were digitized and processed to obtain the best quality images. Rotation of analyzer allowed the image contrast to be adjusted by varying the reflected polarization that was passing to the camera. The measurements were performed during continuous compression and expansion of the monolayer with different shutter speeds ranging from 1/50 to 1/500 s. To measure the relative thickness of the film, a camera calibration was necessary in order to determine the relationship between the gray level (GL) and the relative reflectivity (I) for the different shutter speeds.15,16 Consequently, the angle of incidence (φ2) was modified in small steps on both sides of the Brewster angle. These deviations caused an increase in the gray level signal from the pure water background. (The plot of the gray level versus incident angle was a parabola with a minimum at the Brewster angle15). The theoretical relative reflectivity for a pure water surface (Rpw) at different incident light angles (φ2) can be calculated from the Fresnel equation

Rpw )

n22 cos Φ2 - (n22 - sin2 Φ2)1/2 n22 cos Φ2 + (n22 - sin2 Φ2)1/2

(1)

where n2 is the refractive index of water. The linear plot of Rpw versus gray level allowed us to obtain the equation that relates the relative reflectivity (I) with the gray level. For the shutter speed 1/500, used in this work, the equation is16 (14) Lheveder, C.; Meunier, J.; He´non, S. In Physical Chemistry of Biological interfaces; Baszkin, A., Norde, W., Eds.; Macel Dekker: New York, 2000; pp 559-575. (15) Rodrı´guez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R. Langmuir 1999, 15, 2484-92. (16) Rodrı´guez Patino, J. M.; Sa´nchez, C. C.; Rodrı´guez Nin˜o, M. R. Food Hydrocolloids 1999, 13, 401-408.

Orientational Changes of Amphotericin B in Monolayers I ) -3.9149 × 10-6 + (6.3326 × 10-8)GL

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(2)

Finally, the relation between relative reflectivity (I) and film thickness (d) was determined from the Fresnel reflection equation.17 At the Brewster angle

I ) Cd2

(3)

where I is the relative reflectivity (I ) Ir/Io; reflected intensity (Ir) and incident intensity (Io)), C is a constant, and d2 is the film thickness. This dependence of I on d can be used to determine the “relative” thickness in different regions of the monolayer.

Results and Discussion The pressure/area (π/A) isotherm of amphotericin B spread as a Langmuir monolayer (Figure 1A), was recorded with 4.25 × 1016 molecules deposited on the surface of pure water at 20 °C and does not include the low surface pressure region. As reported previously,9-13 the π/A isotherm of AmB exhibits a broad plateau from an expanded state of low density to a liquid-condensed phase. This plateau transition was found to be temperaturedependent10 and the surface pressure at which the plateau occurs decreases with increasing temperature. Such a behavior is inverse to the plateaus originating from phase coexistence, observed with Langmuir monolayers formed by model fatty acids or long-chained alcohols.18 However, plateau regions attributed to either collapse of a monolayer into a multilayer state19 or due to orientational changes20,21 exhibit analogous trends with temperature as observed for AmB. Direct visualization of the monolayer with BAM is helpful to distinguish between these possibilities and support previous hypothesis9,10 regarding the orientational changes for the plateau appearance. To obtain BAM images corresponding to the expanded phase, 1.4 × 1016 molecules were deposited onto the surface of Lauda film balance which enabled visualization of the preplateau region at large areas per molecule (from 400 Å2/molecule) (Figure 1B). The corresponding images, presented in Figure 2, illustrate the monolayer morphology at the points a-f designated on the isotherms of Figure 1. Image a was recorded at 323.5 Å2/molecule and corresponds to the expanded region where the surface pressure reads only 0.4 mN/m. Wide areas of gas phase (dark areas) coexisting with condensed phase structures, forming oval domains, are observed. The images of most of the domains are very bright, although a few darker domains are also visible. The domains increase in number and size as the compression proceeds (b). Although each domain has a uniform intensity, the differences in brightness between particular domains can clearly be distinguished. With the analyzer rotated at 60°, the domains change their brightness as shown in photograph b′. The image of the region just preceding the plateau (c) is generally the same as that recorded for the expanded state (b), and the only difference is the appearance of bright grains, filling the gaps between domains. Different brightness of the domains is due to different tilt azimuthal orientation of AmB molecules, which reflects in different polarization. Because the domains look dark or bright on the whole, it is expected that the tilt azimuthal directions of the molecules are almost the same in a particular (17) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized light, 1st ed.; North-Holland: Amsterdam, 1992. (18) Pallas, N. R.; Pethica, B. A. Langmuir 1995, 1, 509. (19) Seitz, M.; Struth, B.; Preece, J. A.; Plesnivy, T.; Brezesinski, G.; Ringsdorf, H. Thin Solid Films 1996, 304, 284/285. (20) Kellner, B. M. J.; Cadenhead, D. A. J. Colloid Interface Sci. 1978, 63, 452. (21) Vogel, V.; Mo¨bius, D. Thin Solid Films. 1985, 132, 205.

Figure 2. Visualization of amphotericin B monolayer upon compression by Brewster angle microscopy: (a) domains at 323.5 Å2/molecule and π ) 0.4 mN/m, shutter speed 1/50; (b) domains at 217.8 Å2/molecule and π ) 1.8 mN/m, shutter speed 1/125; (b′) as above, for a position of the analyzer relative to the plane of incidence of 60°; (c) oval domains in the beginning of the plateau (141.4 Å2/molecule, π ) 6.9 mN/m), shutter speed 1/125; (d) midplateau region (85.7 Å2/molecule, π ) 10 mN/m), shutter speed 1/250; (d′) as above, for a position of the analyzer relative to the plane of incidence of 120°; (e) termination of plateau (52.3 Å2/molecule, π ) 11.6 mN/m), shutter speed 1/125; (f) postplateau region (20.8 Å2/molecule, π ) 38 mN/m) shutter speed 1/500.

domain. The domains having tilt azimuth of the molecules in the p-plane look bright whereas those containing molecules with tilt azimuth orthogonal to the p-plane look dark when the analyzer is in the p-plane. Upon further compression, on reaching the plateau (d), the domains become more regular (circular) with inverted image at the analyzer rotated to 120° with respect to the plane of incidence. The contrast between domains becomes less pronounced in the region corresponding to the termination of the plateau (e). This observation, however, enables us to rule out the multilayer formation for the origin of the plateau and supports the previous conclusion, based solely on the analysis of surface pressure measurements, that the plateau is due to orientational changes of molecules upon compression. Further compression leads to a continuous condensed monolayer phase homogeneously reflecting at microscopic scale at π ) 38 mN/m (f), indicating uniform orientation of molecules in the monolayer in the postplateau region. Upon decompression (Figure 3), a

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Figure 3. Visualization of amphotericin B monolayer upon decompression by Brewster angle microscopy: (g) (24.4 Å2/molecule, π ) 13.9 mN/m) shutter speed 1/500; (h) (85.7 Å2/molecule, π ) 6.5 mN/m) shutter speed 1/500; (i) (200 A2/molecule, π ) 0.6 mN/m) shutter speed 1/50; (j) (200 A2/molecule, π ) 0.6 mN/m, 11 h after the expansion) shutter speed 1/50.

microscopically homogeneous condensed phase remains unchanged without reappearance of the domains (images g and h). After the expansion to the maximum molecular area (200 Å2/molecule), the monolayer undergoes break up of the homogeneous structure to regular domains of condensed AmB molecules (i). The condensed AmB domains, as observed upon monolayer compression (images a-d), can well be characterized by turning the analyzer from 60° to 120° (data not shown here). These domains decrease in number and increase in size with time. In fact, the domains become circular after 11 h of relaxation in the expanded state at the maximum area (j). These results suggest that the AmB molecules recover

the horizontal orientation at the maximum monolayer expansion. For a more quantitative analysis, the relative reflectivity, I, was measured simultaneously with the compression/expansion π/A isotherm, after the camera calibration, according to the procedure described in the experimental part. At the Brewster angle, I is proportional to d2, where d is the film thickness.17 Therefore, the relative thickness of the monolayer regions can be determined even if the optical properties of the film are unknown.15,16,22 In fact here we are not interested in the exact value of the film (22) de Mul, M. N. G.; Mann, J. A., Jr. Langmuir 1998, 14, 2455.

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Figure 4. Relative intensity, I, as a function of surface pressure, π, for amphotericin B monolayer (1.4 × 1016 molecules spread on water subphase at 20 °C) upon compression (A) and decompression (B), and the time evolution of surface pressure and relative reflectivity during a compression-expansion cycle (C).

thickness but rather in the change of relative reflectivity (and the same relative thickness) on the monolayer compression from the expanded to a condensed state. In the case of AmB, we are not expecting formation of any bilayer/multilayer structures along the course of the isotherm. Thus any changes in the relative thickness may be related to different orientation of molecules at the surface. Figure 4 presents the surface pressure dependence

on the relative reflectivity during monolayer compression (A), expansion (B), and the time evolution of surface pressure and relative reflectivity during compression/ decompression (C). As it can be seen, upon monolayer compression (Figure 4A) the relative intensity increases while on decompression (Figure 4B) it decreases, analogically to the observed changes in surface pressure on compression/expansion of the monolayer. This can clearly

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be seen in Figure 4C where time evolution of both parameters (relative reflectivity and surface pressure) during a compression-expansion cycle is plotted on the same graph. The existence of noise peaks in the relative reflectivity during the monolayer compression was noticed (Figure 4A) when condensed domains of AmB pass through the spot where this measurement is performed. The noise peaks were found to disappear during the monolayer expansion up to the maximum molecular area. The noise peaks observed after the monolayer expansion at the maximum molecular area correlates well with the observed domains of AmB molecules (Figure 3i,j). A hysteresis was observed during the compression/expansion cycle in both π-A isotherm and relative reflectivity-surface pressure plots (Figure 4). This phenomenon suggests that the orientation change of AmB molecules during the compression/expansion cycle is time-consuming, as observed with BAM images. However, we cannot reject the loss of

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molecules from a condensed monolayer by dissolution10 as a cause of the hysteresis. As evidenced from Figure 4A, the relative reflectivity changes from 1 × 10-6 to 1.05 × 10-5 (∼10 times), which corresponds to a 3-fold increase in monolayer thickness on compressing the monolayer from expanded to a condensed state. Interestingly, the ratio of the crosssectional area of amphotericin B lying flat at the surface (180 Å2)9 to a vertically oriented molecule (55 Å2)9 is also 3. Again this supports that the plateau reflects gradual orientational changes of amphotericin molecules from a horizontal to a vertical position. Acknowledgment. This work was sponsored by the Consellerı´a of Education of the Xunta de Galicia (Spain) under Project XUGA 203113B96. LA0008380