Amphotericin B−Dipalmitoyl Phosphatidyl Glycerol Interactions

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Amphotericin B-Dipalmitoyl Phosphatidyl Glycerol Interactions Responsible for the Reduced Toxicity of Liposomal Formulations: A Monolayer Study J. Min˜ones Jr.,† O. Conde,† J. Min˜ones,† J. M. Rodrı´guez Patino,‡ and R. Seoane† Department of Physical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain, and Department of Chemical Engineering, Faculty of Chemistry, University of Seville, s/n. 41012 Seville, Spain Received March 26, 2002. In Final Form: July 24, 2002 Mixtures of amphotericin B (AmB) and dipalmitoyl phosphatidyl glycerol (DPPG) were studied as floating Langmuir monolayers at the air/water interface. The films were analyzed using surface pressure-area (π-A) isotherms and compressional modulus-surface pressure (Cs-1-π) plots in addition to Brewster angle microscopy. The film components were found to interact with each other with the strongest interactions occurring at the ca. 2:1 AmB:DPPG mixed film composition (XAmB ) 0.66), suggesting the formation of stable complexes composed of two horizontally oriented AmB molecules and one vertically oriented DPPG molecule. It has been suggested that the interactions between AmB and the phospholipid may compete in vivo with those between the antibiotic molecules and cellular membrane sterols. The toxic effects of AmB formulations containing free AmB (Fungizone) are thought to be due to the presence of free drug molecules that can interact with cellular membrane sterols, forming channels responsible for the observed toxicity. In liposomal AmB formulations on the other hand, AmB molecules are “immobilized” by their interactions with liposomal phospholipids, and therefore, are not as toxic to host cells.

Introduction Amphotericin B (AmB)sthe polyene antibiotic most widely used to treat the majority of systemic fungal infectionsshas an undesirable property of being poorly soluble in water as well as in the majority of solvents used in intravenous drug administration. Consequently, an appropriate vehicle that facilitates its colloidal dispersion in water is necessary. This has been achieved by dispersing the antibiotic in an aqueous medium containing sodium deoxycholate and glucose. Such a solution (fungizone) is suitable for intravenous drug administration; however, its clinical use is considerably limited given its toxicity to host cells that gives rise to secondary side effects such as fever, chills, nausea, vomiting, and general uneasiness.1 Furthermore, it can also cause more severe complications such as nephrotoxicity,2 which can result in serious kidney failure when administered for prolonged treatment periods. It is postulated that the main reason for the high toxicity of this drug is the close similarity of the membranelocated targets of AmBsin fungi (ergosterol) and mammalians (cholesterol). Due to the very small difference of affinity with both targets, AmB induces comparable membrane permeabilizing effects in both organisms, being only a little greater in fungi since the mechanism of action on both types of cells is of similar nature: AmB interacts with membrane-located sterols, and AmB-sterol complexes associate and form transmembrane channels,3,4 through which free diffusion of many components essential for cell life occurs (particularly K+ ions and other small molecules), leading to cell death. Although the molecular * To whom correspondence should be addressed. Fax: +34-981594912. E-mail: [email protected]. † University of Santiago de Compostela. ‡ University of Seville (c/o Prof. Garcı´a Gonza ´ lez). (1) Hoeprich, P. D. J. Clin. Infect. Dis. 1992, 14 (Suppl. 1), S114. (2) Carlson, M. A.; Condon, R. E.. J. Am. Coll. Surg. 1994, 179, 361. (3) Bolard, J. Biochim. Biophys. Acta 1986, 864, 257. (4) Brajtburg, J.; Bolard, J. Microbiol. Rev. 1996, 9, 512.

structure of the channels is still an object of study,5,6 the particular type of membrane sterol is believed to play a role in the channel formation and, consequently, in selective toxicity. Furthermore, the state of the antibiotic in solution (monomeric or aggregate state) is another essential factor influencing its selective toxicity.7 To solve this problem, other less toxic formulations of AmB (maintaining its antifungal potency) have been sought. The strategy was to use other vehicles, such as lipids to form a emulsion, instead of sodium deoxycholate (AmB intralipid emulsion). AmB administered as a fat emulsion in experiments carried out on animals has been reported to be less toxic than conventional forms of administration.8 However, in AIDS patients with cryptococcal meningitis, AmB administered in this form still causes some nephrotoxicity.9 Another form of AmB administration, commercially known as ABLC, was introduced in December of 1995, and it became the first lipid-formulated AmB product approved by the U. S. Food and Drug Administration (FDA) for use in the USA. It contains a high amount of AmB (33%) in addition to two phospholipids (namely, dimyristoyl phosphatidyl choline and dimyristoyl phosphatidyl glycerol) and is rapidly entrapped by the mononuclear phagocytic system (MPS), resulting in considerably lower blood levels than those achieved by fungizone. Against some infections the efficacy of ABLC was equal (5) Millie, P.; Langlet, J.; Berge`s, J.; Caillet, J.; Demaret, J. P. J. Phys. Chem. 1999, 103, 10883. (6) Milhaud, J.; Ponsinet, V.; Takashi, M.; Michels, B. Biochim. Biophys. Acta 2002, 1558, 95. (7) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry 1991, 30, 5707. (8) Chavanet, P. Y.; Gary, I.; Charlier, N.; Caillot, D.; Kisterman, J. P.; D’Athis, M.; Portier, H. J. Br. Med. 1992, 305, 921. (9) Yoli, V.; Geoffray, C.; Reynes, J.; Goujard, C.; Mechali, D.; Yeni Programand, P. Abstracts of the Thirty-Third Interscience Conference of Antimicrobial on Agents and Chemotherapy; American Society for Microbiology: New Orleans, LA, Washington DC, 1993; abstract 814, 268.

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to that of fungizone, while against other infections fungizone was 2- or 4-fold more effective than ABLC.10 Unlike the deoxycholate form of AmB, which cannot be administered at therapeutic levels because of its toxicity, ABLC with its reduced toxicity can be given at high drug levels that generate a therapeutic response. For example, large doses (up to 12.8 mg/kg) cured most mice with severe acute aspergillosis, cryptococcosis, and candidiasis, whereas the responses to the maximum tolerated dose of fungizone (0.8 mg/kg) by these animals were poor.10 Another AmB formulation, Amphocil (ABCD), is a cholesteryl sulfate colloidal dispersion of AmB. It received the FDA approval in December of 1996 and was found to be active against clinical isolates of pathogenic fungi, similar to fungizone, although it is more rapidly metabolized in the liver, and therefore, it is less nephrotoxic.11 For the past decade, investigators have evaluated the use of phospholipid vesicles, known as liposomes, as a target drug delivery system for AmB in an attempt to attenuate its nephrotoxicity and increase its therapeutic potential. The first attempt to use AmB in the form of liposomes was guided by the prior finding of an enhancement of antileishmanial activity of antimony compounds by liposomes. Similar studies demonstrated that liposomeencapsulated AmB is significantly less toxic to the host and has higher therapeutic capabilities than micellar dispersion of free AmB.12 In subsequent studies, liposomes were used as vehicles for AmB in treatment of murine histoplasmosis,13 cryptococcosis,14 and candidiasis.15 In all of these experiments, liposome-encapsulated AmB was as effective as the free drug, whereas its toxicity to the host decreased,16-18 which permits AmB to be administered in much higher doses in long-term treatment. This pattern of response was observed in clinical studies where cancer patients with fungal infections were treated with liposomal AmB;19-21 the tolerance of liposomal AmB was much better than that of fungizone. Until now, the reasons for the reduced acute toxicity of liposomal (lipid emulsion) AmBsin contrast with that of the free drugshave not been completely clarified. It has been postulated that by changing the liposome composition, the toxicity of liposomal AmB can be modulated. Thus, AmB trapped in liposomes of saturated phospholipids was less toxic than in unsaturated lipids,16 which could be explained by greater leakage from the latter. (10) Clark, J. M.; Whitney, R. R.; Olsen, S. J.; George, R. J.; Swerdel, M. R.; Kunselman, L.; Bonner, D. P. Antimicrob. Agents Chemother. 1991, 35, 615. (11) Fielding, R. M.; Singer, A. W.; Wang, L. H.; Babba, S.; Guo, L. S. Antimicrob. Agents Chemother. 1992, 36, 299. (12) New, R. R. C.; Chance, M. L.; Heath, S. J. Antimicrob. Chemother. 1981, 8, 371. (13) Taylor, R. L.; Williams, D. M.; Craven, P. C.; Graybill, J. R.; Drutz, D. J.; Magee, W. E. Am. Rev. Respir. Dis. 1982, 125, 610. (14) Graybill, J. R.; Craven, P. C.; Taylor, R. L.; Willians, D. M.; Magee, W. E. J. Infect. Dis. 1982, 145, 748. (15) Lo´pez-Berestein, G.; Mehta, R.; Hopfer, R. L.; Mills, K.; Kasi, L.; Metha, K.; Fainstein, V.; Luna, M.; Hersh, E. M.; Juliano, R. J. Infect. Dis. 1983, 147, 939. (16) Juliano, R. L.; Grant, C. W. M.; Barber, K. R.; Kalp, M. A. Mol. Pharmacol. 1987, 31, 1. (17) Juliano, R. L.; Lo´pez-Berestein, G.; Hopfer, R.; Mehta, R.; Mehta, K.; Mills, K. Ann. N. Y. Acad. Sci. 1985, 464, 390. (18) Lo´pez-Berestein, G. In Liposomes in Therapy of Infectious Diseases and Cancer; Lopez-Berestein,G., Fidler, I. J., Eds.; Alan R. Liss Inc.: New York, 1989; pp 317-327. (19) Lo´pez-Berestein, G.; Fainstein, V.; Hopfer, R.; Mehta, K.; Sullivan, M. P.; Keating, M.; Rosemblum, M. G.; Mehta, R.; Luna, M.; Herst, E, M.; Reuben, J.; Juliano, R. L.; Bodey, G. P. J. Infect. Dis. 1985, 151, 704. (20) Lo´pez-Berestein, G.; Bodey, G. P.; Frankel, L. S.; Mehta, K. J. Clin. Oncol. 1987, 5 (2), 310. (21) Sculier, J. P.; Coune, A.; Meunier, F.; Brassinne, C.; Laduron, C.; Hollaert, C.; Collete, N.; Heyman, C.; Klastersky, J. Eur. J. Cancer Clin. Oncol. 1988, 24, 527.

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These results allow one to suppose that there is a certain affinity between the drug and the vehicle-forming phospholipids. Therefore, our present study is aimed at verifying the existence of interactions between amphotericin B and a saturated liposome phospholipid, namely, dipalymitoyl phosphatidyl glycerol (DPPG), using the monolayer technique. This method constitutes an in vitro model of liposomes and permits the alteration of the molecules’ orientation at the interface, which is of great importance in interpreting the nature of interactions existing between AmB and the phospholipid. With this end in mind, we have investigated mixed monolayers of both compounds and compared the surface pressure-area (π-A) isotherms of mixed films with those for pure components. This analysis allowed us to determine the strength of the interactions as well as their nature. In addition, the application of Brewster angle microscopy for in situ studies of mixed and pure films allowed us to directly visualize the morphology of monolayers, which was found to be of value in interpreting the obtained results. Experimental Section. Materials. L-R-Phosphatidylglycerol dipalmitoyl (DPPG; 99% purity) was purchased from Sigma. AmB was supplied by Squibb (Bristol-Myers Lab), and its purity was specified as higher than 95%. The phospholipid was dissolved in a chloroform/ethanol mixture (4:1 (v/v)) and AmB in the mixture of dimethylformamide and 1 M HCl (3:1 (v/v)). The spreading mixed solutions 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 Microman Gilson microsyringe (precise to (0.2 µL), was kept constant in all the experiments. After spreading, the monolayers were left alone for 10 min for solvent evaporation and then compression was initiated. Ultrapure water, used as a subphase, was obtained from Milli RO, Milli Q reverse osmosis system (Millipore Corp.) containing two carbon and two ion-exchange columns. Finally, the water was purified through a 0.22 µm Zetapore filter. The resistivity of the purified water was 18 MΩ cm. The purity of the subphase was controlled by surface tension measurements during compression of an uncovered subphase. The surface pressure obtained in these blank tests was less than 0.1 mN/m. The pH of the subphase was controlled periodically and was about 6.0. The subphase temperature was maintained constant at 20 °C. Surface Film Balance. The Langmuir-Blodgett KSV-5000 (Finland) trough, equipped with two symmetrical compartments, 71 × 12 cm2 each, 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 at the same speed from the edge of each compartment toward its center, 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 of each individual monolayer spread in its corresponding compartment. Good reproducibility of the isotherms (accuracy of (0.1 mN/m), 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 2.2 cm/min (26.4 cm2/min), which corresponds to 6.2 Å2/(molecule min). Brewster Angle Microscopy. As for the microscopic observation of the monolayers’ structure, the Brewster angle microscope, BAM 2 (NFT, Go¨ttingen, Germany), equipped with a 30 mW laser emitting p-polarized light at 690 nm wavelength, was used; light was reflected off the air/water interface at approximately 53.1° (Brewster angle). The lateral resolution of the microscope was 2 µm, and the images were digitized and processed

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Figure 1. Surface pressure-area (π-A) isotherm of AmB monolayer spread on water at pH 6 (T ) 20 °C): (A) rectangular domains at π < 10 mN/m (shutter speed, 1/125 s); (B) domains at midplateau region (shutter speed, 1/250 s); (B′) same as above, with the analyzer rotated 120°; (C) homogeneous image recorded above the plateau. (shutter speed, 1/500 s). The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

Figure 2. Surface pressure-area (π-A) isotherm of DPPG monolayer spread on water at pH 6 (T ) 20 °C): (A) liquidcondensed domains dispersed in the expanded phase (shutter speed, 1/50 s); (B) ungrouped domains at π )0.8 mN/m (shutter speed, 1/125 s); (C) formation of condensation nuclei (shutter speed, 1/125 s); (D) monolayer collapse (shutter speed, 1/250 s). The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

in order to obtain high-quality images. The measurements were performed upon monolayer compression with different shutter speeds (within the range of 1/125 to 1/500 s). A camera calibration was necessary in order to determine the relationship between the intensity unit (i.e. “gray level” (GL)) and the relative reflectivity (I) for different shutter speeds.22,23

become more regular (circular shaped) (picture B), with the image inverting as the analyzer is rotated to 120° with respect to the plane of incidence (photo B′), which shows their optical anisotropy. Upon further compression, the monolayer becomes completely homogeneous (image C), indicating uniform orientation of molecules in the postplateau region. DPPG exhibits a condensed-type monolayer with a compressional modulus (Cs-1 ) -A(dπ/dA) of 157.5 mN/m, where (dπ/dA) is the slope of the π-Α isotherm), and a collapse pressure of about 65 mN/m. The area extrapolated to zero surface pressure is 55 Å2/molecule (Figure 2). At molecular areas higher than 62.5 Å2/ molecule, liquid-condensed (LC) domains (bright regions in image A, Figure 2) coexist with the gaseous phase (dark zones). As compression takes place, the LC domains group together, leaving small holes between them which are occupied by the low-density expanded phase (image B in Figure 2, corresponding to the monolayer at π ) 0.8 mN/ m). These domains exhibit optical anisotropy, which can be observed by changing the analyzer angle to 60° with respect to its initial position (image not shown here). In the condensed region, the monolayer is practically homogeneous; however, small condensation nuclei can be seen (image C), which finally arrange themselves into stripes at the monolayer collapse (image D).

Results At the surface pressure of 10 mN/m, the monolayer of AmB shows a phase transition as evidenced by a plateau region in the π-A isotherm that spans over the areas of approximately 115 to 55 Å2/molecule (Figure 1). Below this plateau, the BAM images reveal the existence of rectangular domains (picture A), some of them very bright and others much darker. This difference in brightness demonstrates the different tilt azimuth orientations of AmB molecules: the domains having tilt azimuth of the molecules in the p-plane are bright, whereas those containing molecules with tilt azimuth orthogonal to the p-plane have a darker appearance when the analyzer is in the p-plane. On reaching the plateau, the domains (22) Rodriguez Patino, J. M.; Sa´nchez, C. C.; Rodriguez Nin˜o, M. R. Langmuir 1999, 15, 4884. (23) Rodriguez Patino, J. M.; Sa´nchez, C. C.; Rodriguez Nin˜o, M. R. Food Hydrocolloids 1999, 13, 401.

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Figure 3. Surface pressure-area (π-A) isotherms for AmB-DPPG mixed monolayers: (1, b-b) XAmB ) 0; (2, O-O) XAmB ) 0.1; (3, 9-9) XAmB ) 0.3; (4, 0-0) XAmB ) 0.5; (5, 2-2) XAmB ) 0.7; (6, 4-4) XAmB ) 0.9; (7, [-[) XAmB ) 1. (Inset) Compressional modulus-surface pressure (Cs-1-π) plots: (2, O-O) XAmB ) 0.1; (3, 9-9) XAmB ) 0.3; (4, 0-0) XAmB ) 0.5; (5, 2-2) XAmB ) 0.7; (6, 4-4) XAmB ) 0.9. M: characteristic minimum in Cs-1-π curves corresponding to phase transitions at π ) 21 mN/m. A: characteristic minimum in Cs-1-π curves corresponding to the mixed monolayer (XAmB ) 0.9) phase transition at π ) 10 mN/m.

The general effect of increasing DPPG concentration in mixed films is the condensation of the expanded phase and expansion of the condensed phase of the AmB monolayer (Figure 3 and Table 1). A similar behavior has been observed for other mixed systems.24-27 Moreover, in mixed films containing AmB in the range of mole fractions 0.1-0.7, there exists a phase transition at a surface pressure of 21 mN/m which appears as a pseudoplateau in the π-A isotherms (curves 2-5). With the increase of AmB content in the mixed monolayer, the length of this pseudoplateau increases and the transition becomes flatter. However, the mixed monolayer containing XAmB ) 0.9 shows two phase transitions (curve 6): the first starts at π ) 10 mN/m (similarly to pure AmB monolayer) and the second at a higher surface pressure of ca. 19 mN/ m, which is below the transition observed for all other

mixed films. When the collapse pressure can be precisely determined (i.e. for mixed monolayers containing 50%, or less, of AmB), film collapse can be observed at the same surface pressure of about 65 mN/m, similar to pure DPPG monolayer, which suggests that this component is squeezed out of the film into the subphase. For mixtures with a higher proportion of AmB (>50%), the collapse pressure values cannot be exactly evaluated due to the dissolution of AmB in the subphase.28,29 The above-mentioned phase transition can be more clearly seen in the plots of the compressional modulus as a function of the surface pressure (inset of Figure 3). A characteristic minimum (M) can be clearly seen in the Cs-1-π curves, which is equivalent to the pseudoplateau recorded in the π-A isotherms at 21 mN/m. Such a minimum can be observed in all the graphs corresponding to different monolayer composition, including the mixture of XAmB ) 0.9 in which case a second minimum (A) appears at π ) 10 mN/m. The relative reflectivity-surface pressure (I-π) plots, recorded with BAM, confirm the results commented on above. At the Brewster angle, I is proportional to d2, where d is the film thickness.30 Therefore, the relative thickness of the monolayer regions can be determined even if the optical parameters of the film are unknown.31 In fact, here we are not interested in the exact value of the film thickness, rather in the change of the relative reflectivity (film thickness) upon the monolayer compression from the expanded to the condensed state. Given that there is no bilayer/multilayer formation along the course of the isotherm, the change of relative reflectivity can be

(24) Cadenhead, D. A.; Phillips, M. C. Adv. Chem. Ser. 1968, 84, 131. (25) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Biochim. Biophys. Acta 1998, 1375, 73. (26) Seoane, R.; Min˜ones, J.; Conde, O.; Casas, M.; Iribarnegaray, E. Langmuir 1999, 15, 3570. (27) Seoane, R.; Min˜ones, J.; Conde, O.; Iribarnegaray, E.; Casas, M. Langmuir 1999, 15, 5567.

(28) Rey Go´mez-Serranillos, I.; Dynarowicz-Ła¸ tka, P.; Min˜ones, J., Jr.; Seoane, R. J Colloid Interface Sci. 2001, 234, 351. (29) Rey Go´mez-Serranillos, I.; Min˜ones, J., Jr.; Seoane, R.; Conde, O.; Casas, M. Prog. Colloid Polym. Sci. 1999, 112, 29. (30) Azzam, R. M. A.; Bashara, N. M. Ellipsometry and Polarized Light, 1st ed.; North-Holland: Amsterdam, 1997. (31) De Mul, N. M. G.; Mann, J. A., Jr. Langmuir 1998, 14, 2455.

Table 1. Values of Limiting Areas (A0(expt) and A0(cond) ) and Surface Pressures of Phase Transition (πc) for Mixed AmB-DPPG Monolayers with Different Mole Fractions of AmB (XAmB) Spread on Water at pH 6 (T ) 20 °C) XAmB

A0(expt) (Å2/molecule)

A0(cond) (Å2/molecule)

DPPG 0.1 0.3 0.5 0.7 0.9 AmB

84.7 121.6 151.7 157.4 188.8 169.6

54.9 47.2 43.8 38.2 37.9 36.8 44.4

πC (mN/m) Ab Μa 21.5 21.3 21.6 21.0 18.6

9.8 9.9

a M: second minimum in C -1-π curves. b A: first characteristic s minimum in Cs-1-π curves.

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Figure 4. Relative reflectivity-surface pressure (I-π) plots recorded upon compression of pure monolayers of AmB, DPPG, and mixed AmB-DPPG films containing XAmB) 0.5, 0.7, and 0.9, spread on water at pH 6 (T ) 20 °C).

attributed to the different orientation of the molecules at the interface. Figure 4 shows the I-π curves corresponding to AmB, DPPG, and some selected mixed AmB-DPPG monolayers of XAmB ) 0.5, 0.7, and 0.9. For the pure AmB film, the relative reflectivity increases during film compression with an abrupt increase observed at the transition region (i.e. at 10 mN/m), where the corresponding π-A isotherm pseudoplateau begins. In the case of DPPG, the relative reflectivity gradually increases up to 10 mN/m, and upon further compression its value is maintained practically constant. Relatively low I values and the lack of noise signals for DPPG monolayer result from the fact that the LC domains of DPPG are smaller and more closely packed (Figure 2) than those of AmB (Figure 1). Below 20 mN/m, the relative reflectivity curve for the mixture containing XAmB ) 0.5 resembles that for pure DPPG. The absence of reflectivity signals in the I-π graph indicates that the mixed monolayer structure is governed by the phospholipid. This can be confirmed in the BAM images (Figure 5, image A) which show the presence of small circular domains attributed to DPPG molecules. An important change in mixed monolayer structure occurs at 20 mN/m as the relative reflectivity starts to increase rapidly. The observed reflectivity peaks can be related to the presence of AmB in the mixed film. This increase of relative reflectivity in the I-π curve occurs at the same pressure corresponding to the pseudoplateau in π-A isotherms (21 mN/m, Figure 3) and is related to the change in orientation of AmB molecules which results in the increase of film thickness. As for the mixed films of XAmB of 0.7 or 0.9, the monolayers structure is dominated by AmB molecules, even at surface pressures lower than 10 mN/m where reflectivity signals are clearly visible in the course of the I-π plot. These reflectivity peaks become more pronounced at surface pressures higher than 20 mN/m (see Figure 4). In the low-pressure region (π ) 4.3 mN/m) of the BAM images, there are AmB domains with optical anisotropy that are clearly visible (images B and B′, Figure 5) and that become more compact as the amount of AmB in the mixed monolayer increases (image C). At higher surface pressures (π ) 33 mN/m), the images are homogeneous, without optical anisotropy (image D), similar to the

Figure 5. BAM images corresponding to mixed AmB-DPPG monolayers, spread on water subphase at pH 6: (A) bright circular domains observed for the mixture of XAmB ) 0.5 at 0.7 mN/m (shutter speed, 1/50 s); (B and B′) AmB domains with optical anisotropy (B′ analyzer at 60°) corresponding to the mixture of XAmB) 0.7 at 4.3 mN/m (shutter speed, 1/125 s); (C) increase of domains density for the mixed film of XAmB ) 0.9 at 2.2 mN/m (shutter speed, 1/125); (D) homogeneous image without optical anisotropy for the mixture of XAmB) 0.7 at 33 mN/m (shutter speed, 1/500). The horizontal direction of the image corresponds to 630 µm and the vertical direction to 470 µm.

structure of the pure AmB monolayer recorded above the transition region (image C, Figure 1). According to the above stated results, the presence of the phospholipid in the mixture of XAmB) 0.7 “pulls down” the relative reflectivity signal, as compared to the pure AmB monolayer (Figure 4), to the surface pressure of 21

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Figure 6. Mean molecular area vs composition plots for AmBDPPG mixed films spread on water subphase, pH 6 (T ) 20 °C).

mN/m. This clearly demonstrates the existence of interactions between both components in this mixed monolayer. These interactions seem to be weaker in the mixed film of XAmB ) 0.9, as its reflectivity curve nearly coincides with that for pure AmB between the surface pressures of 0-10 mN/m. However, I starts to increase progressively at higher surface pressures, and its most pronounced change occurs at approximately 18 mN/m. One of the traditional methods used to verify the existence of interactions between components in mixed monolayers was to examine the plots of the mean molecular area (total area of the trough divided by the number of molecules of both components spread on the surface) as a function of the mixed film composition (Α ) f(X)) at various surface pressures. If the two components are immiscible or ideally miscible, the dependence of Α vs X is linear as it results from the additivity of the molecular areas.32 Deviations from linearity indicate miscibility and nonideality, with greater cohesive forces between the unlike molecules than the like components (negative deviations) or greater cohesion between like molecules (positive deviations). Figure 6 shows such dependence for the investigated system. It is evident that below the phase transition, i.e., at low (5 mN/m) and intermediate (15 mN/m) surface pressures, the mixed film shows positive deviations from ideality (ideal behavior is represented by dashed lines), which are more pronounced at higher surface pressures. The highest deviations occur for mixtures of XAmB ) 0.5-0.7. On the other hand, at surface pressures above that of the transition (30 mN/m), the deviations from the ideal behavior are negligible. Discussion AmB exhibits a phase transition at 10 mN/m (Figure 1) which can be attributed to the change of orientation of polyene molecules from a mainly horizontal position to a vertical position upon compression.33,34 The relative reflectivity measurements confirm this hypothesis since the abrupt increase of I observed at π ) 10 mN/m corresponds to a ca. 3-fold increase in the film thickness. This value can be compared with that calculated from the length of AmB molecule in the vertical and horizontal position.35

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Mixed AmB-DPPG films also exhibit a first-order phase transition, which however, occurs at higher surface pressures than in the case of the pure AmB monolayer. This transition appears as a pseudoplateau in the π-A isotherms, as a minimum in the Cs-1-π plots and as an abrupt increase in the relative reflectivity signal observed in the I-π curves. All these characteristic features were observed at a surface pressure of ca. 21 mN/m. To explain the existence of this transition, it might be suggested that the presence of the phospholipid in the mixture hinders the reorientation of AmB molecules as a consequence of its polar interaction36,40 with polyene molecules. As a consequence, the stronger that the interaction between both components is, the higher the surface pressure of the transition. Likewise, the length and the flatness of the pseudoplateau can also be useful in quantifying the strength of the interaction. As can be seen in the π-A isotherms (Figure 3), the length and flatness of the pseudoplateau gradually increase with the addition of AmB to the mixed monolayer, reaching maximum values at XAmB ) 0.7. For this particular mixture, Cs-1 also attains its minimum value. This suggests, as was postulated for mixed films AmBdipalmitoyl phosphatidyl choline36 and AmB-dipalmitoyl phosphatidic acid,40 that the strongest interaction occurs when the film components are in the proportion of 2.33:1 (XAmB ) 0.7), or at approximately 2:1, which corresponds to XAmB ) 0.66. This is an intermediate value between XAmB ) 0.5 and XAmB ) 0.7 for which the highest deviation from the ideal behavior were recorded (Figure 6). We may thus suppose that the mixed monolayer is composed of stable complexes, denoted as C(h-v), formed by two horizontally oriented AmB molecules and one DPPG molecule in a vertical position, through their polar groups via the formation of hydrogen bonds between hydroxyl groups of DPPG and AmB, respectively, and via ionic interactions between the ionized phosphate group of DPPG and the positively charged group of AmB. At surface pressures above the phase transition M (21 mN/m), the molecules of AmB which are embedded in the C(h-v) complex change their orientation. This leads to the segregation of the complex into its components (i.e. DPPG and AmB in the vertical position (AmBv)) and finally results in the formation of two separate phases that are immiscible. The application of the Crisp phase rule37 (p ) c - f + 1) to the phase transition, M, supports this interpretation, namely, that the number of components in the investigated system, c, is 2 (AmB and DPPG) and the number of the degrees of freedom, f, is 0, as the results compiled in Table 1 demonstrate, indicating that the transition pressure is maintained nearly constant independent of the film composition. Therefore, there are three phases, p, that coexist in equilibrium at the transition (i.e. C(h-v) complexes, free AmBv, and DPPG). At surface (32) Gaines, G. L. In Insoluble Monolayers at Liquid-Gas Interface; Prigogine, I., Ed.; Interscience: New York, 1966; p 281. (33) Saint-Pierre-Chazalet, M.; Thomas, C.; Dupeyrat, M.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1988, 944, 477. (34) Min˜ones, J., Jr.; Carrera, C.; Dynarowicz-Latka, P.; Min˜ones, J.; Conde, O.; Seoane, R.; Rodriguez Patino, J. M. Langmuir 2001, 17, 1477. (35) Wo´jtowicz, K.; Gruszecki, W. I.; Walicka, M.; Barwicz, J. Biochim. Biophys. Acta 1998, 1373, 220. (36) Min˜ones, J. Jr.; Min˜ones, J.; Conde, O.; Rodriguez Patino, J. M.; Dynarowiz-Latka, P. Langmuir 2002, 18, 2817. (37) Crisp, D. J. Surface Chemistry Supplemental Research; Butterworth: London, 1949; p 17. (38) Chavez, A.; Pujol, M.; Haro, I.; Alsina, M. A.; Cajal, Y. Langmuir 1999, 15, 1101. (39) Negar, S.; Hyuk, Y.; Zografi, G. Langmuir 1998, 14, 151. (40) Min˜ones, J., Jr.; Min˜ones, J.; Conde, O.; Seoane, R.; DynarowiczLatka, P. Langmuir 2000, 16, 5743.

AmB-DPPG Interactions in Liposomal Formulations

Figure 7. Compression-expansion (π-A) isotherm of mixed AmB-DPPG monolayer of XAmB ) 0.9, spread on water subphase at pH 6. Inset: Relative reflectivity-surface pressure (I-π) plots recorded upon decompression of pure films of AmB, DPPG, and mixed AmB-DPPG monolayer containing XAmB ) 0.9, spread on water at pH 6 (T ) 20 °C).

pressures below the transition M, the mixed monolayer is composed of complexes (Ch-v) of horizontally oriented AmB (AmBh) and DPPG, which are miscible with the excess of either phospholipid molecules (for XAmB < 0.66) or amphotericin B molecules (for XAmB > 0.66). The immiscibility of the monolayer components above 21 mN/m is evidenced by the mean molecular area vs mixed film composition plots (Figure 6). As can be seen, the values of molecular areas recorded at π ) 30 mN/m appear approximately along the line corresponding to the additivity rule. This result, in combination with the fact that the surface pressure values corresponding to the phase transition remain constant for films of different composition (Table 1), evidence that, under these experimental conditions, both components are immiscible. Furthermore, the segregation of the C(h-v) complexes above the phase transition can be additionally confirmed by the results of the compression-expansion curve, shown in Figure 7 for the mixed film of XAmB ) 0.9. During compression, two phase transitions can be distinguished (i.e. A and M). As has already been analyzed above, both transitions are due to the change of the AmB orientation; however, the first transition is related to free AmB molecules in excess, while the other transition corresponds to AmB embedded in the complex. Upon monolayer expansion, the decompressed mixed film has the same characteristics as the pure AmB monolayer, (i.e. it only exhibits the second transition (A) at 10 mN/m), indicating the absence of the complexes at the interface. This can also be evidenced from the relative reflectivity curve corresponding to the mixed monolayer expansion of XAmB (inset of Figure 7), which is similar to the AmB curve, demonstrating that the AmB molecules are not embedded in the complexes but are present in the free form. It is thus evident that the complexes undergo separation upon monolayer compression above the transition pressure. Positive deviations from the ideal behavior (Figure 6) indicate some sort of repulsive interaction38 or greater

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cohesion between like molecules as compared to unlike molecules in mixed film.39 This however, is inconsistent with the idea postulated in this work regarding the formation of complexes as the result of attractive forces between the components. To explain this contradiction, we suggest the following interpretation. It is well-known that pure AmB molecules desorb easily at the air/water interface when they are compressed.28,29 However, the presence of DPPG significantly reduces the extent of polyene molecules loss from the surface as a consequence of attractive interactions between both components. Therefore, at 15 mN/m the partial molecular area of AmB in mixed films of XAmB ranging from 0.1 to 0.66 is 120 Å2/molecule instead of 40 Å2/molecule, which corresponds to pure AmB (Figure 6). Saint-Pierre-Chazalet et al.33 reported values of 92 Å2/molecule for the partial molecular area of AmB in mixed film with ergosterol at 20 mN/m versus 25 Å2/molecule for pure AmB, and ascribed these differences to the effect of ergosterol which prevents the desorption of polyene from the surface. A similar explanation has been applied to mixed films of AmB with sterols,25-27 with DPPC36 and with DPPA,40 and can also be applied to the present results. Therefore, the mean molecular areas of pure AmB in Figure 6 are apparent, rather than actual, as they were obtained assuming that all the AmB molecules spread were confined at the interface (i.e., that A ) At/N0, where At is the total monolayer area at a particular time of compression and N0 is the initial number of spread AmB molecules). The actual area is larger because the number of AmB molecules present at the interface, N, is smaller than N0 due to its solubility. Consequently, if the apparent AmB molecule areas in Figure 6 were replaced with their actual values, the solid lines in the figure would appear below the dotted line, which will be modified to account for the correct AmB mean molecular areas. The existence of attractive interactions between AmB and DPPG may be of importance in understanding the mechanism of action of nontoxic liposomal formulation of amphotericin B. It may be supposed that there are three kinds of interactions that compete with each other in the cellular membrane (i.e. sterol-phospholipid, AmB-sterol, and AmB-phosholipid). In the case of fungi cellular membrane (containing ergosterol), the interactions between the sterol and the phospholipid molecules are very weak41 as the equilibrium:

is shifted to the right-hand side of the equation. Therefore, when amphotericin B is administered in the form of fungizone (free AmB), both ergosterol and phospholipid molecules compete with each other to form complexes with AmB. Since the AmB affinity for ergosterol is higher42-46 than that for the phospholipid,47,48 channels (41) Demel, R. A.; Bruckdorfer, K. R. M.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1972, 255, 311. (42) Kinsky, S. C.; Luse, S. A.; Van Deenen, L. L. M. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1966, 25, 1503. (43) Gruda, I.; Nadeau, P.; Brajtburg, J.; Medoff, G. Biochim. Biophys. Acta 1980, 602, 260. (44) Vertut-Croquin, A.; Bolard, J.; Chambert, M.; Gary-Bobo, C. M. Biochemistry 1983, 22, 2939. (45) Readio, J. D.; Bittmann, R. Biochim. Biophys. Acta 1982, 685, 219. (46) Brutyan, R. A.; McPhie, P. J. Gen. Physiol. 1996, 107, 69. (47) Saka, Y.; Mita, T. J. Biochem. 1998, 123, 798. (48) Demel, R. A.; Van Deenen, L. L. M.; Kinsky, S. C. J. Biol. Chem. 1965, 240, 2749.

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lesterol instead of ergosterol), the interactions between cholesterol and the phospholipid are very strong49,50 and the equilibrium

is shifted to the left-hand side. Consequently, the amount of free cholesterol (and also free phospholipid) is low, and, moreover, there is low affinity of AmB for cholesterol42-46 (Figure 8A). As a result, the interaction that can be established between cholesterol and AmB is not of much importance. Therefore, the extent of cellular damage by the channels formation is significantly higher in the case of fungi cellular membranes as compared to host membranes. In the latter case, however, it may still cause some secondary toxicity. When liposomes are used as vehicles for AmB administration, the interactions between the polyene and the phospholipid molecules of liposomal cover significantly reduce the concentration of free AmB that is able to interact with the components of the cellular membrane. The low equilibrium concentration of free AmB may still be sufficient for induction of toxicity to fungal cells due to the strong interaction with the ergosterol of the membrane (Figure 8B). In the host cells, this equilibrium concentration of AmB is too low for induction of toxicity due to the lesser affinity of AmB to free cholesterol (which is also present in small amounts). Figure 8. Schematic representation of AmB interactions with cellular membrane components of fungi, host cells, and with liposomes: (A) free AmB (fungizone); (B) liposomal AmB.

formation is induced in the fungi membrane which eventually leads to the cells’ death (Figure 8A). In the case of mammalian cellular membrane (containing cho-

Acknowledgment. This work was supported by the Project PGIDT99PXI20302B (Xunta de Galicia). LA020290S (49) Phillips, MC. In Progress in Surface and Membrane Science; Danielli, J. F., Rosemberg, M. D., Cadenhead, D. A., Eds.; Academic Press: New York, 1972; Vol. 5, p 139. (50) Gershfeld, N. L. Biophys. J. 1978, 22, 469.