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Colloids and Surfaces B: Biointerfaces 29 (2003) 205 /215 www.elsevier.com/locate/colsurfb
Interactions of amphotericin B with saturated and unsaturated phosphatidylcholines at the air/water interface J. Min˜ones, Jr a, Patrycja Dynarowicz-La˛tka b,*, O. Conde a, J. Min˜ones a, E. Iribarnegaray a, M. Casas a a
Department of Physical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain b Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krako´w, Poland Received 10 July 2002; accepted 18 December 2002
Abstract Three synthetic phospholipids, differing in the degree of saturation of phosphatidylcholine (PC) acyl chains, namely: dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC) and palmitoyl-oleoyl phosphatidyl choline (POPC) were mixed with the antimycotic polyene antibiotic, amphotericin B (AmB), and investigated as monolayers at the air/water interface. The mixed films were spread on water (pH 6) at room temperature, and the surface pressure /area (p /A ) isotherms were recorded upon compression. The interactions were examined by analysing the phase transitions of the monolayers and calculating the films compressibility. The obtained results indicate the existence of stronger interaction between AmB and the saturated phospholipid as compared with unsaturated ones, and reveal the influence of apolar structure of PC chains on the stoichiometry of AmB /PC complexes. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Interactions; Amphotericin B; Phospholipids; Langmuir monolayers; Air/water interface
1. Introduction Amphotericin B (AmB), a polyene antibiotic, is well known for its potent antifungal properties [1]. Due to its insolubility in most solvents, it is clinically administered as a suspension in sodium deoxycholate (Fungizone). This formulation, however, has a number of serious adverse side effects,
* Corresponding author. Tel.: /48-12-633-6377; fax: /4812-634-0515. E-mail address: [email protected] (P. DynarowiczLa˛tka).
especially nephrotoxicity [1,2]. It has been reported that AmB encapsulated into phospholipid vesicles (liposomes) can significantly reduce the drug toxicity to host cells, without loss of antimycotic properties [3]. The reason for a lesser toxicity of liposomal AmB is still not well understood, even though it seems to exist a certain relationship between vehicle-forming phospholipids and AmB toxicity [4 /6]. A number of studies indicated that AmB-incorporated in saturated phospholipids is not toxic whereas AmB formulations composed of unsaturated phospholipids are almost as toxic as Fungizone [4]. Also, the Langmuir monolayer studies on the influence of AmB on the pressure/
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area (p /A ) isotherms of unsaturated and hydrogenated egg yolk lecithin indicated a pronounced effect of AmB on the shape of the saturated phospholipid isotherm, as compared with unsaturated one [7]. However, one has to take into account that egg yolk lecithin is a mixture of various lipids, differing both in chain length as well as a kind of the headgroup. Therefore, the conclusions derived from these experiments should be treated with caution. In this work we have performed systematic studies on the influence of the degree of saturation of phosphatidylcholine (PC) acyl chains on the characteristics of AmB monolayers to get deeper insight into understanding of the reduced toxicity of liposomal AmB. We have thus chosen three synthetic phospholipids for investigations, having the same (PC) headgroup bound to apolar part composed either by two saturated chains (dipalmitoyl phosphatidylcholine, DPPC), two unsaturated chains (dioleoyl phosphatidylcholine, DOPC) or one saturated and the other unsaturated chain (palmitoyl-oleoyl phosphatidylcholine, POPC). The interactions are examined by analysing the phase transitions that appear in the course of surface pressure/area (p /A ) isotherms.
2. Experimental L-a-Phosphatidylcholine
dipalmitoyl (DPPC), dioleoyl (DOPC), and La-phosphatidyl choline b-palmitoyl-g-oleoyl (POPC) (99% purity) were purchased from Sigma. AmB (purity /95%) was supplied by Squibb (Bristol /Myers Lab). The investigated phospholipids were dissolved in 4:1 (v:v) mixture of chloroform (Merck, p.a.) and absolute ethanol (Merck, p.a.). AmB spreading solution was prepared in the mixture of dimethylformamide and 1M HCl (3:1 v/ v). Mixed solutions were prepared by mixing appropriate volumes of respective stock solutions. The solutions were stored at 4 8C in a desiccator saturated with the spreading solvent. Monolayer compression isotherms were obtained with a KSV (Finland) 5000 LB system, equipped with two symmetrical compartments, 71 /12 cm2 each, placed on an anti-vibration
L-a-phosphatidylcholine
table. Surface pressure was measured to a precision of 9/0.05 mN m 1 with a platinum Wilhelmy plate. Each Langmuir monolayer was prepared by dropping aliquots of approximately 200 ml (4.25 / 1016 molecules) with a Microman Gilson microsyringe, precise to 9/0.2 ml, on an aqueous subphase prepared with ultrapure water (resistivity 18.2 MV cm) obtained using Milli-Q water purification systems. Once spread, monolayers were left for approximately 10 min before the compression was initiated, for solvent evaporation. Monolayer compression was performed at a barrier speed of 30 mm min 1 (36.0 cm2 min 1), which ˚ 2 per molecule min. The corresponds to 8.5 A subphase temperature (20 8C) was controlled thermostatically to within 0.5 8C by a circulating water system.
3. Results 3.1. Pure monolayers Figs. 1/3 show the pressure/area (p /A ) isotherms of AmB in mixtures with the investigated PCs (DOPC, POPC and DPPC, respectively) spread on pure water subphase (pH 6, 20 8C). AmB, which is zwitterionic at pH 6 (pKa values /5.5 and 10.0 [8]), exhibits three distinct regions, as described elsewhere [9]. At low surface pressures, the monolayer is in the liquid expanded state of relatively low compressional modulus (Cs 1 /14 mN m 1 at p /5 mN m 1). Upon compression, the monolayer undergoes a phase transition (at p /10 mN m 1), which is seen as a plateau (A ) in the course of the isotherm, and spans over a large region of molecular areas (100 / ˚ 2 per molecule). Beyond this transition the 60 A surface pressure rises due to the increase of molecular packing. Upon further compression, with the increase of surface pressure, the isotherm shifts to low molecular areas, as a result of dissolution of monolayer material into bulk phase [9,10] and no monolayer collapse can be observed in the course of the isotherm. The plateau transition of AmB monolayer has been attributed by Saint /Pierre /Chazalet et al. [11] to the gradual change in orientation of AmB molecules from the
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207
Fig. 1. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and DOPC spread on water at pH 6, T /208C.
horizontal to vertical position. This hypothesis has been recently confirmed with quantitative BAM studies [12]. At 20 8C, both DOPC and POPC exhibit an expanded monolayer, while DPPC shows a typical LE /LC phase transition (at p /4 mN m 1) evidenced as a plateau (L) in the course of the isotherm. The parameters characteristic of PCs isotherms (such as lift-off area of 120, 110 and 100 ˚ 2 as well as the collapse pressures of 47, 44 and A 65 mN m 1 for DOPC, POPC and DPPC,
respectively) show good agreement with literature data available [13]. 3.2. Mixed films First, let us analyse mixtures of AmB and PCs, which do not show any transition in the course of the isotherms at room temperature (DOPC and POPC). As it can be seen in Figs. 1 and 2, AmB / DOPC and AmB /POPC mixed films (for XAmB ranging from 0.9 to 0.3/0.5) exhibit a phase
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Fig. 2. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and POPC spread on water at pH 6, T /208C.
transition (M) at surface pressures of approximately 12 mN m 1 (AmB /DOPC) and 13.5 mN m 1 (AmB/POPC) which appears as a pseudoplateau region. The length and the flatness of this pseudo-plateau increase as the proportion of AmB in the mixed film is augmented. The longest and the most horizontal plateau is achieved for mixed monolayer of XAmB /0.9. The addition of POPC to AmB provokes film condensation at surface pressures below the transition M, while above this value, film expansion can be observed (Fig. 2B). Similar behaviour has also been described for other mixed systems [14 /16]. However, for AmB /DOPC system, such a behaviour can only be observed when XAmB is less or equal to 0.3. For XAmB /0.3, the addition of DOPC causes always the expansion of AmB monolayer, whatever the surface pressure is. For both investigated mixtures, the plateau surface pressure for AmB monolayer (pA /10 mN m 1) increases with the addition of a phospholipid, and simultaneously the pseudoplateau of the mixtures (M ) becomes gradually more inclined and its length diminishes. Finally, for both investigated systems, at XAmB B/0.3, the transition M practically disappears. Apart from this plateau, for mixed monolayers rich in AmB (XAmB ranging from 0.9 to 0.5 for AmB /DOPC and from 0.9 to 0.7 for AmB / POPC) another ‘kink’ can be distinguished at
higher surface pressures (ca. 35 mN m 1). As it can be seen, the value of surface pressure for this transition remains practically constant with the monolayer composition for the above-mentioned mixtures, and can be attributed to the collapse of AmB (CA). On the other hand, for mixed films containing XAmB ranging from 0.1 to 0.8 (for AmB /DOPC) and from 0.1 to 0.7 (for AmB / POPC) another collapse, corresponding to that of a pure phospholipid (Cp), occurs at the surface pressure of about 45 mN m 1. To get deeper insight into the phase transitions appearing in the course of p/A isotherms, the plots of the compressional modulus (Cs 1 / /A (dp / dA )) as a function of surface pressure (Figs. 4 and 5), has been drawn. Such dependencies are very useful for clear representation of phase transitions, which are seen in the curves as minima A and M . As it can be seen, M , which corresponds to the plateau transition of the mixed monolayers, appears at somehow higher p as compared with the transition for pure AmB (A ). Although the surface pressure corresponding to the transition M hardly changes with the mixed film composition (see also results compiled in Table 1), the values of the compressional modulus, Cs 1, become smaller as the proportion of AmB in the mixed monolayer is increased. These Cs 1 values ‘quantify’ the flatness
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Fig. 3. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and DPPC spread on water at pH 6, T /208C.
of the plateau in the p /A curves: the smaller Cs 1 value, the more horizontal plateau is. For AmB /DPPC system, both pure components exhibit its own phase transition: DPPC shows a typical LE /LC transition at p /4 mN m 1. This plateau gradually disappears when AmB is incorporated into the monolayer (XAmB /0.1 /0.3) and its pressure shifts to higher values (5.7 and 9 mN m 1, respectively) (Fig. 3A). The Cs 1/p plots (Fig. 6A) show that the phase
transition of DPPC, seen as a minimum L , shifts to higher pressures for mixtures with increasing amount of AmB (XAmB /0.1 /0.3), and the increasing values of Cs 1 indicate its progressive disappearance. Simultaneously, another minimum (M ) can be distinguished at about 16 mN m 1, thus demonstrating the existence of a second phase transition. Mixtures of high AmB content (XAmB /0.7) (Fig. 6B), apart from having a transition characteristic of pure AmB (A), exhibit
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Fig. 4. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and DOPC spread on water at pH 6, T /208C.
a second transition, M , which, however, is less defined. Similarly to AmB mixtures with DOPC
and POPC, the surface pressure of this transition, M, changes only slightly with the mixed mono-
4.8 12.2 19.7
12.0 11.9 9.9
6.2 3.6
18.5
8.3 14.4
16.2 16.0
21.5 10.8
3.9 5.9 8.4
42.2
b
8.4
4.3
12.9
12.8 3.6 9.9 3.6 9.9
0 0.1 0.3 0.5 0.6 0.7 0.8 0.9 1
a
23.5 13.5
12.4 12.4 11.4 11.0 11.7 11.3
27.9 13.5 3.9 3.7 2.8 2.7
34.6 32.6 32.5 32.0 34.2
46.4 46.5 46.4 46.4 45.7 45.7 45.7
p p p Cs 1 p Cs 1 p
Plateau transition of AmB (A ), DPPC (L ) and mixed monolayers (M ). Collapse of AmB (CA) and phospholipid (CP).
34.2 34.4
44.2 43.1 42.8 42.3
p p p Cs 1 Cs 1
p
38.0
39.2
66.5 66.0 66.1 65.4
p
211
Cs 1
p
Cs 1
p
Cs 1
p
CP CA La M DPPC A CP CA M POPC A CPb CAb
The analysis of the plateau region appearing in the p /A isotherms enables us to draw conclusions regarding the interaction between molecules in the investigated mixtures. As the transition, M , was observed to be shifted to higher surface pressures as compared with the transition pressure of pure AmB (A ), it can be concluded that the addition of phospholipid molecules hinders the reorientation of AmB from horizontal to vertical position in mixed monolayers. The stronger the interaction between mixture components, the higher the pressure for the transition, M . From the plots representing the values for transition pressure versus monolayer composition (Fig. 7) it can be seen that the transition M occurs at the highest pressure for AmB /DPPC (16 mN m 1), then for
Ma
4. Discussion
DOPC Aa
layer composition. Table 1 compiles the data of transition pressures and compressibility modulus values for all three mixtures. In summary, it can be concluded that the progressive addition of a phospholipid into the AmB monolayer causes the disappearance of the phase transition A , typical of AmB, and provokes the appearance of a new phase transition, M , at higher surface pressures.
XAmB
Fig. 5. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and POPC spread on water at pH 6, T /208C.
Table 1 Transition surface pressures (p , mN m 1) and corresponding compressional modulus (Cs 1, mN m 1) values for AmB /phospholipids mixed monolayers spread on water at pH 6, T /20 8C
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Fig. 6. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and DPPC spread on water at pH 6, T /208C.
mixtures with POPC (13.5 mN m 1) and, finally for AmB /DOPC (12 mN m 1). As it has already been mentioned, besides the values of the transition surface pressure (pM), the length and the flatness of the pseudo-plateau M in the p /A isotherms can also be useful for quantifying the interactions. For the AmB /DPPC system, the plateau M reaches the maximum length at XAmB /0.5 /0.7 (Fig. 3). Also, Cs 1 attains its minimum value (maximum flatness) (Fig. 6) at the above composition range. Consequently, the strongest interaction seems to occur when the film components are in the proportion of approximately 2:1, which corresponds to XAmB /0.66 (an intermediate value between 0.5 and 0.7). Therefore, following our previous studies [17], it can be postulated that the film components form a stable complex, composed by two horizontally oriented AmB molecules and one DPPC molecule in the vertical position. At surface pressures corresponding to phase transition M (16 mN m 1), AmB molecules which are embedded in the complex, change their or-
ientation from horizontal to vertical position, thus provoking segregation of the complex into its components, which are immiscible and form two separate phases. This can be supported by applying the Crisp phase rule [18]: f /c /p/1 to the transition M . Since the number of components, c , is two (AmB and DPPC), and supposing that at the transition there are three phases, p , in equilibrium (i.e. mixed monolayer formed by 2:1 complexes, the monolayer of vertically oriented AmB molecules and the monolayer of DPPC molecules), the Crisp phase rule shows that the number of degrees of freedom, f, is zero. In consequence, the pressure of the transition remains independent on the mixed film composition. The results, presented in Fig. 7A and Table 1, confirm our assumption. Also, the analysis of the course of the isotherms in the collapse region proves the immiscibility of the film-forming components after the complex segregation. The AmB-rich mixtures exhibit an inflection at p /40 mN m 1, which corresponds to the squeezing out of AmB molecules from the monolayer, while mixed film containing high
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213
Fig. 7. Transition surface pressures and collapse pressures vs. mixed films composition for AmB /DPPC (A), AmB /POPC (B) and AmB-DOPC (C).
proportion of DPPC show a collapse occurring at nearly the same surface pressure as it is observed for pure phospholipid monolayer (p /65 mN m 1) (Figs. 3 and 7A). Such a behaviour is typical for monolayers formed by immiscible components [19]. The application of the phase rule to the collapse regions leads to the conclusion that there exist three phases in equilibrium (p /3) since, under these conditions, c /2 and f /0 (the collapse pressures are independent on the monolayer composition). At the first collapse, these three phases are the following: (i) that formed by DPPC molecules, segregated from the complex, (ii) that composed by vertically oriented AmB molecules, also separated from the complex, and (iii) the 3D phase formed by the collapsed AmB monolayer. At the second collapse, the molecules of DPPC, segregated from the complex, are squeezed out from the monolayer and, in consequence, there also exist three separate phases, namely two 3D
phases (collapsed AmB and collapsed DPPC) and the monolayer of DPPC formed by phospolipid molecules segregated from the complex. For the mixed film of XAmB /0.9, two phase transitions can be distinguished in the course of the isotherm: A and M (Fig. 3C, Fig. 7A). The first transition, A, corresponds to the change in orientation of the free AmB molecules which are stoichiometrically in excess and are not embedded in the 2:1 complex formation. In this situation, c / 2 (AmB and DPPC) and f /1, since the transition pressure varies with the mixed film composition (Fig. 7A). Therefore, there are two phases in equilibrium (p): (i) mixed monolayer composed of AmB:DPPC complexes and horizontally oriented AmB molecules in excess, and (ii) mixed monolayer formed by complexes and vertically oriented AmB molecules in excess. The second transition, M, is due to orientational changes of AmB molecules embedded in the complex, as it has been already described above.
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For mixtures containing DPPC in excess (XAmB B/0.66), there are also two phase transition visible in the course of the isotherms (Fig. 7A): the first one, corresponding to the LE /LC transition of the monolayer formed by DPPC molecules that are in excess (L ), and the second one, corresponding to the mixed films composed of 2:1 AmB:DPPC complexes (M ). Since the pressure of the transition L varies with the mixed monolayer composition (f /1), there exist two phases in equilibrium (p /2): the first one, formed by complexes mixed with DPPC in the expanded phase, and the other one, formed by the complexes and the phosphilipid molecules in the condensed state. For the other systems investigated here (AmB / POPC and AmB /DOPC), the interaction between film components seems to be weaker since the transition M appears at lower surface pressures (12 /13 mN m 1) than for AmB /DPPC system (16 mN m 1). Moreover, the stoichiometry of the complexes is seen to be different as well. The fact, that the p/A isotherms of these systems show the longest and the most horizontal plateau at XAmB /0.9 (Fig. 1C, Fig. 2B), suggests that the strongest interaction correspond to the complex formation of 9:1 stoichiometry (AmB:POPC/ DOPC). It is possible that the presence of unsaturated bond(s) in the hydrophobic tails of the phospholipids unable these molecules to attain the vertical orientation at the air/water interface, thus making the polar groups of a phospholipid less accessible to AmB molecules and, in consequence, reducing the strength of interaction between filmforming components and leading to the formation of complexes of different stoichiometry, rich in AmB molecules, in order to attain the possibly closest arrangement with the phospholipid. For mixtures of XAmB B/0.9, the 9:1 complexes exist together with the increasing amount of phospholipid molecules (which are present in excess) while the quantity of AmB is diminishing. Under these conditions, a miscible system is formed wherein the plateau, corresponding to the transition M , becomes gradually smaller and less horizontal due to the decreasing number of complexes in the mixture. For monolayers of AmB below 0.3, this effect is no longer observed.
Definitely, the complexes composed by AmB and either DOPC or POPC, contain higher proportion of AmB, however, are less stable than those formed by AmB and DPPC. The lower toxicity of liposomal formulation of AmB formed by saturated phospholipids, as compared with unsaturated ones, can be explained as being due to the AmB /phospholipid interaction, which are stronger for saturated than unsaturated phospholipids. This leads to the presence of a moderate quantity of AmB in the host cells which is enough to provoke the lysis of fungi cells (fungicidal effect), however, it is not sufficient to damage the host cells. The obtained results confirm the hypothesis regarding the influence of the nature of phospholipid apolar group on the interaction with AmB. Nevertheless, no significant differences regarding the magnitude of interaction have been observed in the investigated systems. This is not surprising since in monolayers the interactions between AmB and a phospholipid occur between polar groups, which is identical for all investigated here PCs.
Acknowledgements This work was supported by the grant PGIDT99PX120302B (Xunta de Cialicia).
References [1] G. Medoff, J. Brajtburg, G.S. Kobayashi, J. Bolard, Annu. Rev. Pharmacol. Toxicol. 23 (1983) 303. [2] W.B. Pratt (Ed.), Chemotherapy of Infections, Oxford University Press, New York, 1977. [3] R.L. Juliano, G. Lopez-Berestein, R. Hopfer, R. Mehta, K. Mehta, K. Mills, Ann. NY Acad. Sci. 446 (1985) 390. [4] R.L. Juliano, C.W. Grant, K.R. Barber, M.A. Kalp, Mol. Pharmacol. 31 (1987) 1. [5] V. Joly, J. Bolard, L. Saint-Julien, C. Carbon, P. Yeni, Antimicrob. Agents Chemother. 36 (1992) 262. [6] M.R. Lance, C. Washington, S.S. Davis, J. Antimicrob. Chemother. 36 (1995) 119. [7] M.R. Lance, C. Washington, S.S. Davis, Pharm. Res. 13 (1996) 1008. [8] D.W. Newton, R.B. Kluza, Drug Intel. Clin. Pharm. 12 (1978) 546.
J. Min˜ones, Jr et al. / Colloids and Surfaces B: Biointerfaces 29 (2003) 205 /215 [9] I. Rey Go´mez-Serranillos, J. Min˜ones, Jr, R. Seoane, O. Conde, M. Casas, Progr. Colloid Polym. Sci. 112 (1999) 29. [10] I. Rey Go´mez-Serranillos, P. Dynarowicz-La˛tka, J. Min˜ones, Jr, R. Seoane, J. Colloid Interf. Sci. 234 (2001) 351. [11] M. Saint-Pierre-Chazalet, C. Thomas, M. Dupeyrat, C.M. Gary-Bobo, Biochim. Biophys. Acta 944 (1988) 477. [12] J. Min˜ones, Jr, C. Carrera Sa´nchez, P. Dynarowicz-La˛tka, J. Min˜ones, O. Conde, R. Seoane, J.M. Rodriguez-Patino, Langmuir 17 (2001) 1477. [13] A.F. Mingotaud, in: C. Mingotaud, L.K. Patterson LK (Eds.), Handbook of Monolayers, vol. 1, no 3, Academic Press, San Diego, CA, 1993, pp. 796 (DPPC), 862 (DOPC) and 892 (POPC).
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[14] D.A. Cadenhead, M.C. Phillips, Adv. Chem. Ser. 84 (1968) 131. [15] R. Seoane, J. Min˜ones, O. Conde, E. Iribarnegaray, M. Casas, Langmuir 15 (1999) 5567. [16] J. Min˜ones, Jr, J. Min˜ones, O. Conde, R. Seoane, P. Dynarowicz-La˛tka, Langmuir 16 (2000) 5743. [17] J. Min˜ones, Jr, J. Min˜ones, O. Conde, J.M. RodriguezPatino, P. Dynarowicz-La˛tka, Langmuir 18 (2002) 2817. [18] D.J. Crisp, Surface Chemistry (Supplimentary Research), Butterworths, London, 1949, pp. 17 /35. [19] G. Gainess, in: I. Prigogine (Ed.), Insoluble Monolayers at Liquid /Gas Interface Interscience, New York, 1966, pp. 281 /300.
Interactions of amphotericin B with saturated and unsaturated phosphatidylcholines at the air/water interface J. Min˜ones, Jr a, Patrycja Dynarowicz-La˛tka b,*, O. Conde a, J. Min˜ones a, E. Iribarnegaray a, M. Casas a a
Department of Physical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain b Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krako´w, Poland Received 10 July 2002; accepted 18 December 2002
Abstract Three synthetic phospholipids, differing in the degree of saturation of phosphatidylcholine (PC) acyl chains, namely: dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine (DOPC) and palmitoyl-oleoyl phosphatidyl choline (POPC) were mixed with the antimycotic polyene antibiotic, amphotericin B (AmB), and investigated as monolayers at the air/water interface. The mixed films were spread on water (pH 6) at room temperature, and the surface pressure /area (p /A ) isotherms were recorded upon compression. The interactions were examined by analysing the phase transitions of the monolayers and calculating the films compressibility. The obtained results indicate the existence of stronger interaction between AmB and the saturated phospholipid as compared with unsaturated ones, and reveal the influence of apolar structure of PC chains on the stoichiometry of AmB /PC complexes. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Interactions; Amphotericin B; Phospholipids; Langmuir monolayers; Air/water interface
1. Introduction Amphotericin B (AmB), a polyene antibiotic, is well known for its potent antifungal properties [1]. Due to its insolubility in most solvents, it is clinically administered as a suspension in sodium deoxycholate (Fungizone). This formulation, however, has a number of serious adverse side effects,
* Corresponding author. Tel.: /48-12-633-6377; fax: /4812-634-0515. E-mail address: [email protected] (P. DynarowiczLa˛tka).
especially nephrotoxicity [1,2]. It has been reported that AmB encapsulated into phospholipid vesicles (liposomes) can significantly reduce the drug toxicity to host cells, without loss of antimycotic properties [3]. The reason for a lesser toxicity of liposomal AmB is still not well understood, even though it seems to exist a certain relationship between vehicle-forming phospholipids and AmB toxicity [4 /6]. A number of studies indicated that AmB-incorporated in saturated phospholipids is not toxic whereas AmB formulations composed of unsaturated phospholipids are almost as toxic as Fungizone [4]. Also, the Langmuir monolayer studies on the influence of AmB on the pressure/
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area (p /A ) isotherms of unsaturated and hydrogenated egg yolk lecithin indicated a pronounced effect of AmB on the shape of the saturated phospholipid isotherm, as compared with unsaturated one [7]. However, one has to take into account that egg yolk lecithin is a mixture of various lipids, differing both in chain length as well as a kind of the headgroup. Therefore, the conclusions derived from these experiments should be treated with caution. In this work we have performed systematic studies on the influence of the degree of saturation of phosphatidylcholine (PC) acyl chains on the characteristics of AmB monolayers to get deeper insight into understanding of the reduced toxicity of liposomal AmB. We have thus chosen three synthetic phospholipids for investigations, having the same (PC) headgroup bound to apolar part composed either by two saturated chains (dipalmitoyl phosphatidylcholine, DPPC), two unsaturated chains (dioleoyl phosphatidylcholine, DOPC) or one saturated and the other unsaturated chain (palmitoyl-oleoyl phosphatidylcholine, POPC). The interactions are examined by analysing the phase transitions that appear in the course of surface pressure/area (p /A ) isotherms.
2. Experimental L-a-Phosphatidylcholine
dipalmitoyl (DPPC), dioleoyl (DOPC), and La-phosphatidyl choline b-palmitoyl-g-oleoyl (POPC) (99% purity) were purchased from Sigma. AmB (purity /95%) was supplied by Squibb (Bristol /Myers Lab). The investigated phospholipids were dissolved in 4:1 (v:v) mixture of chloroform (Merck, p.a.) and absolute ethanol (Merck, p.a.). AmB spreading solution was prepared in the mixture of dimethylformamide and 1M HCl (3:1 v/ v). Mixed solutions were prepared by mixing appropriate volumes of respective stock solutions. The solutions were stored at 4 8C in a desiccator saturated with the spreading solvent. Monolayer compression isotherms were obtained with a KSV (Finland) 5000 LB system, equipped with two symmetrical compartments, 71 /12 cm2 each, placed on an anti-vibration
L-a-phosphatidylcholine
table. Surface pressure was measured to a precision of 9/0.05 mN m 1 with a platinum Wilhelmy plate. Each Langmuir monolayer was prepared by dropping aliquots of approximately 200 ml (4.25 / 1016 molecules) with a Microman Gilson microsyringe, precise to 9/0.2 ml, on an aqueous subphase prepared with ultrapure water (resistivity 18.2 MV cm) obtained using Milli-Q water purification systems. Once spread, monolayers were left for approximately 10 min before the compression was initiated, for solvent evaporation. Monolayer compression was performed at a barrier speed of 30 mm min 1 (36.0 cm2 min 1), which ˚ 2 per molecule min. The corresponds to 8.5 A subphase temperature (20 8C) was controlled thermostatically to within 0.5 8C by a circulating water system.
3. Results 3.1. Pure monolayers Figs. 1/3 show the pressure/area (p /A ) isotherms of AmB in mixtures with the investigated PCs (DOPC, POPC and DPPC, respectively) spread on pure water subphase (pH 6, 20 8C). AmB, which is zwitterionic at pH 6 (pKa values /5.5 and 10.0 [8]), exhibits three distinct regions, as described elsewhere [9]. At low surface pressures, the monolayer is in the liquid expanded state of relatively low compressional modulus (Cs 1 /14 mN m 1 at p /5 mN m 1). Upon compression, the monolayer undergoes a phase transition (at p /10 mN m 1), which is seen as a plateau (A ) in the course of the isotherm, and spans over a large region of molecular areas (100 / ˚ 2 per molecule). Beyond this transition the 60 A surface pressure rises due to the increase of molecular packing. Upon further compression, with the increase of surface pressure, the isotherm shifts to low molecular areas, as a result of dissolution of monolayer material into bulk phase [9,10] and no monolayer collapse can be observed in the course of the isotherm. The plateau transition of AmB monolayer has been attributed by Saint /Pierre /Chazalet et al. [11] to the gradual change in orientation of AmB molecules from the
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207
Fig. 1. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and DOPC spread on water at pH 6, T /208C.
horizontal to vertical position. This hypothesis has been recently confirmed with quantitative BAM studies [12]. At 20 8C, both DOPC and POPC exhibit an expanded monolayer, while DPPC shows a typical LE /LC phase transition (at p /4 mN m 1) evidenced as a plateau (L) in the course of the isotherm. The parameters characteristic of PCs isotherms (such as lift-off area of 120, 110 and 100 ˚ 2 as well as the collapse pressures of 47, 44 and A 65 mN m 1 for DOPC, POPC and DPPC,
respectively) show good agreement with literature data available [13]. 3.2. Mixed films First, let us analyse mixtures of AmB and PCs, which do not show any transition in the course of the isotherms at room temperature (DOPC and POPC). As it can be seen in Figs. 1 and 2, AmB / DOPC and AmB /POPC mixed films (for XAmB ranging from 0.9 to 0.3/0.5) exhibit a phase
208
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Fig. 2. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and POPC spread on water at pH 6, T /208C.
transition (M) at surface pressures of approximately 12 mN m 1 (AmB /DOPC) and 13.5 mN m 1 (AmB/POPC) which appears as a pseudoplateau region. The length and the flatness of this pseudo-plateau increase as the proportion of AmB in the mixed film is augmented. The longest and the most horizontal plateau is achieved for mixed monolayer of XAmB /0.9. The addition of POPC to AmB provokes film condensation at surface pressures below the transition M, while above this value, film expansion can be observed (Fig. 2B). Similar behaviour has also been described for other mixed systems [14 /16]. However, for AmB /DOPC system, such a behaviour can only be observed when XAmB is less or equal to 0.3. For XAmB /0.3, the addition of DOPC causes always the expansion of AmB monolayer, whatever the surface pressure is. For both investigated mixtures, the plateau surface pressure for AmB monolayer (pA /10 mN m 1) increases with the addition of a phospholipid, and simultaneously the pseudoplateau of the mixtures (M ) becomes gradually more inclined and its length diminishes. Finally, for both investigated systems, at XAmB B/0.3, the transition M practically disappears. Apart from this plateau, for mixed monolayers rich in AmB (XAmB ranging from 0.9 to 0.5 for AmB /DOPC and from 0.9 to 0.7 for AmB / POPC) another ‘kink’ can be distinguished at
higher surface pressures (ca. 35 mN m 1). As it can be seen, the value of surface pressure for this transition remains practically constant with the monolayer composition for the above-mentioned mixtures, and can be attributed to the collapse of AmB (CA). On the other hand, for mixed films containing XAmB ranging from 0.1 to 0.8 (for AmB /DOPC) and from 0.1 to 0.7 (for AmB / POPC) another collapse, corresponding to that of a pure phospholipid (Cp), occurs at the surface pressure of about 45 mN m 1. To get deeper insight into the phase transitions appearing in the course of p/A isotherms, the plots of the compressional modulus (Cs 1 / /A (dp / dA )) as a function of surface pressure (Figs. 4 and 5), has been drawn. Such dependencies are very useful for clear representation of phase transitions, which are seen in the curves as minima A and M . As it can be seen, M , which corresponds to the plateau transition of the mixed monolayers, appears at somehow higher p as compared with the transition for pure AmB (A ). Although the surface pressure corresponding to the transition M hardly changes with the mixed film composition (see also results compiled in Table 1), the values of the compressional modulus, Cs 1, become smaller as the proportion of AmB in the mixed monolayer is increased. These Cs 1 values ‘quantify’ the flatness
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209
Fig. 3. Surface Pressure /area (p /A ) isotherms for mixed monolayers of AmB and DPPC spread on water at pH 6, T /208C.
of the plateau in the p /A curves: the smaller Cs 1 value, the more horizontal plateau is. For AmB /DPPC system, both pure components exhibit its own phase transition: DPPC shows a typical LE /LC transition at p /4 mN m 1. This plateau gradually disappears when AmB is incorporated into the monolayer (XAmB /0.1 /0.3) and its pressure shifts to higher values (5.7 and 9 mN m 1, respectively) (Fig. 3A). The Cs 1/p plots (Fig. 6A) show that the phase
transition of DPPC, seen as a minimum L , shifts to higher pressures for mixtures with increasing amount of AmB (XAmB /0.1 /0.3), and the increasing values of Cs 1 indicate its progressive disappearance. Simultaneously, another minimum (M ) can be distinguished at about 16 mN m 1, thus demonstrating the existence of a second phase transition. Mixtures of high AmB content (XAmB /0.7) (Fig. 6B), apart from having a transition characteristic of pure AmB (A), exhibit
210
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Fig. 4. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and DOPC spread on water at pH 6, T /208C.
a second transition, M , which, however, is less defined. Similarly to AmB mixtures with DOPC
and POPC, the surface pressure of this transition, M, changes only slightly with the mixed mono-
4.8 12.2 19.7
12.0 11.9 9.9
6.2 3.6
18.5
8.3 14.4
16.2 16.0
21.5 10.8
3.9 5.9 8.4
42.2
b
8.4
4.3
12.9
12.8 3.6 9.9 3.6 9.9
0 0.1 0.3 0.5 0.6 0.7 0.8 0.9 1
a
23.5 13.5
12.4 12.4 11.4 11.0 11.7 11.3
27.9 13.5 3.9 3.7 2.8 2.7
34.6 32.6 32.5 32.0 34.2
46.4 46.5 46.4 46.4 45.7 45.7 45.7
p p p Cs 1 p Cs 1 p
Plateau transition of AmB (A ), DPPC (L ) and mixed monolayers (M ). Collapse of AmB (CA) and phospholipid (CP).
34.2 34.4
44.2 43.1 42.8 42.3
p p p Cs 1 Cs 1
p
38.0
39.2
66.5 66.0 66.1 65.4
p
211
Cs 1
p
Cs 1
p
Cs 1
p
CP CA La M DPPC A CP CA M POPC A CPb CAb
The analysis of the plateau region appearing in the p /A isotherms enables us to draw conclusions regarding the interaction between molecules in the investigated mixtures. As the transition, M , was observed to be shifted to higher surface pressures as compared with the transition pressure of pure AmB (A ), it can be concluded that the addition of phospholipid molecules hinders the reorientation of AmB from horizontal to vertical position in mixed monolayers. The stronger the interaction between mixture components, the higher the pressure for the transition, M . From the plots representing the values for transition pressure versus monolayer composition (Fig. 7) it can be seen that the transition M occurs at the highest pressure for AmB /DPPC (16 mN m 1), then for
Ma
4. Discussion
DOPC Aa
layer composition. Table 1 compiles the data of transition pressures and compressibility modulus values for all three mixtures. In summary, it can be concluded that the progressive addition of a phospholipid into the AmB monolayer causes the disappearance of the phase transition A , typical of AmB, and provokes the appearance of a new phase transition, M , at higher surface pressures.
XAmB
Fig. 5. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and POPC spread on water at pH 6, T /208C.
Table 1 Transition surface pressures (p , mN m 1) and corresponding compressional modulus (Cs 1, mN m 1) values for AmB /phospholipids mixed monolayers spread on water at pH 6, T /20 8C
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Fig. 6. Compressional modulus /surface pressure (Cs 1 /p ) plots for mixed monolayers of AmB and DPPC spread on water at pH 6, T /208C.
mixtures with POPC (13.5 mN m 1) and, finally for AmB /DOPC (12 mN m 1). As it has already been mentioned, besides the values of the transition surface pressure (pM), the length and the flatness of the pseudo-plateau M in the p /A isotherms can also be useful for quantifying the interactions. For the AmB /DPPC system, the plateau M reaches the maximum length at XAmB /0.5 /0.7 (Fig. 3). Also, Cs 1 attains its minimum value (maximum flatness) (Fig. 6) at the above composition range. Consequently, the strongest interaction seems to occur when the film components are in the proportion of approximately 2:1, which corresponds to XAmB /0.66 (an intermediate value between 0.5 and 0.7). Therefore, following our previous studies [17], it can be postulated that the film components form a stable complex, composed by two horizontally oriented AmB molecules and one DPPC molecule in the vertical position. At surface pressures corresponding to phase transition M (16 mN m 1), AmB molecules which are embedded in the complex, change their or-
ientation from horizontal to vertical position, thus provoking segregation of the complex into its components, which are immiscible and form two separate phases. This can be supported by applying the Crisp phase rule [18]: f /c /p/1 to the transition M . Since the number of components, c , is two (AmB and DPPC), and supposing that at the transition there are three phases, p , in equilibrium (i.e. mixed monolayer formed by 2:1 complexes, the monolayer of vertically oriented AmB molecules and the monolayer of DPPC molecules), the Crisp phase rule shows that the number of degrees of freedom, f, is zero. In consequence, the pressure of the transition remains independent on the mixed film composition. The results, presented in Fig. 7A and Table 1, confirm our assumption. Also, the analysis of the course of the isotherms in the collapse region proves the immiscibility of the film-forming components after the complex segregation. The AmB-rich mixtures exhibit an inflection at p /40 mN m 1, which corresponds to the squeezing out of AmB molecules from the monolayer, while mixed film containing high
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213
Fig. 7. Transition surface pressures and collapse pressures vs. mixed films composition for AmB /DPPC (A), AmB /POPC (B) and AmB-DOPC (C).
proportion of DPPC show a collapse occurring at nearly the same surface pressure as it is observed for pure phospholipid monolayer (p /65 mN m 1) (Figs. 3 and 7A). Such a behaviour is typical for monolayers formed by immiscible components [19]. The application of the phase rule to the collapse regions leads to the conclusion that there exist three phases in equilibrium (p /3) since, under these conditions, c /2 and f /0 (the collapse pressures are independent on the monolayer composition). At the first collapse, these three phases are the following: (i) that formed by DPPC molecules, segregated from the complex, (ii) that composed by vertically oriented AmB molecules, also separated from the complex, and (iii) the 3D phase formed by the collapsed AmB monolayer. At the second collapse, the molecules of DPPC, segregated from the complex, are squeezed out from the monolayer and, in consequence, there also exist three separate phases, namely two 3D
phases (collapsed AmB and collapsed DPPC) and the monolayer of DPPC formed by phospolipid molecules segregated from the complex. For the mixed film of XAmB /0.9, two phase transitions can be distinguished in the course of the isotherm: A and M (Fig. 3C, Fig. 7A). The first transition, A, corresponds to the change in orientation of the free AmB molecules which are stoichiometrically in excess and are not embedded in the 2:1 complex formation. In this situation, c / 2 (AmB and DPPC) and f /1, since the transition pressure varies with the mixed film composition (Fig. 7A). Therefore, there are two phases in equilibrium (p): (i) mixed monolayer composed of AmB:DPPC complexes and horizontally oriented AmB molecules in excess, and (ii) mixed monolayer formed by complexes and vertically oriented AmB molecules in excess. The second transition, M, is due to orientational changes of AmB molecules embedded in the complex, as it has been already described above.
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For mixtures containing DPPC in excess (XAmB B/0.66), there are also two phase transition visible in the course of the isotherms (Fig. 7A): the first one, corresponding to the LE /LC transition of the monolayer formed by DPPC molecules that are in excess (L ), and the second one, corresponding to the mixed films composed of 2:1 AmB:DPPC complexes (M ). Since the pressure of the transition L varies with the mixed monolayer composition (f /1), there exist two phases in equilibrium (p /2): the first one, formed by complexes mixed with DPPC in the expanded phase, and the other one, formed by the complexes and the phosphilipid molecules in the condensed state. For the other systems investigated here (AmB / POPC and AmB /DOPC), the interaction between film components seems to be weaker since the transition M appears at lower surface pressures (12 /13 mN m 1) than for AmB /DPPC system (16 mN m 1). Moreover, the stoichiometry of the complexes is seen to be different as well. The fact, that the p/A isotherms of these systems show the longest and the most horizontal plateau at XAmB /0.9 (Fig. 1C, Fig. 2B), suggests that the strongest interaction correspond to the complex formation of 9:1 stoichiometry (AmB:POPC/ DOPC). It is possible that the presence of unsaturated bond(s) in the hydrophobic tails of the phospholipids unable these molecules to attain the vertical orientation at the air/water interface, thus making the polar groups of a phospholipid less accessible to AmB molecules and, in consequence, reducing the strength of interaction between filmforming components and leading to the formation of complexes of different stoichiometry, rich in AmB molecules, in order to attain the possibly closest arrangement with the phospholipid. For mixtures of XAmB B/0.9, the 9:1 complexes exist together with the increasing amount of phospholipid molecules (which are present in excess) while the quantity of AmB is diminishing. Under these conditions, a miscible system is formed wherein the plateau, corresponding to the transition M , becomes gradually smaller and less horizontal due to the decreasing number of complexes in the mixture. For monolayers of AmB below 0.3, this effect is no longer observed.
Definitely, the complexes composed by AmB and either DOPC or POPC, contain higher proportion of AmB, however, are less stable than those formed by AmB and DPPC. The lower toxicity of liposomal formulation of AmB formed by saturated phospholipids, as compared with unsaturated ones, can be explained as being due to the AmB /phospholipid interaction, which are stronger for saturated than unsaturated phospholipids. This leads to the presence of a moderate quantity of AmB in the host cells which is enough to provoke the lysis of fungi cells (fungicidal effect), however, it is not sufficient to damage the host cells. The obtained results confirm the hypothesis regarding the influence of the nature of phospholipid apolar group on the interaction with AmB. Nevertheless, no significant differences regarding the magnitude of interaction have been observed in the investigated systems. This is not surprising since in monolayers the interactions between AmB and a phospholipid occur between polar groups, which is identical for all investigated here PCs.
Acknowledgements This work was supported by the grant PGIDT99PX120302B (Xunta de Cialicia).
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[14] D.A. Cadenhead, M.C. Phillips, Adv. Chem. Ser. 84 (1968) 131. [15] R. Seoane, J. Min˜ones, O. Conde, E. Iribarnegaray, M. Casas, Langmuir 15 (1999) 5567. [16] J. Min˜ones, Jr, J. Min˜ones, O. Conde, R. Seoane, P. Dynarowicz-La˛tka, Langmuir 16 (2000) 5743. [17] J. Min˜ones, Jr, J. Min˜ones, O. Conde, J.M. RodriguezPatino, P. Dynarowicz-La˛tka, Langmuir 18 (2002) 2817. [18] D.J. Crisp, Surface Chemistry (Supplimentary Research), Butterworths, London, 1949, pp. 17 /35. [19] G. Gainess, in: I. Prigogine (Ed.), Insoluble Monolayers at Liquid /Gas Interface Interscience, New York, 1966, pp. 281 /300.