Interactions of a Fungistatic Antibiotic, Griseofulvin, with Phospholipid

Poincare´ Nancy 1, Faculte´ des Sciences, BP 239, 54506 ... Nutrition, INSERM U-724/UniVersite´ Henri Poincare´ Nancy 1, Faculte´ de Me´decine, BP 184...
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Langmuir 2006, 22, 7701-7711

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Interactions of a Fungistatic Antibiotic, Griseofulvin, with Phospholipid Monolayers Used as Models of Biological Membranes Yohann Corvis,† Wanda Barzyk,†,‡ Gerald Brezesinski,§ Nadir Mrabet,⊥ Mounia Badis,† Sabrina Hecht,† and Ewa Rogalska*,† Groupe d’Etude des Vecteurs Supramole´ culaires du Me´ dicament UMR 7565 CNRS/UniVersite´ Henri Poincare´ Nancy 1, Faculte´ des Sciences, BP 239, 54506 VandoeuVre-le` s-Nancy Cedex, France, Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek, 30-239 Krakow, Poland, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, Am Mu¨hlenberg 1, 14476 Potsdam, Germany, and Laboratoire de Pathologie Cellulaire et Mole´ culaire en Nutrition, INSERM U-724/UniVersite´ Henri Poincare´ Nancy 1, Faculte´ de Me´ decine, BP 184, 54505 VandoeuVre-le` s-Nancy Cedex, France ReceiVed April 12, 2006. In Final Form: June 9, 2006 Griseofulvin (GF) is an oral antibiotic for widely occurring superficial mycosis in man and animals caused by dermaphyte fungi; it is also used in agriculture as a fungicide. The mechanism of the biological activity of GF is poorly understood. Here, the interactions of griseofulvin with lipid membranes were studied using 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-myristoyl-sn-glycero3-phosphoethanolamine (DMPE) monolayers spread at the air/water interface. Surface pressure (Π), electric surface potential (∆V), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) were used for studying pure phospholipid monolayers spread on GF aqueous solutions, as well as mixed phospholipid/GF monolayers spread on pure water subphase. Moreover, phospholipase A2 (PLA2) activity toward DLPC monolayers and molecular modeling of the GF surface and lipophilic properties were used to get more insight into the mechanisms of GFmembrane interactions. The results obtained show that GF has a meaningful impact on the film properties; we propose that nonpolar interactions are by and large responsible for GF retention in the monolayers. The modification of membrane properties can be detected using both physicochemical and enzymatic methods. The results obtained may be relevant for elaborating GF preparations with increased bioavailability.

Introduction Griseofulvin, (1′S,6′R)-7-chloro-2′,4,6-trimethoxy-6′-methylspiro[benzofuran-2(3H),1′-[2]cyclohexene]-3,4′-dione, is a fungistatic antibiotic active against dermatophytes which comes from the mold Penicillium griseofulVum.1,2 Griseofulvin was the first available oral agent for the treatment of dermatophytoses and has now been used for more than 40 years. It treats fungus infections caused by tinea organisms of the skin, hair, and nails. It is not effective against yeasts such as candida or malassezia.3 Presently, it is one of five available oral antifungal agents (griseofulvin, ketoconazole, itraconazole, terbinafine, and fluconazole) in the treatment of onychomycosis.4 On the other hand, griseofulvin could be useful in the treatment of cancer, as it blocks cell-cycle progression at the G2/M phase and induces apoptosis in human tumor cell lines.5 However, water solubility and bioavailability of GF are very low, and more research is needed on GF formulation for enhancing its absorption. * Corresponding author. E-mail: [email protected]. † Groupe d’Etude des Vecteurs Supramole ´ culaires du Me´dicament, Universite´ Henri Poincare´. ‡ On leave from the Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences. § Max Planck Institute of Colloids and Interfaces. ⊥ Laboratoire de Pathologie Cellulaire et Mole ´ culaire en Nutrition, Universite´ Henri Poincare´. (1) Baran, R.; Gupta, A. K.; Pierard, G. E. Expert Opin. Pharmacother. 2005, 6, 609-624. (2) http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/webglimpse.cgi?ID ) 16&whole ) ON&cache ) yes&query ) griseofulvin (accessed April 12, 2006). (3) http://dermnetnz.org/treatments/griseofulvin.html (accessed April 12, 2006). (4) Schlefman, B. S. J. Foot Ankle Surg. 1999, 38, 290-302. (5) Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9878-9883.

Antifungal agents currently utilized for mycoses attack one of four targets in fungal pathogens: fungal cell membrane (plasma membrane), fungal cell wall, DNA and protein synthesis, and fungal mitotic spindles (cell division-mitosis). The precise mechanism of the griseofulvin action is not well-known. Several hypotheses have been put forward, and it might work on different levels. The first mechanism proposed is an interference with the synthesis of certain components of the fungal cell walls such as chitin.6 Another mechanism would be the capacity of griseofulvin to break the structure of the mitotic nuclear envelope (the doublelayered membrane enclosing the nucleus of a cell), thus stopping cell division at the metaphase stage.7,8 It has recently been proposed that the primary mechanism by which griseofulvin inhibits mitosis in human cells is by suppressing mitotic spindle microtubule dynamics in a manner qualitatively similar to that of much more powerful antimitotic drugs, including the vinca alkaloids and the taxanes.5 It is interesting to note that microtubules9 are protein polymers possessing the ability to apply individually an extensive force at a significant distance on the cellular scale. The interactions of microtubules with cell membranes seem to influence cell morphologies. Indeed, a sharp transition between elongated and circular morphologies was observed recently upon microtubule action in unilamellar phospholipid vesicles used as model membranes. This effect was interpreted in terms of mechanical constraints imposed by both microtubule and membrane.10 (6) Anderson, D. W. Ann. Allergy 1965, 23, 103-110. (7) Gull, K.; Trinci, A. P. J. Nature 1973, 244, 292-294. (8) Develoux, M. Ann. Dermatol. Venereol. 2001, 128, 1317-1325. (9) http://www.ruf.rice.edu/∼bioslabs/studies/invertebrates/microtubules.html (accessed April 12, 2006).

10.1021/la060998x CCC: $33.50 © 2006 American Chemical Society Published on Web 08/04/2006

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The available knowledge on the griseofulvin-microtubule and microtubule-membrane interactions, as well as the possible impact of griseofulvin on the mitotic envelope, prompted us to study the griseofulvin-membrane interactions. Indeed, griseofulvin biological activity being also related to its capacity to modify directly cell membranes cannot be excluded; this in turn might influence, among others, microtubule-membrane interactions. Biological membranes are complex structures, of which the major components are phospholipids forming bilayers. Other components of the membranes, such as sterols or proteins, are dispersed in the bilayer. A phospholipid monolayer spread at the air/water interface can be considered to be a model of biological membranes, being in a way a half-membrane. The phase boundaries between a phospholipid bilayer or a monolayer and aqueous solution are similar in terms of lipid structure, if both types of films are compared at the same molecular packing, defined as the surface area, A, per lipid molecule. In terms of electrical properties defined by the dielectric constant, the gas phase at the air/phospholipid monolayer boundary is a substitute of acyl chains of the reversed monolayer added to form the bilayer, i.e., constituting the superstructure. Nonetheless, for the results obtained with such simple model membranes to be extrapolated to much more complex biological systems, the adequate physicochemical properties of the model and its limitations must be established. In this study, the interactions between model membranes and GF were examined using compression isotherms of pure DPPC, DLPC, and DMPE monolayers spread on subphases containing GF, mixed DPPC/GF, DLPC/GF, and DMPE/GF monolayers spread on pure water subphase, and kinetics of adsorption of GF to DPPC monolayers of a given density under stationary diffusion conditions. These phospholipids were chosen because both phosphocholines and phosphoethanolamines are representative lipid components of eukaryote membranes. The interactions of GF with lipid monolayers were monitored via simultaneous measurements of surface pressure (Π) and surface electric potential (∆V). Moreover, the lateral organization and morphology of the monolayers formed in the presence of GF was investigated using grazing incidence X-ray diffraction (GIXD) and Brewster angle microscopy (BAM), respectively. Finally, the quantity of GF incorporated into the monolayer was estimated based on modeling of GF structure and on compression isotherm parameters. The results obtained show that very low quantities of GF present in the film have a meaningful impact on its structure and properties; these changes can be detected in certain conditions using PLA2 activity. These observations support the conjecture that GF biological activity may be related to the interactions with membranes. From an application point of view, it is important to notice that GF does not penetrate easily through the polar head region of the film but stays in the membrane when mixed with the phospholipids. This finding may help in preparing efficient GF formulations.11-13 Experimental Section Chemicals. DPPC, DMPE, DLPC (purity 99%), and GF were from Sigma (purity at least 95%). Calcium chloride (CaCl2) was from Prolabo, purity 97%. Aqueous solutions of 20 µM GF were used in all experiments; GF solubility in water is slightly above 20 (10) Fygenson, D K.; Elbaum, M.; Shraiman, B.; Libchaber, A. Phys. ReV. E 1997, 55, 850-859. (11) Ezz El-Din, M. M.; Abu-Zaid, S. S.; Ghanem, E. H. Afr. J. Mycol. Biotechnol. 1995, 3, 89-99. (12) Sue, M. S.; Liu, K. M.; Yu, H. S. Gaoxiong Yixue Kexue Zazhi 1993, 9, 1-8. (13) Stozek, T.; Borysiewicz, J. Pharmazie 1991, 46, 39-41.

CorVis et al. µM. The solutions were prepared by stirring GF for 24 h in MilliQ, degassed water. MilliQ water used in the experiments contained between 6.1 and 21.7 ng mL-1 (between 0.15 and 0.54 µM) of Ca2+ cations, as determined using a Spectra AA 246 FS graphite furnace atomic absorption spectrometer (GFAAS; Varian, Palo Alto, CA). Spectrophotometric grade chloroform and methanol (Aldrich, ACS) were used for preparing phospholipid solutions. Compression Isotherms and Brewster Angle Microscopy. The surface pressure (Π) and electric surface potential (∆V) measurements were performed using a KSV 5000 Langmuir balance (KSV, Helsinki). A Teflon trough (15 cm × 58 cm × 1 cm) with two hydrophilic Delrin barriers (symmetric compression) was used in compression isotherm experiments. The system was equipped with an electrobalance and a platinum Wilhelmy plate (perimeter 39.24 mm) as a surface pressure sensor and a surface potential measuring head with a vibrating electrode. A platinum plate (4 cm diameter) immersed 4 mm below the water surface was used as a counter electrode. The apparatus was closed in a Plexiglas box, and temperature was kept constant at 20 °C. Before each use, the trough and the barriers were cleaned using cotton soaked in chloroform, gently brushed with ethanol and then with tap water, and finally rinsed with MilliQ water. All solvents used for cleaning the trough and the barriers were of analytical grade. Aqueous subphases for monolayer experiments were prepared with water purified by reverse osmosis, which had a surface tension of 72.8 mN m-1 at 20 °C. Any residual surface-active impurities were removed before each experiment by sweeping and suction of the surface. Monolayers were spread using calibrated solutions (concentration around 1 mg mL-1) of DPPC and DLPC in chloroform and DMPE solution in chloroform/ methanol 3:1 v/v. The stability of the surface potential (∆V) signal was checked before each experiment, after cleaning the subphase surface. After the ∆V signal had reached the constant value, it was zeroed and the film was spread on the subphase. After the equilibration time of 20 min, the films were compressed at the rate of 2.5 mm min-1 barrier-1. A PC computer and KSV software were used to control the experiments. Each compression isotherm was performed at least three times. The standard error was ( 0.5 Å2 with mean molecular area measurements and ( 5 mV with surface potential measurements, respectively. The morphology of the films was imaged with a computerinterfaced KSV 2000 Langmuir balance combined with a Brewster angle microscope (KSV Optrel BAM 300, Helsinki). The Teflon trough dimensions were 6.5 cm × 58 cm × 1 cm; other experimental conditions were as described above. Grazing Incidence X-ray Diffraction. GIXD measurements were performed on pure water using the liquid surface diffractometer (beamline BW1) at HASYLAB, DESY, Hamburg, Germany. The synchrotron beam was made monochromatic by a beryllium (002) crystal. Experiments were performed at an angle of incidence of 0.85Rc (Rc is the critical angle for total external reflection). A linear position sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) with a vertical acceptance 0 < QZ < 1.27 Å-1 was used for recording the diffracted intensity as a function of both the vertical (Qz) and the horizontal (Qxy) scattering vector components. The horizontal resolution of 0.008 Å-1 was determined by a Soller collimator mounted in front of the PSD. The measurements were performed by scanning along the horizontal scattering vector component Qxy ∼ (4π/λ) sin θxy, where 2θxy is the horizontal diffraction angle. The analysis of the in-plane diffraction data yields lattice spacings according to dhk ) 2π/Q hk xy . The in-plane and outof-plane peak positions give information about the tilt angle and the tilt direction.14-17 (14) Jacquemain, D.; Wolf, S. G.; Leveiller, F.; Deutsch, M.; Kjaer, K.; AlsNielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1992, 104, 130-152. (15) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep. 1994, 246, 251-313. (16) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779819. (17) Kjaer, K. Physica B 1994, 198, 100-109.

GriseofulVin/Phospholipid Monolayer Interactions Adsorption Kinetics Experiments. The surface pressure (Π) and electric surface potential (∆V) measurements were performed at 20 °C, using a KSV 5000 Langmuir balance (KSV, Helsinki). Round Teflon trough of the inner diameter of 10 and 1 cm depth was used for the adsorption experiments. In these experiments a Ag,AgCl/Cl- electrode (Radiometer) was used as a counter electrode. The Π-t and ∆V-t changes due to interactions of the penetrant with the phospholipid film were measured under stationary diffusion conditions. The lipid monolayers were formed by depositing a chloroform DPPC solution on top of a fresh 20 µM GF aqueous solution. After reaching the target surface pressure (Πt between 5 and 50 mN m-1) Π and ∆V variations were monitored. Each experiment was done using a fresh GF solution. Enzymatic Lipolysis. Porcine pancreatic phospholipase PLA2 (EC.3.1.1.4; SIGMA, P6534; 1840 units mg-1, concentration of 2.9 mg mL-1) was used in DLPC monolayer lipolysis experiments. The lipolysis conditions were not optimized. The subphase was MilliQ water containing 5 mM CaCl2 (pH 5.8) or 0.55 µM Ca2+ cations (pH 5.6). GF (20 µM) was present in the subphase or in the lipid film spread as an equimolar DLPC/GF mixture. The experiments were performed with a KSV 5000 Langmuir balance (KSV, Helsinki) and a “zero-order trough”18 with a symmetric compression. The trough had a round reaction compartment connected to two reservoir compartments by small surface channels. Enzyme was injected under the film in the reaction compartment only, whereas the substrate film covered all three compartments. The reservoir compartments contained mobile barriers, which were used to compensate for substrate molecules removed from the film in the reaction compartment by enzyme hydrolysis, thereby keeping the surface pressure constant. The latter was measured in the reservoir compartment with a Wilhelmy plate (perimeter 3.94 cm) attached to an electromicrobalance, connected in turn to a computer controlling the movement of the mobile barriers. The subphase was thermostatically maintained at 20 °C and was continuously agitated in the reaction compartment with a 2.5.cm magnetic stirrer moving at 250 rpm. The diameter of the reaction compartment and the width of the reservoir compartments were 10 cm. The surface area of the reaction compartment was 78.5 cm2 (volume 78.5 mL); the volume of the subphase in the reaction compartment was 80 mL. The enzyme solution was injected through the film with a Hamilton syringe (1 µL of the commercial enzyme solution). Final PLA2 concentration in the subphase was 66.7 units L-1. Surface pressure was maintained constant at 10 mN m-1 using the barostat technique. Molecular Modeling. The 3D coordinates of GF were obtained from DrugBank.19 The structure was further optimized on a PC (Windows 2000) using MOPAC (AM1) implemented in the VEGA molecular modeling program.20,21 The 3D coordinates of DPPC, DMPE, and DLPC were derived from the molecular dynamics simulation model, HHPL1.PDB, of Heller et al.,22,23 with the following modifications. Hydrogen atoms were constructed in VEGA, and acyl chains and/or the polar headgroup chemistries were adjusted as required. VEGA was also used to generate molecular lipophilic potentials, represented graphically by projecting the Broto-Moreau lipophilicity atomic constants on a 1.5 Å probe-radius-generated accessible surface,24 and to compute log P and lipole values based on the Broto method.25 GF accessible surface area (ASA) was computed on an SGI O2 R10000 with the SurVol analytical (18) Verger, R.; de Haas, G. H. Chem. Phys. Lipids 1973, 10, 127-136. (19) http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/ getCard.cgi?CARD ) APRD01004.txt (accessed April 12, 2006). (20) Pedretti, A.; Villa, L.; Vistoli, G. J. Comput.-Aided Mol. Des. 2004, 18, 167-173. (21) http://www.ddl.unimi.it (accessed April 12, 2006). (22) Heller, H.; Schaefer, M.; Schulten, K. J. Phys. Chem. 1993, 97, 83438360. (23) http://www.umass.edu/microbio/rasmol/bilayers.htm (accessed April 12, 2006). (24) Gaillard, P.; Carrupt, P. A.; Testa, B.; Boudon, A. J. Comput.-Aided Mol. Des. 1994, 8, 83-96. (25) Broto, P.; Moreau, G.; Vandycke, C. Eur. J. Med. Chem. 1984, 19, 7178.

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Figure 1. Griseofulvin structure (refs 32 and 33): (1′S,6′R)-7chloro-2′,4,6-trimethoxy-6′-methylspiro[benzofuran-2(3H),1′-[2]cyclohexene]-3,4′-dione. Note the presence of the two ketone groups at positions 3 and 4′. procedure26 implemented in the BRUGEL molecular modeling package.27,28 The graphical representation of the accessible surface as a dot surface was generated with the Connolly method29,30 also implemented in BRUGEL. The atomic radii used were adapted from Li and Nussinov; solvent radius was 1.68 Å for all atoms except polar atoms (O) for which a 1.37 Å solvent probe was used instead.31

Results and Discussion Compression Isotherms. To address the question of the interactions between GF and lipid membranes, the interfacial properties of GF were first determined. GF is a small, rigid molecule (Figure 1), which has no structural features in common with lipids and other amphiphilic molecules, such as classical surfactants. Not surprisingly, GF shows no meaningful surface activity. Indeed, values of the equilibrium surface pressure and surface potential obtained with a close to saturation, 20 µM GF water solution were, respectively, 0.5 ( 0.2 mN m-1 and 100 ( 5 mV. Both compression of the free surface of this solution in a rectangular Teflon trough and spreading GF from chloroform solution on pure water subphase followed by compression yielded similar values, indicating that GF does not form films at the air/water interface. The scarce solubility of GF in water and lack of affinity for the air/water interface may suggest that the GF impact on lipid membranes is small. On the other hand, the low hydrophilicity of GF indicates that it may have affinity for hydrophobic systems, such as hydrocarbon interior of lipid bilayers. To check this point, the GF impact on monomolecular films formed with DPPC, DLPC, and DMPE was monitored. The pure phospholipid monolayers were spread on pure water and on 20 µM GF aqueous solution subphases and mixed DPPC/GF, DLPC/GF, and DMPE/ GF were spread on pure water subphase using chloroform solutions of phospholipid and GF mixtures in 1:1 molar proportions. The surface pressure-area (Π-A) and surface potential-area (∆V-A) isotherms obtained upon compression of the monolayers are shown in Figure 2. As shown in Figure 2, parts A-D, the Π-A isotherms of all three pure phospholipids, as well as the DPPC/DMPE equimolar mixture spread on the GF solution subphase (green curves), are shifted to higher molecular areas at low surface pressures compared to those of the pure water subphase (blue curves). This observation suggests that at low surface pressures GF penetrates into the phospholipid film from the subphase. At low surface pressures the impact of GF on the monolayer structure can be observed as well using BAM (Figures 3-5). Indeed, the domains characteristic for the gas-liquid expanded phase transition (26) Alard, P. Ph.D. Dissertation, Universite´ Libre de Bruxelles, Faculte´ des Sciences, Brussels, Belgium, 1991. (27) Delhaise, P.; Bardiaux, M.; Wodak, S. J. Mol. Graphics 1984, 2, 103106. (28) www.algonomics.com (accessed April 12, 2006). (29) Connolly, M. L. Science 1983, 221, 709-713. (30) Connolly, M. L. J. Appl. Crystallogr. 1983, 16, 548-558. (31) Li, A.-J.; Nussinov, R. Proteins: Struct., Funct., Genet. 1998, 32, 111127.

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Figure 2. Compression isotherms of phospholipid monolayers spread in the presence and in the absence of GF. Results obtained with (A) DPPC, (B) DMPE, (C) DLPC, and (D) DPPC/DMPE. Solid lines, Π-A isotherms; dotted lines, ∆V-A isotherms; blue curves, pure lipid film spread on pure water subphase; green curves, pure lipid film spread on 20 µM GF solution subphase; red curves, mixed lipid/GF film spread on pure water subphase. (E-H) Analysis of the GF impact on the film properties presented as molecular area shift in function of surface pressure and surface potential, compared to that of pure water subphase: green curves, shift of the isotherms obtained with GF subphase; red curves, shift of the isotherms obtained with phospholipid/GF films on pure water subphase.

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Figure 3. Brewster angle micrographs of DPPC monolayers spread on (A-D) water and (E-H) 20 µM GF solution. (I-L) BAM micrographs of a DPPC/GF monolayer. The micrographs were taken in the gas-phase region at 162, 169, and 178 Å2 molecule-1 (A, E, I, respectively), 3.0 mN m-1 (B, F, J), 4.0 (C, G, K), and 10.0 mN m-1 (D, H, L). Scale: the width of the snapshots corresponds to 400 µm.

Figure 4. Brewster angle micrographs of DMPE monolayers spread on (A-D) water and (E-H) 20 µM GF solution. (I-L) BAM micrographs of a DMPE/GF monolayer. The micrographs were taken in the gas-phase region at 96, 119, and 126 Å2 molecule-1 (A, E, I, respectively), 6.0 mN m-1 (B, F, J), 8.0 (C, G, K), and 19.0 mN m-1 (D, H, L). Scale: the width of the snapshots corresponds to 400 µm.

observed in the DPPC, DMPE, and DPPC/DMPE mixture monolayers spread on pure water disappear when GF is present in the subphase. The latter effect correlates well with the ∆V-A results (Figure 2, parts A-D), which show lower fluctuations of the surface potential at the large molecular areas;34 this indicates stabilization of the molecule orientation, conformational ordering, (32) http://images.google.fr/images?q ) griseofulvin+structure&ie ) UTF8&hl ) fr&btnG ) Recherche+Google (accessed April 12, 2006). (33) http://www.sigmaaldrich.com/catalog/search/ProductDetail/ SIAL/G2664 (accessed April 12, 2006). (34) Corvis, Y.; Brezesinski, G.; Rink, R.; Walcarius, A.; Van der Heyden, A.; Mutelet, F.; Rogalska, E. Anal. Chem. 2006, 78, 4850-4864.

and absence of domains (Figures 4 and 5). The BAM micrographs indicate that in the presence of GF in the subphase the domains characteristic for the liquid expanded-liquid condensed phase transition in DPPC, DMPE, and in the mixed DPPC/DMPE film coalesce at slightly higher surface pressures compared to those of the pure water subphase, which is in accordance with the shift of the phase transition to higher surface pressures seen in the compression isotherms. It has to be noticed, however, that isotherms obtained on pure water and on GF solution subphases almost superpose in the most condensed phase regions, both in the case of Π-A and ∆V-A dependencies. This effect, which

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Figure 5. Brewster angle micrographs of DMPE/DPPC monolayers spread on (A-D) water and (E-H) 20 µM GF solution. (I-L) BAM micrographs of a DMPE/DPPC/GF monolayer. The micrographs were taken in the gas-phase region at 173, 169, and 171 Å2 molecule-1 (A, E, I, respectively), 5.1 mN m-1 (B, F, J), 6.1 (C, G, K), and 9.5 mN m-1 (D, H, L). Scale: the width of the snapshots corresponds to 400 µm.

is well visualized in the analysis of isotherm shift presented in Figure 2, parts E-H, (green curves) indicates that GF is squeezed out from the film upon compression. The effect of GF observed upon adsorption was compared to that observed in mixed films formed on pure water subphase with equimolar DPPC/GF, DMPE/GF, DLPC/GF, and DPPC/ DMPE/GF chloroform mixtures. As shown in Figure 2 (red curves), the isotherms corresponding to the mixed films formed on pure water subphase have different profiles, compared to the isotherms obtained with GF solutions used as subphases. At low surface pressure the Π-A isotherm shift to higher molecular areas is less important compared to that of the GF solution subphase, indicating lower amounts of GF incorporated in the film. It can be seen from the Π-A isotherm shift that GF is squeezed from the mixed films at higher surface pressures compared to those of the adsorption experiments. The DMPE isotherm shift decreases with the onset of a plateau at 19.4 mN m-1 (51 Å2 molecule-1) indicating a partial expulsion of GF. In the case of the DPPC film, GF is totally squeezed out from the film in a short plateau appearing at 25.9 mN m-1 (47 Å2 molecule-1), above which the isotherms superpose. These experiments show that GF stays at the interface when trapped in the phospholipid monolayers. The fact that GF is trapped both in the phosphocholine and phosphoethanolamine films suggests that it is maintained in the film by interactions with the hydrocarbon chains, and not with the polar heads, as is probably the case in adsorption experiments. Structure of the DPPC Network Formed at the Air/GF Solution Interface. To understand GF-monolayer interactions better, GIXD measurements were performed. These experiments enable the 2D symmetry of the chain lattices on the angstrom scale to be elucidated. Figure 6 shows selected contour plots of the diffracted intensities at different lateral pressures. Starting at low pressures, the monolayer exhibits three low-order diffraction peaks indicative of an oblique chain lattice. The deviation from orthorhombic packing is not very pronounced.

Figure 6. Selected contour plots of the corrected X-ray intensities vs in-plane and out-of-plane scattering vector components Qxy and Qz at 20 °C on a subphase containing 20 µM GF. The results shown were obtained at 10, 20, 30, and 40 mN m-1 (from bottom to top).

Similar behavior is observed with DPPC monolayers spread on pure water. At 40 mN m-1, the diffraction pattern can be described

GriseofulVin/Phospholipid Monolayer Interactions

Figure 7. (A) Tilt angle t of the DPPC aliphatic chains on (2) water and (b) a subphase containing 20 µM GF, as a function of the lateral pressure Π. (B) 1/cos(t) as function of the lateral pressure Π for DPPC on a subphase containing 20 µM GF at 20 °C. The linear extrapolation toward zero tilt angle yields the pressure of the tilting transition, which is Πt ) 47.5 mN m-1. (C) Distortion of the unit cell vs sin2(t), where t is the tilt angle to the normal for DPPC on a subphase containing 20 µM GF. All experiments were performed at 20 °C.

by only two Bragg peaks, the degenerate (1, (1) reflection above the horizon and the nondegenerate (0, 2) reflection at zero QZ indicative of a centered rectangular chain lattice. The chains are tilted in the direction toward nearest neighbors (NN) along the short axis of the orthorhombic in-plane unit cell. The lattice is distorted from hexagonal packing in the NN direction (Q nxy > Q dxy), where Q nxy is the maximum position of the nondegenerate peak. Plotting the tilt angle as a function of the lateral pressure (Figure 7A) shows the expected behavior. The tilt decreases on compression. However, the comparison with the tilt angle data of DPPC on pure water clearly shows a reduction of the tilt angle due to interactions with GF. A linear relationship of the square of the tilt order parameter, η2 ) sin2(t), versus the lateral pressure is expected for a second-order transition, where η is proportional to (Π0 - Π)1/2 and Π0 is the tilting transition pressure.16 The

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Figure 8. Variations of (A) surface pressure and (B) surface potential (∆Π and ∆∆V, respectively; black curves) upon GF adsorption to DPPC monolayers of a given Πi. For comparison, the ∆Π and ∆∆V values obtained from compression isotherms presented in Figure 2A are also given (red curves). The films were formed by spreading DPPC solution on top of 20 µM GF solution. The values of Π and ∆V were taken after 60 min from spreading of DPPC. The experiments were performed at 20 °C.

linear relation is also observed for 1/cos(t) versus lateral pressure. This follows from the simplest approximation which assumes that the packing of the chains in the cross section normal to the chains remains unchanged in the condensed state, independent of the phase structure.35 This is indeed true for the investigated system. The obtained cross-sectional area of DPPC chains is 20.0 ( 0.1 Å2, which corresponds, within the span of the error bar, to the value observed with DPPC on pure water (20.1 ( 0.1 Å2). Figure 7B displays such a linear dependence for DPPC on a GF containing subphase. The value of the tilting transition pressure obtained from the extrapolation to zero tilt is 45.7 mN m-1. This value is much smaller than that of DPPC films spread on pure water (around 66 mN m-1). Plotting the signed unit cell distortion d ) (l12 - l22)/(l12 + 2 l2 ) cos2(β - ω), where l1 and l2 are the major and minor axes of the ellipse passing through all six nearest neighbors of a hydrocarbon chain and β and ω are the tilt and distortion azimuths, respectively, as a function of sin2(t), a linear relation is expected according to the Landau theory prediction.36-38 The data obtained (35) Bringezu, F.; Dobner, B.; Brezesinski, G. Chem. Eur. J. 2002, 8, 32033210. (36) Landau, L. D.; Lifshitz, E. M. Statistical Physics; Pergamon Press: Oxford, 1980. (37) Kaganer, V. M.; Loginov, E. B. Phys. ReV. Lett. 1993, 71, 2599-2602.

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Figure 9. Stick representation of (A) griseofulvin, (B) DPPC, (C) DLPC, and (D) DMPE with carbons in green, chlorine in yellow, oxygens in red, phosphorus in magenta, nitrogen in blue, and hydrogens in white. The molecular lipophilic potential (MLP), computed with VEGA for each molecule, is mapped from orange to blue (MLP scale shown on the left side of the figure) on a transparent solid surface (probe size ) 1.5 Å). The graphics were obtained with VEGA (refs 20 and 21).

on the GF subphase lie on a straight line (Figure 7C). The extrapolation to zero tilt yields a d0 value, which gives information about the different contributions to the distortion (chain tilt, backbone ordering). The observed d0 value is very small and amounts to -0.003, indicating that the lattice distortion is only due to the tilting of the chains. On the basis of these values, the phase expected above the tilting transition should be the 2D hexagonal phase (LS: hexagonal packing of nontilted chains).16 GF Adsorption to the DPPC Monolayer under Stationary Diffusion Conditions. One more method used to study the impact of GF on phospholipid monolayers was the monitoring of Π and ∆V variations upon GF adsorption to DPPC monolayers of a given surface density. In Figure 8 (black curves) the values obtained at equilibrium are plotted, in function of the initial surface pressures (Πi) of the DPPC film. While variations of Π and ∆V indicate that GF penetrates into the monolayer, the net effect is not very important. The comparison of these results with the Π and ∆V shift of the DPPC compression isotherms obtained on pure water and on GF solution (Figure 2A and Figure 8, black curves) suggests that a higher number of GF molecules are incorporated into the DPPC film, when the latter is spread in the gas phase and compressed, compared to the preformed films with a higher initial surface pressure. The same tendency is observed in both types of experiments, that is, a decreasing impact of GF on the monolayer at high surface pressures. These results are in accordance with other studies published in the literature on molecule penetration to monolayers.39,40 Interactions between GF and Phospholipid Monolayers. To understand the interactions between GF and lipid membranes better, the polar/nonpolar properties of the GF surface were analyzed. To this end, molecular modeling of its accessible surface areas (ASA) was performed. Total solvent ASA (Figure 9) is 807.3 Å2, with oxygen atoms providing 15.0%; hence, neglecting the contribution of aromatic rings, the polar ASA is small but (38) Kaganer, V. M.; Loginov, E. B. Phys. ReV. E 1995, 51, 2237-2249. (39) Seelig, A. Biochim. Biophys. Acta 1987, 899, 196-204. (40) Lakhdar-Ghazal, F.; Vigroux, A.; Willson, M.; Tocanne, J. F.; Perie, J.; Faye, J. C. Biochem. Pharmacol. 1991, 42, 2099-2105.

Figure 10. Vectorial representation of the lipole moment of the GF molecule. The lipole moment is shown as a blue arrow pointing toward the fluorine atom. Color code: carbons in green, chlorine in yellow, oxygens in red. The graphic was generated with BRUGEL (refs 27 and 28).

not negligible. It must be noticed that the chlorine atom, being part of an aryl halide, behaves as a nonpolar atom. The presence of two polar ketone groups, at positions 3 and 4′, with respective ASA of 26.4 and 78.2 Å2, in an otherwise mostly nonpolar molecule is expected to have an influence on the interactions between GF and DPPC. On one hand, these electronegative atoms may establish favorable interactions with the positively charged amino group of DPPC, DLPC, and DMPE, hence stabilizing GF in the headgroup zone of the monolayer. On the other hand, their interactions with the negatively charged phosphate group would be rather repulsive and might prevent GF from penetrating deeper into the monolayer, toward the nonpolar hydrocarbon chain zone. Further analysis using VEGA to compute the molecular

GriseofulVin/Phospholipid Monolayer Interactions

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Table 1. Surface Pressure Isotherm Shift and the Estimated GF Content in the Monolayers DPPC adsorbed Π (mN m-1)

∆A (Å2)

10 20 30

2.5 1.0 0.9

DLPC mixed

mol %

∆A (Å2)

4.5 1.9 1.8

10.0 4.7 0.6

adsorbed

mol %

∆A (Å2)

18.5 8.6 1.1

6.5 3.3 2.1

DPPC + DMPE

DMPE mixed

mol %

∆A (Å2)

12.0 6.1 3.9

6.7 5.7 5.0

lipophilicity potential (MLP) surface clearly shows the existence of a lipophilic gradient in GF going down from the nonpolar (orange) chlorine atom to each oxygen of the ketone groups (Figure 9A); furthermore, not unexpectedly, the most polar zone (blue) lies around the most exposed ketone group at the 4′ position of the molecule. For comparison, the MLP surfaces of DPPC, DLPC, and DMPC are shown in Figure 9, parts B-D, respectively. Computation of log P (Broto) yields a value of 1.642 with GF, in accordance with the presence of a few polar groups in an otherwise nonpolar molecule; using VEGA, we could calculate a Broto’s lipole value of 2.109. We define a vector representation of this lipole in Figure 10, the direction of which is defined as follows. The origin of the lipole moment is set such that (i) it lies on a segment connecting the centers of the two ketone oxygens, 3 and 4′ and (ii) its position on this segment is normalized by means of a ratio method that takes into account the respective ASA values of each of these atoms; the head of the lipole vector is then set to correspond to the center of the chlorine atom. Note, however, that with this simplified construction, the amplitude of the vector is not to scale; in particular, the exact positions of both the origin and head of the vector along the direction line should coincide with the lipophilic surface as defined by the chosen probe size. By projecting the GF molecule on a flat layer orthogonal to the direction of this lipole, we could determine a total projection surface equal to 54 Å2. On the basis of the dimensions of GF and on the isotherm shift, the content of GF in the film was estimated. The calculations were done for 10, 20, and 30 mN m-1; the latter value is known to be the internal pressure of biological membranes. As shown in Figure 2 and Table 1, the shift of the phosphocholine isotherms obtained in the presence of GF in the subphase (Figure 2D) is higher compared to that of the DMPE isotherms. This indicates that GF is squeezed out more easily from the DMPE film, compared to the phosphocholine films. This difference between the two types of phospholipids may indicate that the interactions between the nonpolar GF moieties and the phosphocholine quaternary amine methyl groups stabilize GF in the film. Moreover, it cannot be excluded that the expulsion of GF from the headgroup region is sterically hindered by the bulky phosphocholine N-methyl groups, which are absent in the DMPE monolayer. We observed that higher amounts of GF are adsorbed to the mixed DPPC/DMPE films compared to single phospholipid films, indicating that retaining of GF in the monolayer depends on the degree of monolayer organization; this may also be the case in physiological conditions. However, comparison of the mixed phospholipid/GF films shows that at 10 and 20 mN m-1 higher amounts of GF are retained in the DMPE films compared to the phosphocholine films; at 30 mN m-1 the GF amount is higher in the DMPE film compared to the DPPC film but lower compared to the DLPC film (Table 1). These results suggest that in the case of mixed films GF is retained within the hydrocarbon chain region; the retention of GF in the mixed films would depend primarily on the chain organization, decisive for GF accom-

adsorbed

mol %

∆A (Å2)

12.4 10.5 9.2

1.2 0.9 0.7

mixed

mol %

∆A (Å2)

2.2 1.8 1.4

18.5 5.9 2.1

adsorbed

mol %

∆A (Å2)

34.3 11.1 3.9

3.8 1.9 1.6

mixed

mol %

∆A (Å2)

mol %

7.0 3.5 3.0

14.1 8.3 2.8

26.0 15.4 5.2

modation in the lipid molecule network. Indeed, a higher amount of GF present in the mixed DPPC/DMPE/GF film compared to that in DPPC/GF and DMPE/GF films supports the proposal that GF is more easily retained in the less organized films. The fact that, in general, higher amounts of GF are present in the mixed phospholipid/GF films compared to those of adsorption experiments (Table 1) indicates that nonpolar interactions are important for retaining GF in the monolayers. Taking into account the low content of GF in the monolayers (e.g., cholesterol content in the eukaryotic plasma membrane is above 20 w/w %41,42), the impact of GF on the monolayer structure observed in our studies can be considered as significant. This impact could be more important in multicomponent biological membranes retaining possibly higher amounts of GF, even if formation of GF-phospholipid complexes analogical to those formed with cholesterol43,44 is not expected. The inserting of GF in the lipid monolayers is presented schematically in Figure 11, based on experimental results and modeling. Enzymatic Probing of the GF Effect. In physiological conditions phospholipids undergo hydrolytic degradation catalyzed by lipolytic enzymes, phospholipases. Lipolytic enzymes are known to be active on the aggregated forms of their lipid substrates46-50 and to depend in their activity and specificity on the physicochemical properties of the aggregate surface.51-53 Here, PLA2 was used as a probe differentiating between the phospholipid monolayers in the presence and in the absence of GF. In these experiments 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) was chosen as a model substrate, as the reaction products are water soluble.54-56 Since PLA2 is a calciumdependent enzyme,50,57,58 the reactions were performed in the presence of Ca2+ cations in the subphase. To evaluate the influence of Ca2+ on the properties of the DLPC monolayer, the Π-A (41) Jamieson, G. A.; Robinson, D. M. Mammalian Cell Membranes; Butterworth: London, 1977. (42) Raffy, S.; Teissie, J. Biophys. J. 1999, 76, 2072-2080. (43) Radhakrishnan, A.; McConnell, H. M. J. Am. Chem. Soc. 1999, 121, 486-487. (44) Radhakrishnan, A.; McConnell, H. M. Biophys. J. 1999, 77, 1507-1517. (45) HyperChem, v.6.03 HyperCube Inc.: Gainesville, FL. (46) Scott, D. L.; Sigler, P. B. AdV. Protein Chem. 1994, 45, 53-88. (47) Warwicker, J. FEBS Lett. 1997, 404, 159-163. (48) Ferrato, F.; Carriere, F.; Sarda, L.; Verger, R. Methods Enzymol. 1997, 286, 327-347. (49) Gelb, M. H.; Min, J.-H.; Jain, M. K. Biochim. Biophys. Acta 2000, 1488, 20-27. (50) Berg, O. G.; Gelb, M. H.; Tsai, M.-D.; Jain, M. K. Chem. ReV. 2001, 101, 2613-2653. (51) Rogalska, E.; Ransac, S.; Verger, R. J. Biol. Chem. 1993, 268, 792-794. (52) Rogalska, E.; Nury, S.; Douchet, I.; Verger, R. Chirality 1995, 7, 505515. (53) Carlson, P. A.; Gelb, M. H.; Yager, P. Biophys. J. 1997, 73, 230-238. (54) Grainger, D. W.; Reichert, A.; Ringsdorf, H.; Salesse, C.; Davies, D. E.; Lloyd, J. B. Biochim. Biophys. Acta 1990, 1022, 146-154. (55) Signor, G.; Mammi, S.; Peggion, E.; Ringsdorf, H.; Wagenknecht, A. Biochemistry 1994, 33, 6659-6670. (56) Borioli, G. A.; Fanani, M. L.; Caputto, B. L.; Maggio, B. Biochem. Biophys. Res. Commun. 2002, 295, 964-969. (57) Verheij, H. M.; Slotboom, A. J.; de Haas, G. H. ReV. Physiol., Biochem. Pharmacol. 1981, 91, 91-203. (58) Fernandez, M. S.; Mejia, R.; Zavala, E. Biochem. Cell Biol. 1991, 69, 722-727.

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Figure 12. Compression isotherms of DLPC monolayers. Results obtained with water subphase containing 0.55 µM Ca2+ (cyan curve), 5.0 mM CaCl2 (blue curve), 20 µM GF (yellow curve), and 5.0 mM CaCl2 and 20 µM GF (red curve). Solid lines, Π-A isotherms; dotted lines, ∆V-A isotherms.

Figure 11. Insertion of GF in the lipid films: (A, B) DPPC and (C, D) DMPE monolayer. (A, C) Adsorption of GF to the monolayer from the aqueous subphase. (B, D) Mixed GF/lipid monolayers. The molecule models were built using the HyperChem molecular modeling package (version 6) (ref 45). Color code: carbons in green, chlorine in yellow, oxygens in red, phosphorus in magenta, nitrogen in blue, hydrogens in white.

compression isotherms were performed on pure water (i.e., MilliQ water containing trace quantities of Ca2+ cations, as determined with GFAAS), on 20 µM GF solution in pure water, on 5 mM CaCl2 solution, and on a subphase containing both 20 µM GF and 5 mM CaCl2 (Figure 12). Unlike GF, the presence of 5 mM CaCl2 in the subphase can be seen not to influence the isotherm profile. The enzymatic lipolysis of the DLPC monolayer was performed at an arbitrarily chosen surface pressure of 10 mN m-1. As shown in Figure 13, when 5 mM CaCl2 was present in the subphase, the velocities of the reactions performed in the presence and in the absence of GF in the subphase (curves red and blue, respectively) were very close.

Figure 13. Hydrolysis of DLPC catalyzed by PLA2. The concentration of Ca2+ cations measured with GFAAS was, respectively, 21.7 ng mL-1 in the GF solution and 22.5 ng mL-1 in MilliQ water used for dissolving GF. Blue curve, pure DLPC film and subphase containing 5 mM CaCl2; red curve, pure DLPC film and subphase containing 5 mM CaCl2 and 20 µM GF; green curve, pure DLPC film and MilliQ water subphase containing 22.5 ng mL-1 (around 0.55 µM) Ca2+ and 20 µM GF; orange curve, mixed equimolar DLPC/GF film and MilliQ water subphase containing 22.5 ng mL-1 Ca2+; cyan curve, pure DLPC film and MilliQ water subphase containing 22.5 ng mL-1 Ca2+.

However, in the presence of trace amounts of Ca2+ in the subphase (around 22 ng mL-1; 0.55 µM), the reaction rate was higher in the presence of GF (PLA2 specific activity 81.5 × 10-3 µmoles min-1 mg-1) compared to the reaction performed in the absence of GF (PLA2 specific activity 3.5 × 10-3 µmoles min-1 mg-1) (curves green and cyan, respectively). The same tendency was observed when lipolysis was performed with a mixed equimolar DLPC/GF film (orange curve), albeit the phenomenon was less pronounced (PLA2 specific activity 25.7 × 10-3 µmoles min-1 mg-1). It must be noticed that in the latter case the activating effect of GF is much more pronounced relative to the overall quantity of GF in the system. Indeed, GF concentration was less than 0.06 µmoles in the case of the mixed film, compared to 1.6 µmoles when 20 µM GF solution was used as subphase. While at present we cannot account for the mechanism of the observed phenomenon, it can be supposed that the higher activity of PLA2

GriseofulVin/Phospholipid Monolayer Interactions

observed in the presence of GF is due to the modification of film properties,59 which in turn influences the process of the binding of the enzyme to the substrate surface.60 These results suggest that in physiological conditions GF may also have an impact on the functioning of proteins other than microtubules.

Conclusions The results obtained show that GF modifies phospholipid monolayer properties both when it is adsorbed to the film from the aqueous subphase and when it is present in the films formed by spreading mixed phospholipid/GF solutions. However, the effect is different in the two cases. As indicated by the isotherm profiles, GF adsorbed from the subphase is easily squeezed from the film upon compression. Since expulsion is easier in the case of DMPE compared to DPPC and DLPC, it can be supposed that the interactions responsible for retaining GF in the films are predominantly nonpolar. On the other hand, the tilt angle, tilt direction, and tilting transition pressure obtained from GIXD measurements of DPPC monolayers indicate that GF interacts with the film even in the most condensed state; these interactions can be supposed to be established with the DPPC headgroups. In the case of mixed phospholipid/GF films, GF is present in the monolayers even at high surface pressures. These observations (59) Jain, M. K.; Streb, M.; Rogers, J.; de Haas, G. H. Biochem. Pharmacol. 1984, 33, 2541-2551. (60) Scott, D. L.; Mandel, A. M.; Sigler, P. B.; Honig, B. Biophys. J. 1994, 67, 493-504.

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suggest that GF is retained in the mixed films by nonpolar interactions established with the hydrocarbon chains. Molecular modeling supports these conjectures, as the GF accessible surface is predominantly nonpolar. The amount of GF present in the monolayer at 30 mN m-1 calculated from the isotherm shift and molecular modeling is around 1-5 mol %, depending on the lipid used. The enzymatic lipolysis experiments indicate that these low amounts of GF modify the process of surface recognition by PLA2. In summary, our results suggest that the interactions with membranes may be important for the mechanism of the biological activity of GF. Moreover, these results indicate that in encapsulating procedures GF should be incorporated into the lipid, rather than the aqueous phase. Acknowledgment. This work was supported by a Ph.D. Fellowship (Bourse Docteur Inge´nieur) from BioMaDe Technology Foundation and the Centre National de la Recherche Scientifique (Y.C.) and Procope 03163XE bilateral project. N.M. acknowledges the financial support from La Ligue contre le Cancer and l’Institut National pour la Sante´ et la Recherche Me´dicale. We thank Dr. Eric Meux and Dr. Se´bastien De´liberto, Laboratoire d’Electrochimie des Mate´riaux, UMR CNRS 7555, Universite´ de Metz, for trace analysis. We also appreciate helpful discussions with Dr. Beata Korchowiec. We thank Ms. Nicole Marshall for revising the English. LA060998X