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Langmuir 1998, 14, 542-546
Liquid Crystals and Colloids in Water-Amiodarone Systems Y. Bouligand,*,† F. Boury,‡ J.-M. Devoisselle,§ R. Fortune,§ J.-C. Gautier,| D. Girard,‡,| H. Maillol,§ and J.-E. Proust‡ Histophysique EPHE and Biophysique Pharmaceutique, 10, rue A.-Bocquel, Universite´ d’Angers, 49100 Angers, France, and Technique Pharmaceutique Industrielle, Universite´ de Montpellier, Montpellier, France, and SANOFI Recherche, Montpellier, France Received December 2, 1996. In Final Form: October 22, 1997 Amiodarone, a molecule used in treatment of heart arrhythmias, presents several metastable states. Transparent sols (50 mg/mL) and gels (100 mg/mL) are obtained by heating amiodarone crystals in water to 70 °C and cooling to 20 °C. These preparations diluted in water remained transparent above 1.5 mg/mL, but further dilutions resulted in a milky cloud, which was studied by light scattering and photon correlation spectroscopy. A swollen smectic phase appeared at 200 mg/mL and another isotropic gel at 500 mg/mL. This behavior is similar to that of some classical lyotropic liquid crystals.
I. Introduction Amiodarone hydrochloride (Figure 1a), a drug used against severe heart attacks,1 is a nonchiral and highly lipophilic compound, with a partition coefficient above 16 500, when [125I]amiodarone is observed in the presence of cell membranes or phospholipidic bilayers.2 Amiodarone is poorly soluble in water, at a possible maximum of 0.7 mg/mL at 25 °C,3 whereas a lower critical micelle concentration (cmc) of 0.5 mg/mL can be recorded, corresponding to a drop of electric conductance.4 Transparent liquid preparations are obtained at higher concentrations (50 mg/mL and more) after heating (60-80 °C) and remain stable at 20 °C.4 These pseudosolutions with a high amiodarone content show complex sedimentation profiles during ultracentrifugation, suggesting the presence of micelles.4 NMR studies indicated a reversible disruption of micelles at 60 °C.5 These clear pseudosolutions ceased to be transparent and showed a milky opalescence at room temperature, when diluted at concentrations close to the cmc, a process initiated without pH increase (Figure 1b). On the contrary, at higher concentrations, birefringent lamellar phases differentiated and a further isotropic state appeared. Our purpose is to describe these structures and to discuss the origin of transparent preparations concentrated 100 times above the saturated solubility, whereas strong dilutions induce an opalescence below the cmc. II. Material and Methods Pseudosolutions were prepared from pure amiodarone hydrochloride, in the form of a crystalline powder provided by SANOFI (France), with a specific purity of 99.4% on HPLC assay. This compound was stirred in ultrapure water, heated in a †
Histophysique EPHE. Biophysique Pharmaceutique. § Technique Pharmaceutique Industrielle. | SANOFI Recherche. ‡
(1) Gill, J.; Hill, R. C.; Fitton, A. Drugs 1992, 43, 69. (2) Chatelain, P.; Laruel, R. J. Pharm. Sci. 1985, 74, 783. (3) Bonati, M.; Gaspari, F.; d’Aranno, V.; Benfenati, E.; Neyroz, P.; Galletti, F.; Tognoni, G. J. Pharm. Sci. 1984, 73, 829. (4) Ravin, L. J.; Shami, E. G.; Rattie, E. S. J. Pharm. Sci. 1975, 64, 1830. (5) Warren, R. J.; Stedman, R. J.; Shami, E. G.; Rattie, E. S.; Ravin, L. J. J. Pharm. Sci. 1970, 59, 1357.
Figure 1. (a) Chemical structure of amiodarone (Cordarone). (b) Dependence of pH on amiodarone concentration in ultrapure water, at room temperature, at preparation time or 24 h later. thermostated bath at 70 °C for 15 min, to obtain a transparent 50 mg/mL stock preparation at room temperature. For light scattering, concentrations ranging from 0.125 to 50 mg/mL were obtained by dilution of the stock preparation, kept for 72 h in the dark at 20 °C, and measurements were made immediately after dilution. All experiments were performed on a SEMATECH-633-RTG photogoniodiffusometer, with a real time correlator (SEMATECH, Nice, France). Samples loaded into a 10 mm diameter tube were placed in a thermostated chamber at 20 °C. The light of an He-Ne laser source (632.8 nm) was focused on the sample, and the scattered light was detected at an angle of 90°, using a 1 mm photomultiplier aperture.6 (6) Yi, P. N.; MacDonald, R. C. Chem. Phys. Lipids 1973, 11, 114.
S0743-7463(96)02060-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/01/1998
Liquid Crystals and Colloids in Water-Amiodarone Systems
Figure 2. (a) Scattered light intensity plotted against amiodarone concentration. (b) Scattered intensity, in the 4-50 mg/ mL interval (smaller units). (c) Expected diameter distribution from photon correlation. (d) Polydispersity index of diameters (diameter standard deviation/mean diameter). Measurements were repeated ten times, with variations less than 10%, the mean value being considered only. Photon correlation spectroscopy was performed with the same apparatus, with running times of 200 or 2000 s and the sampling time ranging from 5 to 45 µs. The photomultiplier aperture was fixed at 200 µm. Latex beads of different diameters (109, 497, and 2000 nm) were used as size standards. Viscosity, temperature, and refractive index of the preparation were incorporated in the computer software, for the study of a suspension of noninteracting isodiametric particles (RTG20, SEMATECH, Nice, France). Monomodal analysis was applied,7 measuring the hydrodynamic diameter of “particles” and the polydispersity index (assuming spherical particles). Preparations of amiodarone were observed between slides and coverslips with a Zeiss Photomicroscope Axiophot Pol, a polarizing microscope equipped with retardation plates and compensators, to have the local ellipsoid of indices, and also an Orthoplan Pol, Leitz, with a Zernike phase contrast and a thermostated stage (Mettler FP 52). About microscopic preparations, it is worth remembering that the pH between slide and coverslip is sensitively increased by the proximity of the alcaline glass.
III. Results The pH values of stock pseudosolutions and of transparent sols or gels were measured at different concentrations, just after preparation or 24 h later. The pH globally increased with dilution (Figure 1b), but a slight buffer effect occurred between 20 and 60 mg/mL and also between 0.5 and 2 mg/mL with a local minimum of pH. A strong pH increase was observed below 0.5 mg/mL, at dilutions beyond those of the milky opalescence. In all tested amiodarone preparations, the intensity of scattered light was higher than that observed for water itself, considered as a reference. The measurements at an angle of 90° showed a peak of intensities lying in the 0.5-0.75 mg/mL range, corresponding to the “milky opalescence” (Figure 2a). At lower concentrations, the scattering remained high, indicating that the true solubility is possibly lower than 0.125 mg/mL. The intensity was much lower in the 4-50 mg/mL interval, increasing from 0.3 × 10-5 to 3.8 × 10-5 (Figure 2b). (7) Seras, M.; Handjani-Villa, R.-M.; Ollivon, M.; Lesieur, S. Chem. Phys. Lipids 1992, 63, 1.
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The analysis by photon correlation normally gives the particle diameters, but the largest dimensions were more than 10 µm (Figure 2c), a dimension above the maximum measured by this method. Moreover, at these concentrations, about 0.5 mg/mL, the pseudosolutions were not stable, and a sedimentation process occurred for the largest aggregates, as shown by the evolution of the scattered intensities. At higher concentrations (2-8 mg/mL), the pseudosolutions were stable enough and the hydrodynamic dimensions were found in the 100-500 nm interval (Figure 2c). The polydispersity index curve (Figure 2d) shows an analogous profile and, at low amiodarone concentrations (0.125-2 mg/mL), the solution displays a broad peak, indicating a large spectrum of dimensions, from less than 1 µm to more than 10 µm. On the contrary, in the 2-8 mg/mL range, these variations are restricted to the 100 ( 50 nm interval. Transparent sols and gels observed between slides and coverslips were isotropic without any differentiated structures visible in polarizing or phase contrast microscopy, but they can show a brief positive flow birefringence, when the coverslip is displaced or submitted to the pressure of a needle. A drop of the stock preparation (50 mg/mL) was deposited onto a slide and immersed in pure water, to be sandwiched by a coverglass. The central drop was laterally diluted, with a concentration gradient distributed radially, and the milky cloud formed a closed ring, whose diameter increased regularly. Small spherical droplets were observed, agitated by brownian movements, their diameter varying strongly around 1 µm. They were similar to those of diluted sols or gels at about 1.25 mg/mL (Figure 3a) which are responsible for the turbidity, since their dimensions corresponded to those expected from the light scattering study in Figure 2. Some droplets attached and spread onto the glasses, mainly onto the slide, since amiodarone is a heavy molecule, with two iodine atoms. The system looked like a dispersion of one liquid into another, both being optically isotropic, without signs of chromatic polarization between crossed polars. The droplets examined in phase contrast microscopy showed a refraction index higher than that of the surrounding medium. In transparent sols and gels (50 mg/mL or more) kept at room temperature, true crystals grew slowly and sedimented (after weeks or months in stock preparations). They were birefringent and formed elongated parallelograms, with an acute angle close to 72°-73° (Figure 3b). The extinction between crossed polars occurred when the long edges of parallelograms lay at (43° from one of the two polarizers. In the hot stage, these crystals dissolved at about 66 °C and did not reappear immediately after cooling, but only after several days or later. Fluid and birefringent stripes differentiated in the course of dehydration of the sols and gels considered above, and if present, the crystals remained unchanged (Figure 3c). These “stripes” were similar to those observed in lamellar phases of lyotropic systems, as lecithin dispersed in water for instance.8,9 Myelin forms are fingerlike projections, made of cylindrically wrapped bilayers, separated by important water intervals, and they showed a negative uniaxial birefringence for amiodarone (Figure 3d). The loss of water due to evaporation at the edge of the coverslip had two opposite effects: a decrease of (8) Nageotte, J. Morphologie des gels lipoı¨des: Mye´ line, Cristaux Liquides, Vacuoles; Hermann: Paris, 1937; 183 p, 29 plates of micrographs. (9) Rosevear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628.
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Figure 3. (a) Large droplets producing the milky cloud (up to 10 µm in diameter), in the stock pseudosolution diluted at 0.5 mg/mL. (b) Amiodarone crystals from the bottom of an old stock preparation, after weeks at ordinary temperature. (c) The same preparation after evaporation and transition to a smectic phase. (d) Myelin forms. (e, f) Dark areas corresponding to horizontal layers or to polygonal fields, separated by oily streaks, brilliant and cross-striated, corresponding to vertical or strongly oblique bilayers; two different optical sections are shown. (g) A dark zone (IZ) due to birefringence inversion separate the negatively uniaxial lamellar structure (-) from the positively uniaxial one (+); an air bubble B is visible at top left (crossed polarizers, first-order retardation plate added in a-c; bar ) 50 µm).
preparation thickness, or an increase, when air bubbles formed. The first process resulted in horizontal alignment
of bilayers, appearing black between crossed polarizers, whereas the second one produced fluid streams, tilting
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Figure 4. (a) Lamellar phase of amiodarone, with a concentration gradient normal to the parallel bilayers, which lie vertically (normal to slide and coverslip) and extend from top left to bottom right. Irregularities and defect lines visualize the general lamellar orientation. The highest amiodarone concentration is at top right, at the contact of an air interface, whereas the lamellar phase is swollen at bottom left. The orientation of the ellipsoid of indices (or more precisely its elliptic section by the preparation plane) is indicated schematically by two ellipses in the yellow and in the blue zone (left to top right, bar ) 50 µm). (b) Randomly distributed smectic domains form dark areas separating large smectic domains of aligned vertical bilayers (blue), with dark stripes parallel to bilayers. (c) At high concentration, between three air bubbles, the lamellar phase (strong chromatic polarization) transforms into a water-poor isotropic phase (crossed polars and first-order retardation plate; bar ) 50 µm).
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bilayers. Oily streaks correspond to vertical or strongly oblique bilayers extending as brilliant stripes, between crossed polars, separating black domains of horizontal bilayers and are represented for amiodarone in Figure 3e,f. The black domains of horizontal bilayers are often modulated by polygonal fields which assemble numerous “Maltese crosses” (Figure 3e,f), a common pattern in polarizing microscopy, appearing when superimposed horizontal bilayers cease to be planar and transform into domes or basins, nested along vertical axes.10,11 These polygonal textures in amiodarone reproduce exactly those described in phospholipids, associating confocal parabolaes.12 A dark zone often separates a water-poor material at the periphery of the preparation from water-rich regions, which are more central (Figure 3g). Birefringence is zero or very weak along this dark zone, which shows the sensitive tint when a first-order retardation plate is added (Figure 4a). This zone separates the “swollen” smectic liquid, whose birefringence is negatively uniaxial, due to the presence of thick water layers intercalated between bilayers (extrinsic birefringence), and a waterpoor smectic liquid, whose birefringence is positively uniaxial, due to the alignment of elongated molecules (intrinsic birefringence). Along this inversion zone, the ellipsoid becomes locally spherical.13 This zone of birefringence inversion often crossed the polygonal fields or the oily streaks. Some textures showed remarkably the direction of bilayers, underlined by dark stripes (Figure 4a,b), due to local densities of edge dislocations, what was extremely useful to recognize zones of positive or negative uniaxiality. At the end of evaporation, the preparation became isotropic and transparent, with a high viscosity. A sharp interface separated this state from the birefringent lamellar systems (Figure 4c). In the last steps of evaporation, the solution occupied some restricted domains in the preparation. Colors of chromatic polarization reappeared after water reintroduction between slide and coverslip, owing to the formation of lamellar mesophases, and a milky ring of isotropic droplets differentiated at the periphery, in the weak concentration zone, as described above. (10) Demus, D.; Richter, L. Textures of Liquid Crystals, 2nd ed.; VEB; D. Vlg. f. Grundstoffindustrie: Leipzig, 1978; 228 p, 212 plates. (11) Bouligand, Y. In Dislocations in Solids; Nabarro, F. R. N., Ed.; North Holland: Amsterdam, 1980 Vol. 5, p 299; J. Phys. (Paris) 1972, 33, 715. (12) Rosenblatt, Ch.; Pindak, R.; Clark, N.; Meyer, R. B. J. Phys. (Paris) 1977, 38, 1105. (13) Born, M.; Wolf, E. Principles of Optics, 6th ed.; Pergamon Press: Oxford, reprinted 1989.
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IV. Concluding Remarks A lamellar liquid crystal intercalated between two optically isotropic sols or gels along a water concentration axis is a frequent situation in water-lipid systems,14,15 and the corresponding non-birefringent states are known to be cubic phases, made of a regular lattice of cylindrical micelles, joining three by three, four by four, or six by six,16,17 and also sponges, with a topology close to that of cubic phases, but without the 3D periodicity.18,19 For the cubic phases, as for the sponges, there are two inverse structures: the core of the joining micellar segments is either hydrophilic or hydrophobic. Such systems, cubic phases or sponges, can be swollen by water, and this could correspond to the structure of sols and gels in the concentration range from 2 to 100 mg/mL. Preliminary neutron scattering studies of the amiodarone stock pseudosolution, prepared in D2O, suggested cylindrical shapes for micelles, instead of spherical or discoidal, but without indications about the presence or not of branching.20 Networks of cylindrical micelles, with branching points, were observed by transmission electron microscopy in certain vitrified lyotropic sols, obtained by ultrarapid deepfreeze.21 A plausible interpretation of the amiodarone milky cloud is that the micellar networks do not swell indefinitely in water but disrupt into lumps, each one being itself a separated network of branching micelles, with the spherical shape of a droplet. True crystals of amiodarone chlorhydrate can be grown from ethanol solutions22 and are similar in the polarizing microscope to the crystals found in sols and gels or in the lamellar phase. This indicates that the sols, gels, and lamellar phases are metastable, even if these structures can be kept for weeks or months. Long term metastability is frequent in liquid crystals.23 Acknowledgment. We are grateful to Dr. G. Porte for useful discussions and permission to cite his unpublished results in neutron scattering of amiodarone. LA962060H (14) Winsor, P. A. In Liquid Crystals and Plastic Crystals; Gray, G. W., Winsor, P. A., Eds.; Ellis Horwood Ltd.: Chichester, 1975; Vol. 1, p 199. (15) Scriven, L. E. In Micellization, Solubilization and Microemulsions; Mittal, K. L., Ed.; Plenum: New York, 1977; p 877. (16) Luzzati, V. Biol. Membr. 1968, 1, 71. (17) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (18) Helfrich, W. In Physics of Defects; Balian, R., et al., Eds.; North Holland: Amsterdam, 1981; p 715. (19) Porte, G. J. Phys., Cond. Matter 1992, 4, 8649. (20) Porte, G. Personal communication, 1996. (21) Lin, Z. Langmuir 1996, 12, 1724. (22) Cody, V.; Luft, J. Acta Crystallogr. 1989, B45, 172. (23) Kelker H.; Hatz R. Handbook of Liquid Crystals; Verlag Chemie: Weinheim, 1980; p 917, 438 figs and 48 tables.
