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Phase Behavior of the Phytantriol/Water System Justas Barauskas*,† and Tomas Landh‡ Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden, and Camurus Lipid Research Foundation, Ideon, Science Park, SE-223 70, Lund, Sweden Received June 19, 2003. In Final Form: September 18, 2003 Phytantriol, 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol, is frequiently used as a cosmetic ingredient; however, very little is known about its physical and chemical properties. Here, we present the phase behavior of phytantriol in water, as determined by X-ray diffraction. At room temperature, the phase sequence upon increasing the water concentration is reversed micellar, lamellar, cubic phase Q230, and cubic phase Q224. At 44 °C, the cubic liquid crystals are transformed into a reversed hexagonal phase. The temperature-composition phase diagram of phytantriol/water mixtures is, thus, qualitatively similar to that of aqueous glycerol monooleate. The chemical stability of phytantriol makes it an interesting alternative to glycerol monooleate in exploiting various scientific and technical applications of, in particular, the cubic liquid crystalline phases.
Introduction Polar lipids and surfactants show a rich polymorphism.1,2 Among the great variety of liquid crystalline phases they form, the family of cubic phases has some specific properties3 that are attracting both scientific and industrial attention in various fields ranging from biophysics4-6 to drug delivery.7-12 There are, however, only few polar lipids/surfactants known that have a suitable phase behavior such that the cubic liquid crystalline phases can be utilized.13,14 Two features often sought in such technically interesting cubic phases are equilibrium in excess water and their existence over an acceptable temperature range, that is, 4-40 °C. Additional features, such as the safety aspect of the cubic phases and its dispersions (Cubosome®) in drug delivery applications, depend on the application. In fact, the unsaturated monoglycerides,15 such as glycerol monooleate and glycerol monolinoleate, are among the very few binary systems * To whom correspondence should be addressed. Tel.: +46 46 2221439. Fax: +46 46 2224413. E-mail: Justas.Barauskas@ fkem1.lu.se. † Lund University. ‡ Camurus Lipid Research Foundation. (1) Luzzati, V. In Biological Membranes; Chapman, D., Ed.; Academic Press: New York, 1968; Vol. 1, p 71. (2) Small, D. The physical Chemistry of Lipids: From Alkanes to Phospholpids. In Handbook of Lipid Research; Hanahan, D. J., Ed.; Plenum Press: New York, 1986; Vol. 4. (3) Larsson, K. Phys. Chem. 1989, 93, 7304-7314. (4) Pebay-Peyroula, E.; Rummel, G.; Rosenbusch, J. P.; Landau, E. M. Science 1997, 277, 1676-1681. (5) Cherezov, V.; Fersi, H.; Caffrey, M. Biophys. J. 2001, 81, 225242. (6) Cherezov, V.; Clogston, J.; Misquitta, Y.; Abdel-Gawad, W.; Caffrey, M. Biophys. J. 2002, 83, 3393-3407. (7) Nielsen, L. S.; Schubert, L.; Hansen, J. Eur. J. Pharm. Sci. 1998, 6, 231-239. (8) Engstro¨m, S.; Norde´n, T.; Nyquist, H. Eur. J. Pharm. Sci. 1999, 8, 243-254. (9) Chung, H.; Kim, J.; Um, J. Y.; Kwon, I. C.; Jeong, S. Y. Diabetologia 2002, 45, 448-451. (10) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Langmuir 2001, 17, 5748-5756. (11) Siekmann, B.; Bunjes, H.; Koch, M. H. J.; Westesen, K. Int. J. Pharm. 2002, 244, 33-43. (12) Spicer, P. T.; Small, W. B., II; Small, W. B.; Lynch, M. L.; Burns, J. L. J. Nanopart. Res. 2002, 4, 297-311. (13) Drummond, C. J.; Fong, C. Curr. Oppin. Colloid Interface Sci. 2000, 4, 449-456. (14) Larsson, K. Curr. Oppin. Colloid Interface Sci. 2000, 5, 64-69. (15) Caffrey, M. Biophys. J. 1989, 55, 47-52.
Figure 1. Chemical structure of phytantriol (3,7,11,15tetramethyl-1,2,3-hexadecanetriol).
showing these properties that are also regarded as safe to use in, at least, oral drug delivery applications.16 However, monoglycerides are regarded as being exceptions to the common aqueous swelling behavior of polar lipids and surfactants because they do not exhibit the phase sequence L2 (reversed micellar), HII (reversed hexagonal), QII (reversed cubic), and LR (lamellar) expected upon hydration of poorly soluble amphiphilic molecules.17 Here, we present the aqueous temperature-composition phase diagram of 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol, commonly known as phytantriol (Figure 1). It is a well-known active ingredient for the cosmetics industry in hair and skin care.18 It improves the moisture retention properties of skin and hair and acts as a penetration enhancer to increase the effect of panthenol, vitamins, and amino acids. Phytantriol shows the same type of phase sequence as the unsaturated long-chain monoglycerides and might be a complementary alternative in certain applications of, in particular, the cubic liquid crystalline phases. To the best of our knowledge, this is the first publication regarding the aqueous phase behavior of phytantriol. Experimental Section A mixture of 3,7,11,15-tetramethyl-1,2,3-hexadecanetriol optical isomers (>96%), denoted as phytantriol, was kindly provided by Kuraray Co., Ltd. (Japan), and used without further purification. At room temperature, phytantriol is a viscous isotropic liquid with a density of about 0.94 g/cm3. It has a very low solubility in water and a melting point far below 0. The water used was ion-exchanged, distilled, and passed through a milli-Q water purification system. Samples were prepared by weighing appropriate amounts of phytantriol (ca. 0.7-1 g) and water into glass vials (i.d. 14 mm). (16) Norring, T.; Lading, P.; Engstro¨m, S.; Larsson, K.; Krog, N.; Nissen, S. S. J. Clin. Periodontol. 1992, 19, 687-692. (17) Seddon, J. M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 380393. (18) Wagner, E. Parfuem. Kosmet. 1994, 75, 260-267.
10.1021/la0350812 CCC: $25.00 © 2003 American Chemical Society Published on Web 10/08/2003
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Figure 2. (a) Identity and location of each phase and the coexisting phases in the phytantriol/water system, as determined by X-ray diffraction in the heating direction in the 2268 °C temperature and 0-33% (w/w) water composition ranges. The phases are labeled as follows: filled squares, LR; filled circles, Q230; filled diamonds, Q224; filled triangles, HII; crosses, L2; squares, Q230 + L2; circles, Q230 + HII; triangles, HII + L2; reversed triangles, Q230 + Q224. (b) Temperature composition phase diagram of the phytantriol/water system based on the interpretation of both the data in Figure 2a and the Gibbs rule. The vials were immediately sealed and allowed to equilibrate in the dark at 22 °C for at least 8 weeks before measurements. To obtain phase homogeneity, samples were centrifuged up and down at 1500 g for 1 h. If needed, the centrifugation was repeated several times. X-ray diffraction measurements were performed on a Kratky compact small-angle system equipped with an OED 50 M positionsensitive detector (MBraun, Graz, Austria) containing 1024 channels of width 53.1 µm. Cu KR nickel-filtered radiation of wavelength 1.542 Å was provided by a Seifert ID 3000 X-ray generator (Rich Seifert, Ahresburg, Germany) operating at 50 kV and 40 mA. A few milligrams of the samples were mounted between thin mica windows in a steel sample holder at the sampleto-detector distance of 277 mm. Temperature control within 0.1 °C was achieved using a Peltier element. X-ray diffraction patterns were collected in the temperature range 22-68 °C in increments of 3 °C in the heating direction. Samples were preequilibrated at a particular measurement temperature for 10-120 min before making the 10-min exposure. Because no significant difference was observed between different times, 10 min of preequilibration was typically used to collect the X-ray data. The recorded diffraction patterns were evaluated using 3D-View software (Mbraun, Graz, Austria) without any additional data manipulation.
Results and Discussion The phases identified by X-ray diffraction and their location in the temperature-composition diagram of the binary phytantriol/water system are shown in Figure 2a. The phase boundaries and coexistence regions, drawn to conform to both the experimental data and the Gibbs phase rule, are presented in Figure 2b. The determined phase boundaries are reasonably accurate with the exception of the cubic-to-hexagonal transition region, where different phases are squeezed into a relatively small area. The phase
boundaries in this area are, therefore, difficult to locate experimentally, and they are drawn more according to the Gibbs rule. At room temperature, the phase sequence with increasing water content is reversed micellar (L2), lamellar (LR), reversed cubic of crystallographic space group Ia3d (Q230), and reversed cubic of space group Pn3m (Q224). In addition, a reversed hexagonal phase (HII) appears at temperatures 44-60 °C. Figure 3 shows representative X-ray diffractograms of all the identified phases. At room temperature, an LR phase exists between 6 and 13% (w/w) water with a repeat distance varying from 30 to 33 Å. Although only the first 100 Bragg peak is observed for this phase (Figure 3a), a typical birefringence and texture when viewed in a polarized microscope indicate its lamellar structure. By using simple geometric considerations, we are able to calculate structural characteristics for the LR phase. For an ideal one-dimensional swelling, the experimentally measured repeat distance (d) in this phase is related simply to 2l/φ,1 where 2l is the bilayer thickness and φ is the lipid volume fraction. The swelling-law data of the phytantriol LR phase is illustrated in Figure 4, where d is plotted versus 1/φ. A linear fit to the experimental data results in the bilayer thickness 2l ) 28.4 ( 0.4 Å. The calculated thickness is considerably smaller than that for the glycerol monooleate,19 which is not surprising because the hydrocarbon chain of the phytantriol molecule is shorter. At temperatures above 35 °C, the LR phase transforms into a fluid isotropic L2 phase. The latter does not show more than a diffuse diffraction, and the X-ray data seen in Figure 3a indicate a broadening of the first reflection in the LR phase, similar to that observed in the glycerol monooleate/water system. The L2 phase occupies the hightemperature part of the diagram at all the hydration levels, and because pure phytantriol is a viscous liquid, it also exists at room temperature. At about 14% (w/w) water, a stiff isotropic phase is observed. It shows six Bragg peaks (the last four are usually very weak, especially at a low water content) with relative positions in ratios x6, x8, x14, x16, x20, and x22, which can be indexed as hkl ) 211, 220, 321, 400, 420, and 332 reflections (Figure 3b) of a body-centered cubic phase of Ia3d space group (Q230). The calculated lattice parameter for the Q230 phase increases from 86 to close 100 Å at a water concentration of 23% (w/w) and a temperature of 22 °C. At higher concentrations of water, the Q230 phase transforms into another viscous isotropic phase, which can be indexed as the 110, 111, 200, 211, 220, and 221 reflections (Figure 3c). The observed six strong Bragg reflections follow the relationship x2:x3: x4:x6:x8:x9, which is in agreement with a primitive cubic lattice of Pn3m crystallographic space group (Q224). This phase appears at fairly narrow 26-28% (w/w) water content, at which limits the lattice parameters are determined as 64 and 66 Å, respectively. The lattice parameter of the Q224 remains unchanged upon the further addition of water. This fact indicates that the cubic phase is in equilibrium with excess water. As the temperature is increased, the boundary between the cubic phases and Q224/Q224 + water moves to lower hydration. The measured lattice parameter also decreases with temperature. The change in the lattice parameter as a function of temperature is higher for the Q224 (ca. 0.27 Å/°C) than for the Q230 phase (ca. 0.1 Å/°C). The upper limits for the Q230 and Q224 phases are approximately 48 and 43 °C, respectively. At a higher (19) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II (France) 1996, 6, 723-751.
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Figure 3. Representative X-ray diffractograms of (a) the lamellar phase [L; 11% (w/w) water, 25 °C] and the reversed micellar phase [LR; 11% (w/w) water, 41.5 °C], (b) the cubic phase Q230 [22% (w/w) water, 25 °C], (c) the cubic phase Q224 [27% (w/w) water, 25 °C], and (d) the reversed hexagonal phase [HII; 24.15% (w/w) water, 44.8 °C]. In parts b-d, the indexing of the Bragg reflections are indicated with their corresponding Miller indexes.
Figure 4. Repeat distance (d) of the lamellar phase in the phytantriol/water system plotted versus the reciprocal lipid volume fraction (1/φ). The solid line is the linear fit to the data with a 0 intercept (r2 ) 0.942).
temperature, the cubic phases undergo a transition to a HII phase, which is typically characterized by three strong reflections with ratios of 1:x3:x4 (Figure 3d). This phase exists up to ca. 60 °C between 20 and 26% (w/w) water with the lattice parameter ranging from ca. 39 to 41 Å (at 50 °C). At a higher water concentration, the HII phase is in equilibrium with the excess of water; that is, the lattice parameter remains constant. All we have done so far is identify the space group of both cubic phases. Our X-ray structural measurements also provide evidence that they are bicontinuous structures based on G and D minimal surfaces. The first evidence comes from the relationship between the equilibrium lattice parameters. In the two-phase region (23-26% water), the Q230 and Q224 phases are in equilibrium, with lattice parameters 100.9 and 63.6 Å (at 22 °C), respectively. The ratio between these parameters, 1.586, is close to the calculated ratio of 1.11 × x2 ) 1.57, which is obtained theoretically by the Bonnet transformation from a G to a D surface without a change in the curvature.20 This relationship between the lattice parameters of the cubic (20) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallografiya 1984, 168, 213-219.
phases also remains constant along the two-phase region at higher temperatures. Second, the lattice parameter measurements concur with the theoretical swelling law (data not shown), which is based on the differential geometric properties of the underlying minimal surface. The fact that phytantriol/water cubic phases occur between other reversed phases indicates that they are type II (QII) water-in-oil phases, where the surfactant bilayer is centered on the minimal surface. Recently, there has been some debate regarding the authenticity of the type of some well-known nonpolar phospholipid- and glycerol monooleate-based reversed cubic phases.21 Although we believe that the Q230 and Q224 phases of aqueous phytantriol are reversed bicontinuous, mathematical analysis of the X-ray diffraction data would provide useful additional information. More complete results from the phase and microstructure studies from differential scanning calorimetry, NMR techniques, and Fourier transform Raman spectroscopy will be published in the future. Conclusion The phase diagram of the phytantriol/water system shares several features with that of monoglycerides. These systems depart from the generally expected swelling sequence, and the phases are found in seemingly anomalous locations of the phase diagrams. For example, the inverse cubic phases occur at higher hydrations than the lamellar phase. Another important feature is the existence of the cubic Q224 phase in excess water. Besides the phase behavior of monoglycerides and phytantriol, similar behavior was recently reported for a phytanyl-chained alkyl glucoside, namely, for 1-O-phytanyl-β-D-xyloside.22 These findings are likely to improve our understanding of the molecular properties required to form cubic liquid crystals in an excess of the solution phase (water). (21) Garstecki, P.; Holyst, R. Langmuir 2002, 18, 2529-2537. (22) Hato, M.; Minamikawa, H.; Salker, R. A.; Matsutani, S. Langmuir 2002, 18, 3425-3429.
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Cubic liquid crystalline phases are also of technical interest in a number of fields. However, the exploration of their use has hitherto been somewhat restricted by the very limited choice of commercially available polar lipids and surfactants that show suitable aqueous phase behavior. The large similarity of the temperature-composition diagrams in the aqueous glycerol monooleate and phytantriol systems provides the basis for a further exploration of technical applications of cubic liquid crystalline phases.
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Acknowledgment. We are grateful to Kåre Larsson for discussing the manuscript. T.L. thanks Jo¨rg Schreiber and co-workers at Beiersdorf AG in Hamburg for first pointing out the existence of the L’Oreal patent applications concerning the use of phytantriol. This made us to start looking into its phase behavior. This research was made possible through a generous donation made by Camurus Lipid Research Foundation. LA0350812
