Phase Behavior and Aggregate Formation for the Aqueous Monoolein

Nov 6, 2001 - Phase Behavior and Aggregate Formation for the Aqueous Monoolein System Mixed with Sodium Oleate and Oleic Acid. Johanna Borné,*Tommy ...
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Phase Behavior and Aggregate Formation for the Aqueous Monoolein System Mixed with Sodium Oleate and Oleic Acid Johanna Borne´,* Tommy Nylander, and Ali Khan Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received May 2, 2001. In Final Form: August 16, 2001 The phase behavior and microstructure of the two ternary systems monoolein (MO)-sodium oleate (NaO)-water (2H2O) and MO-oleic acid (OA)-2H2O are studied by a combination of optical microscopy, cryo-transmission electron microscopy, small-angle X-ray diffraction, and NMR methods. The results demonstrate significant differences in phase behavior between the two systems. The isothermal phase diagram of the MO-NaO-2H2O system is dominated by a large lamellar liquid crystalline phase that shows an ideal swelling up to high water contents. Stable vesicles are the dominant aggregates at water concentration >90 wt %. The existence of a lamellar phase is, however, absent from the MO-OA-2H2O system, where the largest single-phase region is a reversed hexagonal liquid crystalline phase, HII, at low water content. A similar water-poor HII phase is also identified for the MO-NaO-2H2O system. The two types of bicontinuous cubic structures, gyroid (CG) and diamond (CD), formed by the binary MO-2H2O system are also present in the ternary systems. Part of the single CG phase initially formed by the ternary system with NaO is found to be metastable and becomes destabilized within a few weeks, leaving the rest of the CG phase which is stable like other thermodynamically stable phases for the system. A cubic phase with a reversed micellar type structure is characterized for the oleic acid system. The experimentally determined phase diagrams and the phase structures can be qualitatively understood in terms of the geometry of the lipid molecule in combination with electrostatic effects.

Introduction The present study was undertaken to further increase the knowledge about the aggregation processes and the structures of the phases (vesicles, micelles, emulsions, gels, and liquid crystals) that may form during the lipolysis of triolein. Mixing of the hydrolysis products, like fatty acids and di- and monoglycerides, as well as added surfactant molecules will alter the structure of the lipid aggregates and the characteristics of the lipid-aqueous interface. Knowledge of the phase behavior of the relevant lipid-aqueous systems is required to understand the action of lipases on these “real substrates”. The determined phase diagrams will serve as maps to navigate through the changes of the lipid aggregate structures that occur during the lipolytic process. We have previously studied the aqueous phase behavior of the two metabolites diolein (DO) and monoolein (MO) that form during the lipolysis of triolein.1 The phase equilibria of the MO-DO aqueous system are dominated by a reversed micellar phase at high MO concentration, and a reduction of the MO content favors the formation of a reversed hexagonal phase at a DO content of 4-30% and a water content less than 27 wt %. Below this DO concentration, cubic and lamellar phases appear as observed in the binary monoolein aqueous system. A large multiphase region that features a more distinct and extended emulsion region was also observed. The structures of cubic phases formed by the binary MO-H2O system have been extensively studied.1-7 The * To whom correspondence should be addressed. Fax Int: + 46 46 2224413. Phone Int: + 46 46 2228204. E-mail: johanna.borne@ fkem1.lu.se. (1) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 10044. (2) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forse´n, S. J. Am. Chem. Soc. 1979, 101, 5465.

bicontinuous structures of the cubic phases can be considerably extended to higher water content by introducing various ionic and nonionic amphiphiles.6,8-13 The formation of a micellar cubic phase has been reported for aqueous systems containing MO and oleic acid (OA)14-16 as well as for aqueous mixtures of sodium oleate (NaO) and OA.17 The structure of the micellar cubic phase corresponds to the face-centered lattice with space group Fd3m.16 The liquid crystalline phases identified in this study for the two systems MO-water with NaO as well as with OA have been reported previously.14-16,18 Yet most of these investigations were made with limited composition (3) Larsson, K. Nature 1983, 304, 664. (4) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213. (5) Chung, H.; Caffrey, M. Biophys. J. 1994, 66, 377. (6) Landh, T. J. Phys. Chem. 1994, 98, 8453. (7) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723. (8) Gustafsson, J.; Ora¨dd, G.; Nyden, M.; Hansson, P.; Almgren, M. Langmuir 1998, 14, 4987. (9) Lindell, K.; Engblom, J.; Jonstro¨mer, M.; Carlsson, A.; Engstro¨m, S. Prog. Colloid Polym. Sci. 1998, 108, 111. (10) Engblom, J.; Miezis, Y.; Nylander, T.; Razumas, V.; Larsson, K. Prog. Colloid Polym. Sci. 1999, 112. (11) Gustafsson, J.; Nylander, T.; Almgren, M.; Ljusberg-Wahren, H. J. Colloid Interface Sci. 1999, 211, 326. (12) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2000. (13) Pitzalis, P.; Krog, N.; Larsson, H.; Ljusberg-Wharen, H.; Monduzzi, M.; Nylander, T. Langmuir 2000, 16. (14) Mariani, P.; Luzzati, V.; Delacroix, H. J. Mol. Biol. 1988, 204, 165. (15) Mariani, P.; Luzzati, V.; Rivas, E.; Delacroix, H. Biochemistry 1990, 29, 6799. (16) Luzzati, V.; Vargas, R.; Gulik, A.; Mariani, P.; Seddon, J. M.; Rivas, E. Biochemistry 1992, 31, 279. (17) Seddon, J. M.; Bartle, E. A.; Mingins, J. J. Phys.: Condens. Matter 1990, 2, SA285. (18) Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96.

10.1021/la010650w CCC: $20.00 © 2001 American Chemical Society Published on Web 11/06/2001

Phase Behavior for the Aqueous Monoolein System

ranges. In the present study, we described the complete phase diagrams for the systems, obtained existence regions of thermodynamic equilibrium single phases, and identified the coexisting phases that are required by the phase rules. Using a combination of microscopy, small-angle X-ray diffraction (SAXD), and NMR techniques, the type, location, and stability ranges of different phases, including the heterogeneous regions and the aggregate microstructure, have been determined. It is well-established that a prior knowledge of the complete phase diagrams for colloidal systems is a prerequisite for exploring the detailed self-aggregation processes experimentally and with theoretical modeling. Experimental Section Material. Monoolein (MO), Rylo Mg 90-glycerol monooleate (TS-ED 173) (Lot No. 1876-88), was kindly provided by Danisco Ingredients (Braband, Denmark). This is a commercially available product, and it is important to note that it is not a singlecomponent product, but also contains other polar lipids. The supplier has, however, given us the detailed compositions of the material. The monoolein samples consist of 95.7 wt % monoglycerides, 3.8 wt % diglycerides, 0.4 wt % free fatty acids, and 0.1 wt % free glycerol. The fatty acid composition was 90.0 wt % oleic acid, 5.0 wt % linoleic acid, 2.7 wt % stearic acid, 1.0 wt % palmitic acid, 0.3 wt % linolenic acid, and 1.0 wt % other fatty acids. The phase behavior of the aqueous dispersion of this and similar preparations from the same source is well-known and has been found to be quite similar to that of a pure sample although the exact phase boundaries are slightly different (cf. refs 6, 19). NaO (Lot S-1120-J23-J) was from Nu-Chek-Prep, Inc. (purity >99%), and OA (O1008, 112-80-1) was from SIGMA (purity 99%) with the following fatty acid compositions: C18:1 92%, C18:2 6%, and saturated acids 2%. The deuterated water (>99.8%) was obtained from Dr. Glaser AG (Basel). Sample Preparations and Equilibration. Samples were prepared by weighing appropriate amounts of the lipids and deuterated water in small sample tubes (∼0.5 cm, inside diameter). The sample size was 300-400 mg. After the tubes were flame-sealed, the samples were centrifuged back and forth at 2900g to enhance mixing, equilibration, and separation of different phases. Samples containing OA were centrifuged at 25 °C and those with NaO at 37 °C (above the Krafft temperature for NaO).20 The centrifugation was repeated 40 min every day during 2 weeks. The samples were then left standing to equilibrate at 25 °C prior to the first measurements. The samples were observed regularly between crossed polarizers during 1 year with intermittent centrifugation to ascertain the equilibrium of the system. Methods. Optical Polarizing Microscopy. An Axioplan Universal polarizing light microscope from Carl Zeiss, equipped with a differential interference contrast (DIC) unit, was used to identify anisotropic liquid crystalline phases based on the observed textures.21-23 A short description of the textures relevant to the surfactant phases was given previously.1 Cryo-Transmision Electron Microscopy (Cryo-TEM). The cryo-TEM method24,25 was used for the direct imaging of samples in the dilute corner of the phase diagram. Vitrified samples were prepared and imaged according to a procedure described elsewhere.25 A Philips CM 120 Bio-Twin microscope was used that was specially designed for handling cryo-samples.26 2H NMR. The 2H NMR of deuterated water allows us to determine the phase boundaries of isotropic and anisotropic single (19) Larsson, K.; Fontell, K.; Krog, N. Lipids 1980, 27, 321. (20) Small, D. M. The Physical Chemistry of Lipids: From Alkanes to Phospholipids. Handbook of Lipid Research; Plenum Press: New York and London, 1986; Vol. 4. (21) Rosvear, F. B. J. Am. Oil Chem. Soc. 1954, 31, 628. (22) Rosvear, F. B. J. Soc. Cosmet. Chem. 1968, 19, 581. (23) Ekvall, P. Adv. Liq. Cryst. 1975, 1, 1. (24) Vinson, P. K.: San Francisco, 1987. (25) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. J. Electron Microsc. Tech. 1988, 10, 87. (26) Borne´, J.; Nylander, T.; Khan, A., manuscript in preparation.

Langmuir, Vol. 17, No. 25, 2001 7743 phases as well as to characterize multiphase regions of the binary and ternary amphiphilic systems.1,27,28 2H NMR in liquid crystalline (lc) phases also provides information about the hydration state of the amphiphiles.29 An isotropic micellar solution or an isotropic liquid crystalline phase is characterized by a sharp singlet as the quadrupolar interaction is averaged to zero, while for an anisotropic phase, a 2H (I ) 1) NMR spectrum splits into 2I, giving rise to two peaks with equal intensity. For a multiphase sample, a recorded 2H NMR spectrum is composed of superpositions of spectra arising from individual phases, provided that the deuteron exchange between the different phases is slow. The 2H NMR spectra were recorded at a resonance frequency of 15.371 (2.3 T) MHz on a Bruker DMX 100 superconducting spectrometer, working in the Fourier transform mode. The samples were thermally equilibrated for at least 1 day at the desired temperature, prior to any measurements. They were then transferred to the preheated NMR probe and left to equilibrate for 10 min prior to the recording of the spectra. Two sequential determinations of each specimen were used to confirm that the spectra were recorded at thermal equilibrium. Between 100 and 300 pulses with a pulse length of 10 µs were sufficient to obtain a good-quality spectrum. A variable-temperature control unit was used to regulate the temperature of the airflow in which the samples were located. The accuracy of the measured temperature was within 0.5 °C. 23Na+ NMR. The counterion 23Na+ with I ) 3/ splits into three 2 peaks with a central isotropic peak and two equidistant satellites from the central signal in anisotropic liquid crystalline phases. We have recorded 23Na+ NMR at a resonance frequency of 26.49 MHz on the same Bruker DMX 100 superconducting spectrometer following a similar procedure as for 2H NMR. A spectrum with a good signal-to-noise ratio is obtained with about 4000 pulses with a pulse length of 10 µs. Small-Angle X-ray Diffraction (SAXD). The SAXD data were recorded with a Kratky compact small-angle system equipped with a position sensitive detector (OED 50M from Mbraun, Austria) containing 1024 channels of width 53.0 µm. A monochromator with a nickel filter was used to select the Cu KR radiation (λ ) 1.542 Å) provided by the generator. The generator, a Seifert ID-300 X-ray, was operating at 50 kV and 40 mA. A few milligrams of the sample was enclosed in a stainless steel sample holder with mica windows. The distance between the sample and detector was 277 mm. The diffraction patterns were recorded at 25 and 37 °C. The temperature was maintained constant within 0.1 °C by a Peltier element. The optics and the sample cell were both held under vacuum to minimize the scatter from air. Phase Diagram Determination. The equilibrated samples were examined at regular intervals by visual inspection and between a cross polarizer to verify sample homogeneity and identify anisotropic (birefringent) and/or isotropic phases (nonbirefringent). From these examinations, we acquired an overview of the number of anisotropic and isotropic phases as well as their coexisting regions present in the system. From the appearance of 2H NMR spectra, it is normally possible to establish whether a certain sample consists of a single homogeneous phase or of a multiphase region. Thus, in a system where lamellar and hexagonal phases are formed, NMR is a good technique to identify them. However, in our case, 2H NMR quadrupolar splitting was not obtained in the large lamellar phase for the MO-NaO-2H2O system nor in the hexagonal phase of the MO-OA-2H2O system. For the MO-NaO-2H2O system, we instead used 23Na NMR. As expected, the 23Na NMR quadrupolar splitting was twice as large for the lamellar phase as compared to the one for the hexagonal phase. The liquid crystalline textures observed by polarizing optical microscopy in combination with SAXD data also yield the characterization of different liquid crystalline phases and the phase boundaries. NMR and SAXD methods were also used to obtain further information on microstructures of phases formed in the binary and ternary systems. The ternary phase diagrams were based on the phase behavior of 150 samples for each system (27) Ulmius, J.; Wennerstro¨m, H.; Lindblom, G.; Arvidson, G. Biochemistry 1977, 16, 5742. (28) Khan, A.; Lindman, B. J. Colloid Interface Sci. 1984, 101, 193. (29) Sadaghiani, A. S.; Khan, A.; Lindman, B. J. Colloid Interface. Sci. 1989, 132, 352.

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Figure 1. The ternary phase diagram of the MO-NaO-2H2O system at (a) 25 °C and (b) at 37 °C. Composition is in wt %. Phase notations are as follows: C, cubic phase; LR, lamellar phase; L2, reversed micellar solution phase; hydr lip cryst, hydrated lipid crystals; HII, reversed hexagonal phase; and HI, normal hexagonal phase. prepared over the entire composition range, and the determined phase boundaries are within an accuracy of 2-3 wt %.

Results and Discussion Phase Equilibrium. Our primary aim is to examine the formation and stability of self-assembled aggregates of MO mixed with either OA or the corresponding soap NaO in water by following the phase equilibrium method. The phase behavior of the binary MO-2H2O1-7 and NaO2 H2O30,31 systems has been reported previously, and the main features are summarized below. Binary MO-2H2O System. The aqueous solubility of MO is very low at 25 °C (∼10-7 M). Instead, an isotropic bicontinuous cubic liquid crystalline phase is formed in an excess of water. For the particular sample used in this study, the single-phase regions of the diamond type of cubic phase, CD, the gyroid type of cubic phase, CG, and the lamellar phase, LR, occur at MO concentrations (in wt %) of 63-66, 63-86.5, and 88-92, respectively.1 At a lipid content larger than 92 wt %, a very narrow solution phase, probably a reversed micellar phase (L2), which is followed by hydrated lipid crystals at even higher MO content, has been identified. Binary NaO-2H2O System. For an aqueous solution with 1 wt % NaO, a Krafft temperature of about 23 °C has been reported, and the temperature is reported to increase with the NaO concentration to 33 °C at 35 wt % NaO.32 Above the Krafft point and below about 20 wt % NaO, a clear micellar solution is obtained. The cryo-TEM images of a sample with 95 wt % water exhibit spherical micelles. On increasing the NaO content, the isotropic solution becomes viscous prior to the formation of an anisotropic liquid crystalline phase above 21 wt % NaO (Figure 1). The liquid crystalline phase exhibits a hexagonal type of microscopic texture (Figure 2a), a quadrupolar splitting in 2H NMR spectra, and a SAXD pattern with a Bragg spacing ratio 1:x3:2 typical for a hexagonal liquid crystalline phase. The hexagonal phase is of normal type (HI). Our observations agree with those of earlier studies.30,31 At 37 °C and a further increase of the NaO content to 37 wt % NaO, the HI phase is in equilibrium with (30) Vold, R. D. J. Phys. Chem. 1939, 43, 1213. (31) Luzzati, V.; Mustacchi, H.; Skoulios, A.; Husson, F. Acta Crystallogr. 1960, 13, 660. (32) Small, D. M. Handbook of Lipid Research; Plenum: New York, 1986; Vol. 4.

hydrated crystals. At 45 wt %, only hydrated crystals could be observed. Binary OA-2H2O and OA-MO Systems. Since the solubility of OA in water is very low, the binary OA-2H2O system separates readily into a viscous diffuse oil phase and an aqueous phase (Figure 3). Only dispersed crystals were observed on adding up to 10 wt % of OA to MO. At an even higher OA content, the system separates into a transparent viscous oil phase and crystals. No further examination of the binary MO-OA system has been done. Ternary MO-NaO-2H2O System. The isothermal phase diagrams for the ternary MO-NaO-2H2O system are shown in Figure 1 at 25 °C. The ternary phase diagram is dominated by a large lamellar (LR) liquid crystalline phase. Since two lamellar phases exist, we use the notation LR1 for the lamellar phase originating from the binary MO-2H2O system and LR2 for that formed by mixing three components only. A large reversed hexagonal (HII) liquid crystalline phase is also formed for the ternary system at low water content. The binary cubic phase swells with water on adding a small amount of NaO. Lamellar Phases. The LR2 phase region exists within a large range of water content (13-85 wt % 2H2O) and between 7 and 60 wt % MO at 25 °C. The LR2 phase region with respect to NaO content is more extended at around 50 wt % 2H2O than at either lower or higher water content. The minimum amount of NaO necessary to form the LR2 phase in the ternary MO-NaO-aqueous system is 5 wt % NaO at 85 wt % 2H2O (water-rich part) and 35 wt % NaO at 10 wt % 2H2O (water-poor part). The phase boundary of the single LR2 phase occurs at 40-45 wt % NaO below 60 wt % 2H2O. This region borders the hydrated crystal via a two-phase area. The LR2 liquid crystalline phase is opaque and mucous in consistency. The viscosity and fogginess decrease with increasing water content, and the samples on the waterrich region, for example, with 10 wt % MO and 5 wt % NaO, are slightly bluish. At very high water content, the closed bilayer vesicles imaged by cryo-TEM are expected to be entropically stabilized. The formation of vesicles and their characteristics are under study. Within the single phase, we observe significant changes of morphologies of the lamellar liquid crystalline phase for samples with different compositions, and a few representative micrographs are shown in Figure 2. An increase in water content changes the lamellar texture from maltese crosses, straighted (Figure 2b) and mosaic (figure not shown), at

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Figure 2. Micrographs recorded in the lc phases for the MO-NaO-2H2O system at 25 °C. Composition is in wt %, MO-NaO-2H2O: (a) reversed hexagonal phase (70-10-20), magnification 20×; (b) lamellar phase (46-34-20), 20×; (c) (30-30-40), 20×; (d) (20-20-60), 40×; and (e) (10-5-85); see text.

low water content to maltese crosses (Figure 2c and d) and vesicles at high water content (Figure 2e). A slight decrease in NaO content transforms the maltese crosses observed in Figure 2c to the straighted texture in Figure 2b. Furthermore, a texture that is difficult to identify is observed together with the maltese crosses in Figure 2d. The lamellar liquid crystalline phase, LR1, originating from the binary system can only solubilize ∼0.5 wt % NaO, equivalent to a molar ratio, [NaO]/[MO] ≈ 6 × 10-3, at MO concentrations of 88-92 wt %. It should be noted that the LR1 phase does not coexist with the LR2 phase. Reversed Hexagonal Liquid Crystalline Phase, HII. The hexagonal lc phase appears between the two lamellar phases, LR2 and LR1, and extends over a rather large range of water content (6-26 wt % 2H2O). The phase is formed already at 1 wt % NaO and solubilizes about 27

wt % NaO within its stability range ([NaO]/[MO] ≈ 0.682). At NaO concentrations between 20 and 27 wt %, the hexagonal phase also extends toward the water-rich corner to about 26 wt % 2H2O ([NaO]/[MO] ≈ 0.58). The HII phase was distinguished from both LR phases by their different microscopic textures (Figure 2a-e). SAXD spectra show three reflections for the hexagonal phases, but for lamellar phases, only one reflection is recorded for the LR1 phase and two reflections for the LR2 phase in the considered water region. Both the binary lamellar and ternary hexagonal phases produce quadrupolar splittings in 2H NMR spectra, and the splitting values for the lamellar phase are approximately 2 times larger than those for the hexagonal phase. However, no 2H NMR splitting is recorded for the ternary lamellar phase. To establish an unequivocal identification of phases, we have

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Figure 3. The ternary phase diagram of MO-OA-2H2O at 25 °C. Composition is in wt %. Phase notations: Cmic, cubic micellar phase. For other notations see Figure 1.

also carried out 23Na+ counterion NMR and obtained quadrupolar splitting on the 23Na+ NMR spectrum that consists of an isotropic peak with two equidistant satellite signals, one on each side of the central isotropic peak for both the ternary lamellar and hexagonal phases. The splitting values, measured as a distance in hertz, between the satellite signal and the central peak are, respectively, about 2.5 and 5 kHz for the hexagonal and lamellar phases. Cubic (C) Phase. The cubic phase swells dramatically with water up to about 55 wt % 2H2O in the presence of about 10 wt % NaO, equivalent to a molar ratio [2H2O]/ [MO] ≈ 28. The samples of the cubic lc phase with NaO show similar characteristics regarding flow properties, microscopic appearance, and NMR properties as the binary samples. The indexing of the SAXD spectra reveals that this cubic structure is of the CG type. In fact, the CD phase (with 34-37 wt % 2H2O) can only solubilize about 1 wt % NaO, while a larger content of NaO leads, via a two-phase region, CD + CG, to a single CG phase. At lower water content, about 30 wt % 2H2O, the CG phase can only solubilize about 1 wt % NaO. During the initial observations of the phase equilibria at 25 °C, it was noticed that the stability of the single cubic lc phase shown in Figure 1a was found to change with time. The stability of different phases and the reproducibility of the ternary phase diagram were, therefore, examined by observing the samples regularly for 1 year. The main observations are summarized here. Samples Stored at 25 °C. After about 2 months, the CG phase within the composition range of 37-66 wt % MO and 1-8 wt % NaO (Figure 1a) was transformed into multiphase regions. The exact borders between twoand three-phase regions were not determined, although SAXD analyses indicated the presence of the CG and/or HII phase(s) (Figure 1b). It is also noticeable that the single cubic phase region is shifted to about 2-4 wt % of NaO at a higher concentration (35-48 wt %) of MO. However, the borders of the single lamellar and hexagonal liquid crystalline phases did not change during the period of 2 months and were found to remain unchanged for the whole period of observation (more than 1 year). Even if hydrolysis and peroxidation did take place, they did not seem to affect the phase boundaries of the system with the exception of

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the cubic phase region. Recently, the extent of hydrolysis and acyl migration versus time was investigated with 13C NMR on a similar MO preparation.33 For L2 samples (water content of about 5%), about 2 mol % of the MO was hydrolyzed after 8 weeks. In addition, the ratio between the 1 and 2 isomers of MO was shifted as a function of time. Possibly these effects could explain the changes of the cubic phase boundaries during the first 2 months. The phase transition from a cubic phase to a HII phase was indeed observed as an effect of lipase-catalyzed hydrolyses in monoolein-aqueous-based cubic phases.34 Samples Equilibrated at 37 °C. The destabilization of the cubic lc phase was found to occur faster at 37 °C than at 25 °C, and the rate of destabilization was further increased if the samples were centrifuged at 37 °C. However, after a few days at 37 °C, the phase boundaries of the cubic phase were as shown in Figure 1b, that is, the same as obtained after 2 months at 25 °C, and no further changes were observed with time. Moreover, the other single-phase regions did not change with time when the temperature was increased to 37 °C. Ternary MO-OA-2H2O System. Significant differences in phase behavior are observed between MO-NaO2H O and MO-OA-2H O systems. We will discuss the 2 2 phase equilibrium of the MO-OA-2H2O system (Figure 3) in relation to that of the MO-NaO-2H2O system (Figure 1), as these two systems more or less represent the protonated and fully dissociated form of OA, respectively. Like in the ternary oleate system, a reversed hexagonal liquid crystalline phase is formed in the water-poor part of the MO-OA-2H2O system, and the phases exhibit physicochemical properties identical to those of the oleate system. The HII phase exists within a narrow range of water (6-18 wt %), and the phase extends toward an increased amount of OA (20 wt %), that is, [OA]/[MO] ≈ 0.36. It has limited swelling capacity with water. On the whole, the stability range of the HII phase with OA is much smaller as compared to that of the HII phase of the oleate system. The large lamellar liquid crystalline phase, LR2, obtained in the oleate system is absent with the oleic acid system. Instead, the system forms a stiff isotropic phase in equilibrium with isotopic or anisotropic lc phases depending on sample composition. We did not succeed in obtaining a single phase of the stiff isotropic liquid crystal, despite preparing several samples in the expected one-phase region (Figure 3, Table 1). This indicates that the onephase region is very narrow. However, we expect that the single phase lies within the area labeled as Cmic (Figure 3). The isotropic phase is separated, and its structure is studied with SAXD. The SAXD pattern displayed eight or nine distinct reflections which, after analysis, confirmed that the phase is cubic liquid crystal with a face-centered space group Fd3m (Table 1). The existence of this cubic lc phase in equilibrium with other phases has been reported previously14-16 for the MO-OA-H2O system, and the preparation of the single cubic phase has, to the best of our knowledge, not yet been reported. Thermodynamic equilibrium phases with an extremely limited stability range ( 0.32 and the dimension of the hydrophilic part of the lipid is expected to increase with the water content. Below this value, the increase in water content will not affect the hydration of the polar headgroup. The correct description would involve two (40) Khan, A.; Fontell, K.; Lindblom, G.; Linman, B. J. Phys. Chem. 1982, 86, 4266. (41) Wennerstro¨m, H.; Lindblom, G.; Lindman, B. Chem. Scr. 1974, 6, 97. (42) Glaser, J. A. Water 1972, 1, 215. (43) Persson, N. O.; Lindman, B. J. Phys. Chem. 1975, 79, 1410. (44) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1.

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polar regions with two dividing interfaces: one consisting of the polar headgroup with bound water and the other with free water. However, based on the available techniques, we are not able to independently determine the dimension of the former region as a function of water content. Therefore, we have regarded the water and polar headgroup together as the polar region in all the swelling calculations. The volume fractions of the various compounds were calculated from the composition of the samples as described previously.1 The densities and molecular weights used during these calculations are as follows: The density of MO (FMO ) 0.942 g/cm3 with molecular weight MMO ) 356.55 g/mol), oleic acid (FOA ) 0.894 with molecular weight MOA ) 282.47 g/mol), heptadecene (FHD ) 0.785 g/cm3 with molecular weight MHD ) 238.46 g/mol), glycerol (FGL ) 1.261 g/cm3 with molecular weight MGL ) 92.11 g/mol), NaO (FNaO ) 0.894 g/cm3 with molecular weight MNaO ) 304.46 g/mol), and the density of methanoic acid (FMeta ) 1.22 g/cm3 with MMeta ) 46.024 g/mol) were obtained from the Handbook of Chemistry and Physics.45 SAXD Analysis of the Lr Structures in the MONaO-2H2O and MO-OA-2H2O Systems. The repeat distance of the lamellar phase, aLR, can be divided into the thicknesses of the apolar part of the lipid (hydrocarbon chain) (dhc), the polar part of the lipid (dhg), and the water layer (dw). Thus, the bilayer thickness, dbilayer, equals 2(dhc + dhg) with an average volume per lipid molecule of νlip. For the one-dimensional ideal swelling, the repeat distance is inversely proportional to the volume fraction of the lipid or hydrocarbon chain, Φlip or Φhc:31

dbilayer 2νlip aLR ) ) Φlipid SLRΦlip

or

2dhc 2νhc aLR ) ) (2) Φhc SLRΦhc

where dhc and νhc are the length and volume, respectively, of the hydrocarbon chain, and SLR is the average interfacial area per molecule. LR Structure MO-NaO-Aqueous System. The purpose here is to get more knowledge of the swelling of the lamellar phase. Earlier reports on the swelling of lamellar phases in the MO-cetyltrimethylammonium bromide (CTAB)-aqueous system have indicated the formation of a defective lamellar phase with nonideal swelling.8 The swelling of the large ternary LR2 phase is illustrated in Figure 5, where the interlayer spacing, aLR, is plotted versus 1/φ where φ is either φlip or φhc. The data agree well with eq 2, indicating an ideal swelling and no indication of a defective lamellar phase. Here we note that the MO/NaO molar ratios were between 2.0 and 0.5 (weight ratio 2.2-0.6). Because of the limited resolution of the SAXD equipment at large spacing (∼150 Å), the resolution of data recorded in this range is a limiting factor. Thus, we could not establish the exact swelling limit for this LR2 phase. From the linear fit, we obtained a bilayer thickness, dbilayer ) 37.6 ( 0.6 Å, and a corresponding dhc of 15.4 ( 0.4 Å. Despite the large content of NaO, this value agrees very well with values reported for the lamellar phase of the binary MO aqueous system 5,7 and also with the dbilayer ) 35.0 ( 0.2 Å, dhc ) 13.7 ( 0.2 Å, and SLR1 ) 35.8 ( 0.3 Å2 found for the LR1 obtained from the same ternary system at low NaO content. We also recorded similar values for the LR phase in the MO-OA-2H2O system, namely, dbilayer ) 34.6 ( 0.6 Å, dhc ) 13.6 ( 0.2 Å, and SLR ) 36.3 ( 0.6 Å2. (45) Handbook of Chemistry and Physics; CRC Press: Cleveland, 1974.

Figure 5. The lattice parameter aLR (Å) of the lamellar phase (LR) in the ternary system MO-NaO-2H2O plotted versus the reciprocal of the (a) apolar volume fraction, 1/φhc, and (b) lipid volume fraction, 1/φlipid.

SAXD Analysis of the HII Structure. A detailed and comprehensive description of the calculation of the dimension of HII phases is given by Rand and Fuller.46 The HII phase consists of water cylinders surrounded by a surfactant monolayer, where the hydrocarbon chains of the surfactants are stretched to a length, l1, into the hexagonal corners of the unit cell and have a length, l2, along the hexagonal faces. From the lattice spacing, aHII, obtained from SAXD experiments, these distances, the cross section at radius Rcyl, and the mean polar headgroup area (SHII) can be obtained from simple geometric considerations if ideal swelling is assumed:31,44,46

l1 )

( (

l2 )

)) )) )

x3 1 (1 - Φhc) 2π x3

( ( (

x3 1 (1 - Φhc) 2 2π

SHII )

1 2π (1 - Φhc) Φhc x3

1/2

aHII

(3)

1/2

1/2

aHII

(4)

2νhc aHII

(5)

If we plot aHII versus Φhc, we can assume that either l1, l2, or SHII is constant. Close analyses of the data and our previous work on the MO-DO-2H2O system1 reveal that only minor changes are observed, but the l2 value seems to remain almost constant (data not shown). Therefore, we have chosen to fit eq 4 to our SAXD data on the HII phases. (46) Rand, R. P.; Fuller, N. L. Biophys. J. 1994, 66, 2127.

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Figure 6. The lattice parameter aHII (Å) of the hexagonal phase (HII) in the ternary system MO-NaO-2H2O plotted versus the apolar volume fraction φhc.

Borne´ et al.

these are the same as the average values of the acyl chain length in the LR1 phase (13.7 ( 0.2 Å). It is interesting to note that this value is significantly smaller than the value reported for the swollen LR2 phase (15.4 ( 0.4 Å) in the MO-NaO-aqueous system. It is interesting to compare our results with the extensive study on the swelling of HII phases of DOPE with water by Rand and Fuller.46 They also observed that l2 (dlhc/2 in their notation) does not change with the water content, while l1 (dmhc/2 in their notation) increases from about 13.5 to 15.7 Å with the water content from 6 to 26 wt %. This is indeed observed in the present study, where the corresponding values for the MO-NaO-2H2O system are 13.1-14.4 Å. The calculated average area per molecule of HII in the MO-NaO- and the MO-OA-aqueous systems varies with the water content. For instance, at a NaO content of 20 wt %, the average area increases from 29.4 to 33.4 Å2 when the water content increases from 10 to 30 wt %. The corresponding value for the HII in the MO-OA-aqueous system is 29.9-32.0 Å2 when the water content increases from 12 to 25 wt %. Swelling Behavior of the C Structures. The swelling of the cubic phases in the aqueous monoolein system have been modeled as described previously.48 In this model, a neutral surface is defined at a distance t from the middle of the curved bilayer. At the neutral surface, the area per molecule (Ω(t)) is constant, independent of the swelling that is on the curvature of the bilayer. The thickness of the bilayer and the area per lipid molecule at the polarapolar interface will vary with the water content to maintain a constant area per molecule at the neutral interface. The volume fraction of lipids, Φlip, can under these conditions be related to the lattice parameter, acub (unit cell dimension obtained by SAXD):

Figure 7. The lattice parameter aHII (Å) of the hexagonal phase (HII) in the ternary system MO-OA-2H2O plotted versus the apolar volume fraction φhc. b represents one-phase samples, and O represents two-phase samples not used in the fitting.

Φlip )

c1 c2 + acub a 3

(6)

cub

where HII Structure in the MO-NaO- and MO-OAAqueous Systems. The purpose of studying the swelling of the HII phase was to establish the border of the HII phase to the lipid-rich region. This is particularly difficult as we might expect the presence of an L2 phase, which is hard to detect directly with SAXD unless it is present in large quantities. The swelling of the HII phases for the MO-NaO and MO-OA aqueous systems are illustrated in Figures 6 and 7, respectively, where aHII is plotted versus 1/Φhc. Equation 4, the equation for ideal swelling assuming a constant l2 value, was fit to the data. For the MONaO-aqueous system, the fit (Figure 6) is good. However, for the MO-OA-aqueous system, the data seem to fall into two groups, where eq 4 can be fit to the data obtained at high water content. However, the aHII values at high lipid content ()87%) are lower than expected for ideal swelling. From this we conclude that these samples are not pure HII phase, but contain a small amount of another phase, probably an L2 phase. From the fit of eq 4, we obtained an l2 value of 10.0 ( 0.2 Å in the oleate system and a value of 10.4 ( 0.2 Å in the oleic acid system which are identical to the values we reported for the MO-DO-aqueous system. These values are comparable to the thickness of the acyl chain region, 9.3 Å, of a MO monolayer at the air/water interface, determined by specular X-ray reflectivity measurements.47 The corresponding value for l1 is 13.9 ( 0.6 Å in the oleate system and 14.4 ( 0.3 Å in the oleic acid system, and

c1 )

νlip(-16πχH2)1/3 Ω(t)

and

c2 )

+4πχνlipt2 Ω(t)

νlip is the molecular volume of lipid calculated in analogy, and χ is a topology index of the surface known as the Euler-Poincare´ characteristic per cubic unit cell. The latter parameter equals -8 and -2 for the global shape of the gyroid and diamond surfaces, respectively. H is the homogeneity index that connects the lattice parameter to topology and equals 0.7665 and 0.7498 for the CG and CD type of cubic phases, respectively. C. Structure in the MO-NaO- and MO-OAAqueous Systems. The purpose of studying the swelling of the cubic phase by SAXD was to verify the indexing of the CG cubic phase which was extended toward higher water content than in the MO-water binary system. The solid line in Figure 8 represents a best fit of eq 6 to the experimental data for both the MO-2H2O binary samples and the MO-NaO-2H2O ternary samples in our study. The excellent fit to the data demonstrates that the CG phase from the binary system can swell to rather high (47) Jensen, T. R.; Kjær, K.; Howes, P. B.; Svendsen, A.; Balashev, K.; Reitzel, N.; Bjørnholm, T. In Model Systems for Biological Membranes Investigated by Grazing-Incidence X-ray Diffraction and Specular Reflectivity; Kokotos, G., Constantinou-Kokotou, V., Eds.; Crete University Press: Crete, 1999; p 127. (48) Engblom, J.; Hyde, S. T. J. Phys. II 1995, 5, 171.

Phase Behavior for the Aqueous Monoolein System

Figure 8. Volume fraction of lipid φlip versus the lattice parameter acub of the cubic phase, CG, in the binary MO-2H2O and ternary MO-NaO-2H2O systems. The solid line represents a best fit of the model calculated for the CG cubic phase (eq 6). Symbols: 9 represents MO-2H2O, and 2 represents MONaO-2H2O.

water content if we add NaO. The presence of NaO seems to favor the CG phase over the CD phase. This is logical as the CG phase in the binary MO-water system borders to the LR1 phase, and addition of NaO favors the LR phase. The dimensions of the CG phase in the MO-OA-2H2O system are similar to those in the binary MO-2H2O system (Figure 9). From the fit of eq 6, we obtain the area Ω(t) to be 36.55 Å2 at t ) 10.61 Å for MO-OA-2H2O and 35.37 Å2 at t ) 11.03 Å for MO-NaO-2H2O. These values are close to the values reported previously1 by Chung and Caffrey5 and Engblom and Hyde.48 Concluding Comments. So far, our studies on the relation between the activity of lipolytic enzymes and the lateral organization of the lipid (structure) have been focused on the relevant lipid structures that may form during the lipolytic process of triolein. Lipolysis of triolein leads to the formation of several metabolites of which only MO can form aggregates with water. Other products like DO and oleic acid cannot self-assemble in water, but can form a mixed aggregate such as with MO. However, if oleic acid is partially dissociated, there will be a lamellar type of structure even at very high water content.

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Figure 9. Volume fraction of lipid φlip versus the lattice parameter acub of the cubic phase, CG, in the binary MO-2H2O and ternary MO-OA-2H2O systems. The solid line represents a best fit of the model calculated for the CG cubic phase (eq 6). Symbols: 9 represents MO-2H2O and O represents MO-OA2H O. 2

Lipolysis is normally carried out at very low (few wt %) triolein concentration. The aggregates characterized for the model system MO-2H2O with DO, NaO, and triolein49 at high water content will be relevant for understanding the lipid aggregate structure formed during the lipolytic process. No systems except with NaO are shown to form any single phase at such water concentrations. Only multiphase regions of cubic, hexagonal, and cubic micellar liquid crystalline with water exist. With NaO, the single lamellar phase extends to high water content, and at very high water concentration, stable liposomes are shown to exist. Acknowledgment. This work was financed by the EU Biotech Shared Cost project, Contract No. BIO497-2365. LA010650W (49) Lindstro¨m, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstro¨m, B. Lipids 1981, 16, 749.