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Langmuir 2007, 23, 10020-10025
Cubic, Sponge, and Lamellar Phases in the Glyceryl Monooleyl Ether-Propylene Glycol-Water System Sven Engstro¨m,* Pia Wadsten-Hindrichsen, and Bettina Hernius Pharmacutical Technology, Department of Chemical and Biological Engineering, Chalmers UniVersity of Technology, SE-412 96 Go¨teborg, Sweden ReceiVed April 26, 2007. In Final Form: July 5, 2007 The phase behavior of 1-glyceryl monooleyl ether (GME) in mixtures of propylene glycol (PG) and water was investigated by visual inspection, polarization microscopy, small-angle X-ray diffraction, and conductance measurements. A phase diagram, based on over 200 samples of the ternary system GME-PG-water, was constructed at 20 °C. Without PG, GME forms a reverse micellar phase with up to 10 wt % water and a reverse hexagonal liquid-crystalline phase between 10 and 25 wt % water, a phase that can coexist with excess water. If PG is added in amounts exceeding about 10 wt %, then cubic and lamellar liquid-crystalline phases start to form. A cubic phase, belonging to space group Pn3m, can coexist with excess PG-water mixtures. If even more PG is added, then the cubic phase is transformed into a sponge phase. A lamellar phase forms at water contents between 10 and 15 wt % and with widely differing PG/GME weight ratios. We postulate that the phase behavior is caused by the fact that PG makes the interfacial region between self-assembled GME and PG-water less negatively curved, which in turn allows for the formation of the new phases. The phase behavior obtained for the GME system shows a striking similarity with the phase behavior of the corresponding system in which the GME has been replaced by the ester, 1-glycerol monooleate (GMO), differing only in one extra carbonyl oxygen. The major difference is the lower amount of water present in the GME phases, an effect that is mainly due to the more hydrophobic character of GME compared to that of GMO.
Introduction Glyceryl monooleate (GMO or monoolein (MO)), the glyceryl ester of oleic acid, has received considerable attention in the biotechnology field because of its fascinating phase behavior in water. (For a summary, see Larsson et al.1) GMO forms a reversed bicontinuous cubic liquid-crystalline phase of space group Pn3m in excess water. The cubic phase, consisting of one congruent GMO bilayer surrounded by two water channel systems, is often modeled as an infinite periodic minimal surface (IPMS) of the diamond (D) type.2 The cubic phase has a very stiff and glass-clear appearance to the naked eye. It may incorporate hydro-, amphi-, and lipophilic substances of various sizes into its structure and has therefore been utilized for drug delivery,3 biosensors,4 partition studies,5 and membrane protein crystallization.6 One drawback with GMO in several applications is the presence of the ester bond, which makes the molecule susceptible to hydrolysis at both high and low pH as well as by hydrolytic enzymes.7 An alternative to GMO would be to use its ether analogue, 1-glyceryl monoleyl ether (GME), differing in only one carbonyl oxygen. However, it turns out that this slight difference affects the aqueous phase behavior in a rather dramatic way because GME forms a reverse hexagonal phase (instead of a cubic phase) * Corresponding author. Phone: +46 31 772 2765. Fax: +46 31 16 00 31. E-mail:
[email protected]. (1) Larsson, K.; Quinn, P.; Sato, K.; Tiberg, F. Lipids: Structure, Physical Properties and Functionality; The Oily Press: Dundee, UK, 2006; Chapter 3. (2) Hyde, S.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213. (3) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. AdV. Drug DeliVery ReV. 2001, 47, 229. (4) Razumas, V.; Kanapieniene, J.; Nylander, T.; Engstro¨m, S.; Larsson, K. Anal. Chim. Acta 1994, 289, 155. (5) Engstro¨m, S.; Norden, T. P.; Nyquist, H. Eur. J. Pharm. Sci. 1999, 8, 243. (6) Landau, E. M.; Rosenbusch, J. P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14532. (7) Borne, J.; Nylander, T.; Khan, A. Langmuir 2002, 18, 8972-8981.
in excess water.8 A recent publication shows that with mixtures of GME and GMO in excess water the reverse hexagonal phase seems to dominate the phase behavior over the cubic phase.9 In the present study, the phase behavior of GME in mixtures of water and propylene glycol (PG) was investigated. One reason for the study is the fact that the cubic phase of GMO in excess water is transformed to a sponge phase when PG is added in a sufficient amount.10,11 This phase transformation is thought to be due to a flattening of the interfacial lipid/water layer in the cubic phase, leading to increased swelling of the lipid selfassembly structures and a subsequent loss of long-range order and the formation of a sponge phase (sometimes referred to as a “molten cubic phase”12). The flattening is most probably caused by PG, which is totally miscible with both water and lipid but prefers the former.11 The phase behavior of the GMO-PGwater ternary system at 20 °C is given in Figure 1.13 Therefore, it was interesting to find out if PG could transform the reverse hexagonal phase formed by GME in pure water to a less negatively curved self-assembly structure by the same proposed flattening mechanism. To find out, we constructed a phase diagram of the ternary GME-PG-water system based on over 200 samples. The phase characterization was carried out with a combination of visual inspection, polarization microscopy, small-angle X-ray diffraction (SAXD), and conductivity measurements. A similar phase diagram for the GMO-PG-water system, more extensive than that previously published,11 was constructed for comparison. (8) Barauskas, J.; Svedaite, I.; Butkus, E.; Razumas, V.; Larsson, K.; Tiberg, F. Colloids Surf., B 2005, 41, 49. (9) Popescu, G.; Barauskas, J.; Nylander, T.; Tiberg, F. Langmuir 2007, 23, 496-503. (10) Engstro¨m, S.; Alfons, K.; Rasmusson, M.; Ljusberg-Wahren, H. Prog. Colloid Polym. Sci. 1998, 108, 93. (11) Alfons, K.; Engstro¨m, S. J. Pharm. Sci. 1998, 87, 1527. (12) Andersson, D.; Wennerstro¨m, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243. (13) Wadsten-Hindrichsen, P.; Bender, J.; Unga, J.; Engstro¨m, S. J. Colloid Interface Sci., in press (doi: 10.1016/j.jcis.2007.07.011).
10.1021/la701217b CCC: $37.00 © 2007 American Chemical Society Published on Web 08/31/2007
GME-PG-Water System
Langmuir, Vol. 23, No. 20, 2007 10021 (2) the relative amounts and order (from top to bottom) of the phases in the vial (the order of densities is FPG (1.036 g/cm3) > FW (1.0 g/cm3) > FGME (0.915 g/cm3);9 (3) any signs of birefringence shown by the phases when placed between two light-polarizing sheets (lamellar and hexagonal phases are birefringent and cubic, the sponge and liquid phases are nonbirefringent, and the sponge phases may be shear-birefringent); and (4) the rheological appearance of the phases as judged by the naked eye (cubic and hexagonal phases are very stiff; the lamellar phase is viscous). The characterization for the construction of the phase diagram was undertaken at room temperature (20 ( 1 °C) and atmospheric pressure. The phase behavior of some samples, immersed in a thermostated water bath, was also studied at other temperatures by means of the crossed-polarizer sheets. Moreover, we assumed that the lipid behaved as a single component despite its technical origin. (Our experience tells us that this is often a good assumption.) The Gibbs’ phase rule, which in a ternary system at constant temperature and pressure reads15
Figure 1. Approximate phase diagram of the ternary GMO(MO)PG-water system at 20 °C based on the samples indicated as dots (adapted from ref 13.)
Figure 2. Molecular structures of GME (top) and GMO (bottom).
Experimental Section Materials. GME (95% purity), denoted Selachyl alcohol (Nikko Chemicals Co. Ltd., Japan), was obtained as a gift from Jan Dekker International, France. Monoolein (or glyceryl monooleate, RYLO MG 19 Pharma, lot no. 2202-42 with a monoglyceride content of 95%, a diglyceride content of 3.8%, a free glycerine comtent of 0.6%, a water content of 0.1%, and a fatty acid composition of oleic acid 89%-linoleic acid (C18-2) 5%-saturated C18 3%-saturated C16 1%-linolenic (C18-3) 1%, was kindly provided by Danisco Cultor (Brabrand, Denmark). The molecular structures of the lipids are shown in Figure 2. Propylene glycol (1,2-propanediol), methylene chloride, and sodium chloride were purchased from Sigma-Aldrich (Steinheim, Germany). All chemicals were used without further purification, and the water was of Milli-Q quality (Milli-Q Academic, 18.2 MΩ‚cm at 25 °C, Millipore). Sample Preparation. Over 200 samples were prepared in 0.8 mL (7 × 40 mm) autosampler vials purchased from NTK Kemi (Uppsala, Sweden), giving a total weight of 0.4 g. A Gilson XL 221 liquid handler with a single syringe pump (model 402) delivered from Pretech Instruments (Sollentuna, Sweden) was used for the experiments. Because the robot is made for dispensing relatively low-viscosity liquids, GME was dissolved in methylene chloride. The lipid solution (ca. 50% lipid by weight) was dispensed by the robot, and the samples were left standing for at least a week for the solvents to evaporate. The evaporation was followed by weighing at least 10 of the samples in each set. When there were only trace amounts of solvent left, PG and water were added by the robot, and the vials were sealed and left standing at room temperature (20 ( 1 °C) to reach equilibrium. The robot method for sample preparation has been described elsewhere.14 Phase Characterization. To construct the phase diagrams, each sample vial was thoroughly investigated in an iterative manner over time (up to 4 months) by means of visual inspection to determine (1) the number of phases present; (14) Imberg, A.; Engstro¨m, S. Colloids Surf., A 2003, 221, 109.
f)3-p where f is the number of degrees of freedom and p is the number of phases at equilibrium, is also a good guide in the construction of the phase diagram. Despite the large number of samples made, the uncertainty in the positions of the phase borders is about 2%. Finally, we assumed the chemical degradation of all components to be negligible during the time of our phase observations because no visible change in phase behavior took place in any of the samples after equilibrium was reached. Polarizing Microscopy. Some samples, assumed to be lamellar phases, were studied in the polarizing microscope (Olympus BH-2, Japan) at room temperature. Small-Angle X-ray Diffraction. Measurements were performed on a Kratky camera (Hecus X-ray systems, Graz, Austria) with a position-sensitive detector (MBraun, Garching, Germany) containing 1024 channels. An X-ray generator operated at 50 kV and 40 mA (Philips, PW 1830/40, The Netherlands) was used to produce Cu KR radiation (λ ) 0.1542 nm). The sample-to-detector distance was 275 mm, and the exposure time was typically 900 s for each sample. The temperature in the sample cell was kept at 20 °C and was regulated by a Peltier element (accuracy (0.1 °C). The camera and the sample cell were held under vacuum to minimize the scattering from air. The diffractograms were evaluated using 3D-View software (MBraun, Graz, Austria). Calibration of the instrument was made with solid tristearin, which shows a lamellar diffraction pattern with a lattice parameter of 4.50 nm. Powder synchrotron SAXD measurements of some liquidcrystalline and sponge phases were performed at beam line I711 at MAX-lab (Lund University, Sweden) using a Marresearch 165 mm CCD detector mounted on a Marresearch desktop beam line base plate.16 The samples were placed into a 1 mm (i.d.) glass capillary at a sample-to-detector distance of 1433 mm, and diffractograms were recorded under high vacuum at room temperature (22 °C with a wavelength of 0.1082860 nm). The exposure times varied between 30 and 180 s. The resulting CCD images were integrated using Fit2D software provided by Dr. A. Hammersley (http://www.esrf.fr/ computing/scientific/FIT2D). Calibrated (with silver behenate as a standard) wavelengths and detector positions were used. Conductivity Measurements. Conductivity measurements were carried out with a WTW Cond 315i (Wilheim, Germany) at 21 °C on one of the samples that was assumed to be a sponge phase (sample marked no. 3 in Figure 3b). Because the components used in this study are essentially non-ionic, the sponge phase was made with a 155 mM NaCl aqueous solution instead of water. Using this electrolyte solution had no visible effect on the phase behavior. (15) Atkins, P. W. Physical Chemistry, 6th ed.; Oxford University Press: Oxford, U.K., 1998; Chapter 8. (16) Cerenius, Y.; Stahl, K.; Svensson, L. A.; Ursby, T.; Oskarsson, A.; Albertsson, J.; Liljas, A. J. Synchrotron Radiat. 2000, 7, 203.
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Figure 4. Three representative X-ray diffractograms recorded at 20 °C with synchrotron radiation. (a) Reverse hexagonal phase with lattice parameter 5.17 nm (three reflections, sample 1, [GMEPG-W] ) [20:15:65] wt %), (b) cubic phase of Pn3m symmetry with lattice parameter 9.22 nm (six reflections, sample 2, [20:30: 50]), and (c) sponge phase with one diffuse Bragg peak at 9.4 nm (sample 3, [35:40:25]). Sample numbers are shown in Figure 3b.
Figure 3. Phase behavior of the ternary system GME-PG-water at 20 °C. (a) Phase map showing the prepared samples. Symbols: liquid phase, L (O); lamellar phase, La (+); reverse hexagonal phase (4); cubic phase (0); and sponge phase (]). Combined symbols indicate two- or three-phase samples, respectively, with larger symbols for the more dense phases. (b) Approximate phase diagram drawn on the basis of the samples shown in part a considering the Gibbs’ phase rule.
Results and Discussion General Phase Behavior. The phase behavior of the GMEPG-water system at 20 °C is summarized in Figure 3. In Figure 3a, the phase behavior of each individual sample is shown, and in Figure 3b, we have tried to draw one-phase boundaries and the corresponding two- and three-phase areas as well on the basis of the samples in Figure 3a. The phase diagram reveals five one-phase regions surrounded by a large number of two- and three-phase areas. The one-phase regions consist of two liquid phases and three liquid-crystalline phases. The most uncertain part of the phase diagram is two- and three-phase areas above the lamellar phase, which turned out to be very temperaturesensitive (see below). Representative X-ray diffraction patterns, obtained from one of MAXLAB’s synchrotron radiation beam lines, are given in Figure 4 for three of the samples, representing the reverse hexagonal phase (curve a), the cubic phase (curve b), and the
sponge phase (curve c). The hexagonal- and cubic-phase samples were taken from two-phase regions where the phases are formed in excess PG-water solutions whereas the sponge-phase sample was taken from the one-phase region (Figure 3b). Below, we will describe the phase behavior with regard to structural parameters in more detail for the various phases formed. One way to do so is to follow the fate of a small amount of GME added to excess PG-water solutions. One then finds that the lipid solubility does not increase to any appreciable extent (
