Liquid Crystalline Phases and Their Dispersions in Aqueous

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Langmuir 2007, 23, 496-503

Liquid Crystalline Phases and Their Dispersions in Aqueous Mixtures of Glycerol Monooleate and Glyceryl Monooleyl Ether Georgeta Popescu,†,§ Justas Barauskas,*,†,‡ Tommy Nylander,† and Fredrik Tiberg†,‡ Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund UniVersity, P. O. Box 124, SE-221 00 Lund, Sweden, and Camurus AB, So¨lVegatan 41, Ideon Science Park Gamma 1, SE-223 70 Lund, Sweden ReceiVed August 8, 2006. In Final Form: October 9, 2006 The aqueous phase behavior of mixtures of 1-glycerol monooleate (GMO) and its ether analogue, 1-glyceryl monooleyl ether (GME) has been investigated by a combination of polarized microscopy, X-ray diffraction, and NMR techniques. Three phase diagrams of the ternary GMO/GME/water system have been constructed at 25, 40, and 55 °C. The results demonstrate that the increasing amount of GME favors the formation of the reversed phases, evidenced by the transformation of the lamellar and bicontinuous cubic liquid crystalline phases of the binary GMO/water system into reversed micellar or reversed hexagonal phases. For a particular liquid crystalline phase, increasing the GME content has no effect on the structural characteristics and hydration properties, thus suggesting ideal mixing with GMO. Investigations of dispersed nanoparticle samples using shear and a polymeric stabilizer, Pluronic F127, show the possibility of forming two different kinds of bicontinuous cubic phase nanoparticles by simply changing the GMO/ GME ratio. Also NMR self-diffusion measurements confirm that the block copolymer, Pluronic F127, used to facilitate dispersion formation, is associated with nanoparticles and provides steric stabilization.

Introduction Knowledge of the phase behavior of aqueous dispersions of monoglycerides, such as glycerol monooleate (GMO), is of great interest due to their widespread use in, for example, food and pharmaceutical products.1-4 Depending on the concentration and temperature, GMO can, in aqueous dispersion, associate into a range of phases: reversed micellar, lamellar, reversed hexagonal, and two reversed bicontinuous cubic phases, Q230 and Q224.5-7 The latter phase is in equilibrium with excess aqueous solutions. This phase behavior has stimulated the use of aqueous GMO systems in various scientific contexts, ranging from biophysics8-13 to the making of bioelectrode and biosensor constructs.14-17 Because of their nanoscopic structure and intriguing phase * To whom correspondence should be addressed. Tel.: +46 46 222 8175. Fax: +46 46 222 4413. E-mail: [email protected]. † Lund University. ‡ Camurus AB. § Present address: Faculty of Industrial Chemistry and Environmental Engineering, Polytechnics University, Timisoara, Romania.

(1) Larsson, K. Lipids - Molecular Organization, Physical Functions and Technical Applications; The Oily Press: Dundee, U.K., 1994. (2) Krog, N. In Food Emulsions; Larsson, K., Friberg, S., Eds.; Marcel Dekker: New York, 1997; p 141. (3) Clogston, J.; Rathman, J.; Tomasko, D.; Walker, H.; Caffrey, M. Chem. Phys. Lipids 2000, 107, 191-220. (4) Mele, S.; Murgia, S.; Caboi, F.; Monduzzi, M. Langmuir 2004, 20 52415246. (5) Lutton, E. S. J. Am. Oil Chem. Soc. 1965, 42, 1068-1070. (6) Hyde, S.; Andersson, S.; Eriksson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (7) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (8) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acta 1983, 729, 23-27. (9) Razumas, V.; Larsson, K.; Miezis, Y.; Nylander, T. J. Phys. Chem. 1996, 100, 11766-11774. (10) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte¨, Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476-5483. (11) Razumas, V.; Talaikyte¨, Z.; Barauskas, J.; Nylander, T.; Miezis, Y. Prog. Colloid Polym. Sci. 1998, 108, 76-82. (12) Barauskas, J.; Razumas, V.; Nylander, T. Chem. Phys. Lipids 1999, 97, 167-179. (13) Barauskas, J.; Razumas, V.; Nylander, T. Prog. Colloid Polym. Sci. 2000, 116, 16-20. (14) Razumas, V.; Kanapieniene¨, J.; Nylander, T.; Engstro¨m, S.; Larsson, K. Anal. Chim. Acta 1994, 289, 155-162.

structure, the GMO liquid crystalline phases and nanoparticle dispersions have also found use in drug delivery.18-20 An important issue, especially from an application point of view, is the chemical stability of the liquid crystalline phases and their colloidal dispersions. Some recent studies have shown that the phase behavior and long-term stability of the GMO-based materials can be strongly affected by lipid hydrolysis and acyl migration within a few months from sample preparation.21,22 In this respect, the appearance of hydrolysis productssglycerol and oleic acidsmay not be desirable since they can alter the phase equilibrium as well as the functional properties of liquid crystalline material. The hydrolysis effect can only slightly be decreased by dispersing GMO-based liquid crystalline phases in the presence of triblock copolymers (e.g., Pluronic F127).23 It is noteworthy that the hydrolysis of GMO can be significantly accelerated in the presence of hydrolytic enzymes such as lipases.24 Here we present a way to use the particular features of GMObased liquid crystalline phases while decreasing the susceptibility of the system against hydrolysis and thus increasing the chemical stability. It is based on admixing of 1-glyceryl monooleyl ether (GME), which is significantly more stable against hydrolysis compared to the corresponding ester (GMO). The choice of the (15) Rowinski, P.; Bilewicz, R.; Stebe, M.-J.; Rogalska, E. Anal. Chem. 2002, 74, 1554-1559. (16) Barauskas, J.; Razumas, V.; Talaikyte¨, Z.; Bulovas, A.; Nylander, T.; Tauraite¨, D.; Butkus, E. Chem. Phys. Lipids 2003, 123, 87-97. (17) Rowinski, P.; Bilewicz, R.; Stebe, M.-J.; Rogalska, E. Anal. Chem. 2004, 76, 283-291. (18) Engstro¨m, S.; Norde´n, T.; Nyquist, H. Eur. J. Pharm. Sci. 1999, 8, 243254. (19) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456. (20) Larsson, K. Curr. Opin. Colloid Interface Sci. 2000, 5, 64-69. (21) Pitzalis, P.; Krog, N.; Larsson, K.; Ljusberg-Wahren, H.; Monduzzi, M.; Nylander, T. Langmuir 2000, 16, 6358-6365. (22) Murgia, S.; Caboi, F.; Monduzzi, M.; Ljusberg-Wahren, H.; Nylander, T. Prog. Colloid Polym. Sci. 2002, 120, 41-46. (23) Monduzzi, M.; Ljusberg-Wahren, H.; Larsson, K. Langmuir 2000, 16, 7355-7358. (24) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2002, 18, 8972-8981.

10.1021/la062344u CCC: $37.00 © 2007 American Chemical Society Published on Web 11/21/2006

Aqueous Phase BehaVior of GMO and GME

Figure 1. Molecular structures of GMO (a) and GME (b).

corresponding ether lipid ensures high miscibility of the two components (Figure 1). Nonpolar ether lipids of the 1-O-alkyl-2,3-diacyl-sn-glycerol type are present in most animal tissues and are the main constituents in the lever oils of various species of elasmobranch fish such as dogfish and shark.25 1-O-alkyl-sn-glycerols are obtained by the hydrolysis of lever oils, where the most abundant glyceryl ether is GME with a C18:1 alkyl moiety.26 GME is also known as selachyl alcohol, named after the Selachoidei family of fishes, as it was first isolated from the lever oil of the fish. In addition to biological effects, such as antibacterial and antifungal action, immunological stimulation, and antitumor properties,27 glyceryl ethers are amphiphilic.28 A recent phase behavior study has shown that synthetic GME forms a reversed micellar solution and reversed hexagonal phase at low and high hydration, respectively.29 The hexagonal phase exists over a wide temperature range and is in equilibrium with excess solution. In the present study, the temperature-dependent aqueous phase behavior of the mixtures of GMO and GME was investigated by means of X-ray diffraction (XRD) and 2H NMR. As a result, three ternary phase diagrams were constructed and discussed. In addition, bicontinuous liquid crystalline cubic phase nanoparticle dispersions was prepared from the mixed GMO/GME system and examined by XRD and 1H NMR self-diffusion techniques. Experimental Section Materials. RYLO MG 19 GMO (96.7% monoglycerides) was produced and provided by Danisco Ingredients (Brabrand, Denmark) with the following fatty acid composition (lot no. 2119/65-1): 89.3% oleic, 4.6% linoleic, 3.4% stearic, and 2.8% palmitic acid. GME (95% purity), denoted as Selachyl alcohol, was purchased from Nikko Chemicals (Tokyo, Japan). The poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblock copolymer with the trade name Pluronic F127 and approximate formula of PEO98PPO57PEO98 (average molecular weight, 12 600 g/mol) was obtained from BASF Svenska AB (Helsingborg, Sweden). Heavy water, D2O (purity 99.8%), was obtained from Armar Chemicals (Do¨ttingen, Switzerland). Millipore water was taken from a Milli-Q water purification system. Sample Preparation. Samples were prepared by weighing appropriate amounts of GMO and GME into 14 mm (i.d.) glass ampules (total lipid amount, ∼0.5-1 g), co-melting lipids at 40 °C, and then adding water. For the 2H NMR quadrupolar splitting experiments, D2O was used instead of water at the same solvent/ lipid molar ratio. The vials were immediately sealed and allowed to equilibrate at 25 °C for at least 4 weeks before measurements. To obtain phase homogeneity, samples were centrifuged at 1500 g for 10 min. The vials were then place upside-down, and the centrifugation was repeated. The samples were investigated between (25) Snyder, F.; Lee, T.-C.; Wykle, R. In Biochemistry of Lipids, Lipoproteins and Membranes; Vance, D. E., Vance, J. E., Eds.; Elsevier: Amsterdam, 2002; p 233. (26) Bordier, C. G.; Seller, N.; Foucault, A. P.; Goffic, F. L. Lipids 1996, 31, 521-528. (27) Weber, N. In Progress in Biochemical Pharmacology; Braquet, P., Mangold, H. K., Vargaftig, B. B., Eds.; Karger: Basel, Switzerland, 1988; p 48. (28) Gopinath, D.; Ravi, D.; Rao, B. R.; Apte, S. S.; Rambhau, D. Int. J. Pharm. 2002, 246, 187-197. (29) Barauskas, J.; Sˇ vedaite¨, I.; Butkus, E.; Razumas, V.; Larsson, K.; Tiberg, F. Colloids Surf., B 2004, 41, 49-53.

Langmuir, Vol. 23, No. 2, 2007 497 crossed polarizers to check for sample homogeneity and the presence of birefringent phases. If needed, the centrifugation cycles were repeated several times. For measurements at higher temperatures, the samples were preequilibrated for 1 day at a given temperature. Dispersion Preparation. Dispersions were prepared by adding appropriate amounts of melted GMO or GMO/GME lipid mixture into an aqueous F127 solution (for the 1H NMR self-diffusion measurements, a corresponding molar amount of D2O was used instead of pure water). In all experiments, the lipid/polymer ratio was 9/1 (wt/wt), and the total amphiphile (lipid + polymer) concentration was 5 wt %. The sample volume was usually 50-100 mL. The samples were immediately sealed, hand-shaken, and mixed for 24-48 h on a mechanical mixing rocking table at 350 rpm and room temperature. The resulting coarse dispersions were homogenized by passing 5-8 times through a Microfluidizer 110S (Microfluidics Corp., Newton, MA) at 345 bar and 25 °C. To prepare nanoparticles with a high degree of order, low polydispersity, and low contamination of metastable vesicular aggregates, homogenized dispersions were subjected to a heat treatment procedure. Heat treatment of the dispersions was performed using a bench-type autoclave (CertoClav CV-EL, Certoclav Sterilizer GmbH, Traun, Austria) operated at 125 °C and 1.4 bar. The samples were filled into Pyrex glass bottles (100-200 mL) and put into the autoclave. A period of about 12 min was required to vent the entrapped air and to heat up the autoclave. The samples were then subjected to heat treatment for 20 min at 125 °C. After the heat treatment, the samples were allowed to cool to room temperature before analysis. XRD. Measurements of bulk samples 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. The samples were filled into either a 1 mm (i.d.) quartz capillary in a steel sample holder or mounted between thin mica windows at a sample-to-detector distance of 277 mm. The typical exposure time was 10-60 min. To minimize scattering from air, the camera volume was kept under vacuum during the measurements. Temperature control within 0.1 °C was achieved using a Peltier element. The recorded slit-smeared diffraction patterns were desmeared and evaluated using 3D-View software (MBraun, Graz, Austria). Powder synchrotron XRD measurements of liquid crystalline nanoparticle dispersions were performed at beamline I711 at MAXlab (Lund University, Sweden), using a Marresearch 165 mm CCD detector mounted on a Marresearch Desktop Beamline baseplate.30 The samples were filled into a 1 mm (i.d.) glass capillary at a sampleto-detector distance of 1456 mm. Diffractograms were recorded under high vacuum at room temperature (22 °C) with a wavelength of 1.079616 Å. The exposure time was 10 min. The resulting CCD images were integrated using the Fit2D software provided by Dr. A. Hammersley (http://www.esrf.fr/computing/scientific/FIT2D). Calibrated wavelengths and detector positions were used. 2H Nuclear Magnetic Resonance (2H NMR). Birefringent H 2 liquid crystalline phases were characterized by deuterium NMR. This homogeneous anisotropic phase is characterized by a single splitting (Pake-pattern) in the 2H NMR spectrum. The spectra were recorded at a resonance frequency of 15 (2.3 T) MHz on a Bruker DMX 100 superconducting spectrometer, working in the Fourier transform mode. The quadrupolar splittings of the samples were measured with a 90°-acquire sequence. Between 100 and 300 pulses with a pulse length of 10 µs were used to obtain spectra. The 2H NMR quadrupole splitting value ∆(2H) was measured as the peakto-peak distance in hertz. Particle Size Measurements. Particle size distributions were measured using a Coulter LS230 laser diffraction particle size analyzer (Beckman-Coulter, Inc., Miami, FL), which operates on the principles of Fraunhofer diffraction for large particles (0.4-2000 µm) and uses the polarization intensity differential scattering method for small (30) Cerenius, Y.; Ståhl, K.; Svensson, L. A.; Ursby, T.; Oskarsson, Å.; Albertsson, J.; Liljas, A. J. Synchrotron Radiat. 2000, 7, 203-208.

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particles (0.04-0.5 µm). The instrument was fitted with a 125 mL volume module. Data were collected during 90 s. A standard model based on homogeneous oil spheres with a refractive index (RI) of 1.46 was used for the particle size calculations. A change in RI to either side only shifts the obtained particle size distributions within a few percent. Note that the model is based on spherical particles, and the measured mean particle size is therefore an apparent size. 1H NMR Self-Diffusion Measurements. All experiments were performed at 25 (( 0.5) °C on a Bruker DMX-200 spectrometer, with a 1H resonance frequency of 200 MHz, equipped with a Bruker field gradient probe unit. Diffusion measurements were performed using pulsed magnetic field gradient spin-echo proton NMR, as previously described.31 The experiments were carried out by varying the duration of the gradient pulse (δ) while keeping the gradient strength (G) and the time between the leading edges of the gradient pulses (∆) constant. The self-diffusion coefficient (D) was obtained by fitting the obtained NMR data to the following equation: I ) I0 exp{-(γGδ)2D(∆ - δ/3)}

(1)

where I denotes the observed echo intensity, I0 is the echo intensity in the absence of field gradient pulses, and γ is the magnetogyretic ratio.

Results and Discussion 1. Phase Diagrams. The phase equilibrium of the binary GMO/ water and GME/water systems has been previously reported.5-7,29 However, the composition of commercial GMO and GME samples may vary from batch to batch. We therefore reexamined the binary isothermal (25 °C) GMO/water and temperaturecomposition GME/water phase diagrams and compared them with those of previous studies. About 20 samples were used to map out the binary GMO/ water phase diagram. The equilibrium phase boundaries were therefore determined within an accuracy of about 2 wt %. With increasing water content, GMO exhibits the phase sequence and boundaries as follows: dispersion of crystals (LC phase, up to 4 wt % of water) f lamellar liquid crystalline phase (LR phase, 7-12 wt % of water) f reversed bicontinuous cubic phase Q230 of space group type Ia3hd (13-32 wt % of water) f reversed bicontinuous cubic phase Q224 of space group type Pn3hm (3338 wt % of water). The latter phase coexists with excess water. The obtained sequence of phases and transition boundaries agree well with the previously determined phase behavior of a GMO sample of similar purity.12 Figure 2 shows the temperature-composition phase diagram determined for the GME/water system. At room temperature and up to about 9% (w/w) water, a fluid isotropic reversed micellar solution (L2 phase) is formed. At higher water content, the L2 phase transforms into a reversed hexagonal liquid crystalline phase (H2) which swells up to about 25 wt % of water. The phase is in equilibrium with excess solution. As the temperature is increased, the H2/H2 + water boundary slightly moves to lower hydration. The upper limit for the H2 phase is close to 80 °C. At higher temperatures, only the L2 phase is present. The phase diagram is very similar to that previously obtained for a highpurity synthetic.29 The only difference is that the temperature stability of the H2 phase for the present GME preparation is higher, which is probably due to the presence of considerable amounts of the saturated alkyl chains. Figure 3a shows the experimentally determined ternary GMO/ GME/water phase diagram at 25 °C, where the phases were identified by polarized microscopy and XRD. The samples investigated were prepared in the water content region of 5-40 (31) So¨derman, O.; Stilbs, P. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 445-482.

Figure 2. (a) Location of the phases in the GME/water system as determined by XRD in the heating direction. The phases are labeled as follows: reversed triangles, L2; filled circles, H2; open circles, L2+H2. (b) Temperature-composition phase diagram of the GME/ water system drawn to conform to both the data in panel a and the Gibbs rule.

wt % in steps of 3-5 wt %. Eight dilution lines at a constant GMO/GME ratio but different water concentration were examined. The corresponding phase diagram with the one-phase boundaries drawn to conform to the experimental data is presented in Figure 3b. In addition, Figure 3c,d shows the phase diagrams obtained at 40 and 55 °C, respectively. As evident from the phase diagrams presented in Figure 3, the addition of GME as well as the increase in temperature promotes the phase transition toward more reversed phases. All the liquid crystalline phases originating from the binary GMO/water mixtures ultimately transform to either the L2 or H2 phase upon the addition of GME, as is obvious when comparing with the phase behavior for the two-component aqueous GME system. Different phases, however, demonstrate different sensitivity to the amount of GME added. At room temperature, the LC and LR phases are destabilized even at a GMO/GME ratio of about 90/ 10 (w/w), while the cubic phases can accommodate much larger amounts of GME. A GMO/GME ratio of about 75/25 is required to destabilize the Q224 phase, whereas the Q230 phase prevails up to almost 60/40. This effect can be explained by the fact that, in a binary GMO/water system, the Q230 phase is more stable with temperature over the Q224, phase which is most probably due to the better structural packing within the phase.6 Since GME favors reversed phases and induces the same effect as temperature, the Q224 phase is destabilized much faster in a mixed GMO/GME/water system. Earlier investigations of the phase behavior of aqueous GMO have also shown the Q230 phase to be more stable than the Q224 phase in the presence of weakly polar substances such as vitamin K110 and alkylated ferrocene derivatives.16 As expected, with increasing temperature, reversed phase regions dominate in the ternary GMO/GME/water phase diagram

Aqueous Phase BehaVior of GMO and GME

Langmuir, Vol. 23, No. 2, 2007 499

Figure 3. Phase diagram showing the prepared and investigated sample dilution lines (a) and the resulting ternary GMO/GME/water phase diagram (b) at 25 °C. Symbols in panel a denote L2 (filled circles), LR (open circles), Q230 (filled reversed triangles), Q224 (open reversed triangles), H2 (filled squares). (c,d) Schematic phase diagrams obtained at 40 and 55 °C, respectively.

(Figure 3c). At even higher temperatures, the ternary phase diagram is composed of only L2 and H2 phases (Figure 3d). 2. Swelling of the Liquid Crystalline Phases. XRD data were further used to investigate the structural characteristics of the liquid crystalline phases formed in the ternary GMO/GME/ water system. The application of swelling laws requires the definition of the dividing interface between volume fractions of polar and nonpolar regions. Here we assume that the nonpolar part consists of the GMO and GME, with the polar fraction being only water. The total lipid volume fraction for the ternary mixtures can then be calculated as

wGMO wGME + FGMO FGME φlip ) wGMO wGME + + wwater FGMO FGME

(2)

where w is the weight fraction of the component and F is the density. The density of GMO, FGMO ) 0.942 g/cm3, was obtained from the literature,32 whereas the density of GME was measured to be 0.915 g/cm3 by using a pycnometer. Assuming one-dimensional ideal swelling (constant bilayer thickness), the measured repeat distance for the LR phase, alam, is inversely proportional to φlip: (32) Handbook of Chemistry and Physics; CRC Press: Cleveland, OH, 1974.

alam )

2l φlip

(3)

where l is the lipid length. The swelling behavior of the cubic phases was also analyzed assuming constant bilayer thickness. It is generally accepted that the reversed bicontinuous cubic phases consist of a curved bilayer where the mid-plane surface mimics an infinite periodic minimal surface (IPMS). Three types of IPMSs, described by different cubic space groups, have been shown to be important in lipid systems:33-35 (i) the diamond (D) type of IPMS corresponding to the Pn3hm primitive lattice (Q224); (ii) the gyroid (G) type of IPMS corresponding to the Ia3hd body-centered lattice (Q230), and (iii) the primitive (P) type of IPMS, which corresponds to the Im3hm body-centered lattice (Q229). The monolayer on each side of the mid-plane has a constant thickness, l, which can be determined by solving the following equation:36,37 (33) Andersson, S.; Hyde, S. T.; Larsson, K.; Lidin, S. Chem. ReV. 1988, 88, 221-242. (34) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (35) Hyde, S. T.; Andersson, S.; Larsson, K.; Blum, Z.; Landh, T.; Lidin, S.; Ninham, B. The Language of Shape. The Role of CurVature in Condensed Matter: Physics, Chemistry and Biology; Elsevier: Amsterdam, 1997. (36) Anderson, D, M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5364-5368. (37) Turner, D. C.; Wang, Z.-G.; Gruner, S. M.; Mannock, D. A.; McElhaney, R. N. J. Phys. II 1992, 2, 2039-2063.

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( )

Popescu et al.

( )

l 4πχ l + acub 3 acub

φlip ) 2A0

(4)

where A0 (3.09 for the Q230, and 1.92 for the Q224) and χ (-8 and -2 for the Q230 and Q224, respectively) are the constants for the given minimal surface, and acub is the lattice parameter for a given cubic phase. Equations 3 and 4 were fitted to the experimentally measured lattice parameters versus φlip plots for the LR and cubic phases of the ternary GMO/GME/water system at 25 °C (Figure 4). As shown in Figure 4, the theoretical swelling law describes well the experimental data and fitting results in the following values of the lipid length (monolayer thickness): 17.2 ( 0.2, 16.8 ( 0.1, and 17.0 ( 0.1 Å for the LR, Q230, and Q224 phases, respectively. The calculations show that all three of these liquid crystalline phases are characterized by the same dimensions of the bilayer. The calculated lipid layer thicknesses are in excellent agreement with those previously determined for the liquid crystalline phases of the binary GMO/water system.12,16,38 The results also clearly demonstrate that the data points fall into universal lines for all investigated mesophases independently of whether GME is present or not. Aside from the tendency to promote phase transitions to more reversed phases, the incorporation of GME has practically no effect on the structural characteristics of a particular crystalline phase based on aqueous GMO. This is not surprising since GME and GMO are homologous, having a similar acyl chain. XRD data were also used to evaluate the effect of lipid composition and temperature on the water channel dimensions of the H2 phase formed in the GMO/GME/water system. On the basis of simple geometric considerations, the obtained lattice parameter, aH2, allows calculations of the radius of the water cylinders (RW):39

(

R W ) a H2

)

x3(1 - φlip) 2π

1/2

(5)

As seen from Figure 5, the calculated RW increases from about 6 to 16 Å with increasing water content in the H2 phase of the ternary GMO/GME/water system. The obtained dimensions are practically the same as those for the binary GME/water system,29 showing that the GME content has no effect on the structural characteristics of the H2 phase since the data points from different GMO/GME ratios fall on the same line. The data presented in Figure 5 also give an indication that H2 phases are characterized by slightly smaller RW values at elevated temperatures. 3. 2H NMR of the H2 Phases. The hydration properties of birefringent GMO/GME phases were further investigated by 2H NMR quadrupole splitting. Since the lamellar phase occupies only a narrow region of the diagram, the experiments were only performed on H2 phases at different hydrations. The magnitude of a deuteron quadrupole splitting ∆(2H) contains information on the hydration of the amphiphilic aggregates in terms of the fraction of water molecules appreciably oriented by the aggregate surfaces. The recorded ∆(2H) for H2 phases were analyzed following the procedure described previously.40 The quadrupole splitting can be expressed as41 (38) Chung, H.; Caffrey, M. Biophys. J. 1994, 66, 377-381. (39) Rand, R. P.; Fuller, N. L. Biophys. J. 1994, 66, 2127-2138. (40) Khan, A.; Fontell, K.; Lindman, B. J. Colloid Interface Sci. 1984, 101, 193-200. (41) Khan, A.; Fontell, K.; Lindblom, G.; Lindman, B. J. Phys. Chem. 1982, 86, 4266-4274.

Figure 4. Lattice parameter (a) of the lamellar (LR) and bicontinuous cubic phases (Q230 and Q224) as a function of lipid volume fraction (φlip) at 25 °C. The GMO/GME (w/w) ratios are 100/0 (filled circles), 90/10 (open circles), 80/20 (filled reversed triangles), 70/30 (open reversed triangles), and 60/40 (filled squares). The lines represent the best fits according to eqs 2 and 3.

Figure 5. Water channel radius (RW) of the H2 phase as a function of lipid volume fraction (φlip) at 25 and 55 °C. The GMO/GME (w/w) ratios are 80/20 (filled circles), 70/30 (open circles), 60/40 (filled reversed triangles), 50/50 (open reversed triangles), 40/60 (filled squares), 20/80 (open squares), and 10/90 (filled diamonds). The lines are drawn to guide the eye.

∆(2H) )

( )

Xlip nVQS Xw

(6)

where S is the order parameter of bound water molecules, VQ is the quadrupole coupling constant (220 kHz), n is the average hydration number of GMO and GME, and Xlip and Xw are the molar fractions of lipid (GMO+GME) and water, respectively. The hydration properties of the lipid molecules in the H2 phase are analyzed by plotting ∆(2H) versus the molar ratio of total amphiphile to water (Figure 6). The results show that the values of 2H quadrupole splittings decrease linearly with increasing water content, as predicted by eq 6. This shows that, at least in the investigated water content region, the splitting follows the simple “two-site” model with constant hydration proposed for the water binding to amphiphile aggregates.42 Thus, an increase in the water concentration does not change the hydration of the amphiphiles, but only the amount of free water. Such an ideal swelling behavior has also been observed for H2 phases in aqueous mixtures of GMO/glycerol dioleate43 and GMO/sodium oleate.44 The obtained data also indicate that the GMO replacement by GME has no effect on the overall hydration properties of a particular phase since the data for different GME/GMO ratios follow the same line. (42) Persson, M. O.; Lindman, B. J. Phys. Chem. 1975, 79, 1410-1418. (43) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 10044-10054. (44) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 7742-7751.

Aqueous Phase BehaVior of GMO and GME

Figure 6. The quadrupole splitting values of the H2 phases for the ternary GMO/GME/water system as a function of the molar ratio of total amphiphile (GMO+GME) (Xlip) to water (Xw) at 25 °C. The GMO/GME (w/w) ratios are 20/80 (filled circles), 40/60 (filled reversed triangles), 50/50 (open reversed triangles), and 60/40 (filled squares).

Figure 7. Particle size distributions of prepared cubic phase nanoparticle dispersions in a quaternary GMO/GME/F127/water system. The GMO/GME (w/w) ratios are 95/5, 90/10, 85/15, and 80/20. All dispersions have lipid/polymer ratios of 9/1 and a water content of 95 wt %.

4. Dispersions. Recent studies have shown that GMO cubic phase nanoparticles can be prepared by the mechanical agitation of the cubic phase in excess water in the presence of small amounts of triblock copolymeric stabilizer, F127.45,46 The efficient steric stabilization provided by the copolymer has been attributed to its preferential localizing (adsorption) at the surface of the formed liquid crystalline nanoparticles. Cubic phase nanodispersions for the mixed GMO/GME/F127 system in water were prepared according to a recently developed method that allows the formation of liquid crystalline nanoparticles with high structure and size uniformity.47,48 The bulk phase diagram of aqueous GMO/GME (Figure 2) shows that the bicontinuous Q224 phase is formed up to GMO/GME ratios of 70/30 (w/w). We chose to investigated GMO/GME mixtures with up to an 80/20 (w/w) ratio to ensure that the composition corresponds to a cubic phase region. On the basis of previous results, the lipid-to-polymer ((GMO+GME)/F127) ratio was fixed to 9/1 (w/w) to prevent the formation of vesicular aggregates.49 Figure 7 shows the particle size distributions of the prepared dispersions as a function of GME content. As seen from Figure (45) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (46) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (47) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Langmuir 2005, 21, 2569-2577. (48) Barauskas, J.; Johnsson, M.; Tiberg, F. Nano Lett. 2005, 5, 1615-1619. (49) Landh, T. J. Phys. Chem. 1994, 98, 8453-8467.

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7, all dispersions are characterized by monomodal size distributions with a polydispersity index (the ratio between the standard deviation of the distribution and the mean value) of about 0.2. There is also no detectable effect of the lipid composition. All nanoparticles are characterized by distributions with a mean size of about 370 nm. Note that the GMO/F127 cubic phase nanoparticles previously prepared according to the same procedure and containing the same amphiphile concentration have similar size distributions, with a mean size of about 400 nm.47,48 Figure 8 shows high-resolution synchrotron powder X-ray diffractograms of the cubic nanoparticle dispersions as a function of GME content. At low GME content, the dispersions clearly show at least eight Bragg diffraction peaks, which are spaced in ratios of x2:x4:x6:x10:x12:x14:x16:x18 (Figure 8a,b). The peak positions can be indexed as hkl ) (110), (200), (211), (310), (222), (321), (400), and (411) reflections of a body-centered cubic lattice of the Im3hm space group (Q229). The characteristic peak intensity distribution and the absence of the (220) peak (x8) are consistent with the structure factor calculations for this cubic phase50 and observations in other lipid systems.51 From the slopes of plots of d-1 versus (h2+k2+l2)1/2, where d is the measured interlayer distance and h, k, and l are the Miller indices, lattice parameters of 137 and 134 Å are calculated for dispersions with GMO/GME ratios of 95/5 and 90/10, respectively. At higher GME content, the nanoparticle structure transforms into a different cubic phase structure, the reflections of which can be indexed as hkl ) (110), (111), (200), (211), (220), (221), and (310) (Figure 8d). These Bragg peaks follow the relationship x2:x3:x4:x6: x8:x9:x10, which is in agreement with a primitive cubic lattice of Pn3hm space group (Q224). The calculated lattice parameter for the nanoparticles of this composition is 93 Å. A very similar effect of the admixing of a more hydrophobic substance into GMO/F127 cubic phase nanoparticles was recently observed with oleic acid.47 In that case, oleic acid also induced an intercubic phase transition from Q229 to Q224. Figure 8c also shows the X-ray diffractogram of the nanoparticle sample where both of the cubic phase structures are in equilibrium. The lattice parameters for coexisting Q229 and Q224 phases are 128 and 95.5 Å, respectively. The ratio between these parameters, 1.34, is close to the theoretically predicted value of 1.28 for coexisting bicontinuous cubic phases based on P and D IPMS.52 We therefore conclude that, within the range of GMO/GME compositions investigated (up to a GMO/GME ratio of 80/20 (w/w)), two kinds bicontinuous cubic phase nanoparticles based on P and D IPMS are formed at lower and higher GME content, respectively. On the basis of nondispersed sample phase behavior, the higher GME concentration will most likely induce the formation of reversed hexagonal nanoparticles. GMO/GME/F127 cubic phase dispersions at different GMO/ GME compositions were further investigated by 1H NMR. Typical 1H NMR spectra recorded for F127 solutions and GMO/GME/ F127 cubic phase nanoparticles are presented in Figure 9. Selfdiffusion NMR experiments were performed on the four peaks marked in Figure 9. These correspond to the proton signals for the PEO and PPO blocks (3.62 and 1.06 ppm, respectively) of F127 and for the CH2 (1.2 ppm) and CH3 (0.8 ppm) groups of the GMO and GME fatty acid residue. Although the proton peak for the PPO block due to low intensity is overlapped by lipid signals in the dispersion spectrum, it was still possible to follow (50) Anderson, D. M.; Davis, H. T.; Scriven, L. E.; Nitsche, J. C. C. AdV. Chem. Phys. 1990, 77, 337-396. (51) Templer, R. H.; Seddon, J. M.; Warrender, N. A.; Syrykh, A.; Huang, Z.; Winter, R.; Erbes, J. J. Phys. Chem. B 1998, 102, 7251-7261. (52) Templer, R. H.; Seddon, J. M.; Warrender, N. A. Biophys. Chem. 1994, 49, 1-12.

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Figure 8. High-resolution synchrotron X-ray powder diffractograms of prepared cubic phase nanoparticle dispersions in a quaternary GMO/GME/F127/water system. The GMO/GME (w/w) ratios are 95/5 (a), 90/10 (b), 85/15 (c), and 80/20 (d). All dispersions have lipid/ polymer ratios of 9/1 and a water content of 95 wt %. The Miller indices in panel c stand for Q224 structure, whereas the arrows denote the peak positions of the Q229 phase. Table 1. Results Obtained from Fitting the Self-diffusion NMR Data to Eq 1 for a 0.5 wt % F127 Solution and 5 wt % GMO/ GME/F127 (lipid/F127, 9/1 w/w) Cubic Phase Dispersions for Different GMO/GME Weight Ratios at 25 °C DF127 × 1012/m2 s-1 sample F127 solution

Figure 9. 1H NMR spectra of 0.5 wt % F127 solution (1) and cubic phase nanoparticle dispersions in a quaternary GMO/GME/F127/ water system (the GMO/GME (w/w) ratio is 90/10, and the water content is 95 wt %) (2). Note that the total F127 concentration in both cases is equal. The four peaks that are followed with selfdiffusion NMR are marked (PEO, PPO, -CH2-, -CH3) in addition to that of water (denoted H2O).

the F127 diffusion in the cubic phase nanoparticle preparation by the strong PEO signal located at 3.62 ppm. The results obtained from single-exponential fits (eq 1) of the intensity decays for a F127 solution, and the different nanoparticle dispersions are presented in Table 1. An immediate observation from the results shown in Table 1 is that the self-diffusion for F127 molecules in solution is much faster than that in the GMO/GME/F127 cubic phase nanoparticle dispersion. As pointed out in earlier studies,53 a critical micelle concentration for F127 is strongly temperature dependent and extends over a large interval in the concentration range compared to short-chain surfactants. This means that the coexistence region of monomers and micelles is broad. The reason for this is that triblock copolymers like F127 are likely to contain impurities (53) Alexandridis, A.; Hatton, T. A. Colloids Surf., A 1995, 96, 1-46.

GMO/GME (100/0) GMO/GME (95/5) GMO/GME (92.5/7.5) GMO/GME (90/10) GMO/GME (85/15) GMO/GME (80/20)

PEO

PPO

52

51

GMO/GME/F127 disp. 1.2 1.2 1.4 1.0 1.2 1.1

Dlip × 1012/m2 s-1 -CH2-

-CH3

1.1 1.2 1.2 1.0 0.9 1.2

1.1 1.2 1.2 1.0 1.0 1.2

such as PEO, PPO, and other copolymers.54 In addition, the blocks in the polymers are polydisperse. It has been also shown that, for 0.5 wt % F127 solution, the critical micelle temperature is about 27.5 °C.55 Therefore, it is most likely that the determined DF127 in Table 1 represents some kind of an average diffusion of a mixture of various size monomers and micelles (note, that we performed only single exponential analysis of the intensity decay). The obtained value also agrees well with the previously determined diffusion coefficient for the 0.5 wt % F127 micellar solution at 40 °C (30 × 10-12 m2 s-1).56 The lower value of DF127 (by a factor of about 45) for the cubic nanoparticles compared to that of free F127 solution (Table 1) shows that, in this case, the polymer is no more in the “free” monomer or micellar state. The determined DF127 and Dlip values in nanoparticle dispersions are almost the same, which indicates the correlation and gives strong evidence that the polymer and (54) Wang, G.; Olofsson, G. J. Phys. Chem. 1995, 99, 5588-5596. (55) Li, Y.; Xu, R.; Bloor, D. M.; Holzwarth, J. F.; Wyn-Jones, E. Langmuir 2000, 16, 10515-10520. (56) Jansson, J.; Schille´n, K.; Olofsson, G.; da Silva, R. C.; Loh, W. J. Phys. Chem. B 2004, 108, 82-92.

Aqueous Phase BehaVior of GMO and GME

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lipids diffuse together. The results in Table 1 also show that diffusion coefficients do not depend on the GMO/GME ratio used to prepare the dispersions. As determined in previous studies, the GMO diffusion coefficient in the fully swollen bulk cubic phase varies between 10 and 30 × 10-12 m2 s-1.10,57,58 The broad variation in the determined coefficient values is most likely due to the purity of the GMO preparation. Nevertheless, as seen from Table 1, our values for the dispersed cubic phase are at least 10 times lower. Considering the fact that our dispersed cubic phase consists of particles with size of about 400 nm (Figure 7), this is not surprising. According to the experimental setup (∆ ) 250 ms and more), the possible molecular displacement during the experiment, 〈r2〉 ) 2∆D, is on the order of one micron and more. The particles are simply too small to measure molecular diffusion, and therefore the measured diffusion coefficient represents the aggregate diffusion instead.59 From the measured diffusion coefficient, we can calculate the corresponding hydrodynamic radius of the particle (RH) by the Stokes-Einstein relation:

RH )

kBT 6πη0D

(7)

where kB is Boltzmann’s constant, T is the absolute temperature, and η0 is the viscosity of the solvent. By taking an averaged diffusion coefficient from Table 1 for the cubic phase nanoparticle (1.11 × 10-12 m2 s-1), the calculated RH is about 220 nm. This gives a particle size of about 440 nm, which is in excellent agreement with the particle size measurements presented in Figure 7. (57) Eriksson, P. O.; Lindblom, G. Biophys. J. 1993, 64, 129-136. (58) Caboi, F.; Borne´, J.; Nylander, T.; Khan, A.; Svendsen, A.; Patkar, S. Colloids Surf., B 2002, 26, 159-171. (59) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forse´n, S. J. Am. Chem. Soc. 1979, 101, 5465-5470.

In conclusion, NMR self-diffusion experiments clearly show that F127 is associated with the dispersed cubic phase nanoparticles, thus providing final evidence for the adsorption/ incorporation and steric stabilization effect of the polymer.

Conclusions In this work we demonstrate that, by admixing GMO with its ether analogue GME, it is possible to combine improved chemical stability characteristics with retained structural and morphological property features to those of the corresponding pure GMO-based liquid crystalline phases. At higher GME contents, phase transitions are observed to phases with increased negative lipid monolayer curvature with increasing fractions of nonlamellar phase-forming GME. The phase behavior results show the total miscibility of two lipids and provide the possibility to also form different liquid crystalline phases by only changing the lipid ratio, in addition to enhancing the physical/chemical stability by increasing the GME content in the system. We also show the possibility of preparing two different stable cubic phase dispersions using the mixed GMO/GME/F127 system, and, finally, the results confirm that F127 is associated with the nanoparticles providing steric stabilization of the dispersions. Acknowledgment. We are grateful to Yngve Cerenius for assistance with the synchrotron XRD measurements at the beamline I711 at MAX-lab (Lund University, Sweden). This work was supported by the EU Marie Curie program “Surface and colloid technology - self-assembled structures of biological and technological relevance” (contract no. HPMT-CT-200000150) and EU-STREP FP6 project BIOSCOPE (contract no. NMP4-CT-2003-505211). LA062344U