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Dispersions of Liquid Crystalline Phases of the Monoolein/ Oleic Acid/Pluronic F127 System Minoru Nakano,† Takashi Teshigawara,† Atsuhiko Sugita,† Warunee Leesajakul,† Atsuhiko Taniguchi,† Tomoari Kamo,† Hideki Matsuoka,‡ and Tetsurou Handa*,† Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, and Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Received July 24, 2002. In Final Form: September 18, 2002 Aqueous dispersions of lyotropic liquid crystalline phases (cubosomes and hexosomes) were prepared using lipid mixtures, monoolein (MO) and oleic acid (OA), and emulsifier Pluronic F127 by changing their composition. The size and internal structure of the prepared particles were characterized by dynamic light scattering and small-angle X-ray scattering, respectively. At the weight ratio of MO:OA ) 5:5 and 8 wt % F127 to the total lipid mixtures, particles with a diameter of ca. 140 nm and including an inverted hexagonal (HII) phase were formed. With an increase in the F127 concentration, the particle size decreased, but the internal structure (lattice constant) did not change, suggesting that F127 absorbs at the particle surface with little incorporation in the HII phase. The lipid ratios and solvent pH strongly affected the morphology of the internal structure of the particles. By increasing the weight fraction of OA in the lipid mixtures, the internal structure transformed in the order of bicontinuous cubic-inverted hexagonal-inverted cubic. In addition, transformation from the cubosome to the hexosome was observed by decreasing the pH, suggesting that the interior of the nanoparticles is responsive to the outer environment.
Introduction Surfactants and lipids form a variety of assemblies such as micelle or lyotropic liquid crystalline phases by hydration. These assembly types are determined by packing parameter or spontaneous curvature, both of which denote the balance of the effective size of hydrophilic and hydrophobic groups. Monoolein (MO, Figure 1a) is an intriguing lipid for many researchers since it forms bicontinuous cubic phases by just adding water. Two different types, diamond type (CD) and gyroid type (CG), of the cubic phases are formed depending on water content. In the presence of excess water the CD phase separates from the water phase.1,2 The addition of amphiphiles with higher hydrophobicity transforms to an inverted hexagonal (HII) phase,3,4 and amphiphiles having positive spontaneous curvature induce phase transition from CD to primitive type cubic phase (CP) and to lamellar phase.5,6 Oleic acid (OA, Figure 1b) is a fatty acid with the same acyl chain as MO. It has been found that the effect of OA on the phase behavior of hydrated MO/OA mixtures is sensitive to pH and salt concentration.7 That is, electrostatic repulsion between OA headgroups plays an important role in determining the phases. A recent study on * Corresponding author: e-mail
[email protected]. † Graduate School of Pharmaceutical Sciences. ‡ Department of Polymer Chemistry, Graduate School of Engineering. (1) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (2) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-34. (3) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2001, 109, 47-62. (4) Borne, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 1004410054. (5) Li, S. J.; Yamashita, Y.; Yamazaki, M. Biophys. J. 2001, 81, 98393. (6) Pitzalis, P.; Monduzzi, M.; Krog, N.; Larsson, K.; Ljusberg-Wahren, H.; Nylander, T. Langmuir 2000, 16, 6358-6365. (7) Aota-Nakano, Y.; Li, S. J.; Yamazaki, M. Biochim. Biophys. Acta 1999, 1461, 96-102.
Figure 1. Chemical structures of monoolein (a) and oleic acid (b).
the phase diagrams of MO/OA/water and MO/sodium oleate/water showed that sodium oleate has a positive spontaneous curvature and induces a phase transition from the cubic to lamellar phase, whereas the OA possesses a negative curvature leading to a cubic-HII phase transition.8 In addition, at a higher OA fraction the MO/OA mixtures form an inverted micellar cubic phase (CMIC) of space group Fd3m.9-11 Pluronic F127 is an amphiphilic triblock copolymer with a structure formula of PEO99-PPO67-PEO99, where PEO and PPO denote poly(ethylene oxide) and poly(propylene oxide), respectively. The gelation and micellization behaviors of Pluronics have been thoroughly investigated12-14 and applied as vehicles for percutaneous and parenteral administration of drugs.15-17 Gustafsson et al. demon(8) Borne, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 77427751. (9) Luzzati, V.; Vargas, R.; Gulik, A.; Mariani, P.; Seddon, J. M.; Rivas, E. Biochemistry 1992, 31, 279-85. (10) Luzzati, V.; Vargas, R.; Mariani, P.; Gulik, A.; Delacroix, H. J. Mol. Biol. 1993, 229, 540-51. (11) Cribier, S.; Gulik, A.; Fellmann, P.; Vargas, R.; Devaux, P. F.; Luzzati, V. J. Mol. Biol. 1993, 229, 517-25. (12) Malmsten, M.; Lindman, B. Macromolecules 1992, 25, 54405445. (13) Wanka, G.; Hoffmann, H.; Ulbricht, W. Colloid Polym. Sci. 1990, 268, 101-117. (14) Mortensen, K. J. Phys.: Condens. Matter 1996, 8, A103-A124. (15) Kabanov, A. V.; Batrakova, E. V.; Alakhov, V. Y. J. Controlled Release 2002, in press. (16) Miyazaki, S.; Tobiyama, T.; Takada, M.; Attwood, D. J. Pharm. Pharmacol. 1995, 47, 455-7.
10.1021/la026297r CCC: $22.00 © 2002 American Chemical Society Published on Web 10/25/2002
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strated that F127 works as a stabilizer of submicron particles with the bicontinuous cubic phase (cubosomes).18,19 Dispersions of the inverted hexagonal phase (hexosomes) have been also prepared by adding small amounts of triolein or retinyl palmitate.19-21 Previously, we investigated the internal structure of cubosomes formed in the MO/F127/buffer system by smallangle X-ray scattering and 13C NMR.22 In the present study, it was demonstrated that several liquid crystalline phases of MO/OA/F127 mixtures can be dispersed into the submicron scale. The dependence of the internal structure of particles on the composition and solvent pH is discussed. Experimental Section Materials. Monoolein (1-monooleoyl glycerol, MO, purity >99%) was supplied from NOF Corp. (Tokyo). Oleic acid (OA, purity >99%) was purchased from ICN Biomedicals Inc. (Aurora, OH). Pluronic F127 (PEO99-PPO67-PEO99) was provided by BASF Japan Ltd. (Osaka). These materials were used without further purification. Other materials described later without notation were purchased from Wako Pure Chemical Industries Ltd. (Osaka). Sample Preparation. Aqueous dispersions containing various compositions of MO, OA, and F127 were prepared. In the typical case with the weight ratio of MO:OA ) 5:5 and 8 wt % F127 to the total lipid mixtures, MO (150 mg), OA (150 mg), and F127 (24 mg) were weighed and mixed in chloroform. After the solvent was evaporated, the mixture was dried in a vacuum. Then 30 mL of phosphate-buffered saline (PBS, 50 mM phosphate, 100 mM NaCl, pH 7.0) was added, and the mixture was heated to 80 °C and roughly dispersed using a homogenizer (Microtec Co. Ltd., Chiba, Japan). The decrease in the solution’s pH induced by OA was compensated by adding small amounts of 1 M NaOH aqueous solution. Further size reduction was performed using a high-pressure emulsifier (nanomizer system YSNM-1500-5, Yoshidakikai Co. Ltd., Nagoya) under a pressure of 35 MPa for 30 min at 60 °C. Nondispersed samples were also prepared. To the mixtures of lipids (MO and OA, 100 mg) and F127 (0 or 8 mg), PBS (10 mL) was added, and the solution pH was adjusted to 7.0 using NaOH aqueous solution. After heating to 80 °C and vortexing the mixtures for 30 s, they were kept at room temperature for more than 1 day. Floating coagulates were gathered and used for X-ray scattering experiments. Dynamic Light Scattering. The particle size of the dispersions was determined from dynamic light scattering (DLS) measurements (Photal LPA-3000/3100; Otsuka Electronic Co., Osaka). The dispersions were diluted (more than 40 times) by buffer and put into a quartz cell. At this concentration, it can be assumed that the interparticle interaction is negligible. Measurements were performed at 25 °C. The mean hydrodynamic diameter was evaluated by the cumulant method. In the diameter calculation, the viscosity and the refractive index of the solvent (buffer) were assumed the same as pure water (0.890 cP and 1.331, respectively). We confirmed with these parameters that the measurements of silica colloidal particles in buffer and in pure water gave the same diameter. Small-Angle X-ray Scattering. The dispersions were concentrated (to more than 20 wt % lipids) by ultrafiltration (Millipore) to obtain adequate intensity on small-angle X-ray scattering (SAXS) experiments. These samples and the nondispersed samples were put into a glass capillary (W. Mu¨ller, Berlin, (17) Guzman, M.; Garcia, F. F.; Molpeceres, J.; Aberturas, M. R. Int. J. Pharm. 1992, 80, 119-127. (18) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (19) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (20) Monduzzi, M.; Ljusberg-Wahren, H.; Larsson, K. Langmuir 2000, 16, 7355-7358. (21) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. B 1999, 103, 3896-3899. (22) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917-3922.
Figure 2. X-ray diffraction patterns of the nondispersed liquid crystalline phases of MO/OA mixtures in PBS (pH 7) in the absence and presence of 8% F127. MO:OA ) 10:0 (a), 5:5 (b), and 0:10 (c). Germany; 1.5 mm o.d., 1/100 mm wall thickness). SAXS measurements were performed using a Kratky type camera (Rigaku Co., Tokyo) with Ni-filtered Cu KR radiation (wavelength λ ) 1.54 Å) generated by a Rigaku RU-200 rotating anode X-ray generator (50 kV, 200 mA). The slit-smeared diffraction patterns were detected by a position-sensitive proportional counter and desmeared by a standard procedure. Scattering intensities were plotted against reciprocal spacing s ) 2(sin θ/2)/λ, where θ is the scattering angle. Three hours or 30 min of exposure time was taken for the dispersed or nondispersed samples, respectively.
Results Small-Angle X-ray Scattering of Nondispersed MO/OA/Buffer and MO/OA/F127/Buffer Systems. Diffraction patterns were obtained for nondispersed liquid crystalline phases in MO/OA/buffer and MO/OA/F127/ buffer systems. The concentrations of total lipids (10 g/L to buffer) and pH ()7.0) were fixed, and the effect of F127 (0 or 8% w/w to total lipids) on the diffraction pattern at three lipid weight ratios (MO:OA ) 10:0, 5:5, and 0:10) was investigated (Figure 2). At MO:OA ) 10:0 and in the absence of F127, diffraction peaks were observed with the reciprocal spacing ratio of x2:x3:x4:x6:x8:x9 corresponding to the Pn3m space group, which suggested the formation of CD type bicontinuous cubic phase. In the presence of 8% F127 diffraction peaks of the Im3m space group with the reciprocal spacing ratio of x2:x4:x6:x10: x12:x14 (x8 could not be seen) was observed, which indicated the formation of the CP type bicontinuous cubic phase. At MO:OA ) 5:5 and in the absence of F127, three diffraction peaks with the reciprocal spacing ratio of x1:x3:x4 were observed, representing the formation of the HII phase. In addition, the diffraction pattern was identical to that in the presence of 8% F127. At MO:OA ) 0:10 the diffraction patterns were also independent of F127. The hydrated OA with both 0% and 8% F127 gave diffraction peaks with the reciprocal spacing
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Langmuir, Vol. 18, No. 24, 2002 9285 Table 1. Characteristics of the Phases of the Nondispersed Samples and the Internal Structures of the Dispersed Samples Determined from X-ray Diffraction Data nondispersed MO/OA
phase
10/0 9/1 8/2 7/3 6/4
CP CP CP CP CP/HII
5/5 4/6 3/7 2/8 1/9 0/10
HII HII HII CMIC CMIC CMIC
lattice constanta (Å) 129.6 ( 1.9 156.1 ( 1.7 171.0 ( 1.9 162.2 ( 2.9 157.9 ( 2.6/ 66.8 ( 0.4 64.2 ( 0.3 62.0 ( 0.4 58.0 ( 0.2 162.5 ( 0.5 153.2 ( 0.7 142.6 ( 0.9
dispersed phase CD/CP CP CP CP CP/HII HII HII HII HII CMIC CMIC
lattice constanta (Å) 97.5 ( 0.4c/130.1b 147.6 ( 3.6 158.9 ( 3.7 151.3 ( 2.6 150.1 ( 1.6c/ 67.6 ( 0.6 66.7 ( 0.5 63.8 ( 0.5 61.3 ( 0.3 56.5 ( 0.2 152.4 ( 0.8 142.8 ( 1.4
a Mean ( S.D. was determined from positions of more than three peaks in diffraction data, except for b and c where only one or two peaks were available.
Figure 3. X-ray diffraction patterns of the nondispersed liquid crystalline phases of MO/OA/F127 mixtures in PBS (pH 7) with 8% F127.
ratio of x3:x8:x11:x12:x16:x19, which was in accordance with the Fd3m space group, suggesting the formation of an inverted micellar cubic (CMIC) phase. The diffraction patterns of the MO/OA/F127/buffer nondispersed system with a different lipid weight ratio is shown in Figure 3. The concentration of F127 (8% w/w to total lipids) and pH ()7.0) were fixed, and the effect of the lipid weight ratio on the diffraction pattern was investigated. The SAXS data suggested that the mixtures transformed their liquid crystalline phase from CP to HII and then to CMIC with an increase in the OA weight fraction. The lipid ratio of MO:OA ) 6:4 was the phase boundary between CP and HII where diffraction from both phases was observed. The HII-CMIC phase boundary was between 3:7 and 2:8. The lattice constant of the CP phase slightly increased with an increase in the OA fraction and showed a maximum at MO:OA ) 8:2. In contrast, the lattice constant of both the HII and CMIC phases decreased with an increase in the OA fraction (Table 1). Particle Size and Stability of Dispersions. Highpressure emulsification of the mixtures of MO, OA, and F127 produced milklike dispersion. The mean diameter of the particles was obtained by DLS within 2 h after the preparation and plotted as a function of the weight fraction of OA in the total lipids, as shown in Figure 4. With an increase in the OA fraction the particle size first decreased and then increased with a minimum around 40% of OA. The particle diameter was measured again 3 weeks after the preparation. The particle size did not change for the dispersions with the OA fraction up to 70%. When the OA fraction was 80% or greater, however, an increase in the size (20-100 nm) was observed, indicating less stability against aggregation. Small-Angle X-ray Scattering of Dispersions. SAXS experiments were performed for the dispersions, which were concentrated by ultrafiltration. Figure 5 shows SAXS patterns of the particles with different MO:OA weight ratios. Diffraction peaks were ambiguous compared with
Figure 4. Mean hydrodynamic diameter of the particles as a function of the weight fraction of OA in the total lipids. The DLS measurements were performed within 2 h after the preparation. The solid curve was drawn for ocular clarity to show a parabolic profile.
those from the nondispersed samples due to the comminution of the phases into the submicron scale. However, adequate intensity to discern the phases could be obtained. At MO:OA ) 10:0 the intensity of the first diffraction peak at s ∼ 0.011 Å-1 was relatively weaker than that of the second one at ∼0.015 Å-1, suggesting the coexistence of CD and CP phases, which we previously reported.22 As expected, sequential variation of the internal structure of the particles from the bicontinuous cubic to hexagonal into inverted micellar cubic was observed when the OA fraction was increased. However, the lattice constants obtained did not coincide with those of the nondispersed system. They were about 10 Å smaller in CP phases and a few angstroms larger in HII phases (Table 1). In addition, the phase transition from HII to CMIC took place when the MO:OA ratio was between 2:8 and 1:9 for the dispersion, while it was between 3:7 and 2:8 for the nondispersed system. F127 Concentration Dependence of the HII Phase. At the weight ratio MO:OA ) 5:5, both the nondispersed and dispersed samples showed the diffraction patterns of the HII structure. We prepared nanoparticles with this lipid ratio and different F127 concentrations and evaluated the effect of F127 on the structure of the particles. The mean diameters of the particles were determined by DLS to be 207 and 136 nm for 4% and 8% F127, respectively. As shown in Figure 6, the particles with different F127 concentrations produced similar diffraction patterns. The
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Figure 7. X-ray diffraction patterns of the dispersions of MO/OA/F127 mixtures in PBS with 8% F127. The dispersions were prepared using PBS at pH 7 (a) or pH 6 (b), or the pH was changed from 7 to 6 after the preparation (c). MO:OA ) 8:2.
Figure 5. X-ray diffraction patterns of the dispersions of MO/OA/F127 mixtures in PBS (pH 7) with 8% F127.
Figure 6. X-ray diffraction patterns of the dispersions of MO/OA/F127 mixtures in PBS (pH 7) with 8% (a) and 4% (b) F127. MO:OA ) 5:5.
diffraction peaks were less clear at 8% than those for 4% F127 probably due to the fractionization of the HII phases as mentioned above. The lattice constants were 66.0 and 66.8 Å for 4% and 8% F127, respectively, suggesting the independence from the F127 concentration. Effect of pH on Internal Structure. Since the OAcontaining phases were considered to be pH-sensitive, we prepared dispersions with MO:OA ) 8:2 using PBS at a different pH. Indeed, the diffraction pattern of Im3m observed for the dispersed sample at pH 7 changed to that of HII at pH 6 as shown in Figure 7. The mean particle diameters of the dispersions were 140 nm at pH 7 and 204 nm at pH 6. In addition, when the pH was changed from 7 to 6 after preparation, the mean diameter was decreased to 125 nm, and the diffraction pattern of HII was observed. Discussion Liquid Crystalline Phases Formed in the Nondispersed System. The nondispersed mixtures investigated in this study were shown to form several nonlamellar liquid crystalline phases. The structures of these phases are represented in Figure 8. The bicontinuous cubic phases consist of two intercrossing water channels partitioned by lipid bilayers with zero mean curvature at the bilayer center (minimal surface).23,24 The HII phase also
Figure 8. Schematic representation of the nonlamellar liquid crystalline phases: primitive type cubic (CP, (a)), diamond type cubic (CD, (b)), inverted hexagonal (HII, (c)), and inverted micellar cubic (CMIC, (d)) phases. The membrane curvature becomes more negative in this order.
has water channels, which form hexagonally arranged rods surrounded by a lipid monolayer.25 The CMIC phase comprises two kinds of reversed micelle of different size, packed in a three-dimensional array.26 The hydrated MO forms the bicontinuous cubic phases, CP and CD, in the presence and absence of F127, respectively. F127 is a highly hydrophilic amphiphile so that it should give a positive curvature to the MO membrane. The transition from CD to CP by F127 is reasonable since the membrane of the latter phase has less negative curvature than that of the former one.27 In HII and CMIC phases, however, F127 did not influence the structure. This suggests that F127 is not incorporated into the MO/OA membrane in the HII and CMIC phases and is distributed into the aqueous phase. The low compatibility of the amphiphilic copolymer with lipid phases is presumably due to the packing constraint.28 This property of the copolymer is practically important for the preparation (23) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (24) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221256. (25) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1-69. (26) Seddon, J. M.; Robins, J.; Gulik-Krzywicki, T.; Delacroix, H. Phys. Chem. Chem. Phys. 2000, 2, 4485-4493. (27) Stroem, P.; Anderson, D. M. Langmuir 1992, 8, 691-709.
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of the dispersion of the liquid crystalline phases. Since if the amphiphile used as a dispersion stabilizer has high affinity to the lipid phase, it may transform or destroy the phase. F127 has little affinity to the HII and CMIC phases and even to the CP phase, because further addition of F127 to the CP phase brought little change.22 Thus, the amphiphilic polymer, F127, is considered to work efficiently as a stabilizer. The successive formation of the CP, HII, and CMIC phases by the increasing OA fraction (Figure 3) is ascribed to a change in the membrane spontaneous curvature. The effect of OA on the curvature is, however, rather complicated because it is a weak acid. Since pKa of the carboxyl group of long-chain fatty acid is about 5,29 OA is negatively charged at neutral pH. Thereby, OA gives a positive curvature to the membrane at low OA fractions (OA < 20%), leading to an increase of the lattice constant in the CP phase (Table 1). However, as the OA fraction in MO/OA membrane increases, the polyanionic surface formed has increased attraction to cations, which increases a protonated (noncharged) portion in the total OA. Consequently, OA provides a negative curvature at higher fractions, which decreases the lattice constant in CP and induces the phase transition to HII and CMIC. Formation of Particles Including Liquid Crystalline Phases: Cubosomes and Hexosomes. By highpressure emulsification of MO/OA/F127 mixtures in PBS (pH 7) with 8% F127, particles with the diameter of 100-300 nm were obtained. The X-ray diffraction results suggest that the particles have liquid crystalline phases in their interior, namely, the formation of cubosomes and hexosomes. The particle diameter was dependent on the lipid composition and first decreased and then increased with the increasing weight fraction of OA (Figure 4). This suggests the effect of OA differs at low and high OA fractions. It is due to a change in the noncharged portion in total OA as mentioned above. At low OA fractions, negatively charged OA decreases the hydrophobicity of lipid mixtures, and the hydrophobicity increases with the increase in noncharged OA. Consequently, the particle size showed a parabolic profile against the weight fraction of OA, suggesting that the particle size is sensitive to the hydrophobicity of core lipids. X-ray diffraction (Figure 5) shows that several nanoparticles with different internal structures can be prepared by modifying the lipid composition (MO:OA). At low OA fractions (OA < 40%), the number and the intensity of the reflections obtained were not sufficient to assign the space groups unambiguously. However, with the help of the diffraction data of nondispersed samples (Figure 3), it could be interpreted that they indicated the existence of the cubic structures. The successive transformation of the internal structure (CP-HII-CMIC) was observed similar to the nondispersed samples. However, the phase structure of the dispersed and nondispersed samples with the same composition did not necessarily coincide with each other, as their diffraction profiles were compared (Figures 3 and 5). This is attributed to the change in the distribution of molecules in the dispersion process, especially, F127 and OA. At MO:OA ) 10:0 the nondispersed sample formed the CP phase, but the dispersion showed the coexistence of CD and CP. This is ascribed to the inhomogeneous distribution of F127; that is, F127 preferably localizes to (28) Kunieda, H.; Uddin, M. H.; Furukawa, H.; Harashima, A. Macromolecules 2001, 34, 9093-9099. (29) Ptak, M.; Egret-Charlier, M.; Sanson, A.; Bouloussa, O. Biochim. Biophys. Acta 1980, 600, 387-397.
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Figure 9. Fits of the experimental diffraction patterns of MO/buffer (a), MO/F127/buffer (b) nondispersed samples, and MO/F127/buffer dispersion (c). In (a) and (b), the experimental patterns (open circles) were fitted by the theoretical curves (solid lines) of CD with L ) 25.3 Å and CP phases with L ) 33.0 Å, respectively. In (c), the experimental pattern could not be fitted by the curve of CP (dotted line, L ) 92.9 Å, shifted downward to make clearly visible) and was well reproduced by the coexisting model of both CD with L ) 25.0 Å and CP with L ) 33.2 Å (solid line).
the particle surface by the dispersion process, which creates F127-depleted regions (i.e., CD phases) in the particles.22 The coexistence of two cubic phases was further confirmed by model fitting of the diffraction patterns using the simplified model which assumes a flat contrast density profile according to Garstecki and Holyst.30,31 Using eq 10 of ref 31, the diffraction patterns of the nondispersed and dispersed samples of MO/F127 system were fitted with varying the width of the layer decorating the base minimal surface (L, corresponding to the lipid bilayer thickness32) as the fitting parameter. The results are shown in Figure 9. The experimental patterns from the nondispersed MO and MO/F127 were well fitted by the theoretical curves of the simplified model of CD and CP, respectively (Figure 9a,b). On the other hand, for the MO/F127 dispersion (Figure 9c), the diffraction pattern could not fitted by the curve of CP, and the obtained value of L (92.9 Å) was too large considering the lipid length. However, the pattern was well reproduced by coexisting model of both CD and CP, and the obtained values of L (25.0 and 33.2 Å, respectively) were less than twice the length of MO acyl chain (∼22 × 2 Å). In the presence of OA up to 40%, however, the coexistence of both cubic phases was not observed, and the CP phase was shown to solely exist. This was also suggested by the model fitting, although the fitting quality with the theoretical curves of CP was not so high (data not shown). This is presumably because small amounts of OA increased the affinity of the lipid mixtures to F127, which prevented the creation of the F127-depleted regions, and/ (30) Garstecki, P.; Holyst, R. Langmuir 2002, 18, 2519-2528. (31) Garstecki, P.; Holyst, R. Langmuir 2002, 18, 2529-2537. (32) In ref 31, the authors fitted the diffraction from MO/F127/water systems and concluded that their cubic phases were of a direct type by comparing the lipid volume fraction and the surface area per molecule. However, in the direct cubic phase, lipids have to occupy the region between two layers decorating minimal surfaces, and the maximum half distance between the layers given by 0.457 × (lattice constant) 0.5L becomes well over the lipid length. Thus, we regard these cubic phases as an inverse type and L as the lipid bilayer thickness.
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or because the F127-deminished regions (if created) also formed the CP phase. The MO/OA mixtures of these composition ranges (without F127) form the CP phase at low ionic strength and transform to CD with increasing NaCl concentration.7 We confirmed that the MO/OA mixture (8:2) in PBS (100 mM NaCl) formed the CD phase (data not shown). However, it is clear that the membrane curvature of the mixture is less negative than that of MO alone, and the smaller amount of F127 could be adequate to transform to CP. In the case of the hexosomes (MO:OA ) 5:5), the change in the F127 concentration had no influence on the internal structure (Figure 6). As mentioned above, this is because the F127 is not very compatible to the MO/OA membrane in the HII phase. However, the dispersion process slightly increased the lattice constant (64.3 and 66.8 Å for nondispersed and dispersed samples, respectively). This change would be derived not by F127 but by OA, which is discussed below. The dispersed samples have smaller lattice constant values for CP and larger for HII than the corresponding nondispersed samples (Table 1). It is suggested that OA has a little higher affinity to the particle surface than MO. It decreases the OA fraction in the interior of the particles. Since the effect of OA changes at low and high fractions, a decrease in the amount of OA, contributive to the formation of liquid crystalline phases, makes the membrane curvature more/less negative and results in the decrease/increase in the lattice constants of the cubosomes/hexosomes, respectively. The difference in the HII-CMIC phase boundary between nondispersed and dispersed samples can also be explained by the same reason. At higher OA fractions (1:9 or 0:10), where the decrease in the OA quantity brings little or no change to the internal lipid composition, the dispersions had an identical phase to the corresponding nondispersed samples (Table 1). It was demonstrated that the size and the internal structure were strongly dependent on the pH (Figure 7). The decrease in pH increases the protonated portion in total OA, which increases the negativity of the curvature and hydrophobicity of the membrane. For 90% MO/10%
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OA mixture in the buffer containing 0 M NaCl phase transition from CP to CD and from CD to HII occurs at pH 5.8 and 5.3, respectively.7 In the present study, the internal structure of the particles was already HII at pH 6. This was due to the difference in the OA fraction (20%) and salt concentration (100 mM NaCl). Interestingly, when the pH was changed from 7 to 6 after the preparation, transformation from cubosome to hexosome was observed. This suggests that the structure inside the particles is sensitive to the pH of the outer aqueous phase. In a previous study, we demonstrated by 13C NMR with a paramagnetic shift reagent that the ions added from the outer aqueous phase can penetrate to the inside of the cubosomes.22 The hexosomes prepared in Tris-HCl buffer also permitted easy access of the ions (data not shown). These results agree with the finding that the transformation of the particles takes place by changing the pH of the dispersions. The cubosome-to-hexosome transition diminished the particle size (from 140 to 125 nm), and the resultant was stable and considerably smaller than the hexosomes prepared using PBS with pH 6 (204 nm). It is suggested that the exclusion of water from the interior decreased the size on the transition. In addition, the difference in the size of the hexosomes with an identical internal structure suggests that such kinetically stabilized dispersions strongly depend on the method of preparation. The highly stable nanoparticles with the liquid crystalline core responsive to the environment could be useful to explore the phase transition and be applied to drug delivery systems. Acknowledgment. This study was supported by various foundations, grants-in aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 12470488 and 13771354) and funds from the Japan Health Sciences Foundation, the Mochida Memorial Foundation, and the Cosmetrogy Research Foundation. We gratefully acknowledge these agencies for making this work possible. LA026297R
