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Nonlamellar Liquid Crystalline Phases and Their Particle Formation in the Egg Yolk Phosphatidylcholine/Diolein System Tomoari Kamo,† Minoru Nakano,*,† Warunee Leesajakul,† Atsuhiko Sugita,† 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 19, 2003. In Final Form: August 25, 2003

The phase behavior of fully hydrated egg yolk phosphatidylcholine (EPC)/diolein (DO) mixtures was investigated by small-angle X-ray scattering and 31P NMR. EPC formed a lamellar phase, whereas DO gave a negative curvature to the lipid membranes, leading to nonlamellar phase formation. At an EPC/DO molar ratio of 8:2, the mixture formed a bicontinuous cubic phase of primitive type. A bicontinuous cubic phase of diamond type was also observed at the ratio of 7:3, but it coexisted with an inverted hexagonal (HII) phase. The mixture with higher DO fractions formed the HII phase. We further investigated if these nonlamellar phases could be dispersed by high-pressure emulsification with Pluronic F127, to form cubosomes or hexosomes, as have been obtained in monoolein-based cubic and HII phases (Langmuir 2001, 17, 39173922; Langmuir 2002, 18, 9283-9288). As a result, cubosomes could not be obtained and vesicles were formed instead, presumably due to the existence of F127, which gave a positive curvature to the negatively curved membranes. However, the HII phase, which was less compatible with F127, could be dispersed to form hexosomes.

Introduction Diacylglycerols (DGs) are important cellular membrane second messengers, produced from phospholipids such as phosphatidylcholine1 and phosphatidylinositol 4,5-bisphosphate2 by enzymatic action of phospholipase C. DGs activate protein kinase C,2,3 increase the susceptibility of phospholipids in bilayers against phospholipase attack,4,5 and promote fusion.6 The latter two functions are considered to closely correlate with the fact that DG in phospholipid bilayers perturbs the membranes and induces a packing stress,7 because of its high hydrophobicity and negative spontaneous curvature. DG/phosphatidylcholine lipid mixtures with relatively high DG contents form nonlamellar phases, bicontinuous cubic phases,8 inverted hexagonal (HII) phases,9,10 and inverted micellar cubic phases.8,11,12 * Corresponding author. E-mail: [email protected]. † Graduate School of Pharmaceutical Sciences, Kyoto University. ‡ Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University. (1) Pelech, S.; Vance, D. E. Trans. Biochem. Soc. 1989, 14, 28-30. (2) Nishizuka, Y. Nature 1984, 308, 694-698. (3) Bell, R. M. Cell 1986, 45, 631-632. (4) Dawson, R. M. C.; Hemington, N. L.; Irvine, R. F. Biochem. Biophys. Res. Commun. 1983, 117, 196-201. (5) Dawson, R. M. C.; Irvine, R. F.; Bray, J.; Quinn, P. J. Biochem. Biophys. Res. Commun. 1984, 125, 836-842. (6) Siegel, D.; Banschbach, J.; Alford, A.; Ellens, H.; Lis, L. J.; Quinn, P. J.; Yeagle, P. L.; Bents, J. Biochemistry 1989, 28, 3703-3709. (7) Bezrukov, S. M. Curr. Opin. Colloid Interface Sci. 2000, 5, 237243. (8) Takahashi, H.; Hatta, I.; Quinn, P. J. Biophys. J. 1996, 70, 14071411. (9) Leikin, S.; Kozlov, M. M.; Fuller, N. L.; Rand, R. P. Biophys. J. 1996, 71, 2623-2632. (10) Semmler, K.; Meyer, H. W.; Quinn, P. J. Chem. Phys. Lipids 1999, 99, 155-167. (11) Seddon, J. M. Biochemistry 1990, 29, 7997-8002. (12) Oradd, G.; Lindblom, G.; Fontell, K.; Ljusberg-Wahren, H. Biophys. J. 1995, 68, 1856-1863.

We have shown that the monoolein-based bicontinuous cubic, HII, and inverted micellar cubic phases can be dispersed into nanoparticles (cubosomes and hexosomes) by high-pressure emulsification with Pluronic F127.13,14 Although the cubosomes are stable for several months in buffer solution, they show poor stability and easily collapse in rat plasma, mainly by the process of monoolein uptake by serum albumin (unpublished data). This process must be avoided if these liquid-crystal-containing nanoparticles are intended for use as drug carriers by intravenous administration. Serum albumin has binding sites for monoolein15,16 and fatty acid17 but not for lipids with two acyl chains. Thus, phosphatidylcholine and DG can be candidate constituents of novel liquid crystalline particles. In the present study, we demonstrated that fully hydrated egg yolk phosphatidylcholine (EPC)/diolein (DO) mixtures form nonlamellar phases at 25 °C. We attempted to form nanoparticles including these nonlamellar phases (cubosomes and hexosomes) using F127 and discussed our findings in connection with the compatibility of F127 with lipid phases. Experimental Section Materials. EPC was supplied by Asahi Kasei Co. (Tokyo). The purity (>99%) was detected by thin-layer chromatography. The acyl chain composition was 14:0 (0.2%), 16:0 (34.9%), 16:1 (0.3%), 18:0 (11.9%), 18:1 (30.4%), 18:2 (15.0%), 20:2 (0.2%), 20:3 (13) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917-3922. (14) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (15) Thumser, A. E. A.; Buckland, A. G.; Wilton, D. C. J. Lipid Res. 1998, 39, 1033-1038. (16) Duff, S. M.; Kalambur, S.; Boyle-Roden, E. J. Nutr. 2001, 131, 774-778. (17) Brecher, P.; Saouaf, R.; Sugarman, J. M.; Eisenberg, D.; LaRosa, K. J. Biol. Chem. 1984, 259, 13395-13401.

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(0.3%), 20:4 (3.2%), 22:6 (3.3%), and others (0.3%), according to the supplier. DO (purity > 99%, mixed isomers with ca. 85% 1,3and 15% 1,2-isomer) was purchased from Sigma Chemical Co. (St. Louis, MO). 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 of the highest purity available. Sample Preparation. Aqueous dispersions containing various compositions of EPC, DO, and F127 were prepared. EPC and DO (total of 300 mg) and F127 (8 wt % of total lipids) were weighed and mixed in chloroform. After the solvent was evaporated, the mixture was dried in a vacuum. Then, 30 mL of Tris-buffered saline (TBS, 10 mM Tris-HCl, 150 mM NaCl, pH 7.0) was added, and the mixture was repeatedly frozen and thawed using liquid nitrogen and water, respectively, to ensure full hydration. After rough dispersion using a homogenizer (Microtec Co. Ltd., Chiba, Japan), further size reduction was performed using a high-pressure emulsifier (nanomizer system YSNM-15005, Yoshidakikai Co. Ltd., Nagoya) under a pressure of 30 MPa for 30 min at 40 °C. Nondispersed samples were also prepared. To the mixtures of lipids (EPC and DO), an excess amount of TBS was added. The mixtures were subjected to repeated freeze-and-thaw cycles and kept at room temperature for 1 week before small-angle X-ray scattering (SAXS) or 31P NMR 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) with buffer and put into a quartz cell. Measurements were performed at 25 °C. The mean hydrodynamic diameter was evaluated by the cumulant method. To evaluate the stability of dispersions, their particle size was measured after the samples were kept at room temperature for several days. Small-Angle X-ray Scattering. The dispersions were concentrated (to more than 20 wt % lipids) by ultrafiltration (Millipore, Bedford, MA) to obtain an adequate intensity in SAXS experiments. These samples and the nondispersed samples were put into a glass capillary (W. Mu¨ller, Berlin, Germany; 1.5 mm o.d., 1/100 mm wall thickness) and flame-sealed. SAXS measurements were performed at 25 °C 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. For the dispersion, solvent scattering was subtracted. 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 used for the dispersed or nondispersed samples, respectively. 31P NMR Spectroscopy. 31P NMR measurements were performed at 25 °C on a Varian Gemini 300 spectrometer at 121.5 MHz in the presence of a proton-decoupling field. The dispersions were put into NMR tubes (5 mm o.d.). D2O was added to gain a lock and shim signal. A paramagnetic-shifting reagent, praseodymium(III) nitrate, was added, and the variance of the spectrum was traced. For measurements of the nondispersed systems, the same samples used for SAXS experiments were utilized. The capillaries of the nondispersed samples were put into NMR tubes containing D2O.

Results Characterization of the Nondispersed Samples by SAXS and 31P NMR. The liquid crystalline samples of the EPC/DO mixtures in TBS with different lipid compositions were investigated by SAXS and 31P NMR at 25 °C to identify their phases. Figure 1 shows the SAXS profiles of the lipid mixtures. Pure EPC and the EPC/DO mixture with a molar ratio of 9:1 gave lamellar reflections up to second order. The 8:2 mixture showed a diffraction pattern with a space group of Im3m, a typical diffraction for the bicontinuous cubic phase of primitive type (CP), and the diffraction profile was satisfactorily reproduced by the theoretical curve of the simplified model of CP, which

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Figure 1. SAXS profiles of the nondispersed EPC/DO mixtures in TBS. The dotted curves in EPC/DO ) 8:2 and 7:3 are the theoretical curves of the simplified model of CP and CD, respectively.

assumes a flat contrast density profile18,19 with a lattice constant of 192 Å. For the 7:3 mixture, peak positions corresponded to those of the bicontinuous cubic phase of diamond type (CD, space group of Pn3m); however, the theoretical curve of the CD phase (with a lattice constant of 183 Å) had lower intensity at the peaks of s ) 0.016 and 0.028 Å-1, which suggests the coexistence of the inverted hexagonal (HII) phase. At higher DO fractions (6:4 and 5:5), the diffraction patterns of the HII phase were observed. The same lipid mixtures were also investigated by 31P NMR to identify the phases from the chemical shift anisotropy. Figure 2 shows 31P NMR spectra of these lipid mixtures. The pure EPC and the 9:1 mixture showed an asymmetric peak with a maximum at ca. -10 ppm and a lower-field shoulder, suggesting a lamellar phase. The 8:2 and 7:3 mixtures provided a sharp, symmetric peak, which suggests the formation of an isotropic phase, most probably the bicontinuous cubic phase according to SAXS results. The spectra of the 6:4 and 5:5 mixtures gave an asymmetric peak with a maximum at ca. 8 ppm and a higher-field shoulder, suggesting the formation of an HII phase. However, a symmetric peak was also found in these spectra in addition to the asymmetric peak. Different results between SAXS and NMR were observed for the 7:3, 6:4, and 5:5 mixtures. SAXS showed the existence of the HII phase for the 7:3 mixture, which was not shown by NMR. In contrast, the existence of the isotropic phase (cubic phase) in the 6:4 and 5:5 mixtures, suggested by NMR, was not observed by SAXS. It is conceivable that these two methods have different sensitivities to the cubic and HII phases. That is, the HII phase gives stronger diffraction than the bicontinuous cubic phase, and the cubic phase provides a sharper 31P NMR signal than the HII phase. Other isotropic phases, which cannot be detected by SAXS (e.g., reversed micelles), might coexist with the HII phase in the 6:4 and 5:5 mixtures. Phosphatidylcholine-DG mixtures with relatively high DG fractions are (18) Garstecki, P.; Holyst, R. Langmuir 2002, 18, 2519-2528. (19) Garstecki, P.; Holyst, R. Langmuir 2002, 18, 2529-2537.

Behavior of a Phosphatidylcholine/Diolein System

Figure 2. 5:5 (f).

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P NMR spectra of the nondispersed EPC/DO mixtures in TBS. EPC/DO ) 10:0 (a), 9:1 (b), 8:2 (c), 7:3 (d), 6:4 (e), and

31

known to form the reversed micellar cubic phase with a space group of Fd3m,8,11 although this phase should be detectable by SAXS. Size and Structure of the Dispersions. The dispersions of the nonlamellar-forming lipid mixtures with 8 wt % F127 were prepared by high-pressure emulsification. The size and stability of the particles were dependent on the composition of the lipid mixtures as shown in Figure 3. The dispersions were stable for at least 3 days but started to aggregate after longer storage. The mean diameter of the products and the aggregation rate increased with an increase in the mole fraction of DO, due to the increase in the hydrophobicity of the lipid mixtures. 31 P NMR with a paramagnetic shift reagent can reveal whether a system contains vesicles or not, because of little membrane permeability of the ions, by which the paramagnetic ions cannot access inner layers of the vesicles.20 We have shown by 13C NMR that ions can access cubosomes consisting of monoolein, oleic acid, and F127.13 Hexosomes also showed high accessibility of ions (unpublished data). According to these findings, it is possible to check whether the dispersions prepared consist primarily of nanoparticles of nonlamellar phases or vesicles. (20) Saito, H.; Nishiwaki, K.; Handa, T.; Ito, S.; Miyajima, K. Langmuir 1995, 11, 3742-3747.

Figure 3. The mean particle diameter of the dispersions of the EPC/DO mixtures with 8 wt % F127 as a function of the number of days after preparation. EPC/DO ) 8:2 (open circles), 7:3 (closed circles), 6:4 (open squares), and 5:5 (closed squares).

Figure 4 shows 31P NMR spectra of the dispersions of the lipid mixtures with 8 wt % F127. For the dispersion of the 8:2 mixture, a shifted peak at ca. 10 ppm was observed in addition to a peak near 0 ppm by the addition of Pr3+. The similarity in amplitude of these two peaks suggests

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Discussion

Figure 4. 31P NMR spectra of the dispersions of the EPC/DO mixtures with 8 wt % F127 before (lower) and after (upper) addition of Pr3+. EPC/DO ) 8:2 (a), 7:3 (b), 6:4 (c), and 5:5 (d).

Figure 5. SAXS profiles of the dispersions of the EPC/DO mixtures with 8 wt % F127.

that the vesicles account for the greater part in the dispersion. The peak near 0 ppm in the presence of Pr3+ diminished as the DO fraction increased, suggesting a decrease in the vesicle content. Figure 5 shows SAXS profiles of the dispersions. The samples were concentrated by ultrafiltration in order to obtain adequate diffraction intensities. The dispersion of the 8:2 mixture showed no indication of the existence of nonlamellar phases but gave only a broad peak with a maximum at ca. 0.015 Å-1, presumably denoting vesicle formation as suggested by 31P NMR. In the SAXS profile of the dispersion of the 7:3 mixture, diffractions from both CD and HII were observed, similar to those of the nondispersed sample. Considering the NMR observation, this system could be a mixture of vesicles, cubosomes, and hexosomes. At 6:4 and 5:5, diffraction patterns of the HII phase were observed. Thus, the 5:5 mixture could form hexosomes with only a small amount of vesicles contained, taking the NMR observation into account.

Most examples reported regarding the formation of bicontinuous cubic phases are those of the monooleinbased phase.21-28 Monoolein forms the bicontinuous cubic phases of CD and CG (gyroid type), depending on water content, and forms the CD phase in equilibrium with an excess of water.29 The cubic phases of monoolein can be dispersed in water using Pluronic F127 as an emulsifier, to form cubosomes.13,30,31 There are two important properties prerequisite for the formation of liquid crystalline particles. First, the liquid crystalline phase to be dispersed should be equilibrated with the water phase. For example, if a bicontinuous cubic phase of a certain system transforms to a lamellar phase by adding water, the production of cubosomes is not possible. Second, the emulsifier (F127) should be less compatible with the liquid crystalline phase; otherwise, it transforms or destroys the phase. The 31P NMR and SAXS results revealed that the EPC/ DO lipid mixture with the molar ratio of 8:2 forms the CP phase. Takahashi et al. observed the diffraction pattern of Im3m in addition to that of the HII phase from a fully hydrated 1:1 dipalmitoylphosphatidylcholine-dipalmitoylglycerol mixture at 65 °C.8 However, in the present study, it was found that the EPC/DO mixture forms the CP phase, solely equilibrating with the aqueous phase at room temperature. The EPC/DO mixture with the molar ratio of 7.5:2.5 also provided a similar diffraction pattern, suggesting CP phase formation (data not shown). At the ratio of 7:3, the CD phase was observed; however, the curve fitting with the simplified model revealed that the cubic phase coexists with the HII phase. The formation of the pure CP phase and the coexistence of the CD phase with the HII phase have also been reported for fatty acid/ phosphatidylcholine mixtures at high temperature.32 Further addition of DO led to the formation of the HII phase with a smaller lattice constant. The successive formation of the lamellar, CP, CD, and HII phases by the increase in the DO fraction is in line with the increase in the negative curvature of the lipid-water interface induced by the nonlamellar-forming lipid. Since we obtained the CP phase equilibrating with the aqueous phase at EPC/DO ) 8:2, which is a prerequisite for the cubosome formation as described above, we then examined if the cubosomes can be formed by high-pressure emulsification using 8 wt % Pluronic F127. However, cubosomes could not be formed for the 8:2 mixture and vesicles were obtained instead. Cubosomes were not obtained even at a lower F127 concentration (3 wt %). This could be because EPC has a propensity to form (21) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (22) Razumas, V.; Larsson, K.; Miezis, Y.; Nylander, T. J. Phys. Chem. 1996, 100, 11766-11774. (23) Razumas, V.; Talaikyte, Z.; Barauskas, J.; Larsson, K.; Miezis, Y.; Nylander, T. Chem. Phys. Lipids 1996, 84, 123-138. (24) Barauskas, J.; Razumas, V.; Nylander, T. Chem. Phys. Lipids 1999, 97, 167-179. (25) Engstro¨m, S.; Norde´n, T. P.; Nyquist, H. Eur. J. Pharm. Sci. 1999, 8, 243-254. (26) Borne, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 1004410054. (27) Caboi, F.; Amico, G. S.; Pitzalis, P.; Monduzzi, M.; Nylander, T.; Larsson, K. Chem. Phys. Lipids 2001, 109, 47-62. (28) Borne, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 77427751. (29) Hyde, S. T.; Ericsson, B.; Andersson, S.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (30) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (31) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (32) Winter, R.; Erbes, J.; Templer, R. H.; Seddon, J. M.; Syrykh, A.; Warrender, N. A.; Rapp, G. Phys. Chem. Chem. Phys. 1999, 1, 887-893.

Behavior of a Phosphatidylcholine/Diolein System

vesicles and because F127 is compatible with the bicontinuous cubic phases to some extent, which leads to the vesicle formation with lipids since F127 gives positive spontaneous curvature and high water-solubility. We performed SAXS measurements for the nondispersed 8:2 mixture in the presence of 8 wt % F127 (data not shown) and found that it also forms the CP phase, but the lattice constant significantly increased from 192 Å (without F127) to 255 Å (with F127). In the case of the dispersion consisting of monoolein and F127, an increase in the F127 concentration changes the internal structure of the cubosomes from CD to CP13 and increases the population of vesicles.31 In addition, the lattice constant of the CP phase (192 or 255 Å) is rather large compared with that of the monoolein-based CP phase (e.g., 117 Å for the monoolein-15% F127 mixture13), which may lead to a higher propensity for vesicle formation. The SAXS patterns of the dispersions of the nonlamellarforming lipid mixtures with 8 wt % F127 were similar to those of the corresponding nondispersed mixtures without F127, at the EPC/DO molar ratios from 7:3 to 5:5. This is because F127 is not very compatible with these nonlamellar phases, and it preferably localizes to the

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particle surface by the dispersion process.14 Thus, F127 is considered an effective emulsifier of the nonlamellar phases, especially the HII phase. The dispersion of the 5:5 mixture with 15 wt % F127 provided a SAXS pattern identical to that of 8 wt % F127, suggesting F127 does not influence the internal structure of the particles. Ultimately, the production of hexosomes in the EPC/ DO/F127 system was possible, although cubosomes could not be obtained. This is because the HII phase has more negative membrane curvature and less compatibility with F127 than the bicontinuous cubic phases. The components of this hexosome were not taken up by serum albumin (data not shown), so this dispersion system could be a novel drug carrier with higher stability after intravenous administration than the monoolein-based cubosomes. Acknowledgment. This study was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (No. 15790026 and 12470488) and the Japan Health Science Foundation. LA035313X