pubs.acs.org/Langmuir © 2009 American Chemical Society
Useful Modified Cellulose Polymers as New Emulsifiers of Cubosomes Makoto Uyama,† Minoru Nakano,*,† Jun Yamashita,‡ and Tetsurou Handa† †
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan , and ‡CytoPathfinder, Inc., Kiba KI Bldg. 4F, 6-4-2 Kiba, Koto-ku, Tokyo 135-0042, Japan Received January 31, 2009. Revised Manuscript Received March 11, 2009
This report introduces modified cellulose polymers as new emulsifiers of cubosomes. We prepared novel nanoparticles containing cubic-phase-forming lipids using hydroxypropyl methylcellulose acetate succinate (HPMCAS). Small-angle X-ray scattering showed a much lower incorporation of HPMCAS into the cubic structure of monoolein than did a conventional emulsifier, Pluronic F127, which is known to modify the cubic structure. Cubosomes prepared with HPMCAS showed roughly equal stability as nanoparticles with Pluronic F127. These results suggest that HPMCAS can be a novel emulsifier of cubosomes, which brings about no internal structure modification.
Introduction Among a series of lipid-based self-assembled systems, bicontinuous cubic phases have attracted a great deal of attention because of their physicochemical properties in various practical applications. Monoolein (1-monooleoyl glycerol, MO, Figure 1a) is well known to form cubic phases in equilibrium with excess water at ambient temperature.1-4 1-O-(5,9,13,17-Tetramethyloctadecanoyl)erythritol (EROCO C22, Figure 1b) and 1-O-(5,9,13,17-tetramethyloctadecyl)-β-D-xylopyranoside (β-XP, Figure 1c) also form cubic phases.5-8 Nanoparticles with cubic phases in their interiors are termed cubosomes. (The word cubosome is a USPTO registered trademark of GS Development AB Corp., Sweden.) Because of their structural matter, an emulsifier is indispensable to the stable dispersion of cubosomes. Pluronic F127, a triblock copolymer consisting of two poly (ethylene oxide) blocks as hydrophilic parts and a poly(propylene oxide) block as a hydrophobic part (Figure 1d), has been widely used in many reports.9-15 F127 is thought to adsorb onto the particle surface; however, this polymer affects the internal structure of cubosomes with MO, changing from Pn3m (corresponding to the diamond type of infinite periodic minimal surfaces (IPMS), CD) to Im3m (corresponding to the primitive *Corresponding author. E-mail:
[email protected]. (1) Ljusberg-Wahren, H.; Hersloef, M.; Larsson, K. Chem. Phys. Lipids 1983, 33, 211–214. (2) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213–219. (3) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223–224. (4) Clogston, J.; Rathman, J.; Tomasko, D.; Walker, H.; Caffrey, M. Chem. Phys. Lipids 2000, 107, 191–220. (5) Yamashita, J.; Shiono, M.; Hato, M. J. Phys. Chem. B 2008, 112, 12286– 12296. (6) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Langmuir 2002, 18, 3425–3429. (7) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Prog. Colloid Polym. Sci. 2004, 123, 56–60. (8) Hato, M.; Yamashita, I.; Kato, T.; Abe, Y. Langmuir 2004, 20, 11366–11373. (9) Landh, T. J. Phys. Chem. 1994, 98, 8453–8467. (10) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611–4613. (11) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964–6971. (12) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917–3922. (13) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254–5261. (14) Yaghmur, A.; de Campo, L.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2006, 22, 9919–9927. (15) Dng, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 9512– 9518.
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type of IPMS, CP),9-12 In addition, the cubic phase is transformed to a lamellar phase (vesicles) at higher F127 compositions.9 Regarding problems in practical use, F127 has been said to be a skin or eye irritant and is hazardous in the case of ingestion or inhalation. Polysaccharide polymers have also been assessed for cubosome preparation, although they are not amphiphilic block copolymers. It has been reported that cubosomes are stabilized with modified starch, dextran,16 or hydrophobically modified ethyl hydroxyethyl cellulose (HMEHEC).17 Spicer et al.16 presented a pseudoternary phase diagram of MO with hydrophobically modified starch in water and prepared cubosomes by the rehydration of spray-dried starch-MO mixtures. In their system, starch was mixed 3-fold higher than the weight of MO, and the particle size was 600 nm on average, which is too large for injection. Almgren et al.17 applied HMEHEC to the MO-based cubic phase. Although cubosomes were formed, HMEHEC interacted so strongly with lipids that it was transformed into lamellar and reversed hexagonal phases. In this study, we applied hydroxypropyl methyl cellulose acetate succinate (HPMCAS, Figure 1e) to the preparation of cubosomes with MO and other cubic-phase-forming lipids. Cellulose products are widely used in the cosmetics, food, and pharmaceutical industries, such as in eye drops and inhalants. HPMCAS is a commercially available enteric coating agent and widely used in dry coating or solid dispersion systems,18-20 but it has never been applied as a dispersing agent of lipidic nanoparticles. We demonstrate here that HPMCAS can act as a suitable emulsifier of cubosomes, which brings about sufficient dispersion stability without any internal structure modification.
Experimental Section Materials. MO was supplied by NOF Corp. (Tokyo, Japan) with >99% purity of the acyl group and >97% purity of the ester. EROCO C22 and β-XP were synthesized as described previously.5,8,21 Pluronic F127 (PEO99-PPO67-PEO99) (16) Spicer, P. T.; Small, W. B.; Lynch, M. L.; Burns, J. L. J. Nanopart. Res. 2002, 4, 297–311. (17) Almgren, M.; Borne, J.; Feitosa, E.; Khan, A.; Lindman, B. Langmuir 2007, 23, 2768–2777. (18) Luo, Y.; Zhu, J.; Ma, Y.; Zhang, H. Int. J. Pharm. 2008, 358, 16–22. (19) Kablitz, C. D.; Kappl, M.; Urbanetz, N. A. Eur. J. Pharm. Biopharm. 2008, 69, 760–768. (20) Konno, H.; Handa, T.; Alonzo, D. V.; Taylor, L. S. Eur. J. Pharm. Biopharm. 2008, 70, 493–499. (21) Minamikawa, H.; Murakami, T.; Hato, M. Chem. Phys. Lipids 1994, 72, 111–118.
Published on Web 3/18/2009
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Letter
Figure 3. SAXS pattern from cubosomes of MO/AS-LF with 10 wt % AS-LF.
Figure 1. Chemical structures of MO (a), EROCO C22 (b), β-XP (c), Pluronic F127 (d), and HPMCAS (e).
Figure 2. SAXS patterns from the liquid-crystalline phase formed in the MO/water binary system (a) and MO/AS-LF/water ternary systems containing 10 wt % (b) and 20 wt % AS-LF (c). was provided by BASF Japan Ltd. (Osaka, Japan). HPMCAS was provided by Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Two kinds of HPMCAS (AS-LF and AS-MF) were used. According to the manufacturer, AS-LF/AS-MF contains 20-24/21-25% methoxy group, 5-9/5-9% hydroxypropyl group, 5-9/7-11% acetyl group, and 14-18/10-14% succinyl group. Water was purified by a Milli-Q reagent water system (Millipore Co., Bedford, MA) containing a carbon filter cartridge and a 0.22 μm filter. All materials were used as received. Sample Preparation. Lipids (MO, EROCO C22, or β-XP) dissolved in methanol were placed in a flask, the solvent was evaporated and dried in vacuum, and an aqueous solution of polymers (F127, AS-LF, and AS-MF) was added. For all samples, the weight ratio of polymers to lipids was 1:10. After several cycles of freeze-thawing, the mixtures were roughly dispersed using a bath sonicator. Further size reduction was carried out using a high-pressure emulsifier (nanomizer system YSNM-1500; Yoshidakikai Co. Ltd., Nagoya) under 35 MPa pressure at room temperature for 30 min. Small-Angle X-ray Scattering (SAXS). X-ray diffraction experiments for nondispersed samples were carried out with Cu KR radiation generated by a Rigaku RU-200 rotating anode X-ray generator (Rigaku Co., Tokyo; 40 kV, 150 mA) with a graphite monochromator (λ(wavelength) = 1.5418 A˚) and a double pinhole collimator (0.3 mm φ 0.3 mm φ). The exposure Langmuir 2009, 25(8), 4336–4338
time was 1 to 2 h at a sample-to-film distance of 300 mm at room temperature. The diffraction pattern was recorded with an imaging plate (Fuji Photo Films), which was digitized and analyzed by a Rigaku R-AXIS system. Dispersions were concentrated by ultrafiltration using a regenerated cellulose membrane filter (100 kDa NMWL, Millipore, MA). SAXS measurements for dispersions were performed on a SAXSess camera (Anton Paar) attached to a PW3830 X-ray generator (40 kV and 50 mA) with a long fine focus sealed glass X-ray tube (λ = 1.542 A˚). The scattering intensity of each sample was measured at 25 °C for 24 h with a Cyclone imaging plate detection system (Perkin-Elmer) and analyzed by SAXSQuant software (Anton Paar). Dynamic Light Scattering (DLS). The particle size was determined from dynamic light scattering measurements (Photal FPAR-1000; Otsuka Electronic Co., Osaka). Measurements were performed at 25 °C. The mean hydrodynamic diameter was evaluated by the cumulant method.
Results and Discussion X-ray Scattering from Liquid-Crystalline Phases and Dispersed Particles. Figure 2a shows the SAXS pattern of a liquid crystalline phase formed in a MO/water binary system. Diffraction peaks were observed up to sixth order ((110), (111), (200), (211), (220), (300), and/or (221)), in accordance with the Pn3m space group, indicating the CD phase. For the MO/ASLF/water ternary systems with 10 and 20 wt % AS-LF, similar diffraction patterns were observed, corresponding to the Pn3m space group (CD phase), as shown in Figure 2b,c. Previously, we reported that liquid-crystalline phases of MO were converted from the CD to CP phase in the presence of F127 (5-15 wt %).12 The present result suggests that HPMCAS does not modify the cubic structure as a result of the lower compatibility with this phase. Diffraction peaks were slightly shifted to wider angles in the presence of AS-LF, which could be ascribed to an osmotic effect of the polymer that cannot be incorporated into water tubes of the cubic phases. The SAXS pattern of the dispersed system of MO/AS-LF/ water is shown in Figure 3. Good accordance with the pattern from the nondispersed bulk phase in Figure 2b was observed, suggesting that cubosomes with the CD phase were obtained. AS-LF and AS-MF have basically the same structure, so differences between the two polymers hardly exist in liquidcrystalline phases. EROCO C22 and β-XP are a new lipid family that forms bicontinuous cubic phases in equilibrium with excess water at ambient temperature,2-5 and a dispersion has been reported for the β-XP/F127 system.22,23 SAXS patterns from (22) Abraham, T.; Hato, M.; Hirai, M. Colloids Surf., B 2004, 35, 107–117. (23) Abraham, T.; Hato, M.; Hirai, M. Biotechnol. Prog. 2005, 21, 255–262.
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Figure 4. SAXS patterns from cubosomes of EROCO C22 dispersed using F127 (a), AS-LF (b), and AS-MF (c) as emulsifiers (10 wt % lipids). Downward arrows indicate cubic Im3m peaks.
Figure 5. SAXS patterns from cubosomes of β-XP dispersed using F127 (a), AS-LF (b), and AS-MF (c) as emulsifiers (10 wt % lipids). Downward arrows indicate cubic Im3m peaks.
dispersions with these lipids with HPMCAS indicated the formation of cubosomes with the CD phase (Figures 4 and 5). It is noteworthy that the diffraction corresponding to the Im3m space group (CP phase) was not observed: it has been reported that F127 affects the cubic phases of β-XP as well as MO, changing from the CD to the CP phase.22,23 Dispersion with EROCO C22 and F127 also showed the coexistence of the CP phase (Figure 4a). The AS-LF and AS-MF results show that these polymers are novel, promising emulsifiers that do not affect the internal structures of cubosomes. Size and Longevity of Particles. We investigated the longevity of cubosomes by periodically measuring the sizes of dispersions, which were stored at ambient temperature. The particle sizes of dispersions prepared with various lipids and polymers (10 wt % lipids) were characterized by DLS and traced for 30 days, as shown in Figure 6. The mean particle sizes of dispersions were all in the approximate range from 100 to 200 nm. This size range would be acceptable for an intravenous injection, different from the case of starch (ca. 600 nm).16 The particle size of dispersions did not differ among polymers. In addition, the aggregation of cubosomes was not observed in preservation, even when AS-LF or AS-MF was used. This suggests that HPMCAS has roughly equal stability to F127. Ten percent HPMCAS was indeed necessary to stably disperse cubosomes: at lower polymer concentration (5 wt % lipids), however, the dispersion with MO and AS-LF was unstable, and aggregates were observed (data not shown). We tried to prepare 4338
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Figure 6. Particle diameter of the dispersions as a function of the number of days after preparation. Dispersions were prepared with MO (a), EROCO C22 (b), and β-XP (c) using 10 wt % F127 (b), AS-LF (0), and AS-MF (2), respectively.
nanoparticles with methylcellulose or hydroxypropyl methylcellulose; however, these nanoparticles were unstable and aggregated within 30 days. The negative charge of the succinyl group in HPMCAS could be involved in the stabilizing effect of the polymer. Indeed, cubosomes with HPMCAS could not be prepared at pH 3.4, which is lower than the pKa of succinic acid (ca. 4.2), whereas stable dispersions were obtained at higher pH (>5.4) (data not shown).
Conclusions SAXS findings suggest that HPMCAS can be used to disperse cubic-phase-forming lipids to form cubosomes. Different from F127, HPMCAS does not change the internal structure of cubosomes at all, suggesting that this polymer is simply adsorbed on the particle surface. The data indicate that HPMCAS has roughly equal ability to F127 to stabilize cubosomes. Taken together, HPMCAS is considered to be a promising agent as an emulsifier of cubosomes. Acknowledgment. This study was supported by grants-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (nos. 17390011, 20050017, and 20790032). Langmuir 2009, 25(8), 4336–4338
