Langmuir 2007, 23, 1659-1663
1659
Aqueous-Phase Behavior of Natural Glycolipid Biosurfactant Mannosylerythritol Lipid A: Sponge, Cubic, and Lamellar Phases Tomohiro Imura,† Yusuke Hikosaka,‡ Wannasiri Worakitkanchanakul,‡ Hideki Sakai,‡ Masahiko Abe,‡ Masaaki Konishi,† Hiroyuki Minamikawa,§ and Dai Kitamoto*,† Research Institute for InnoVation in Sustainable Chemistry and Nanoarchitectonics Research Center, National Institute of AdVanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, and Faculty of Science and Technology, Tokyo UniVersity of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan ReceiVed July 18, 2006. In Final Form: October 25, 2006 The aqueous-phase behavior of mannosylerythritol lipid A (MEL-A), which is a glycolipid biosurfactant produced from vegetable oils by yeast strains of the genus Pseudozyma, was investigated using polarized optical microscopy, small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). MEL-A was found to self-assemble into a variety of distinctive lyotropic liquid crystals including sponge (L3), bicontinuous cubic (V2), and lamella (LR) phases. On the basis of SAXS measurements, we determined the structure of the liquid crystals. The estimated lattice constant for LR was 3.58 nm. DSC measurement revealed that the phase transition enthalpies from the liquid crystal to the fluid isotropic phase were in the range of 0.22-0.44 kJ/mol. Although the present MEL-A phase diagram closely resembled that obtained from relatively hydrophobic poly(oxyethylene) or fluorinated surfactants, the MEL-A L3 region was spread considerably over a wide temperature range (20-65 °C) compared to L3 of those surfactants: this is probably due to the unique structure which is molecularly engineered by microorganisms. In this paper, we clarify the aqueous phase diagram of the natural glycolipid biosurfactant MEL-A, and we suggest that the obtained lyotropic crystals are potentially useful as novel nanostructured biomaterials.
1. Introduction Lyotropic liquid crystalline phases based on self-assembly of surfactants in aqueous media have been extensively studied not only for their fundamental interest but also for practical applications including cosmetics, pharmacy, and dispersion technology.1-3 Many kinds of ionic and nonionic surfactants can self-assemble into three-dimensional ordered lyotropic liquid crystals including sponge, cubic, lamella, and hexagonal phases at high surfactant concentrations.4-6 In particular, sugar-based nonionic surfactants have great advantages compared to the usual surfactants because they are highly biodegradable, environmentally friendly, and less toxic.7,8 Moreover, the biological functions of the sugar moieties, such as their molecular recognition or protein stabilization effects, make their lyotropic liquid crystals attractive as new materials for biotechnology.9,10 Alkyl glucosides (AGs) are well-known sugar-based surfactants that are usually synthesized from fatty alcohols and sugars.11-15 * To whom correspondence should be addressed: Phone: +81-29-8614664. Fax: +81-29-861-4660. E-mail:
[email protected] † Research Institute for Innovation in Sustainable Chemistry, AIST. ‡ Tokyo University of Science. § Nanoarchitectonics Research Center, AIST. (1) Angelov, B.; Angelova, A.; Ollivon, M.; Bourgaux, C.; Campitelli, A. J. Am. Chem. Soc. 2003, 125, 7188-7189. (2) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (3) Rodriguez-Abreu, C.; Carcia-Roman, M.; Kunieda, H. Langmuir 2004, 20, 5235-5240. (4) Kaneko, D.; Olsson, Ulf.; Sakamoto, K. Langmuir 2002, 18, 4699-4703. (5) Nishizawa, M.; Saito, K.; Sorai, M. J. Phys. Chem. B 2001, 105, 29872992. (6) Strey, R.; Winkler, J.; Magid, L. J. Phys. Chem. 1991, 95, 7502-7507. (7) Sadtler, V. M.; Guely, M.; Marchal, P.; Choplin, L. J. Colloid Interface Sci. 2004, 270, 270-275. (8) Ferrer, M.; Comelles, F.; Plou, F. J.; Cruces, M. A.; Fuentes, G.; Parra, J. L.; Ballesteros, A. Langmuir 2002, 18, 667-673. (9) Abraham, T.; Hato, M.; Hirai, M. Colloids Surf., B 2004, 35, 107-117. (10) Vill, V.; Hashim, R. Curr. Opin. Colloid Interface Sci. 2002, 7, 69-80. (11) Minamikawa, H.; Hato, M. Chem. Phys. Lipids 2005, 134, 154-160.
Unlike typical synthetic surfactants, they can self-assemble into specific lyotropic liquid crystalline phases that are stabilized by a hydrogen-bonding network between the sugar moieties.16-20 The chirality of the sugar moieties also plays an important role in their lyotropic and thermotropic phase behaviors.21,22 However, the number of reports on the lyotropic behavior of sugar-based surfactants is still relatively limited because the stereo- and regioselective synthesis of complicated sugar-based surfactants is generally difficult. Although the use of “natural” sugar-based amphiphiles such as gangliosides seems to have the potential to overcome this difficulty, the preparation of a sufficient amount of these membrane glycolipids is also crucial. These facts make it difficult to clarify the whole manner of structure-property relationships for complicated sugar-based surfactants. To this end, we focused our attention on biosurfactants that are abundantly produced from renewable resources by a variety of microorganisms.21 The typical hydrophilic groups of microbial (12) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S. Langmuir 2002, 18, 3425-3429. (13) Coppola, L.; Gordano, A.; Procopio, A.; Sindona, G. Colloids Surf., A 2002, 196, 177-187. (14) Templer, R. H.; Turner, D. C.; Harper, P.; Seddon, J. M. J. Phys. II 1995, 5, 1053-1065. (15) Jeffrey, G. A.; Yeon, Y. Carbohydr. Res. 1992, 237, 45-55. (16) Von Minden, H. M.; Brandenburg, K.; Seydel, U.; Koch, M. H. J.; Garamus, V.; Willumeit, R.; Vill, V. Chem. Phys. Lipids 2000, 106, 157-179. (17) Hato, M.; Yamashita, I.; Kato, Y.; Abe, Y. Langmuir 2004, 20, 1136611373. (18) Bonicelli, M. G.; Ceccaroni, G. F.; La, Mesa, C. J. Colloid Polym. Sci. 1998, 276, 109-116. (19) Boyd, B. J.; Krodkiewska, I.; Drummond, C. J.; Grieser, F. Langmuir 2002, 18, 597-601. (20) Nilsson, F.; Soderman, O.; Johansson, I. J. Colloid Interface Sci. 1998, 203, 131-139. (21) Kitamoto, D,; Toma, K.; Hato, M. Glycolipid-Based Bionanomaterials. In Handbook of Nanostructured Biomaterials and Their Applications in Nanobiotechnolgy; Nalwa, H. S., Ed.; American Scientific: Los Angeles, 2005; Vol. 1, pp 239-271. (22) Kitamoto, D.; Ghosh, S.; Nakatani, Y.; Ourisson, G. Chem. Commun. 2000, 861-862.
10.1021/la0620814 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/11/2007
1660 Langmuir, Vol. 23, No. 4, 2007
Imura et al.
Figure 1. Structure of MEL-A.
biosurfactants are complicated carbohydrates that are stereo- and regioselectively synthesized by enzymatic reactions, and thus, most of them are chiral compounds with a unified molecular configuration. Mannosylerythritol lipid A (MEL-A; see Figure 1) is one of the most promising glycolipid biosurfactants known.22 It is abundantly produced by yeast strains of the genus Pseudozyma from soybean oil or n-alkane at a yield of up to 140 g/L.23,24 We have previously reported that MEL-A exhibits not only excellent surface activities25 but also various biological activities such as antimicrobial activity25 and molecular recognition ability toward human immunoglobulin G (HIgG).26 More recently, a cationic liposome including MEL-A was found to drastically increase the efficiency of gene transfection via membrane fusion.27 Also, we found that the single component of MEL-A self-assembles into a sponge phase (L3 phase) composed of randomly connected bilayer networks at extremely low concentrations in aqueous media.28,29 These results imply that MEL-A can form a variety of lyotropic liquid crystals over a wide concentration range which are potentially useful as sugarbased lyotropic liquid crystals with various biological activities. In this paper, we report the aqueous-phase behavior of the glycolipid biosurfactant MEL-A revealed by polarized optical microscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and so forth. 2. Experimental Section 2.1. Materials and Sample Preparation. 4-O-(4′,6′-Di-O-acetyl2′,3′-di-O-alkanoyl-β-D-mannopyranosyl)-D-erythritol (MEL-A) was obtained by the following procedure. A seed culture (1.5 mL) of the yeast strain of Pseudozyma antarctica was transferred to a 300 mL Erlenmeyer flask containing 30 mL of a fermentation medium [4% (v/v) soybean oil, 0.2% NaNO3, 0.02% MgSO4‚7H2O, 0.02% KH2PO4, 0.1% yeast extract, and distilled water] and incubated on a rotatory shaker (220 rpm) at 30 °C for one week. The culture broth (30 mL) was then extracted twice with 30 mL portions of ethyl acetate. The layer was separated, and the organic layer was washed with brine and concentrated under reduced pressure. The resulting yellow oil (555 mg) was dissolved in chloroform (5 mL) and placed on a column (3 × 40 cm) of silica gel (50 g). The mixture of MEL was then chromatographed with a close gradient elution of chloroform-acetone (10:0 to 0:10). Each fraction was collected and again chromatographed as above to give pure MEL-A. The lipid has the fatty acid composition of n ) 6 (18%), n ) 8 (71%), and n ) 10 (11%). The characterization of MEL-A is given in the Supporting Information of ref 22. The purified MEL-A was dissolved in acetone, and the stock solution was transferred into a test tube. After the solvent was removed by a rotary evaporator, distilled water was (23) Kitamoto, D.; Ikegami, T.; Suzuki, T.; Sasaki, A.; Takeyama, Y.; Idemoto, Y.; Koura, N.; Yanagishita, H. Biotechnol. Lett. 2001, 23, 1709-1714. (24) Morita, T.; Konishi, M.; Fukuoka, T.; Imura, T.; Kitamoto, D. Appl. Microbiol. Biotechnol. 2006, 73, 305-313. (25) Kitamoto, D.; Isoda, H.; Nakahara, T. J. Biosci. Bioeng. 2002, 94, 187201. (26) Im, J. -H.; Yanagishita, H.; Ikegami, T.; Kitamoto, D. J. Biomed. Mater. Res. 2003, 65, 379-385. (27) Igarashi, S.; Hattori, Y.; Maitani, Y. J. Controlled Release 2006, 112, 362-368. (28) Imura, T.; Yanagishita, H.; Kitamoto, D. J. Am. Chem. Soc. 2004, 126, 10804-10805. (29) Imura, T.; Ohta, N.; Inoue, K.; Yagi, N.; Negishi, H.; Yanagishita, H.; Kitamoto, D. Chem.sEur. J. 2006, 12, 2434-2440.
Figure 2. Binary phase diagram of the MEL-A/water system at 25 °C: L3, sponge phase; V2, cubic phase, LR, lamellar phase; FI, fluid isotropic phase. then introduced to the test tube. The MEL-A solutions were obtained by vortexing the test tube for 1 min at room temperature (25 °C). The obtained solutions were temperature-cycled several times between 25 and 70 °C and then equilibrated at 25 °C for at least one week. 2.2. Cross-Polarized Visual Inspection and Polarized Optical Microscopy. A polarized optical microscope (ECLIPSE E600, Nikon, Japan) with crossed polarizing filters equipped with a charge-coupled device camera (DS-SM, Nikon, Japan) was used to observe the lyotropic liquid crystalline phase of the biosurfactants. A 100 W halogen lamp was used as a light source. The pictures were obtained with a 100× objective lens with a numerical aperture of 0.3. Birefringent textures from the optical microscopy allowed the assignment of the particular lyotropic phase types to the samples. 2.3. Small-Angle X-ray Scattering. SAXS experiments were performed with Ni-filtered Cu KR radiation (wavelength (λ) 0.145 nm) generated by a Rigaku RU-200 X-ray generator (40 kV, 100 mA) with a double-pinhole collimator (0.5 mm L × 0.3 mm L). The lyotropic liquid crystals were flame-sealed into a quartz capillary (Glas, Berlin, 1.5 mm outer diameter). The sample temperature was controlled at 25 °C with a Mettler FP82HT hot stage. The diffraction patterns were recorded with an imaging plate (Fuji Photo Films, HR IIIN) in a flat camera. The exposure time was 45 min for the L3 and lamella (LR) phases and more than 8 h for the cubic phase (V2). The scattering vector q ) (4π/λ) sin θ, where 2θ is the scattering angle. 2.4. Differential Scanning Calorimetry. A Seiko Instruments Inc. model DSC6200 (Tokyo, Japan) was used for DSC measurements. Each liquid crystal (20 mg) was placed in an aluminum pan and sealed. A sealed pan containing distilled water was used as a reference. A heating/cooling cycle was repeated several times at the rate of 1 °C/min. A good reproducibility in the DSC thermograms was obtained by the repeated scan (mostly the second run is shown in the figure). Measurements were carried out in the temperature range 5-95 °C. The melting of liquid crystalline phases was accompanied by an endothermic peak which was automatically recorded.
3. Results and Discussion 3.1. Binary MEL-A/Water Phase Diagram at Room Temperature (25 °C). The binary phase diagram of the MELA/water system was obtained by combining cross-polarized visual inspection, polarized optical microscopy, and SAXS. A combination of the different methods is very useful to establish an accurate phase diagram; for example, cross-polarized visual observation constitutes a quicker tool to identify and assess the boundaries of the phase regions. SAXS provides further insight into the characteristic parameters of the structures. To complete an accurate phase diagram, we prepared 1 wt % samples. Figure 2 shows the binary phase diagram of the MEL-A/water system at 25 °C. The typical visual appearance of each phase is also shown in Figure 2. At low MEL-A concentrations (
