2768
Langmuir 2007, 23, 2768-2777
Dispersed Lipid Liquid Crystalline Phases Stabilized by a Hydrophobically Modified Cellulose Mats Almgren,*,† Johanna Borne´,‡ Eloi Feitosa,§ Ali Khan,‡ and Bjo¨rn Lindman‡ Department of Physical and Analytical Chemistry, Uppsala UniVersity, P.O. Box 579, SE-751 23 Uppsala, Sweden, Physical Chemistry 1, Lund UniVersity Center for Chemistry and Chemical Engineering, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden, and Physics Department, Sao Paulo State UniVersity, Sao Jose do Rio Preto, Sao Paulo, Brazil ReceiVed August 23, 2006. In Final Form: NoVember 24, 2006 Aqueous dispersions of monoolein (MO) with a commercial hydrophobically modified ethyl hydroxyethyl cellulose ether (HMEHEC) have been investigated with respect to the morphologies of the liquid crystalline nanoparticles. Only very low proportions of HMEHEC are accepted in the cubic and lamellar phases of the monoolein-water system. Due to the broad variation of composition and size of the commercial polymer, no other single-phase regions were found in the quasi-ternary system. Interactions of MO with different fractions of the HMEHEC sample induced the formation of lamellar and reversed hexagonal phases, identified from SAXD, polarization microscopy, and cryogenic TEM examinations. In excess water (more than 90 wt %) coarse dispersions are formed more or less spontaneously, containing particles of cubic phase from a size visible by the naked eye to small particles observed by cryoTEM. At high polymer/MO ratios, vesicles were frequently observed, often oligo-lamellar with inter-lamellar connections. After homogenization of the coarse dispersions in a microfluidizer, the large particles disappeared, apparently replaced by smaller cubic particles, often with vesicular attachments on the surfaces, and by vesicles or vesicular particles with a disordered interior. At the largest polymer contents no proper cubic particles were found directly after homogenization but mainly single-walled defected vesicles with a peculiar edgy appearance. During storage for 2 weeks, the dispersed particles changed toward more well-shaped cubic particles, even in dispersions with the highest polymer contents. In some of the samples with low polymer/MO ratio, dispersed particles of the reversed hexagonal type were found. A few of the homogenized samples were freeze-dried and rehydrated. Particles of essentially the same types, but with a less well-developed cubic character, were found after this treatment.
Introduction Stable aqueous dispersions of liquid crystalline phases in the form of liquid crystalline nanoparticles (LCNP) have several potential applications in drug delivery, food industry, and consumer products.1-3 Early on, the bicontinuous cubic phase of glycerol monoolein was found to be easily dispersible in sodium cholate and in casein solutions.4 Stable dispersions of cubosomes5 were prepared using a block copolymer, poloxamer 407 (EO99PO67EO99) also called Pluronic F1276 and imaged by cryoTEM.7,8 The most extensively studied polar lipid for LCNP formation has remained glycerol monoolein, although other lipids have been tried as well.9-11 Monoolein (MO) in water exhibits two types of bicontinuous cubic structures, the gyroid (CG) and the diamond (CD).12-14 The former is thermodynamically stable * Correspondingauthor.Phone: +46184713649.E-mail:
[email protected]. † Uppsala University. ‡ Lund University. § Sao Paulo State University. (1) Larsson, K. Curr. Opin. Colloid Interface Sci. 2000, 4, 64. (2) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449. (3) Spicer, P. T. Curr. Opin. Colloid Interface Sci. 2005, 10, 274. (4) Larsson, K. J. Phys. Chem. 1989, 93, 7304. (5) Cubosomes and hexosomes are USPTO registered trademarks of Camurus AB, Lund, Sweden. (6) Landh, T. J. Phys. Chem. 1994, 98, 8453. (7) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611. (8) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964. (9) Larsson, K.; Tiberg, F. Curr. Opin. Colloid Interface Sci. 2005, 9, 365. (10) Barauskas, J.; Johnsson, M.; Tiberg, F. Nano Lett. 2005, 5, 1615. (11) Yaghmur, A.; de Campo, L.; Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Glatter, O. Langmuir 2006, 22, 517. (12) Larsson, K. Nature 1983, 304, 664. (13) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723. (14) Qui, H.; Caffrey, M. Biomaterials 2000, 21, 223.
in contact with excess water, whereas the diamond exists at lower water content. Both cubic phases comprise curved bilayers organized to form two unconnected systems of water channels.4,15,16 The most swollen gyroid cubic phase contains about 40% water, so that the volume of each of the two water domains is about one-third of the volume of the lipid bilayer that separates them. Bicontinuous cubic phases have attracted considerable interest, both in terms of fundamental aspects within biochemistry and physical chemistry and for applications. The features that make them attractive as carriers in drug delivery2,17 and in food industry18 are the ability to encapsulate hydrophilic, lipophilic, and amphiphilic materials; the high internal surface area; and their bioadhesive and biocompatible nature.19 The physical properties of the cubic lc phases, in particular their high viscosity, are problematic, hence the interest for stable dispersions. Associating water-soluble polymers have received broad applications for the stabilization of disperse systems, in particular suspensions and emulsions.20,21 They have also been used for the stabilization of dispersions of lipid liquid crystalline particles in water. The most important types of associating polymers are water-soluble amphiphilic polymers, notably block or graft copolymers, with hydrophobic blocks or grafts. Both types of (15) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forse´n, S. J. Am. Chem. Soc. 1979, 101, 5465. (16) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989,988, 221. (17) Ganem-Quintanar, A.; Quintanar-Guerro, D.; Buri, P. Drug DeV. Ind. Pharm. 2000, 26, 809. (18) Krog, N.; Riisom, T. H.; Larsson, K. In Encyclopedia of Emulsion Technology; Becher, P., Ed.; Marcel Dekker: New York, 1983; Vol. 2, p 321. (19) Nielsen, L. S.; Schubert, L.; Hansen, J. Eur. J. Pharm. Sci.1998, 6, 231. (20) Holmberg, K.; Jo¨nsson, B.; Kronberg, B.; Lindman, B. In Surfactants and Polymers in Aqueous Solution, 2nd ed.; Wiley: London, 2002. (21) Handbook of Applied Surface and Colloid Chemistry; Holmberg, K., Ed.; John Wiley: Chichester, U.K., 2001.
10.1021/la062482j CCC: $37.00 © 2007 American Chemical Society Published on Web 01/17/2007
MO and Hydrophobically Modified EHEC
copolymers interact strongly with surfactants and lipids, but because of polymer architecture they react very differently in the two cases. Block copolymers associate with surfactants and lipids in a way resembling the way of a second surfactant/lipid. For the graft polymers, also referred to as hydrophobically modified water-soluble polymers (HMP), larger equilibrium surfactant/ lipid structures are hindered by the hydrophilic polymer backbone. Therefore, they form less intimate aggregates with surfactants than block copolymers; a typical example is the synergistic thickening effect in mixed solutions of a HMP and a surfactant due to the formation of micellar type cross-links between polymer chains. For the case of cubosomes, previous work of stabilizing the dispersions has focused mainly on block copolymers, such as poloxamers and also polyoxyethylene grafted lipids (PEG lipids) of the types used for stabilization of liposomes.22-24 However, it seems that HMPs could offer an interesting alternative for many applications.25 In contrast to the block copolymers, they would associate more externally to the lipidic particles and exert less influence on the internal structure than block copolymers. Several HMPs are commercially available and have found broad applications inter alia in paint, hair care, and pharmaceutical applications. In this work, we have investigated the possibility of using a commercially available hydrophobically modified ethyl hydroxyethyl cellulose (HMEHEC) to stabilize lipid dispersions in the MO-water system. Indeed it is found that they behave very differently from previously used stabilizers with respect to both particle structure and stability and thus add a new dimension in the area of the dispersion of cubic and other liquid crystalline particles. Materials and Methods Materials. Monoolein (MO), Ryol Mg 19-glycerol monooleate, no. 98-027, JR 1876/88 was kindly provided by Danisco Ingredients (Braband, Denmark). This is a commercially available product. It is important to note that it is not a single component product but contains several polar substances. The sample consisted of 98.8% monoglycerides, 0.1% glycerol, 1.0% diglycerides, and 0.1% free fatty acids. The fatty acid composition was C16 0.5%, C18 2.0%, C18:1 92.3%, C18:2 4.3%, C18:3 trace, C20:4 0.5%. Water was deionized using a Millipore water system. Deuterated water (99.8% 2H2O) was obtained from Dr. Glaser AG, Basel, Switzerland. The hydrophobically modified polymer HMEHEC was supplied by Akzo Nobel AB, Stenungsund, Sweden. HMEHEC is an ethyl hydroxyethyl cellulose ether (Figure 1) with an average molecular weight of 100 000 Da. The degree of ethyl and hydroxyethyl substitutions were DSethyl ) 0.6-0.7 and MSEO ) 1.8, respectively. The DS and MS values correspond to the average number of ethyl and hydroxyethyl groups per repeating anhydroglucose unit of the polymer. The MW, DSethyl, and MSEO were all given by the manufacturer. The hydrophobic modification consists of a low number of nonylphenol groups grafted onto the polymer backbone. The degree of HM modification was measured by UV absorbance of aromatic ring at 275 nm using phenol in aqueous solution as a reference. It was determined to 0.64 mM in a 1w/w% polymer solution. This number suggests a HM modification of 1.7 mol % and corresponds to an average of about 6.5 HM groups per polymer chain. Before use, the polymer was purified as described elsewhere.26 Sample Preparation and Equilibration. Samples were prepared by weighing appropriate amounts of lipid, polymer, and aqueous (22) Rangelov, S.; Almgren, M. J. Phys. Chem. B 2005, 109, 3921. (23) Almgren, M.; Rangelov, S. J. Dispersion. Sci. Technol. 2006, 27, 599. (24) Barauskas, J., Johnsson, M.; Joabsson, F.; Tiberg, F. Langmuir. 2005, 21, 2569. (25) Spicer, P. T.; Small, W. B.; Lynch, M. L.; Burns, J. L. J. Nanopart. Res. 2002, 4, 297. (26) Thuresson, K.; Karlstro¨m, G.; Lindman, B. J. Phys. Chem. 1995, 99, 3823.
Langmuir, Vol. 23, No. 5, 2007 2769
Figure 1. Principal structure of HMEHEC. solution in sample tubes (φ ) 1.5 cm). The sample size was about 10 g. The samples were thoroughly mixed on a rocking shaker for at least 7 days at 25 °C. To certify that the formation of dispersed structures did not depend on the path of preparation, samples were prepared along different preparation lines: (i) all three components were mixed at the same time and then left to equilibrate, (ii) aqueous HM-EHEC samples were prepared and left to equilibrate before addition of MO, (iii) samples with a water content of 80 wt % were prepared and then diluted. Since it was impossible to obtain a homogeneous dispersion by this gentle mixing, a microfluidizer (M-110S) was used for homogenization of the samples. The samples were circulated 5 times at a pressure of 5000 psi; 6 mL of the homogeneous sample was freeze-dried and then rehydrated after freeze-drying to investigate if and how the microstructure would be affected by a drying-rehydration step. Before being freeze-dried, the samples were frozen for 2 min in a mixture of dry ice and acetone. The freeze-drying was performed at -97 °C and a pressure of
