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Cubic Phase Nanoparticles (Cubosome†): Principles for Controlling Size, Structure, and Stability Justas Barauskas,*,‡,§ Markus Johnsson,‡,§ Fredrik Joabsson,§ and Fredrik Tiberg‡,§ Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden, and Camurus AB, Ideon, Science Park, SE-223 70 Lund, Sweden Received September 29, 2004. In Final Form: January 14, 2005 Methods and compositions for producing lipid-based cubic phase nanoparticles were first discovered in the 1990s. Since then a number of studies have been presented, but little is known about how to control key properties such as particle size, morphology, and stability of cubic phase dispersions. In the present work we give examples of how these properties can be tuned by composition and processing conditions. Importantly we show that stable particle dispersions with consistent size and structure can be produced by a simple processing scheme comprising a homogenization and heat treatment step.
Introduction The aqueous phase behavior of unsaturated monoglycerides (uMGs), such as glycerol monooleate (GMO) and glycerol monolinoleate (GMLO), has been thoroughly investigated due to their extensive polymorphism and widespread use in industrial products, e.g., food.1,2 Depending on various molecular and ambient conditions, uMGs can form four liquid crystalline mesophases, lamellar, reversed hexagonal, and two reversed bicontinuous cubic phases, Q230 and Q224.3-5 Although other lipids and surfactants self-assemble into cubic phases, a particularly attractive feature of uMGs is that they form the cubic phase in equilibrium with excess aqueous solution. This is a key property of great importance in applications where it is desirable that the integrity of the lipid mesophase structure is kept in diluted media. This exceptional physical chemical behavior has stimulated the use of aqueous uMGs as a model for biomembranes in a broad range of structural and functional investigations of proteins,6-8 peptides,9-11 and other biomolecules12-14 entrapped in the uMG cubic phase. The uMGs cubic phases * To whom correspondence should be addressed. Tel.: +46 46 2228155. Fax: +46 46 2224413. E-mail: Justas.Barauskas@ camurus.ideon.se. † Cubosome is a USPTO registered trademark of Camurus AB, Sweden. ‡ Lund University. § Camurus AB. (1) Krog, N. In Food Emulsions; Larsson, K., Friberg, S., Eds.; Marcel Dekker: New York, 1997; p 141. (2) Clogston, J.; Rathman, J.; Tomasko, D.; Walker, H.; Caffrey, M. Chem. Phys. Lipids 2000, 107, 191-220. (3) Lutton, E. S. J. Am. Oil Chem. Soc. 1965, 42, 1068-1070. (4) Hyde, S.; Andersson, S.; Eriksson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (5) Qiu, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (6) Ericsson, B.; Larsson, K.; Fontell, K. Biochim. Biophys. Acta 1983, 729, 23-27. (7) Razumas, V.; Larsson, K.; Miezis, Y.; Nylander, T. J. Phys. Chem. 1996, 100, 11766-11774. (8) Barauskas, J.; Razumas, V.; Nylander, T. Prog. Colloid Polym. Sci. 2000, 116, 16-20. (9) Lee, J.; Kellaway, I. W. Int. J. Pharm. 2000, 204, 137-144. (10) Angelova, A.; Ollivon, M.; Campitelli, A.; Bourgaux, C. Langmuir 2003, 19, 6928-6935. (11) Masum, S. Md.; Li, S. J.; Tamba, Y.; Yamashita, Y.; Tanaka, T.; Yamazaki, M. Langmuir 2003, 19, 4745-4753. (12) Caboi, F.; Nylander, T.; Razumas, V.; Talaikyte˘ , Z.; Monduzzi, M.; Larsson, K. Langmuir 1997, 13, 5476-5483.
have also been suggested for use in practical/technical applications such as bioelectrodes,15,16 biosensor construction,17,18 and protein19-23 and metal nanoparticle24,25 crystallization. Because of the three-dimensional nanostructure with hydrophobic and hydrophilic domains, cubic liquid crystalline phases have also found application in pharmaceutical drug delivery applications. The large interfacial area can provide complex diffusion pathway for sustained release of entrapped drug molecules, whereas lipid constituents are biocompatible, bioadhesive, and digestible.26-29 A limitation of cubic phases in some practical applications, such as oral, intravenous, or nasal drug delivery, is the macroscopic size and high viscosity of the bulk materials. Among other things this imposes in vivo transport problems in, e.g., intravenous, oral, and nasal drug delivery applications. It was soon realized that in order to use lipid cubic liquid crystalline materials in (13) Razumas, V.; Talaikyte˘ , Z.; Barauskas, J.; Nylander, T.; Miezis, Y. Prog. Colloid Polym. Sci. 1998, 108, 76-82. (14) Barauskas, J.; Razumas, V.; Nylander, T. Chem. Phys. Lipids 1999, 97, 167-179. (15) Rowinski, P.; Bilewicz, R.; Stebe, M.-J.; Rogalska, E. Anal. Chem. 2002, 74, 1554-1559. (16) Rowinski, P.; Bilewicz, R.; Stebe, M.-J.; Rogalska, E. Anal. Chem. 2004, 76, 283-291. (17) Razumas, V.; Kanapieniene˘ , J.; Nylander, T.; Engstro¨m, S.; Larsson, K. Anal. Chim. Acta 1994, 289, 155-162. (18) Barauskas, J.; Razumas, V.; Talaikyte˘ , Z.; Bulovas, A.; Nylander, T.; Tauraite˘ , D.; Butkus, E. Chem. Phys. Lipids 2003, 123, 87-97. (19) Landau, E. M.; Rosenbusch, J. P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14532-14535. (20) Pebay. Peyroula, E.; Rummel, G.; Rosenbusch J. P.; Landau, E. M. Science 1997, 277, 1676-1681. (21) Cherezov, V.; Fersi, H.; Caffrey, M. Biophys. J. 2001, 81, 225242. (22) Cherezov, V.; Clogston, J.; Misquitta, Y.; Abdel-Gawad, W.; Caffrey, M. Biophys. J. 2002, 83, 3393-3407. (23) Katona, G.; Andreasson, U.; Landau E. M.; Andreasson, L. E.; Neutze, R. J. Mol. Biol. 2003, 331, 681-692. (24) Puvvada, S.; Baral, S.; Chow, G. M.; Qadri, S. B.; Ratna, B. R. J. Am. Chem. Soc. 1994, 116, 2135-2136. (25) Yang, J. P.; Qadri, S. B.; Ratna, B. R. J. Phys. Chem. 1996, 100, 17255-17259. (26) Norling, T.; Lading, P.; Engstro¨m, S.; Larsson, K.; Krog, N.; Nissen, S. S. J. Clin. Perodontol. 1992, 19, 687-692. (27) Appel, L.; Engle, K.; Jensen, J.; Rejewski, L.; Zentner, G. Pharm. Res. 1994, 11, S217. (28) Nielsen, L. S.; Schubert, L.; Hansen, J. Eur. J. Pharm. Sci. 1998, 6, 231-239. (29) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456.
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delivery of drugs, nutraceuticals, and diagnostic agents and still take advantage of the cubic nanostructure, it is often necessary to use small fragments, preferably nanoparticles, of the bulk cubic phase. The preparation of stable colloidal dispersions of the cubic liquid crystalline phase, Cubosome,30,31 has opened exciting new opportunities for application of cubic phase materials. The first versions of a fragmented cubic phase were recognized almost two decades ago when it was proposed that the uMG cubic phase may be dispersed into micrometer-sized particles by mechanical breakup in the presence of micellar solutions of bile salts or caseins. The action of these agents was explained in terms of a formation of a lamellar envelope on the surface of the cubic phase particle.32,33 Later it was discovered that amphiphilic block copolymers can provide very powerful stabilization for the uMG cubic phase dispersions.34 A number of studies clearly demonstrate that poly(ethylene oxide)-based stabilizers, such as Lutrol F127, are suitable candidates for efficient stabilization of the GMO cubic phase dispersions while simultaneously preserving the inner cubic structure of the particles.35-40 The formation of colloidally stable cubic phase particles was related to the preferential location of F127 on the surface of the particles. Furthermore, the presence of vesicular structures in the dispersions indicated further stabilization of the cubic phase particles by a coexisting lamellar (LR) phase. The simplest method of producing cubic phase particles is the agitation of the GMO cubic phase with a magnetic stirrer in the presence of F127 resulting in a coarse dispersion with a particle size in the range of 1-100 µm.41 Further reduction in size can be achieved by use of highshear energy input techniques such as ultrasonication, homogenization, and emulsification of the coarse dispersion.40,42-44 However, these methods result not only in a desirable size reduction of cubic phase particles but also in the formation of vesicles which in terms of number density typically dominate over the cubic phase particles. Another method for producing GMO/F127 cubic phase dispersions was also introduced.45 It is based on mixing the GMO with ethanol in miscible proportions and dilution of the system with aqueous solution containing stabilizer, i.e., Lutrol F127. If the dilution trajectory falls into a cubic phase region, the cubic phase particles are formed (30) Landh, T.; Larsson, K. U.S. Patent 5,531,925, 1993. (31) Larsson, K. Curr. Opin. Colloid Interface Sci. 2000, 5, 64-69. (32) Bucheim, W.; Larsson, K. J. Colloid Interface Sci. 1987, 117, 582-583. (33) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (34) Landh, T. J. Phys. Chem. 1994, 98, 8453-8467. (35) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (36) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (37) Siekman, B.; Bunjes, H.; Koch, M. H. J.; Carlson, T.; Westesen, K. Proc. Int. Symp. Controlled Release Bioact. Mater. 1997, 24, 943944. (38) Neto, C.; Aloisi, G.; Baglioni, P.; Larsson, K. J. Phys. Chem. 1999, 103, 3896-3899. (39) Monduzzi, M.; Ljusberg-Wahren, H.; Larsson, K. Langmuir 2000, 16, 7355-7358. (40) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2001, 17, 3917-3922. (41) Ljusberg-Wahren, H.; Nyberg, L.; Larsson, K. Chim. Oggi 1997, 14, 40-43. (42) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (43) Siekman, B.; Bunjes, H.; Koch, M. H. J.; Westesen, K. Int. J. Pharm. 2002, 244, 33-43. (44) Esposito, E.; Eblovi, N.; Rasi, S.; Drechsler, M.; Di Gregorio, G. M.; Menegatti, E.; Cortesi, R. AAPS Pharm. Sci. 2003, 5, A30. (45) Spicer, P. T.; Hayden, K. L. Langmuir 2001, 17, 5748-5756.
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spontaneously due to molecular diffusion difference at the liquid/liquid interface. Minor modifications to the above preparation procedures have also been introduced.46,47 However, also with this preparation method, a substantial amount of vesicular material was obtained and typically the particle size distributions were rather broad. In fact, judging from the published cryo-TEM images, the majority of the particles displayed vesicular morphology.45 Although several methods have been developed for the manufacturing of cubic phase dispersion, there is still no established process available for controlling the properties and quality of cubic phase particle dispersions. Here we present new methodological protocols for manufacturing of GMO-based cubic phase dispersions of consistent structure and size. Importantly the particle dispersions of the method exhibit an excellent colloidal stability during storage and dilution. The method comprises use of highenergy microfluidization followed by heat treatment which facilitates preparation of the cubic phase dispersions containing negligible amounts of vesicles with good control of the particle size distributions in the range of 100-500 nm. The formation of well-organized cubic phase particle dispersions in mixtures of GMO/F127 and GMO/Oleic acid (OA)/F127 are investigated and discussed in terms of particle size distribution, nanostructure, and stability. Experimental Section Materials. A mixture of mono- and diglycerides (44:1 by weight), denoted as RYLO MG19 glycerol monooleate (GMO), was produced and provided by Danisco Ingredients (Brabrand, Denmark) with the following fatty acid composition (lot no. 2119/ 65-1): 89.3% oleic, 4.6% linoleic, 3.4% stearic, and 2.7% palmitic acid. The poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-poly(ethylene oxide) triblock copolymer with the trade name Lutrol F127 and an approximate formula of PEO98PPO57PEO98 (average molecular weight of 12600 g/mol) was obtained from BASF Svenska AB (Helsingborg, Sweden). Oleic acid (OA) was purchased from Apoteket (Sweden). Sterile water from B. Braun Medical AB (Bromma, Sweden) was used for all experiments. All chemicals were used as received. Sample Preparation. Coarse dispersions were prepared by adding appropriate amounts of melted (40 °C) GMO or GMO/OA mixture (95/5 w/w) into F127 solution. In all experiments the lipid/polymer ratio was 9/1 (w/w) unless otherwise stated. The sample volume was usually 50-200 mL. The samples were immediately sealed, hand-shaked, and allowed to vortex for 1248 h on a mechanical mixing table at ca. 300 rpm and room temperature. The resulting coarse dispersions were homogenized by passing five to eight times through a Microfluidizer 110S (Microfluidics Corp., Newton, MA) at 345 bar and 25 °C. The heat treatment of the homogenized samples was performed using a bench-type autoclave (CertoClav CV-EL, Certoclav Sterilizer GmbH, Traun, Austria) operated at 125 °C and 1.4 bar vapor pressure. The samples were filled into Pyrex glass bottles (50500 mL) and put into the autoclave. A period of about 12 min was required to vent the entrapped air and to heat up the autoclave. The samples were then subjected to heat treatment for 20 min at 125 °C unless otherwise indicated. After the heat treatment, the samples were allowed to cool to room temperature before analysis. In some cases more concentrated dispersions were optionally filtered through 5 µm Acrodisc syringe filter (Pall Corp., Ann Arbor, MA) to remove small amounts of large precipitates formed during cooling at the air/liquid interface. Acid Level Determination. To determine the extent of hydrolysis of GMO during heat treatment, the acid value (IA) of heat-treated dispersions was determined using a standard method comprising titration of a lipid sample with potassium (46) Chung, H. C.; Kim, J.; Um, J. Y.; Kwon, I. C.; Jeong, S. Y. Diabetologia 2002, 45, 448-451. (47) Um, J. Y.; Chung, H.; Kim, K. S.; Kwon, I. C.; Jeong, S. Y. Int. J. Pharm. 2003, 253, 71-80.
Cubic Phase Nanoparticles hydroxide (KOH). Briefly, the dispersions were sampled before and after heat treatment and the aqueous phase was removed by freeze-drying overnight. A 50/50 (v/v) mixture of ethanol (95%) and diethyl ether was added to the lipid residue, and the lipids dissolved. A few drops of phenolphthalein were added, and the sample was titrated with 0.01 M KOH until the pink color of the solution persisted for at least 15 s. A solution (ethanol/diethyl ether; 50/50 v/v) without lipid was used as a reference, and the amount of KOH needed to neutralize the reference solution was subtracted from the final values for the sample solutions. The acid value was calculated as the amount of KOH (in milligrams) required to neutralize the free acids present in 1 g of the lipid sample. Particle Size Measurements. Particle size distributions were measured using a Coulter LS230 laser diffraction particle size analyzer (Beckman-Coulter, Inc., Miami, FL) which operates on the principles of Fraunhofer diffraction for large particles (0.4-2000 µm) and uses the polarization intensity differential scattering (PIDS) method for small particles (0.04-0.5 µm). The instrument was fitted with a small volume module (125 mL). Data were collected during 90 s. The particle sizes were calculated by using a homogeneous oil sphere model with a refractive index (RI) of 1.46. The change in RI to either side does not change the obtained results dramatically (only within a few percent). X-ray Diffraction. Measurements were performed on a Kratky compact small-angle system equipped with an OED 50 M position-sensitive detector (MBraun, Graz, Austria) containing 1024 channels of width 53.1 µm. Cu KR nickel-filtered radiation of wavelength 1.542 Å was provided by a Seifert ID 3000 X-ray generator (Rich Seifert, Ahresburg, Germany) operating at 50 kV and 40 mA. The samples were filled into a 1 mm (i.d.) quartz capillary in a steel sample holder and the sample-to-detector distance was 277 mm. To minimize scattering from air, the camera volume was kept under vacuum during the measurements. Temperature control within 0.1 °C was achieved using a Peltier element. The recorded slit-smeared diffraction patterns were desmeared and evaluated using 3D-View software (MBraun). Cryogenic Transmission Electron Microscopy (cryoTEM). The samples were prepared in a controlled environment vitrification system. The climate chamber temperature was 2528 °C, and the relative humidity was kept close to saturation to prevent evaporation from the sample during preparation. A 5 µL sample drop was placed on a carbon-coated holey film supported by a copper grid and gently blotted with filter paper to obtain a thin liquid film (20-400 nm) on the grid. The grid was then rapidly plunged into liquid ethane at -180 °C and transferred into liquid nitrogen (-196 °C). The vitrified specimens were stored in liquid nitrogen and transferred into a Philips CM120 BioTWIN microscope equipped with a postcolumn energy filter (Gatan GIF 100) using an Oxford CT 3500 cryo-holder and its workstation. The acceleration voltage was 120 kV, and the working temperature was kept below -180 °C. The images were recorded digitally with a CCD camera (Gatan MSC 791) under low-dose conditions with an underfocus of approximately 1 µm.
Results and Discussion Earlier studies of the GMO/F127 bulk system and its dispersions have shown that the choice of the lipid-topolymer ratio is crucial to produce colloidally stable cubic phase particles at high dilutions. The previously reported ternary GMO/F127/water phase diagram is extremely complicated.34 With increasing F127 concentration the cubic phase region (Q224 and Q230 phases) originating from the binary GMO/water system is transformed into the cubic Q229 phase that exists over a large composition range with respect to both F127 (up to 20 wt %) and water (swelling limit about 65 wt %). At an approximate GMO/ F127 ratio of 70/30 (w/w) and at a water content of 70 wt %, an LR phase appears that transforms into a sponge phase (L3) at even higher dilution. These data indicate that in order to produce colloidally stable cubic phase dispersions the GMO/F127 ratio should be kept in the range of about 94/6-80/20 (w/w) where the Q229 phase or Q229 + L3 (or LR) phase is formed in equilibrium with excess
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Figure 1. Particle size distributions of spontaneously formed GMO/F127 (9/1 w/w) dispersions after 0 (curve 1) and 12 (curve 2) h of mechanical mixing. The water content is 95 wt %.
solution at lower and higher polymer concentrations, respectively. Lower concentrations of polymer will simply lead to unstable dispersions due to nonsufficient density of sterically stabilizing polymer F127 at the particle surfaces. Higher concentrations will shift the system into a L3 (or LR) + solution phase region and induce the formation of vesicular structures. In this study we have fixed the GMO/F127 ratio at 90/10 (w/w) to ensure that the composition corresponds or is close to an equilibrium three-phase region (Q229 + L3 (or LR) + solution phase) at dilutions higher than 90 wt % water. In the case of ternary GMO/OA/F127 mixtures, the ratios of GMO/OA and (GMO + OA)/F127 were fixed to 95/5 and 90/10 (w/w), respectively. As can be seen from Figure 1, coarse GMO/F127 dispersions can be prepared by simple mixing of the components followed by mechanical shaking for prolonged periods of time (g12 h). Although the particle size measurements indicate that the dispersion consists of particles in the size range of 0.2-2 µm, there is also a fraction of larger visually observable macroscopic particles of diameter g10 µm that tend to accumulate at the surface of the dispersion. Moreover, mechanical shaking does not allow control of the dispersion process, and the quality of the obtained dispersion depends on a number of factors, such as initial condition of the material, dispersion volume, treatment time, mixing intensity, etc. Similar results were also observed for coarse dispersions of GMO/OA/F127. It is hence generally necessary to homogenize GMO-based dispersions in order to get rid of macroscopic particle contaminants and further narrow the particle-size distribution before further processing can be performed. As pointed out in earlier studies,40,42-44 high shear energy homogenization is a very powerful technique for converting large particles into smaller. However, the attempts to prepare cubic phase dispersions with narrow particle size distributions and mean particle sizes of a few hundred nanometers usually lead to dispersions consisting of almost only noncubic structures. The high shear forces applied break down the particles with cubic structure into nonequilibrium vesicular structures which later typically fuse into colloidally unstable larger particles. Results from recent work has shown that heat cycling of homogenized dispersions results in improved properties of cubic phase dispersions such as more uniform size distributions and/ or particle morphologies as well as improved colloidal stability.48 In the present study we have performed a (48) Wo¨rle G.; Bunjes, H.; Tiberg, F.; Siekmann, B. Johnsson, M.; Barauskas, J. Method for Improving the Properties of Amphiphile Particles. International Patent Application No. PCT/GB2004/003387.
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Figure 2. Particle size distributions of homogenized (curve 1) and then heat treated (125 °C) (curve 2) dispersions: (a) GMO/ F127 (b) GMO/OA/F127. Both dispersions have lipid/polymer ratios of 9/1 and a water content of 95 wt %.
systematic investigation of the interrelation between processing conditions including the effect of heat treatment on the properties of the resulting colloidal dispersions. To investigate the heat treatment effect in detail, aqueous mixtures of GMO/F127 and GMO/OA/F127 were first coarsely dispersed, homogenized, and then autoclaved. In each preparation step dispersions were analyzed by means of particle size distribution, X-ray diffraction, and cryo-TEM. The results obtained are presented in Figures 2-5. By use of microfluidizer, homogenization of the GMO/ F127 sample containing 95 wt % water results in slightly translucent homogeneous dispersions characterized by monomodal particle size distributions with mean sizes ∼100 nm (Figure 2a). The homogenized dispersion displays only one weak X-ray reflection (Figure 3a, curve 2) indicating the presence of only some slightly ordered structure. In agreement with the particle size distribution and X-ray diffraction measurements, cryo-TEM images show that the sample consists of a relatively large fraction of small vesicles and a smaller fraction of particles with inner cubic structure (panels a and b of Figure 4). Heat treatment transforms the homogenized dispersion into a milky fluid with a mean particle size of about 450 nm (Figure 2a). X-ray diffraction measurements on this dispersion reveal three distinct Bragg peaks with relative positions in ratios x2, x4, and x6, which can be indexed as hkl ) 110, 200, and 211 reflections characteristic of a body-centered cubic phase of Im3m space group (Q229) with a lattice parameter of 130.2 ( 0.4 Å (Figure 3a, curve 3). The same type of the cubic structure and a similar value of the unit cell dimension were previously determined for the corresponding bulk and dispersed GMO/F127/water
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Figure 3. X-ray diffractograms of coarse (curve 1), homogenized (curve 2), and then heat treated (125 °C) (curve 3) dispersions: (a) GMO/F127 and (b) GMO/OA/F127. All dispersions have lipid/polymer ratio of 9/1 and the water content is 95 wt %. Miller indexes stand for Q229 structure, whereas values shown in rectangles denote the Q224 organization.
mixtures.34,36 Moreover, the position of the first 110 reflection of the heat-treated sample coincides with the position of the weak diffraction of coarsely dispersed and homogenized samples mentioned above. This clearly indicates that the latter dispersions in addition to vesicular disordered structures also contain small amounts of cubic phase particles with Im3m space group. More importantly, it shows that despite the heat treatment, the dispersions retain their corresponding equilibrium cubic structure. As can also be seen from the cryo-TEM images (panels c and d of Figure 4), the heat-cycled dispersion mainly contains aggregates of highly ordered cubic inner structure with sizes similar to that observed by laser diffraction. In Figure 4c, the cubic phase particle is viewed along the [111] direction, whereas in Figure 4d the electron micrograph shows two domains of the cubic phase particle which are aligned along the [111] and [001] viewing directions. The fast Fourier transformation (FFT) of the domains reveals four, {110}, {200}, {211}, and {220}, reflections of the Q229 structure with average spacings of 90.0, 64.7, 51.5, and 45.4 Å, respectively. From the FFT results, the calculated lattice parameter equals 127 ( 0.7 Å, in good agreement with the X-ray diffraction data. Homogenization of the GMO/OA/F127 system containing 95 wt % water results in a dispersion with similar properties as for the corresponding GMO/F127 system, i.e., formation of dispersions with a mean size of about 100 nm showing almost no X-ray diffraction (Figures 2b, 3b (curve 2), 5a, and 5b). After the heat treatment the dispersions become milky with a narrow, monomodal particle size distribution around 200 nm (Figure 2b). The mean particle size of the heat-treated sample is however
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Figure 4. cryo-TEM images of the homogenized (a, b) and then heat treated (125 °C) (c, d) GMO/F127 (9/1 w/w) dispersions containing 95 wt % water.
considerably smaller than that measured for the sample without added OA (see discussion below). A clear effect of the addition of OA is also seen from the X-ray diffraction results of the heat-treated GMO/OA/ F127 dispersion (Figure 3b, curve 3). From the eight Bragg peaks observed, the first and third are at the same positions as the corresponding reflections from the GMO/ F127 sample and can be indexed as hkl ) 110 and 211 reflections of the Q229 phase with the lattice parameter of 128 ( 0.2 Å (Figure 3b, curve 3). The further six reflections follow the relationship x2, x3, x4, x6, x8, and x9 which can be indexed as hkl ) 110, 111, 200, 211, 220, and 221 peaks of a primitive cubic lattice of the Pn3m crystallographic space group (Q224). The calculated unit cell dimension for this phase is 86.1 ( 0.4 Å. The observation of the Q224 phase in GMO/OA/F127 dispersions is not surprising. As shown by Borne and co-workers,49 OA induces the formation of liquid crystalline phases with negative curvatures in mixtures with GMO. Thus, in our system F127 and OA act in opposite directions, the polymer shifting the phase equilibrium toward the Q229 phase whereas OA tends to shift the phase equilibrium in the opposing direction back to the initial Q224 observed in the GMO-water system. In this respect it is important to emphasize that the pH of the dispersions was about 5.5 and the dissociation degree of OA is therefore predicted to be low.50 Most of the particles visible in the cryo-TEM (49) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2001, 17, 77427751. (50) Kanicky, J. R.; Shah, D. O. J. Colloid Interface Sci. 2002, 256, 201-207.
images are highly ordered cubic phase aggregates of size 100-200 nm (parts c and d of Figure 5). In both figures the particles are aligned along the [111] viewing direction. FFT analyses of the seven particles shown in parts c and d of Figure 5 display only {110} reflections with the spacing in the range of 59.9-60.9 Å. The calculated lattice parameter varies from 84.7 to 86.1 Å and is consistent with that determined by X-ray diffraction. As we only observe aggregates with Q224 structure, the question arises as to whether there are two groups of particles with different cubic structure or, if the particles are polymorphic, contain both the Q224 and the Q229 cubic phase structures within the same aggregate. As of yet we have no firm answer to this question. Nevertheless, the results above reveal that the combination of high-pressure microfluidization and the heat treatment is a very powerful method for producing cubic phase dispersions with consistent size and structural properties. Starting from the homogenized dispersions of small, predominantly vesicular-like particles, the heat treatment produces cubic phase nanoparticles with welldefined particle size distributions and inner morphology. The fraction of small vesicular particles is effectively converted to cubic phase particles; generally of larger size. It is worth noting that the heat treatment process can also result in a transformation of larger vesicles or vesicular-like particles into smaller cubic phase nanoparticles as observed from particles size distributions before and after heat treatment.48 This is not surprising considering that the lipid content of a large vesicle is low whereas the lipid content of cubic phase nanoparticles or
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Figure 5. cryo-TEM images of the homogenized (a, b) and then heat treated (125 °C) (c, d) GMO/OA/F127 dispersions containing 95 wt % water. The (GMO + OA)/F127 ratio is 9/1, and the water content is 95 wt %.
L2 phase particles (at high temperature, see below) is much higher. Accordingly, when a large vesicle is transformed into a cubic phase nanoparticle or L2 phase particle, the water content of the particle is decreased. Thus, the heat treatment process can be used to effectively narrow the particle size distribution of the cubic phase nanoparticle dispersion obtained after cooling. However, to achieve the desired result reproducibly, it is necessary to homogenize the dispersions before the heat treatment. The change in particle size and morphology on heat treatment requires transfer of lipid material from smaller to larger (or larger to smaller) aggregates. This can occur by molecular transport of aggregation/dissociation mechanisms. Here we will concentrate on the particle growth and transformation of vesicular to cubic phase particles observed after the heat treatment. Taking into account the lipid material needed to form the larger cubic aggregates from the vesicles, it seems reasonable to postulate that particle fusion occurs during the heat treatment process. One hypothesis is that fusion is related to the reduced solubility and stabilizing efficiency exhibited by poly(ethylene oxide)-based substances at elevated temperatures. The block copolymer F127 displays a cloud point (CP) in dilute aqueous solutions at temperatures just above 100 °C.51 Considering that the particles are sterically stabilized by adsorbed F127, very slow or even negligible fusion is expected below the CP. Only after reaching the CP a significantly fast particle fusion will
occur. Support for this hypothesis is found in the observation that that prolonged heating (2 h) of the GMO/F127 homogenized dispersion at 95 °C, i.e., below the CP of F127, did not induce the transformation of the vesicular fraction to larger ordered particles (data not shown). In addition to the decreased interparticle repulsion above CP, the lateral interactions between the tethered poly(ethylene oxide) chains of F127 will decrease resulting in change to more negative curvature and consequently providing a further driving force for the transformation of lamellar particles to particle structures with more negative curvature. The nature of the aggregates formed at high temperature (125 °C) has not been established, but it is wellknown that GMO forms an L2 phase in water at temperatures above 90 °C. It is likely that the dispersion observed at 125 °C consists of fragments of L2 phase. This is supported by a recent study of the closely related GMLO/ F127 system where dispersed L2 phase particles were identified at elevated temperatures.52 GMO displays a phase behavior very similar to GMLO with the L2 phase being formed in the GMO/H2O bulk system above ca. 90 °C.4,5 As shown in previous studies,35,36,40,44 homogenization of GMO/F127 L2 phase at higher temperatures followed by cooling can be also used to produce cubic phase dispersions. However, as seen from Figure 6, direct homogenization of dispersed L2 phase at 80-90 °C yields much broader particle size distribution and higher poly-
(51) Pandit, N.; Trygstad, T.; Croy, S.; Bohorquez, M.; Koch, C. J. Colloid Interface Sci. 2000, 222, 213-220.
(52) De Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254-5261.
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Figure 6. Particle size distributions of the directly homogenized at 80 (curve 1) and 90 °C (curve 2) GMO/F127 (9/1) dispersions. Particle size distribution of the homogenized (at room temperature) and then heat treated (125 °C) GMO/F127 (9/1) dispersion. The water content is 95 wt % in all cases.
dispersity. These results clearly show that the method presented in the present communication provides better control of dispersion quality (e.g., size, mesophase structure, shape, and stability) than that obtained by other means of preparation. As is shown in the present work cubic phase particles are identified when the dispersion has been cooled to room temperature (RT), representing a stable colloidal nanodispersion of the thermodynamically stable parent cubic phase (+ solution phase) at RT. The heat treatment process can thus be viewed as a means of changing from one metastable state, represented by lamellar particles, to an
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energetically more favorable state represented by cubic phase nanoparticles. Interestingly, the particle growth observed as a consequence of the heat treatment terminates at a particular size range, which is shown to depend on several factors, particularly the concentration of amphiphile (lipid + polymer), presence of charged lipids, and the ionic strength of the solution. Figure 7 shows the dependence of the mean particle size on the total amphiphile (lipid + polymer) concentration. With increasing amphiphile concentration (from 1 to 5 wt %), the particle size of the heat treated dispersion increases almost linearly from about 200 to 450 nm and from 120 to 200 nm for the GMO/F127 and GMO/OA/F127 systems, respectively. This dependence is also observed in the cryo-TEM images. Figure 8 shows cryo-TEM images of heat treated GMO/F127 (88/12 w/w) dispersions containing 2 wt % of amphiphile. Although in the latter example the dispersion is prepared at slightly lower lipid/polymer ratio, the particle size measurements show practically the same result as with the corresponding GMO/F127 (9/1) dispersions. It is clear from Figure 8 that the cubic phase particles made from 2 wt % of amphiphile are considerably smaller than those prepared in the presence of 5 wt % (panels c and d of Figure 4). It is very important to note that once prepared, the particles in the dispersions do not change the size with time or dilution thus enabling the use of amphiphile concentration as a tool for tuning the size distribution of the cubic phase particles. In view of these results it seems that the particles at high temperatures transform by fusion and structure transition processes to a quasi-equilibrium size. Not even
Figure 7. Particle size distributions as a function of total amphiphile concentration (1, 2, 3, 4, and 5 wt %) of the homogenized and then heat treated (125 °C) GMO/F127 (a) and GMO/OA/F127 (b) dispersions. (c) Dependencies of the obtained mean particle size of the heat treated GMO/F127 (1) and GMO/OA/F127 (2) dispersions on the total amphiphile concentration. Error bars represent the mean value ( standard error of one to five separate experiments. Linear approximations are drawn to guide the eye.
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Barauskas et al. Table 1. Acid Values (IA) before and after Heat Treatment (125 °C) of GMO/F127 (9/1 w/w) Dispersions Containing 98 wt % Water dispersion
IA (mg of KOH/g of lipid)
reference; homogenized heat treatment; 125 °C, 20 min heat treatment; 125 °C, 40 min
1.1 ((0.3)a 1.2 ((0.3)a 1.4 ((0.3)a
a
Figure 8. cryo-TEM images of homogenized and then heat treated (125 °C) GMO/F127 (88/12 w/w) dispersions containing 98 wt % water.
Figure 9. The dependence of the heat treatment time on the mean particle size of GMO/F127 (9/1 w/w) dispersions containing 0.5 (curve 1) and 2 wt % (curve 2) of amphiphile (GMO + F127). Error bars represent the mean ( standard error of the particle size distributions. The lines are drawn to guide the eye.
prolonged heat treatment time causes the particles to grow after reaching the quasi-equilibrium state (Figure 9). We also show that electrostatic interactions play an important role in this process. At the conditions in the present study, the particles composed of GMO/OA/F127 possess some negative charge, a feature that evidently results in smaller particles compared to the GMO/F127 system. Assuming that the charges are preferentially located at the outermost surface (segregation of charged components in the L2 phase
Estimated accuracy of the acid value determination.
particles at elevated temperatures), the dependence of particle size on the amount of charged amphiphile can be partly understood in terms of surface charge density. The smaller the particles, the higher the surface area for a fixed volume fraction of amphiphile. Thus, the surface charge density is lowered by the formation of smaller particles. If the electrostatic interactions are important in determining the final size of the heat treated particles, it is reasonable to assume that the surface charge density should remain close to constant for the GMO/F127 and GMO/OA/F127 particle systems. To obey this condition, it is obvious that smaller particles have to be formed when more of the charged amphiphile is added to the mixture (again assuming segregation of charges to the surface of the L2 phase particles at high temperature). For simplicity we may also assume that the L2 particles formed at 125 °C are spherical. As shown in Figure 7, the diameter of the GMO/F127 particles formed at 5 wt % is about 460 nm and the corresponding diameter in the GMO/OA/F127 system is about 195 nm. In terms of surface area this gives a 2.4 times larger surface area (based on spherical particles) for the GMO/OA/F127 system. Knowing that the GMO/F127 system also contains some OA (about 1 wt %, see below) and that the GMO/OA/F127 system has been fortified with 5 wt % OA (with respect to GMO), a factor of 2.4 seems to indicate that the assumed trend toward nearly equal surface charge density for the two types of particle systems is correct. Thus, this effect probably explains why the heat treated dispersions with OA are characterized by smaller particle sizes. Since the GMO batch used in this study has admixed small amounts of free fatty acids (see below), electrostatic interactions are clearly important also in the case of the GMO/F127 system. This is shown by an attempt to autoclave the homogenized GMO/F127 dispersion in 10 mM NaCl which led to millimeter sized aggregates (data not shown). In this case the salt screens the particle surface charge and induces a rapid growth and macroscopic phase separation. Such interesting effects of the total amphiphile concentration, the amount of charged species, and salt content provide very simple and elegant ways for controlling the characteristics of the cubic liquid crystalline phase dispersions. Indeed, salt can be added to the dispersion once the steric repulsion between particles is operating below the CP of the stabilizing component. An important issue, especially from an application point of view, is the chemical stability of the components during heat treatment and the colloidal stability (storage stability) of a processed dispersion. The main concern is the possible hydrolysis of GMO giving oleic acid and glycerol as products. Table 1 summarizes the results obtained from a determination of the acid value (IA) before and after heat treatment for 20 and 40 min. As may be seen, the temperature exposure does not cause any significant hydrolysis of GMO and there is no significant increase of IA with time up to at least 40 min of heat treatment. In this respect it may be noted that the acid value of the pure GMO batch used for the experiments was 0.4 as given by
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are on the same order as the pure GMO batch. Assuming that only oleic acid is formed by hydrolysis of the GMO, we can estimate that the acid values of 1.1-1.4 correspond to 0.55-0.70 wt % of OA with respect to GMO. Finally, the heat treated GMO/F127 and GMO/OA/F127 dispersions are extremely stable to shelf-storage (quiescent conditions) at RT (Figure 10). The corresponding colloidal stability of homogenized cubic phase dispersions of GMO/ F127 and GMO/OA/F127 that have not been heat treated is on the other hand limited. This further signifies the importance of the heat treatment principle. Conclusions A combination of high shear energy homogenization and heat treatment provides a powerful and scalable way for producing GMO-based cubic phase nanoparticles. The heat treatment of the homogenized dispersions consisting of predominantly vesicular-like particles results in their effective and reproducible conversion to cubic phase particles with narrow particle size distribution and welldefined inner morphology. The process is simple and results in Cubosome nanoparticles containing minimal amounts of lamellar aggregates and possessing good colloidal stability. In addition, the amphiphile concentration, the amount of charged species, and salt content provide an elegant way of further controlling dispersion particle size and nanostructure.
Figure 10. Particle size distributions (a) and the mean particle sizes (b) as a function of time of the homogenized and then heat treated (125 °C) GMO/F127 (1) and GMO/OA/F127 (2) dispersions.
the manufacturer (Danisco, Brabrand, Denmark). Thus, the acid values obtained for the heat treated dispersions
Acknowledgment. We are grateful to Gunnel Karlsson for help with cryo-TEM instrumentation and Professors Heike Bunjes and Kåre Larsson for valuable contributions. This work is performed in the “New Principles for Oral Delivery of Peptides and Petidomimetics” program sponsored by the Swedish Foundation for Strategic Research, Vinnova, and Camurus Lipid Research Foundation. LA047590P
