Langmuir 2005, 21, 5159-5165
5159
Aqueous Phase Behavior and Dispersed Nanoparticles of Diglycerol Monooleate/Glycerol Dioleate Mixtures Markus Johnsson,*,†,‡ Yee Lam,†,‡,§ Justas Barauskas,†,‡ and Fredrik Tiberg†,‡ Physical Chemistry 1, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and Camurus AB, So¨ lvegatan 41, Ideon Science Park Gamma 1, SE-223 70 Lund, Sweden Received January 21, 2005. In Final Form: March 17, 2005 The first part of this study concerns the aqueous phase behavior of mixtures of diglycerol monooleate (DGMO) and glycerol dioleate (GDO) examined by X-ray diffraction (XRD). The ternary phase diagram displays a multitude of liquid crystalline phases (polymorphism). With increasing GDO content the following phase sequence was observed: lamellar (LR); two reversed bicontinuous cubic phases (Q230 and Q224); reversed hexagonal (HII); the reversed micellar (L2) phase. The second part deals with the preparation and characterization of aqueous dispersions of the reversed hexagonal phase in the presence of the nonionic triblock copolymer Pluronic F127. Submicrometer-sized monocrystalline HII phase particles were obtained, as evidenced by cryo-transmission electron microscopy (cryo-TEM), laser diffraction, and XRD, by use of a simple and reproducible preparation method including a heat-treatment step. Moreover, the particle size distributions of the HII phase nanoparticle dispersions were narrow as determined by laser diffraction measurements. Using XRD, we show that the polymeric stabilizer is depleted from the core of the hexagonal particles and preferentially located at the surface. It is concluded that the preferential distribution of stabilizing agents at particle surfaces is a prerequisite for the formation of structurally well-defined and kinetically stable HII phase particles (Hexosome).
Introduction Investigation of lipid phase behavior is stimulated by the multidisciplinary nature of the subject connecting biology, chemistry, and physics. Self-assembly of lipids in water gives rise to a multitude of liquid crystalline (LC) phases, where the lamellar phase (LR) is the most important phase in common lipid systems. Reversed phases have gained an increasing interest due to their fascinating structural architecture, potential uses, and possible biological relevance.1,2 The past decades have seen a new interest in lipid self-assembly structures for delivery of active substances through different portals of the body and thereby extended the application to pharmaceutical and medical science. The application of self-assembly objects in drug delivery3-5 is a major motivation for this study. Perhaps the most well-characterized lipid system forming reversed LC phases is the unsaturated monoglyceride (uMG) system.6,7 In particular, the phase behavior of glycerol monooleate (GMO) has attracted attention.6-9 A relatively unique feature of the GMO/water system is the * To whom correspondence should be addressed. E-mail:
[email protected]. † Lund University. ‡ Camurus AB. § Current address: Mechanical Engineering and Materials Science, Duke University, 144 Hudson Hall, Box 90300, Durham, NC 27708. (1) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661-668. (2) Landh, T. FEBS Lett. 1995, 369, 13-17. (3) Shah, J.; Sadhale, Y.; Chilikuri, D. M. Adv. Drug Delivery Rev. 2001, 47, 229-250. (4) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456. (5) Engstro¨m, S.; Ericsson B.; Landh, T. Proc. Int. Symp. Control. Relat. Bioact. Mater. 1996, 23, 89-90. (6) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (7) Qui, H.; Caffrey, M. Biomaterials 2000, 21, 223-234. (8) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forse´n, S. J. Am. Chem. Soc. 1979, 101, 5465-5470. (9) Larsson, K. Nature 1983, 304, 664.
coexistence of a bicontinuous cubic phase (space group Pn3m) with a dilute water phase. This behavior allows the formation of colloidal cubic phase particles (Cubosome)10 by use of a suitable dispersion method and fragmenting/stabilizing agents. The most frequently used stabilizing agent is Pluronic F127 (or Poloxamer 407).11-13 HII phases have also been shown to coexist with water at high dilution.12,14,15 However, forming colloidally stable HII nanoparticles (Hexosome)10 has proven to be difficult. The use of high-energy input has been necessary to produce colloidal size particles of limited kinetic stability.12,14 Here we present a system where such particles can be prepared using a minimum of energy input and still reproducibly producing highly stable HII nanoparticle dispersions. The prerequisite for making such preparations is the identification of a suitable well-characterized lipid system. With guidance of preliminary examinations we chose to investigate in detail the phase behavior of mixtures of diglycerol monooleate (DGMO) and glycerol dioleate (GDO) (Figure 1). DGMO is a lipid that forms an LR phase in water16,17 whereas GDO only forms an isotropic reversed micellar solution (L2 phase) that absorbs only a few % of water.18 When these two lipids are combined, several LC phases were found as predicted, including two cubic phases (10) Cubosome and Hexosome are USPTO registered trademarks of Camurus AB, Lund, Sweden. (11) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (12) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971. (13) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Langmuir 2005, 21, 2569-2577. (14) Kamo, T.; Nakano, M.; Leesajakul, W.; Sugita, A.; Matsuoka, H.; Handa, T. Langmuir 2003, 19, 9191-9195. (15) Rand, R. P.; Fuller, N. L. Biophys. J. 1994, 66, 2127-2138. (16) Holstborg, J.; Pedersen, B. V.; Krog, N.; Olesen, S. K. Colloids Surf., B 1999, 12, 383-390. (17) Pitzalis, P.; Monduzzi, M.; Krog, N.; Larsson, H.; LjusbergWahren, H.; Nylander, T. Langmuir 2000, 16, 6358-6365. (18) Borne´, J.; Nylander, T.; Khan, A. Langmuir 2000, 16, 1004410054.
10.1021/la050175s CCC: $30.25 © 2005 American Chemical Society Published on Web 04/26/2005
5160
Langmuir, Vol. 21, No. 11, 2005
Figure 1. Molecular structures of glycerol dioleate (GDO) (1) and diglycerol monooleate (DGMO) (2). Note that the lipid samples are mixtures of different isomers and that the molecular structures shown represent the dominating species in the respective samples.
and the reversed HII phase. The next question was to develop a method suitable for producing stable colloidal particles without destroying the inner phase structure. It was natural to use a comparably high molecular weight nonionic triblock copolymer (Pluronic or Poloxamer) as this might be excluded from the interior phase structure and thereby avoid unwanted mesophase structure transitions while adsorbing on particle surfaces and thereby effectively stabilizing them. Earlier studies supported this notion.12,14 In contrast, additions of rather small amounts of a conventional surfactant to a HII phase forming lipid mixture led to the transition to LR phase.19,20 Experimental Section Materials. Glycerol dioleate (GDO) and diglycerol monooleate (DGMO) were obtained from Danisco (Brabrand, Denmark). The composition of the GDO sample as given by the manufacturer was the following: diglycerides 87.5%; monoglycerides 11%; triglycerides 1.5%. The main fatty acid component was oleoyl (C18:1) constituting 91% of the acyl chains. The composition of the DGMO sample was as follows: diglycerol monoester 89%; free glycerol and polyglycerol 5.1%. The balance comprises diglycerol diester and small amounts of mono- and diglycerides. The fatty acid distribution was oleic (C18:1) 90%, saturated 4.7%, and linoleic and linolenic 5.3%. The poly(ethylene oxide) (PEO)poly(propylene oxide) (PPO)-poly(ethylene oxide) triblock copolymer with the trade name Pluronic F127 and approximate formula of PEO98PPO57PEO98 (average molecular weight of 12 600 g/mol) was obtained from BASF Svenska AB (Helsingborg, Sweden). Sterile water from B. Braun Medical AB (Bromma, Sweden) was used for all experiments. All chemicals were used as received. Sample Preparation. Samples were prepared by weighing appropriate amounts of DGMO and GDO into 14 mm (i.d.) glass ampules (total lipid amount of ca. 0.5- 1 g), comelting lipids at 50 °C, and then adding water. The vials were immediately sealed, vortexed, and centrifuged up and down at 1500g for 10 min. The samples were allowed to equilibrate at 25 °C for at least 3 weeks, with intermittent centrifugation, before measurements. To obtain phase homogeneity, samples were centrifuged up and down at 1500g for 10 min. If needed, the centrifugation was repeated several times. The samples were investigated between crossed polarizers to check sample homogeneity and the presence of birefringent phases. Dispersion Preparation. Dispersions were prepared by adding appropriate amounts of melted lipid mixture (DGMO/ GDO ) 60/40 or 50/50 wt/wt) into an aqueous F127 solution. In all experiments the lipid/polymer ratio was 9/1 (wt/wt) and the total amphiphile (lipid + polymer) concentration was generally between 2 and 10 wt %. The sample volume was usually 25-200 mL. The samples were immediately sealed, hand-shaken, and mixed for 12 h on a mechanical mixing table at 350 rpm and (19) Thurmond, R. L.; Lindblom, G.; Brown, M. F. Biophys. J. 1991, 60, 728-732. (20) Johnsson, M.; Bergstrand, N. Colloids Surf., B 2004, 34, 69-76.
Johnsson et al. room temperature. This procedure resulted in homogeneous milky white dispersions. Heat treatment of the dispersions was performed using an bench-type autoclave (CertoClav CV-EL, Certoclav Sterilizer GmbH, Traun, Austria) operated at 125 °C and 1.4 bar. The samples were filled into Pyrex glass bottles (50-500 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. After the heat treatment, the samples were allowed to cool to room temperature before analysis. 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. A standard model based on homogeneous oil spheres with a refractive index of 1.46 was used for the particle size calculations. Note that the model is based on spherical particles and that the measured mean particle size therefore is an apparent size. X-ray Diffraction. XRD experiments were performed on a Kratky compact small-angle system equipped with a positionsensitive wire detector (OED 50M from MBraun, Graz, Austria) containing 1024 channels of width 53.6 µm. Cu KR radiation of wavelength 1.542 Å was provided by a Seifert ID 3000 X-ray generator operated at 50 kV and 40 mA. Bulk LC samples were mounted between mica sheets in a steel body whereas dispersed samples were filled into a 1 mm (i.d.) quartz capillary in a steel sample holder. 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, Graz, Austria). 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 a 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 -182 °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 1. Phase Behavior. The phase equilibrium of the binary diglycerol monooleate (DGMO)/water and glycerol dioleate (GDO)/water systems has previously been reported.16-18 According to Holstborg et al.,16 DGMO exhibits two phases, the reversed micellar phase (L2) up to 8 wt % of water and a lamellar LC phase (LR) in the water content range of 8-45 wt %. At higher hydrations the latter phase is in equilibrium with excess water. Alternatively, Pitzalis et al.17 reported only the formation of LR phase in the water content region of 0-40 wt %. These slightly different results are not surprising because the exact composition of commercial DGMO preparations may vary. On the other hand, it is well-known that the binary GDO/water system does not show any of the liquid crystalline phases.18 Only a fluid isotropic oil phase (L2) is formed with a swelling limit of few wt % of water. Figure 2a shows the phases identified by polarized microscopy and XRD and their location in the ternary
Diglycerol Monooleate/Glycerol Dioleate Mixtures
Figure 2. Diagram showing the prepared and investigated sample dilution lines (a) and the resulting ternary phase diagram (b) (T ) 25 °C). Symbols in (a) denote L2 (triangles), HII (inverted triangles), cubic Ia3d (Q230) (squares), cubic Pn3m (Q224) (diamonds), and LR (circles). Crosses denote 2- or 3-phase samples.
phase diagram of the DGMO/GDO/water system. The samples were prepared and examined in the region of 5-50 wt % water. Ten dilution lines were examined (10 samples/dilution line) starting from the pure DGMO/water system and ending with the DGMO/GDO mixtures of 30/ 70 (wt/wt) composition. A schematic phase diagram indicating the one-phase boundaries drawn to conform to the experimental data are presented in Figure 2b. At room temperature, the phase sequence with increasing GDO concentration and starting from the binary DGMO/water system is the folowing: LR; two reversed cubic phases with crystallographic space groups of Ia3d (Q230) and Pn3m (Q224) at low and high hydration, respectively; reversed hexagonal (HII); L2 phase. All phases except Q230 are found to be in equilibrium with excess solution phase. The obtained results show that the binary DGMO/water mixtures form only the LR phase with a swelling limit of about 45 wt % water. Typical birefringence when viewed between crossed polarizers and three strong reflections with a Bragg spacing ratio of 1:2:3 observed by X-ray diffraction prove the lamellar structure. The ternary DGMO/GDO/water mixtures form the LR phase up to a DGMO/GDO ratio of about 78/22 (wt/wt). With increasing GDO concentration the swelling limit decreases to about 35 wt % water which is likely related to the pronounced hydrophobic nature of GDO. At the DGMO/GDO ratio of 73/27 (wt/wt), a narrow region of a stiff isotropic phase is observed. Between 25 and 35 wt % water this phase shows four Bragg peaks with relative positions in ratios x6:x8:x14:x16, which can be indexed as hkl ) (211), (220), (321), and (400) reflections of a body-centered cubic phase of Ia3d space group (Q230). The calculated lattice parameter (a) for the Q230 phase increases from 138 to 166 Å when moving from
Langmuir, Vol. 21, No. 11, 2005 5161
25 to 35 wt % water. At higher concentration of water, the Q230 phase transforms into another cubic phase. The observed Bragg reflections for this phase follow the relationship x2:x3:x4:x6:x8:x9, which can be indexed as the (110), (111), (200), (211), (220), and (221) reflections of a primitive cubic lattice of Pn3m crystallographic space group (Q224). This phase is found between 40 and 50 wt % water with the lattice parameter varying between 115 and 140 Å at low and high water contents, respectively. The Q224 phase is in equilibrium with excess water since the lattice parameter remains unchanged upon further addition of water. The obtained X-ray data also provide strong evidence that the observed Q230 and Q224 phases correspond to bicontinuous structures on the basis of the G and D minimal surfaces. First, the phases are located between LR and HIIsthe usual position for the reversed bicontinuous cubic phases.21,22 Second, the determined lattice parameters close to the phase transition between the cubic phases are 166.3 and 114.7 Å for the Q230 (35 wt % water) and Q224 (40 wt % water) phases, respectively. The ratio between these parameters obtained theoretically by the Bonnet transformation from a G to a D surface with constant average and Gaussian curvature is 1.57.23 Although the calculated experimental ratio obtained in the present case, 1.45, is slightly lower than the theoretical, it is still in reasonable agreement with theory. The reason is that the observed lattice parameters are not the values at the phase transition (cubic phase coexistence) at which the theoretical value is obtained. Moving closer to the phase transition region between the cubic phases will result in an increase of the Q230 and a decrease of the Q224 lattice parameter. Therefore, the real ratio will be higher than 1.45 and closer to the theoretical value. The modeling described below provides further evidence that the cubic phases in the present system conform to the theoretical minimal surface model. Further addition of GDO induces a transition from lamellar and cubic phases into a reversed hexagonal liquid crystalline phase (HII) which is found in the DGMO/GDO ratio range of approximately 70/30-45/55 (wt/wt). The HII phase is characterized by three strong X-ray diffraction peaks with spacing ratios of x1:x3:x4 proving the twodimensional hexagonal arrangement. The HII phase swells up to about 25 wt % water and remains unchanged upon further addition of water. This indicates the reversed nature of the hexagonal structure.24 Upon further addition of GDO, a fluid isotropic L2 phase is observed which shows only a diffuse diffraction. At the DGMO/GDO ratio of 30/ 70 (wt/wt) the L2 phase swells to about 10 wt % water. Overall, increasing the fraction of the nonlamellar phase forming lipid (GDO) increases the negative curvature of the lipid/water interface. The constructed DGMO/GDO/ water phase diagram shares several features with that of other aqueous mixtures of lamellar phase/reversed micellar phase forming lipids. A similar phase behavior was obtained for aqueous mixtures of soybean phosphatidylcholine (SPC)/diacylglycerol (DAG).25 Starting from SPC/ water lamellar phase, a phase sequence of LR f HII f reversed micellar cubic (III) f L2 was observed with increasing DAG concentration.25 Furthermore, successive formation of LR, bicontinuous cubic, and HII phases by the increase of the GDO content has recently been reported for the egg yolk PC/GDO system.14 The obtained X-ray diffraction data were used to investigate the structural characteristics of the liquid crystalline phases formed in the ternary DGMO/GDO/ (21) Fontell, K. Colloid Polym. Sci. 1990, 268, 264-285. (22) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1-69.
5162
Langmuir, Vol. 21, No. 11, 2005
Johnsson et al.
water system. The application of the swelling laws requires the definition of the dividing interface between polar and nonpolar regions. In the calculations to follow, we assume that the nonpolar fraction consists of the DGMO and GDO and the polar fraction being only water. A total lipid volume fraction for the ternary mixtures is therefore calculated as
wDGMO wGDO + FDGMO FGDO φlip ) wDGMO wGDO + + wwater FDGMO FGDO
(1)
where w is the weight fraction of the component and F is the density. The density of GDO, FGDO ) 0.919 g/cm3, was obtained from the manufacturer. Since the density of DGMO is not known, we used as a reasonable approximation the same value as for the glycerol monooleate (GMO) equal to 0.942 g/cm3.26 Assuming one-dimensional ideal swelling (constant bilayer thickness), the measured repeat distance for the LR phase, alam, is inversely proportional to φlip:
alam )
2l φlip
(2)
Here l is the lipid length. The swelling behavior of the cubic phases was also analyzed by assuming constant bilayer thickness. As briefly discussed above, it is generally accepted that the reversed bicontinuous cubic phases consist of a curved bilayer with a midsurface mimicking an infinite periodic minimal surface (IPMS). Once the monolayer thickness is constant, l is determined by solving the following equation:27,28
( )
φlip ) 2A0
l
acub
+
Figure 3. Lattice parameters of the lamellar (LR) and bicontinuous cubic phases (Q230 and Q224) as a function of lipid volume fraction. The DGMO/GDO weight ratio was 80/20 for the LR samples and 73/27 for the cubic phase samples. Fully drawn lines represent best fits according to eqs 2 and 3 (see text).
( )
4πχ l 3 acub
3
(3)
Here A0 (3.09 for the Q230 and 1.92 for the Q224) and χ (-8 and -2 for the Q230 and Q,224 respectively) are the constants for the given minimal surface. Equations 2 and 3 were used to fit the experimentally measured lattice parameters versus φlip plots for the LR and cubic phases of the ternary DGMO/GDO/water system (Figure 3). The data points for the LR phase correspond to the mixtures having a DGMO/GDO ratio of 80/20 (wt/ wt), whereas the data points for the cubic phases are taken from the dilution line at the fixed DGMO/GDO ratio of 73/27 (wt/wt). As shown in Figure 3, the theoretical swelling law fits agree well with experimental data and result in the following values of the lipid length (monolayer thickness): 18.8, 18.4, and 18.5 Å for the LR, Q230, and Q224 phases, respectively. The calculated monolayer thicknesses are in excellent agreement with the previously determined bilayer thickness of 37 Å for the LR phases formed in the binary DGMO/water system.16 Although (23) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (24) Seddon, J. M.; Templer, R. H. In Handbook of Biological Physics; Hoff, A. J., Ed.; Elsevier: Amsterdam, 1995. (25) Ora¨dd, G.; Lindblom, G.; Fontell, K.; Ljusberg-Wahren, H. Biophys. J. 1995, 68, 1856-1863. (26) Handbook of Chemistry and Physics; CRC Press: Cleveland, OH, 1974. (27) Anderson, D. M.; Gruner, S. M.; Leibler, S. Proc. Natl. Acad. Sci. U.S.A 1988, 85, 5364-5368. (28) Turner, D. C.; Wang, Z.-G.; Gruner, S. M.; Mannock, D. A.; McElhaney, R. N. J. Phys. II 1992, 2, 2039-2063.
Figure 4. Water channel radius of the reversed hexagonal phase (HII) as a function of total water content. Filled and open symbols represent samples with DGMO/GDO ) 50/50 and 70/ 30 wt/wt, respectively. The line is drawn only to guide the eye.
the number of data points used for the fitting is limited, the obtained results indicate that the incorporation of 20 wt % of GDO into the LR phase of aqueous DGMO has practically no effect on the bilayer thickness. The calculations also show that all these three liquid crystalline phases are characterized by the same dimensions of the bilayer. The X-ray diffraction data were also used to evaluate the effect of GDO on the water channel dimensions of the HII phase. On the basis of simple geometric considerations, the obtained lattice parameter, aHII, allows calculations of the radius of the water cylinders (RW):15,29
RW ) aHII
(
)
x3(1 - φlip) 2π
1/2
(4)
The calculated RW increases from about 6 to close to 14 Å with increasing water content in the HII phase of the DGMO/GDO/water system as shown in Figure 4. The results also show that GDO has practically no effect on the structural characteristics since the data points from different DGMO/GDO ratios fall on the same line (Figure 4). 2. Dispersion Formation. From the ternary phase diagram displayed in Figure 2 we conclude that the HII phase exists at DGMO/GDO weight ratios between approximately 70/30 and 45/55 (wt/wt). Thus, dispersions of the HII phase were prepared at DGMO/GDO ) 50/50 or 60/40 (weight ratios) and at 90-98 wt % water. As (29) Luzzati, V. In Biological Membranes, Physical Facts and Function; Chapman, D., Ed.; Academic Press: New York, 1968.
Diglycerol Monooleate/Glycerol Dioleate Mixtures
Langmuir, Vol. 21, No. 11, 2005 5163
Figure 6. X-ray diffractogram of a dispersed sample with DGMO/GDO ) 50/50 wt/ wt and lipid/Pluronic F127 ) 9/1 wt/ wt. The total amphiphile (lipid + polymer) concentration was 5 wt %, and the sample was subjected to the heat-treatment process (see text) before XRD. The peaks correspond to the (10), (11), and (20) reflections of the hexagonal structure.
Figure 5. Size distributions obtained from dispersions of DGMO/GDO ) 50/50 (a) and 60/40 (b) wt/wt. The lipid to polymer (F127) weight ratio was 9/1, and the total amphiphile (lipid + polymer) concentration was 5 wt %. The apparent mean particle size after heat treatment was 194 and 213 nm for the 50/50 (a) and 60/40 (b) sample, respectively.
fragmentation and dispersion stabilizing agent, we used Pluronic F127 such that the lipid/polymer weight ratio was kept constant at 9/1. Importantly, the dispersions could be prepared without the use of high shear/energy techniques but by only shaking the samples for 12 h at 350 rpm. Typical size distributions of the dispersions are shown in Figure 5. It is clear from the data in Figure 5 that well-defined dispersions of submicrometer-sized particles with a polydispersity index (PI) of about 0.4 (expressed as the ratio between the standard deviation of the distribution and the mean value) can easily be obtained by shaking the samples at moderate speed. Thus, in comparison with the previously published preparation procedures of reversed phase dispersions where high shear techniques are employed,12,14 the present systems may be termed “self-dispersing”. Furthermore, we have previously shown that GMO-based cubic phase dispersions can be improved in terms of the width of the size distribution and overall dispersion quality by performing a heat treatment process13 (see Experimental Section). As shown in Figure 5, this treatment caused a slight increase in the mean particle size but also narrowed the particle size distribution (PI ) 0.2-0.3) of the HII phase dispersions. XRD measurements show that the hexagonal structure is retained in dispersion as exemplified in Figure 6. The mean hexagonal spacing (dhex) was determined to be 47.6 and 52.4 Å corresponding to lattice parameters of 55 and 60.5 Å for the dispersed samples at DGMO/GDO ) 50/50 and 60/40 wt/wt, respectively. These values are close to the values obtained for the corresponding DGMO/GDO bulk samples in excess water (50 wt % water) without the polymer for which we measured unit cell dimensions of 55.5 Å (50/50) and 63.3 Å (60/40), respectively. This clearly
indicates that the large F127 polymer is predominantly excluded from the core of the particles and therefore necessarily is situated at the surface of the particles. Such a size-exclusion of the polymeric stabilizer has been observed before in similar types of systems.12 Note that, without the presence of the F127 polymer (or other polymeric additive), the formation of colloidally stable HII phase dispersions is not possible. Direct structural evidence for Hexosome formation was obtained using cryo-TEM as shown in Figures 7 and 8. The samples observed in Figure 7a,b were prepared by shaking at a weight ratio of DGMO/GDO ) 50/50 (lipid/ F127 ) 9/1 wt/wt), and essentially spherical particles with dark seemingly disordered inner textures can be observed. In some cases, the particles display inner curved striations indicating a deformation of the cylinder axes. By performance of the heat treatment process on the shaken dispersions, the appearance of well-defined essentially monocrystalline hexagonal phase particles was observed as shown in Figure 7c,d. The hexagonal packing of the cylinders is clearly observed in Figure 7d. It is also noteworthy that the sample having a higher content of DGMO (DGMO/GDO ) 60/40 wt/wt), besides the hexagonal phase particles, also contains a fraction of vesicles as shown in Figure 8a, whereas vesicles were rarely seen in the micrographs of the 50/50 sample (Figure 7). This probably reflects the fact that the 60/40 sample is closer to the lamellar phase (for which liposomes are obtained in dispersion) compared to the 50/50 sample according to the phase diagram (Figure 2). Thus, the vesicles in the 60/40 dispersion represent highly metastable structures, formed during shaking of the dispersion and kinetically stabilized by adsorbed F127. Nevertheless, during the heat-treatment process, the vesicular structures are effectively converted into hexagonal phase particles although most of those particles had lamellar or vesicular coatings as shown in Figure 8b. The lattice parameter of the hexagonal structure was estimated from fast Fourier transforms to be 57 Å for the 50/50 sample (Figure 7c,d) in good agreement with the results obtained from XRD (see above). Interestingly, the hexagonal crystallinity is also reflected in the particle shape, in particular after the heat-treatment process (Figure 7c,d), because most of the particles viewed in 2D projection are clearly well-defined hexagons. We also note that the particles obtained after heat treatment are exclusively observed from a “top-view” in the cryo-TEM images (Figure 7c,d). This indicates that the hexagonal phase nanoparticles observed in Figure 7c,d align themselves in the relatively thin vitrified film of the cryo-TEM
5164
Langmuir, Vol. 21, No. 11, 2005
Johnsson et al.
Figure 7. Cryo-TEM images of samples with DGMO/GDO ) 50/50 wt/wt and lipid/Pluronic F127 ) 9/1 wt/wt, dispersed by shaking 12 h at 350 rpm (a, b) and after heat treatment (c, d). Note reversed hexagonal phase particles with dark seemingly disordered structure in (a) and particles with internal striations in (b). After heat treatment, the internal hexagonal structure is more evident and most of the particles are observed in a top-view as hexagons (c, d).
Figure 8. Cryo-TEM images of samples with DGMO/GDO ) 60/40 wt/wt and lipid/Pluronic F127 ) 9/1 wt/wt, dispersed by shaking 12 h at 350 rpm (a) and after heat treatment (b). Note the small fraction of vesicles present in (a) and the lamellar coating of the hexagonal phase particles in (b).
experiment due to a high aspect ratio; i.e., the width of these hexagons is much larger than the height (coinlike structures). Further studies are currently ongoing to clarify this issue. The effect of the heat-treatment procedure on particle morphology is likely explained by a transition of the system into a dispersed L2 phase at high temperatures (>100 °C).
From the presumably spherical L2 droplets, the hexagonal structure crystallizes upon cooling to room temperature resulting in well-defined colloidal dispersions of the parent thermodynamically stable HII phase. It is also important to note that the dispersion quality in terms of minimal “contamination” of vesicular structures is significantly enhanced by the heat treatment process. The removal of
Diglycerol Monooleate/Glycerol Dioleate Mixtures
Langmuir, Vol. 21, No. 11, 2005 5165
Figure 10. Size distributions obtained from a dispersion with DGMO/GDO ) 50/50 wt/wt stored at 25 °C for 2 months. The lipid to polymer (F127) weight ratio was 9/1, and the total amphiphile (lipid + polymer) concentration was 5 wt %.
Figure 9. Magnified cryo-TEM image of a hexagonal phase nanoparticle and schematic illustrations of the reversed hexagonal phase structure and cross-sectional views, top-view (left) and side-view (right), of the hexagonal phase nanoparticle. The illustration should be viewed as horizontal (left) and vertical (right) cuts through the reversed micellar cylinders and is not drawn to scale.
vesicular structures (in particular for the DGMO/GDO ) 60/40 wt/wt sample) is related to vesicle fusion upon heating above the cloud point (CP) of the Pluronic F127, which is slightly above 100 °C.30 When the temperature is above the CP of the polymer, vesicles fuse into larger aggregates subsequently forming dispersed L2 phase droplets as discussed above. A similar effect has been observed recently for dispersions of cubic phase particles formed in the GMO/Pluronic F127/water system.13 On the basis of the results obtained on the dispersed reversed hexagonal phase by use of XRD and cryo-TEM, a simplified model of the termination of the hexagonal structure with respect to the surrounding aqueous phase is displayed in Figure 9. We conclude that the stability of the dispersed particles can be attributed to monolayer (or bilayer) capping of the particles minimizing exposure of hydrophobic chains to water and the presence of the Pluronic F127 at the surface conferring steric stabilization of the particles. Notably, the particles are not kinetically stable in the absence of the polymer and it is therefore clear that the main dispersion stabilizing effect originates from the polymer-induced steric stabilization. Finally, the colloidal stability of the Hexosome dispersions is excellent as exemplified in Figure 10. Neither changes in mean particle size and/or size distribution nor any visually detectable changes of the dispersion homo(30) Pandit, N.; Trygstad, T.; Croy, S.; Bohorquez, M.; Koch, C. J. Colloid Interface Sci. 2000, 222, 213-220.
geneity could be observed after several months of storage at 25 °C. The physicochemical stability and the fact that the dispersions could be prepared easily with a minimum of energy/shear input indicate a great potential of these dispersions in drug delivery applications. Indeed, the systems should be useful for loading/formulation of labile pharmaceutical actives such as proteins and peptides. Note also that the volume fraction of lipid in the Hexosome particles is about 70-75% meaning that the particles are ideally suited for loading of small molecule lipophilic and amphiphilic drugs. Indeed, preliminary drug loading studies using a number of sparingly soluble and amphiphilic drug molecules indicate that the Hexosome nanoparticles exhibit much better drug loading capacities compared to conventional lipid- and/or surfactant-based vehicles such as liposomes and micelles (our own unpublished work). Conclusions The aqueous phase behavior of DGMO/GDO is rich featuring a range of phases, including one reversed hexagonal phase and two bicontinuous cubic phases. The observed phase sequence agrees with the expectation of an increased negative lipid monolayer curvature with increasing fractions of the nonlamellar phase forming GDO. We also found that kinetically stable and structurally well-defined colloidal dispersions of the reversed hexagonal phase could be produced by use of a polymeric stabilizer and a simple and reproducible preparation method. The obtained Hexosome dispersions were characterized by XRD and cryo-TEM. The results show extremely well-ordered particles with inner monocrystalline hexagonal phase structure. Forthcoming studies will be aimed at the characterization of the reversed hexagonal phase particles in terms of dimensions (thickness), structural transitions during heat treatment, behavior at interfaces, and application as drug carriers. Acknowledgment. We are greateful to Gunnel Karlsson for help with cryo-TEM instrumentation. This work was 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. LA050175S
