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Langmuir 1997, 13, 6964-6971
Submicron Particles of Reversed Lipid Phases in Water Stabilized by a Nonionic Amphiphilic Polymer Jonas Gustafsson,*,† Helena Ljusberg-Wahren,‡ Mats Almgren,† and Kåre Larsson‡ Department of Physical Chemistry, Uppsala University, Box 532, S-751 21 Uppsala, Sweden, and Camurus Lipid Research, Ideon, Gamma 1, So¨ lvesgatan 41, S-223 70 Lund, Sweden Received May 30, 1997. In Final Form: October 6, 1997X The present study concerns the formation and structure of aqueous dispersions of lipid-based lyotropic liquid crystalline phases, namely reversed types of hexagonal and bicontinious cubic phases. As stabilizer a nonionic triblock polymer proves to be efficient for dispersions of both phases. We demonstrate that these dispersions contain submicron particles with a preserved inner periodicity. The morphology and inner structure of the dispersed particles are characterized by means of SAXS (small-angle X-ray scattering) and cryo-TEM (cryo-transmission electron microscopy). The particle shape is shown to reflect the crystallinity of the lipid structure. Thus the morphology of particles from cubic phases differs from that of the ones obtained from the hexagonal phase. Furthermore, an interesting difference is found in the partitioning of the polymer in the two types of dispersions. The polymer is localized within the core as well as at the surface of the dispersed particles of the cubic phase, whereas the core of the particles from the hexagonal phase seems depleted of polymer at the same polymer concentration.
Introduction The self-assembly of amphiphilic molecules gives rise to a series of lyotropic liquid crystalline phases. The lamellar phase is the one which has attracted most interest for its dispersed state. Lamellar phases in coexistence with an aqueous solution are often easily dispersed due to the ability of the lamellae to bend into the closed structures well-known as vesicles or liposomes. In principle it should be possible to form aqueous dispersions also with liquid crystalline phases of reversed curvature (cf. ref 1), since many of them may be found in coexistence with a dilute aqueous phase. Dispersions of reversed amphiphilic phases have recieved less attention, as they usually, unlike the lamellar phase, show a very limited stability in aqueous dispersions. Besides the usual parameters governing colloidal stability, there is an additional problem inherent to these dispersions, originating from the boundary conditions imposed on the fragmented liquid crystal. The particle inner structure, of two- or three-dimensional crystallinity, must be terminated with respect to the fact that the surrounding media is water; i.e., an exposure of hydrophobic domains must be avoided. The suggested biological relevance2-6 of lipid structures with reversed curvatures is a major motivation to learn more about the nature of fragmented versions of reversed phases. Colloidal particles of a reversed cubic or hexagonal phase with inside aqueous zones provide also certain advantages in technical applications compared to the droplets of common oil-in-water emulsions. One field of application is drug delivery.7,8 †
Uppsala University. Camurus Lipid Research. X Abstract published in Advance ACS Abstracts, November 15, 1997. ‡
(1) Reversed curvatures refer here to situations where the amphiphilic layers are curved toward the polar media. (2) Landh, T. Thesis, Lund University, 1997. (3) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (4) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221256. (5) Petty, H. R. Molecular biology of membranes: structure and function; Plenum Press: New York, 1993. (6) Huang, L.; Zhou, X. Biochim. Biophys. Acta 1994, 1189, 195203. (7) Ljusberg-Warhen, H. Thesis, Royal Institute of Technology, Stockholm, 1982.
S0743-7463(97)00566-0 CCC: $14.00
In the present work dispersions have been made from two types of reversed lipid phases: the reversed hexagonal phase of closed packed water cylinders covered by a lipid monolayer and the bicontinuous cubic phase built up by bilayers periodically curved in three dimensions. In both cases glycerolmonooleate (GMO) is the major amphiphilic component of the crystalline phases. GMO in water forms a bicontinous cubic phase,3,4,9,10 which on addition of a lipophilic substance may transform into a reversed hexagonal phase.11 The choice of dispersion agent or stabilizer for the lipid liquid crystals is crucial. A surface active component added to facilitate dispersion will also be inclined to participate in, and possibly change, the lipidbased liquid crystal structure. As dispersion agent we have used a nonionic triblock polymer, PEO98PPO67PEO98 (Polaxamer 407). Polymers of this architecture have earlier been employed for the stabilization of a variety of different colloids,12-14 from vesicles to carbon black. The general view is that the triblock polymer provides steric stabilization by having its less water soluble middle part adsorbed at, or anchored in, the surface of the stabilized particle, while its more hydrophilic ends extend into the surrounding solution.14,15 In a similar way, steric stabilization may also be given by hydrophilic polymers that are covalently bound to lipid molecules.16 We note that, for mixtures of Polaxamer 407 and GMO in water, a part of the ternary phase diagram has been characterized by Landh.19 The phase behavior described by Landh clearly demonstrates that the structure of the lipid-based cubic phase changes on addition of the amphiphilic polymer, probably due to the incorporation of the polymer into the (8) Ljusberg-Warhen, H.; Nyberg, L.; Larsson, K. Chim. Oggi 1997, 14, 40-43. (9) Lindblom, G.; Larsson, K.; Johansson, L.; Fontell, K.; Forsen, S. J. Am. Chem. Soc. 1979, 101, 5465-5470. (10) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219. (11) Lindstro¨m, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgstro¨m, B. Lipids 1981, 16, 749-754. (12) Kostarelos, K.; Luckham, P. F.; Tadros, T. T. J. Liposome Res. 1995, 5, 117-130. (13) Washington, C.; King, S. M.; Heenan, R. H. J. Phys. Chem. 1996, 100, 7603-7609. (14) Benita, S.; Levy, M. Y. J. Pharm. Sci. 1993, 82, 1069-1079. (15) Alexandridis, P. Curr. Opin. Colloid Interface Sci. 1997, 1, 490501. (16) Stealth Liposomes Lasic, D., Martin, F., Eds.; CRC Press: London, 1995.
© 1997 American Chemical Society
Submicron Particles of Reversed Lipid Phases
lipid bilayers. For the same reason also a lamellar phase forms at high ratios of polymer to GMO. The present study may be regarded as following up a recent short communication17 and is directed mainly to the structure of the stabilized particles. The particle structure is characterized by combined small-angle X-ray scattering (SAXS) and cryo-TEM studies. The former technique provides information on the liquid crystallinity of the particles, whereas electron micrographs are used mainly to reveal particle morphology. By comparison of results from dispersed samples to what is found in nondispersed (equilibrated) mixtures, we also obtain some information on how the polymer is distributed in the dispersions. In addition to the role played by the polymer, the liquid crystalline structure itself will also be important for the resulting morphology of the particles. Experimental Section The glyceryl monooleate (GMO) was a distilled monoglyceride (RYLO MG 90, Danisco Ingridients, Brabrand, Denmark) with the following fatty acid composition: C18:1 92%, C18:2 6%, saturated acids 2%. The triglyceride (TG) was a high monosaturated sunflower oil (Trisun 80 SVO, Eastlake, OH). Retinyl palmitate (VAP) was a gift from Beiersdorf AG (Hamburg, Germany). Polaxamer 407 (PEO98PPO67PEO98) was obtained from BASF (Germany). Dispersion Procedure. A homogeneous melt of lipids and poloxamer 407 was added dropwise into water during stirring to form a coarse dispersion. Further size reduction by homogenization was performed with a Microfluidizer 110 S (Microfluidics, Newton, MA) using pressures of 5000 psi at 80 °C. All dispersions had a water content of 95 wt %. The homogeneity of the dispersion was controlled in a light microscope. The stability toward flocculation and coalescence was checked by visual inspection during the first weeks and after 5 months. SAXS. Small-angle X-ray diffractograms were obtained using a Kratky compact small-angle system, linear collimated and equipped with a position sensitive detector (OED 50M from MBraun, Graz, Austria) with 1024 channels of width 51.3 µm. The radiation (Cu KR) was provided by a Seifert IF 300 X-ray generator operating at 50 kV and 40 mA. The camera length was 277 mm. Samples were poured into quartz capillaries mounted on a steel body that were sealed with screw caps. Slitsmeared spectra were desmeared according to a standard procedure. All measurements were performed at 25 °C. Cryo-TEM. Dispersions studied by cryo-TEM were diluted to a water content of 99 wt %. Electron microscopy investigations were performed with a Zeiss 902 A instrument, operating at 80 kV. Specimens were prepared by a blotting procedure performed in a chamber with controlled temperature (25 °C) and humidity (maximum). A drop of the sample solution was placed onto a copper EM-grid coated with a perforated polymer film.18 Excess solution was thereafter removed with a filter paper, leaving a thin film of the solution on the EM-grid. Vitrification of the thin film was achieved by rapid plunging of the grid into liquid ethane held just above its freezing point. The vitrified specimens were then transferred in the cold state to the microscope and examined at about 100 K.
Results GMO forms in water two bicontinuous cubic phases which have been extensively studied.9,10,19 At water contents above 40 wt % a cubic phase exist in equilibrium with an aqueous phase. In mixtures of GMO with TG in water a reversed hexagonal phase is formed at room temperature, which also can exist in equilibrium with an aqueous phase.11 VAP influences the phase behavior of GMO in a way similar to that of TG. In mixtures of GMO and TG at high water content, the hexagonal phase (17) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (18) Fukami, A.; Adachi, K. J. Electron Microsc. 1965, 14, 112-118. (19) Landh, T. J. Phys. Chem. 1994, 98, 8453-8467.
Langmuir, Vol. 13, No. 26, 1997 6965 Table 1. X-ray Diffraction Data for Two Nondispersed (Equilibrated) Samples with Cubic Phases in Excess Solvent (80 wt % water): (a) GMO in water; (b) GMO with 8 wt % Polaxamer 407 in Watera d (Å) (I)
hkl
(a) GMO in Water: Pn3m, a ) 97 Å 67.7 (vs) 110 56.6 (s) 111 48.3 (m) 200 39.5 (w) 211 34.2 (w) 220 32.3 (w) 221 30.8 (vw) 310
a (Å) 95.8 98.0 96.6 96.7 96.7 96.9 97.4
(b) GMO with 8 wt % Polaxamer 407 in Water: Im3m, a ) 130 Å 90.6 (s) 110 128.1 64.3 (m) 200 128.6 52.7 (s) 211 129.1 46.3 (vw) 220 131.0 41.5 (w) 310 131.2 37.5 (w) 222 128.2 34.9 (w) 321 130.6 a d is the observed Bragg spacing and the corresponding reflection intensities are listed as follows: vs, very strong; s, strong; w, weak; vw, very weak. h k l and a denote the lattice indices and the corresponding unit cell parameter according to the given designation.
appears at a weight ratio of 96:4 (GMO/TG) at room temperature. In order to form an aqueous dispersion of these phases, an additional component acting as a stabilizer must be added. Polaxamer 407 was added as a dispersing agent in weight ratios of polymer to lipid of 2-12%. It was found that 2% of the polaxamer was sufficient to facilitate formation of a coarse dispersion, but homogenized samples with this composition did not show long term (weeks) stability. In these samples phase separation occurred more rapidly for the cubic phase dispersions than for the corresponding hexagonal ones. At polymer concentrations between 4 and 12 wt %, milklike dispersions are produced that are stable for several months. In the following, results from SAXS and cryoTEM investigations of the stable dispersions will be presented along with some measurements and comments on the state of corresponding nondispersed samples. GMO-Based Cubic Phases. According to the phase diagram of GMO-Polaxamer 407-water determined by Landh,19 the GMO-based cubic phase can accommodate more than 30 wt % polymer to lipid of the triblock polymer. The polymer incorporation induces both a transition between different cubic structures and a further swelling of the cubic phase region toward the water corner. At high polymer to lipid content the diagram of Landh also features a rather solvent-rich lamellar phase. Our SAXS measurements on nondispersed samples with 80 wt % solvent and 0-12 wt % polymer to lipid have detected only the cubic liquid crystalline phase. At polymer to lipid concentrations below 2 wt % the scattering profiles were similar to the ones obtained from pure GMO in water. This cubic phase is known from previous studies as CD.10 In the description of reversed cubic phases as periodic minimal surfaces, the center of the bilayer in this structure is found to mimic the D-surface, which can be associated with the Pn3m space group. For excess solvent samples with polymer to lipid concentrations between 4 and 12 wt % a different type of diffraction pattern is recorded; from which at least seven reflections may be found. Indexing of the diffraction lines (see example in Table 1a) shows that they are in accord with the body-centered cubic structure Im3m, which is the structure that Landh19 suggested in this region of the phase diagram. The corresponding minimal surface for the bilayers in this
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Figure 1. (a) SAXS profiles from (lower curve) the CD phase of a nondispersed sample of GMO in excess water and (upper curve) a dispersed sample with 4 wt % polymer to lipid (magnified two times). (b) SAXS profiles from (lower curve) a nondispersed sample with 7.4 wt % polymer to lipid and excess solvent, (middle curve) a dispersion with 7.4 wt % polymer (magnified two times), and (upper curve) a dispersion with 10 wt % polymer (magnified four times). Table 2. Lattice Parameters Derived from SAXS Studies of the Cubic Phase Structures in Dilute Nondispersed Samples and in Dispersed Samples wt % polymer to lipid
lattice parameter (Å) in excess solventa (type of structure)
lattice parameter (Å) in dispersion (type of structure)
0 2 4 7.4 10
96 (CD) 130 (CP) 130 (CP) 130 (CP) 130-140 (CP)
b c 96 (CD) 130 (CP) probably 130 (CP)
a A nondispersed sample with >80 wt % water. b Not possible to disperse. c Limited stability.
cubic phase (CP) is the P-surface. It is important to note in this connection that the lattice parameter of the bodycentered structure was about 130-140 Å, with very little variation at increased polymer concentration in these solvent-rich samples with 2-12 wt % polymer to lipid. Dispersed samples with 95 wt % solvent gave scattering profiles with usually two to four maxima. These reflections were significally broadened. The line broadening may be due to disorder, but it may also be a consequence of the small particle size; as we will see, many particles are well below 1 µm in size. The limited number of lines does not permit unambiguous indexing, but some conclusions may be drawn from superimposing the scattering profiles onto diffractograms of better quality from nondispersed samples. The outcome of such comparisons is illustrated in Figure 1 and summarized in Table 2. Indexing of the diffractograms used in Figure 1 is shown in Table 1. Note that the cubic structure in the nondispersed samples represents only one phase in a multiphase system (i.e., an additional liquid crystalline structure may have been overlooked). The dominating features of a scattering
profile from dispersions with 4 wt % polymer correlate well, in both position and intensity distribution, to the set of diffraction lines found from the CD phase of pure GMO in excess water (Figure 1a). For a dispersion with 7.4 wt % polymer the diffraction pattern is instead similar to what is found from nondispersed samples with the same amphiphilic composition, i.e., indicative of an CP structure with lattice parameter 130 Å (Figure 1b). In addition to the reflections that are shared with the CP structure, some weak peaks appear for which we currently have no explanation. They could in fact originate from a CD structure with a lattice parameter of the order of what was found earlier. Increasing the polymer content to 10 wt % results in a scattering profile with less distinct features (Figure 1b lower curve) but still reminiscent of the profile from the 7.4 wt % sample. The results from electron microscopy on the dispersions with the GMO-based cubic phase have in part been described earlier in a short communication.17 It was demonstrated there that samples with 8 wt % polymer consist mainly of faceted particles of close to cubic shape. The appearance of these particles is recalled in Figure 2a. From the dominance of particles showing cubic shape it was concluded that the particles are preferentially terminated along the principal directions of the cubic unit cell. Furthermore, the periodicity displayed by the inner structure of the particles was attributed to the planes with lattice index (110) of the Im3m structure. Unfortunately this spacing was somewhat overestimated in the previous study, where it was claimed to be 120 Å.17 As judged from a larger collection of particles, this periodicity is mainly in the range 90-100 Å, which is in line with the position (92 Å) of the 110 reflections in the scattering profiles. We note that a direct estimation of these periodicities from the electron micrographs may be made with an accuracy of at most 10-15%. The micrograph of Figure 2a from a dispersion with 7.4 wt % polymer was chosen, since it illustrates the stabilization of the faceted particles so clearly. Being squeezed together on the EMgrid during the film formation, the particles still seem to resist coalescence. A minor fraction of vesicles is observed in coexistence with the particles in all samples with the dispersed cubic phases. Some of them appear to be attached to the surface of the particles. Those of the particles that are closer to spherical shape show lamellar features more clearly in their surface regions, along with a somewhat disordered inner structure. The fraction of vesicles, and the lamellar features of the particles, is increased in the sample with 10 wt % polymer (Figure 2b). The particles of Figure 2b also exhibit a larger spread in the repeat distances of their textures. It is likely that some particles of Figure 2b display other sets of crystal planes, but a larger spread in periodicities is also what one would expect, considering the scattering profile given by this sample. With 12 wt % polymer to lipid the fraction of vesicles is further increased at the expense of the population of well ordered particles. An increased fraction of vesicles mirrors probably the presence of a lamellar phase in the phase diagram at high ratios of the polymer. From the stable dispersion with the lowest polymer content (4 wt %) we may, on the basis of the SAXS results, expect a different inner structure of the particles. In electron micrographs (Figure 2c) most particles are seen to exhibit a fine hexagonal texture, where the periodicity may be estimated to be about 60 Å. If the particles are assumed to be viewed along the 〈111〉 direction, where the projection of the D-surface shows a hexagonal symmetry, this periodicity can be attributed to the (111) planes of the CD structure. SAXS measurements gave a spacing for these planes of 57-58 Å. The particle shape seems
Submicron Particles of Reversed Lipid Phases
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Figure 2. Cryo-TEM micrograph showing dispersed particles from the cubic phase of GMO and Polaxamer 407 in water. Bar equals 100 nm. (a) 7.4 wt % polymer to lipid. Faceted mostly cubic particles showing a periodicity of 90-100 Å. Vesicles appear along with the particles and at their surface. (b) 10 wt %. Faceted particles of a decreased average size, showing a less ordered surface structure with lamellar features. (c) 4 wt %. Particles dominated by a texure of hexagonal symmetry with a periodicity of about 60-65 Å. Vesicles appear again at the particle surface, but particle shape is now less well defined.
less well defined in these dispersions. Cubic particles like the ones formed from the CP structure are not observed. Instead some particles display edges that suggest a preference for higher polyhedras. It should be added that particle scattering at low q-values was observed from most dispersions, but it was
poorly resolved due to the limitation of the Kratky camera in this q-range. Macroscopically, the dispersions showed some tendency to creaming with time. It is therefore probable that phase separation eventually occurs through coalescence in the creaming layer. Reversed Hexagonal Phases. We will focus on two
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Discussion
Figure 3. SAXS profiles from (lower curve) the reversed hexagonal phase of a nondispersed mixture of GMO/VAP (84: 16) in excess water and (upper curve) a dispersion of the corresponding lipid mixture with 5 wt % Polaxamer 407 (magnified two times).
HII-forming (in water) lipid mixtures with Polaxamer 407 added in up to 12 wt % polymer to lipid: GMO and TG in the weight ratio 88/12 and GMO and VAP in the ratio 84/16. The outcome parallels in part the results from the preceding section with cubic phases. As mentioned above, more than 2 wt % the polymer is needed to produce a stable dispersion. The SAXS results on these dispersions are easily summarized. One rather broad Bragg reflection is dominating the scattering profiles of all the stable HII dispersions. For each of the investigated lipid mixtures, it is found that this peak remains at the same position, irrespective of polymer content in the examined region. Equilibrated HII phases from the pure lipid mixtures in excess water show typically three reflections, of which the first is much stronger than the others. When comparing, it is evident that, for each lipid mixture, a direct correlation is found between the position of the first reflection from the equilibrated lipid/water samples and the single Bragg peak of the dispersed samples. This is illustrated for the GMO/VAP mixture in Figure 3. The observed spacing of this (10) reflection is for the GMO/VAP mixture (84/16) 52 Å, corresponding to a hexagonal lattice parameter of 60 Å. The GMO/TG mixture shows a lattice parameter of 59 Å for the 88/12 blend. The quality of the diffraction patterns is typical of the kind shown in Figure 3 for samples with 4-9 wt % polymer. For polymer contents above 9 wt % a further increase in line broadening is observed. In cryo-TEM investigations submicron particles were observed with some variation in appearance. Figure 4(a,b) shows examples from a GMO/VAP mixture with 5 wt % polymer. Faceted particles appear, many of which show curved striations, while others show textures of hexagonal symmetry. When such a texture is displayed, the particles often give a faceted (more or less hexagonal) geometry in projection. An overall or average particle shape is, however, not easily deduced from the micrographs. Small particles (
