© Copyright 2007 American Chemical Society
DECEMBER 4, 2007 VOLUME 23, NUMBER 25
Letters Self-Assembled Geometric Liquid-Crystalline Nanoparticles Imaged in Three Dimensions: Hexosomes Are Not Necessarily Flat Hexagonal Prisms Ben J. Boyd,*,† Shakila B. Rizwan,‡ Yao-Da Dong,† Sarah Hook,‡ and Thomas Rades‡ Department of Pharmaceutics, Victorian College of Pharmacy, Monash UniVersity, 381 Royal Pde, ParkVille, Victoria 3052, Australia, and School of Pharmacy, UniVersity of Otago, P.O. Box 913, Dunedin 9054, New Zealand ReceiVed September 25, 2007. In Final Form: October 28, 2007 Attempts to understand the complex 3D morphology of non-lamellar liquid-crystalline nanostructured particles, formed by the dispersion of a reversed hexagonal phase (hexosomes) and bicontinuous cubic phase (cubosomes) in water, have been limited by the lack of suitable 3D imaging techniques. Using cryo-field emission scanning electron microscopy, we show that whereas the structure of cubosomes generally reflects that anticipated from modeling approaches, hexosomes, which were previously proposed to be flat hexagonal prisms, in fact often possess a “spinningtop-like” structure, which is likely to influence their interactions with surfaces.
Introduction Cubosomes and hexosomes are truly nanostructured hierarchical materials, with structure on the molecular, internal mesophase, and nanoparticle length scales. The complexity of their internal nanostructure, their high internal surface area,1 and the ability to tailor the nanostructure by subtle changes in lipid composition2 are driving the efforts of a number of research groups to gain a better understanding of these materials.3-9 Although it is * Corresponding author. Phone: +61 3 99039112. Fax: +61 3 99039583. E-mail:
[email protected]. † Monash University. ‡ University of Otago. (1) Lawrence, M. J. Chem. Soc. ReV. 1994, 23, 417-423. (2) Dong, Y. D.; Larson, I.; Hanley, T.; Boyd, B. J. Langmuir 2006, 22, 95129518. (3) Spicer, P. T. Cubosomes: Bicontinuous Cubic Liquid Crystalline Nanostructured Particles. In Encyclopedia of Nanoscience and Nanotechnology; Marcel Dekker: New York, 2004; pp 881-892. (4) Andersson, S.; Jacob, M.; Lidin, S.; Larsson, K. Z. Kristallogr. 1994, 210, 315-318. (5) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1996, 12, 4611-4613. (6) Boyd, B. J.; Whittaker, D. V.; Khoo, S. M.; Davey, G. Int. J. Pharm. 2006, 318, 154-162.
important to understand the impact of composition and preparation methods on the internal structure of liquid-crystalline particles for most applications, if we aim to utilize these particles as a platform technology for the delivery of active agents via interaction with a biological surface,10 then it is essential also to understand the particles’ surface and 3D structure. Liquid-crystalline particles have been prepared most often by the dispersion of glyceryl monooleate (GMO) and a mixture of GMO with other lipids in the presence of a stabilizing polymer such as Pluronic F127,11 although other amphiphilic lipids such as phytantriol have been receiving increasing attention.2,9 The particles can be prepared by a number of different methods, including high-temperature mechanical dispersion,12 room(7) Monduzzi, M.; Ljusberg-Wahren, H.; Larsson, K. Langmuir 2000, 16, 7355-7358. (8) Nakano, M.; Teshigawara, T.; Sugita, A.; Leesajakul, W.; Taniguchi, A.; Kamo, T.; Matsuoka, H.; Handa, T. Langmuir 2002, 18, 9283-9288. (9) Barauskas, J.; Johnsson, M.; Tiberg, F. Nano Lett. 2005, 5, 1615-1619. (10) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4, 449-456. (11) Landh, T. J. Phys. Chem. 1994, 98, 8453-8467. (12) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13, 6964-6971.
10.1021/la7029714 CCC: $37.00 © 2007 American Chemical Society Published on Web 11/08/2007
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Figure 1. SAXS pattern for the cubosome dispersion (prepared using phytantriol alone as the lipid base) confirming the internal structure of the particles to be the QPn3m space group. (Inset) Derived plot for D spacing of the lattice from the peak positions vs the reciprocal spacing ratio for the x2:x3:x4:x6 peaks corresponding to the Pn3m structure.27
Figure 2. (A to D) Images of phytantriol and F127 QII Pn3m cubosome dispersions obtained using cryo-TEM (A and B) and cryo-FESEM images (C and D) from ref 17 reproduced with permission from Elsevier. (E, F) Models reproduced from ref 3 previously proposed in the literature for QII Pn3m cubosomes based on the differential geometry approach. (Permission for reproduction obtained from Taylor & Francis, www.informaworld.com.)
temperature fragmentation,11 dilution of hydrotropic solvent solutions of the amphiphile in water,13 and the dilution of mixed micelles of the amphiphilic lipids and surfactants.14 Depending on the composition and method of manufacture, the particles are typically 50-250 nm in diameter. Small-angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM) are typically used to probe the internal structure of cubosomes and hexosomes. The internal lattice of cubosomes is based on an infinite periodic minimal surface, with the diamond and primitive mesostructures (providing the QII Pn3m and QII Im3m phases, respectively) having been observed, depending on the composition of the dispersion. Although the phase structure of bulk nondispersed GMO in excess water is QII Pn3m, the internal structure of GMO-based cubosomes stabilized with Pluronic F127 is of the QII Im3m type.12 This finding is in contrast with that of monolinolein-15 and phytantriol-based2 cubosomes, which display the QII Pn3m structure even at high Pluronic F127 content. The cubosomes in this report have been prepared using phytantriol and Pluronic F127 in a mass ratio of 9:1 and an overall phytantriol concentration of 2% w/w of the total dispersion. (All experimental details are provided in Supporting Information.) The particles’ QII Pn3m internal mesostructure is confirmed by the relative peak positions obtained by SAXS in Figure 1 (as evidenced by the linear relation between D spacing and the reciprocal relationship to the Miller indices for the Pn3m cubic phase, shown in the inset). The internal structure had a lattice parameter of 64.3 Å, consistent with previous reports for this system.2 Many 2D images of cubosomes using cryo-TEM have been published in the literature, and this subject has recently been reviewed.16 The cubosomes prepared in this study were often found to possess an approximately square morphology (Figure 2, panels A and B), although coexisting vesicles were also observed, typical of these systems. Combined SAXS and cryo-TEM results confirmed that the dispersion comprised cubosome particles, providing confidence in our subsequent assessment of 3D structure. We have recently employed cryo-field emission scanning electron microscopy (cryo-FESEM) to obtain new 3D information about the structure of cubosomes that has not been available
using other techniques.17 The advantage of this technique lies in the fact that it allows vitrified samples to be imaged in the same manner as with cryo-TEM, but a sublimation of the surface water at -90 °C and coating with platinum reveal the 3D structure of particles present in the sublimation plane. Panels C and D of Figure 2 show 3D images of phytantriol-based cubosomes by cryo-FESEM. The structures are similar in morphology to models of cubosome structures generated using differential geometry.4 The internal structure of the nanoparticles is consistent with the QII Pn3m structure when studied using SAXS (Figure 1). Panels A and B in Figure 2 illustrate cubosomes imaged in two dimensions using cryo-TEM and display the cubic morphology with some coexisting vesicular structures. This is consistent with findings on similar systems and with previously published cryoTEM images for phytantriol cubosomes stabilized using vitamin E-TPGS instead of Pluronic F127.9 Panels C and D illustrate representative cubosome structures imaged in three dimensions using cryo-FESEM. The much smaller white particles coexisting with the cubosomes are believed to be Pluronic F127 particles because of their presence in identical studies without phytantriol. Many structures of similar, approximately spherical and angular/ cubic morphologies were observed in the sample, and all possess the distinctive nobbled surface texture predicted in the cubosome model structures represented in panels E and F. It is tempting to consider the dimples in the surface to be the openings of the water channels from the interior of the matrix; however, these features are too large to correlate with the internal channel diameters whose radii are known to be much smaller than 10 nm.18 However, there is also a strong resemblance of the images obtained in this study to QII Pn3m cubosome structures determined using an alternate modeling approach based on the construction of 3D superstructures from nanosized vesicle components in which such nodes are present at the surface and represent the regions of intersection of individual nanovesicles.19 When compared with cubosomes, hexosomes have been less well studied in the literature. Although the hexagonal, closepacked, rodlike micelle arrangement that comprises the internal aqueous domains of the reverse hexagonal phase lacks the complexity of the internal bicontinuous cubic phase structure, the small, closed aqueous compartments confer an ability to
(13) Spicer, P. T.; Hayden, K. L. Langmuir 2001, 17, 5748-5756. (14) Abraham, T.; Hato, M.; Hirai, M. Colloids Surf., B 2004, 35, 107-117. (15) de Campo, L.; Yaghmur, A.; Sagalowicz, L.; Leser, M. E.; Watzke, H.; Glatter, O. Langmuir 2004, 20, 5254-5261. (16) Spicer, P. T. Curr. Opin. Colloid Interface Sci. 2005, 10, 274-279.
(17) Rizwan, S. B.; Dong, Y. D.; Boyd, B. J.; Rades, T.; Hook, S. Micron 2007, 38 478-485. (18) Briggs, J.; Chung, H.; Caffrey, M. J. Phys. II 1996, 6, 723-751. (19) Angelov, B.; Angelova, A.; Papahadjopoulos-Sternberg, B.; Lesieur, S.; Sadoc, J. F.; Ollivon, M.; Couvreur, P. J. Am. Chem. Soc. 2006, 128, 5813-5817.
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Figure 3. Images of hexosomes prepared by the dispersion of phytantriol + 10% vitamin E acetate in 1% Pluronic F127 solution. (A) Representative image obtained using cryo-TEM. (B-F) Representative images obtained using cryo-FESEM showing the 3D structure of hexosomes with the spinning top (B-D) and spine (E and F) structure on the upper surface. (F) Large and small hexosome structure in the field of view.
sustain the release of water-soluble active agents from these structures more effectively than does the bicontinuous cubic phase structure.20 It has not yet been established whether controlled release on a useful time scale can be achieved using hexosomes (or indeed cubosomes21); however, the slower release rate from hexagonal-phase structures indicates that they may in fact be a more promising candidate than cubosomes as a novel controlledrelease delivery system. The structure of hexosomes has also been investigated using cryo-TEM, typically revealing hexagonally facetted particles when viewed in two dimensions.12,15,22 The hexosomes in this study were prepared using the same materials and methods as for the cubosomes in this study by substituting 10% of the phytantriol with vitamin E acetate as we have reported previously.2 We observed the same hexagonal morphology using cryo-TEM for the phytantriol + vitamin E acetate hexosomes as those reported previously for other systems (Figure 3, panel A). The hexagonal facetted particles coexist with particles of less-welldefined morphologies, including hexagonal particles with curved striations reported previously15 and vesicles. The presence of the HII structure is confirmed by the SAXS result in Figure 4, in which the peak positions are consistent with the hexagonalphase structure with a lattice parameter of 49.3 Å.2 To the best of our knowledge, the 3D structure of HII nanoparticles has not been directly observed previous to this report. Barauskas et al. and Sagalowicz et al. have attempted to shed light on hexosome structure using tilt-angle cryo-TEM in dispersed monoglyceride + diglyceride and monoglyceride + oil mixed lipid systems, respectively.9,23 Through an analysis of the change in particle silhouette dimensions with changing angle of incidence of the electron beam and using the assumption of hexagonal disk structure, Barauskas et al. deduced a very flat, high aspect ratio particle morphology for hexosomes. It was therefore claimed that the “unambiguous characterization of the structure” of hexosomes was effectively flat hexagonal prisms. (20) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharm. 2006, 309, 218-226. (21) Boyd, B. J. Int. J. Pharm. 2003, 260, 239-247. (22) Fong, C.; Krodkiewska, I.; Wells, D.; Boyd, B. J.; Booth, J.; Bhargava, S.; McDowall, A.; Hartley, P. G. Aust. J. Chem. 2005, 58, 683-687. (23) Sagalowicz, L.; Michel, M.; Adrian, M.; Frossard, P.; Rouvet, M.; Watzke, H. J.; Yaghmur, A.; De Campo, L.; Glatter, O.; Leser, M. E. J. Microsc. (Oxford, U.K.) 2006, 221, 110-121.
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Figure 4. SAXS pattern for the hexosome dispersion (prepared using phytantriol containing 10% w/w vitamin E acetate as the lipid base) confirming the internal structure of the particles to be the HII space group. (Inset) Derived plot for D spacing of the lattice from the peak positions vs the reciprocal spacing ratio for the 1:x3:x4 peaks corresponding to the HII structure.27
However, using the cryo-FESEM technique applied to cubosomes, we have obtained the first 3D images of hexosome particles. Some representative images of hexosomes are presented in Figure 3 (panels B-F). In contrast to the findings of an essentially flat, hexagonal prism shape, we have found that the HII particles actually possess distinctive 3D structures. In some cases, the HII particles adopted a “spinning-top”-like structure (panels B-D) perhaps best described as a short cylinder capped with a cone at either end. This morphology, when viewed side-on, provides the hexagonally shaped silhouette observed in cryo-TEM images. One could also imagine that if these particles were observed from conical apex to apex the cross section would be circular. Circularly shaped particles observed in cryo-TEM images of hexosomes have often been assumed to be vesicles; perhaps there is an argument that in some instances they may in fact represent spinning-top hexosomes viewed parallel to their major axis, although the presence of internal structural elements would be required to demonstrate this possibility conclusively. In roughly equal proportions to the spinning-top-like structures, there were particles that appear closer to the hexagonal prism description of previous reports but possess a prominent central raised “spine” structure running between opposing apexes (panels E and F). Again, when viewed from the top, these particles provide the hexagonal silhouette expected of hexosomes in cryo-TEM images, but when viewed at an angle with the cryo-FESEM technique, they clearly possess structure in all three dimensions. Notably, no “flat” hexagonal prism structures were observed in any of the cryo-FESEM images of HII particle dispersions nor did the particles resemble the visual representation of a previously proposed model for hexosome morphology (Figure 4D).24 It is unlikely that the differences between these studies and the results presented here are a consequence of the different chemical composition of the nanostructured dispersions in this study, considering the cryo-TEM and SAXS confirmation that we also report. Although our findings do not concur with previous tiltangle cryo-TEM, dynamic light scattering studies by Neto et al. have suggested that a globular structure is likely for hexosomes.25 (24) Larsson, K. J. Dispersion Sci. Technol. 1999, 20, 27-34. (25) Neto, C.; Berti, D.; Aloisi, G. D.; Baglioni, P.; Larsson, K. In Situ Study of Soft Matter with Atomic Force Microscopy and Light Scattering. In Trends in Colloid and Interface Science XIV; Buckin, V., Ed.; Springer, Berlin, 2000; pp 295-299.
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Figure 5. (A-C) Cartoons illustrating the likely micellar arrangements observed for a cross section through a hexosome structure. (The cartoon is purely schematic; the cross section of a true hexosome would comprise many more micellar structures than illustrated in this diagram.) (A) An essentially flat hexagonal prism structure as implied from previous cryo-TEM studies.9 (B) A cross section is taken along the spine of a particle, such as those represented in Figure 3E,F. (C) Cross section of a spinning-top-type particle with a sharp apex or indeed a cross section taken through the same particle as in Figure 3C, in which the HII particle was rotated by 90°. (D) A reproduction of a model HII particle proposed in ref 24 (with permission from Taylor & Francis, www.informaworld.com).
Furthermore, the same authors have also argued that the flat disk-like hexosomes observed using AFM contact imaging are likely to be an artifact of the imaging technique and that the particles should possess a more pronounced structure in three dimensions. Various cartoon representations for the cross-sectional configuration of the internal micelles that might satisfy the morphology of hexosomes imaged by cryo-FESEM are represented in Figure 5, panels A-C. When compared to the flat hexagonal prism arrangement in panel A, the internal arrangement of the micelles for a spinning-top-type particle would most likely resemble the pointed apex (panel B), whereas for the more prismlike morphology the apex would be more rounded (panel C). It should be noted that the micelles comprising the HII phase are of the reversed type such that the amphiphile tails (and in this case, vitamin E acetate) constitute the continuous phase, and it is not yet known exactly how the amphiphiles are arranged at the HII-bulk solution interface. It has been proposed that the
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reverse micelles fold over at the extremities of the particles and/ or are joined end to end to form a coating of cylindrical tubes.23 Alternatively, termination of the hexagonally packed micelles at a swollen nodule, previously proposed for the satisfactory packing of lipids at an HII/LR interface,26 may satisfy the geometric curvature constraints at the hexosome surface by maintaining the average curvature close to that required for the lipids comprising the HII phase. Offset packing of the nodular ends of the micelles would then be anticipated to produce the conical structures exhibited by the spinning top particles. Our findings in this study have implications for the potential use of these materials involving the interaction with surfaces because the potential for multilayer packing of materials at the surface may be influenced by the 3D morphology of the particles. It remains to be determined whether the 3D structure of the particles is significant in applications such as drug delivery. However, as their 3D structures become better elucidated, our approach of using surface structure to modify interaction with a target surface and controlling the interior structure to modify agent release is envisaged to lead to the adoption of liquidcrystal particles as a platform for new and exciting applications. It should also be noted that unlike cryo-TEM, cryo-FESEM provides no information on the internal phase structure of the particles, only surface and overall particle morphology information. Hence, there is room for the use of both techniques in concert to better understand the true structure of nanostructured particles in the future. Acknowledgment. We thank the NANO-TAP program for funding and Dr. Jamie Riches and Associate Professor Alasdair MacDowell for assistance with cryo-TEM studies. We thank Dr. Tracey Hanley for assistance in obtaining SAXS data and Liz Girvan for her assistance with establishing the cryo-FESEM technique. We also thank Dr. Michael Rappolt for his insight into potential packing arrangements at the surface of hexosome structures. Supporting Information Available: Materials and experimental methods. This material is available free of charge via the Internet at http://pubs.acs.org. LA7029714 (26) Rappolt, M.; Hickel, A.; Bringezu, F.; Lohner, K. Biophys. J. 2003, 84, 3111-3122. (27) Hyde, S. T. Identification of Lyotropic Liquid Crystalline Mesophases. In Handbook of Applied Science and Colloid Chemistry; Holmberg, K., Ed.; John Wiley & Sons Ltd.: New York, 2001; pp 299-332.
