NANO LETTERS
Self-Assembled Lipid Superstructures: Beyond Vesicles and Liposomes
2005 Vol. 5, No. 8 1615-1619
Justas Barauskas,* Markus Johnsson, 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 April 12, 2005
ABSTRACT A unique set of nanoparticle dispersions of self-assembled lipid mesophases with distinctive reversed cubic, hexagonal, and sponge phase structures has been prepared by use of original lipid combinations and a simple, generally applicable and scalable method. All key properties, particle size distributions, shape, phase structure, and stability, are controlled predictably and reproducibly. The results suggest the crossdisciplinary use of nonlamellar particle structures in science and technology as, for instance, biomimetics, in vivo drug delivery vehicles for diagnostic and therapeutic agents, protein crystallization matrices, and soft nanoporous materials.
Nonlamellar nanoparticles formed by dispersion of selfassembled lipid mesophases have many potential uses in science and technology due to their encapsulating and spacedividing nature, featuring both hydrophilic and hydrophobic domains forming mono- or bi-continuous networks. Suitable applications include biomimetics,1,2 matrices for protein crystallization,3,4 and delivery vehicles of therapeutic agents.5,6 A particular lipid can form various liquid crystalline (LC) phases (polymorphism) where small changes in molecular or ambient properties can lead to morphology and phase changes.7-9 It is well-known that such LC phases can be dispersed into biomimicking particle structures provided, the occurrence of a multiphase region that comprises the liquid crystalline phase and a dilute aqueous phase. The simplest and most studied of such structures are the lamellar phase dispersions - vesicles and liposomes discovered by Bangham and co-workers in the 1960s.10 A key application of liposomes is in pharmaceuticals as drug delivery vehicles of anticancer and antifungal therapeutics.11,12 A milestone in the understanding of lyotropic LC phases is the work by Luzzati and co-workers,13 which clearly demonstrated that lipids can self-assemble into different nonlamellar phase structures. With decreasing spontaneous curvature, these include L3 (or “sponge”), reversed bicontinuous cubic (QII), reversed hexagonal (HII), and reversed micellar cubic (III) phases. In analogy with the liposomal dispersions, nonlamellar phase particles can be prepared by dispersion formation in a multiphase region comprising a liquid crystal in coexistence with a dilute aqueous phase. Such particles were first observed in a study of fat digestion,14 * Corresponding author: Phone +46 462228175, Fax +46 462224413, E-mail:
[email protected]. 10.1021/nl050678i CCC: $30.25 Published on Web 04/30/2005
© 2005 American Chemical Society
and it was later found that that these structures had a bicontinuous cubic structure and could be prepared by dispersing glycerol monooleate (GMO) in water in the presence of surfactant and polymeric stabilizers.15,16 However, the full potential of nonlamellar colloidal particles has not yet been realized due to the lack of a predictive and reproducible manufacturing method. A further hinderance was the fact that that only a limited number of lipid constituents were available for making such particle structures, most notably unsaturated monoglycerides (uMGs).17,18 Here we demonstrate that it is possible to produce different self-assembled lipid superstructures with a high degree of internal order and structure uniformity. All key particle properties - size, shape, and internal mesophase structure - can be tuned by exploiting suitable lipid combinations and by including an optional heat treatment step (125 °C, 20 min) in the preparation process. Representative sets of cryogenic transmission electron microscopy (cryo-TEM) images of nanoparticles with different internal mesophase structures are shown in Figure 1. These molecular superstructures are of unusual quality and illustrate the diversity and beauty of self-assembled lipid architectures. Images a-d in Figure 1 show examples of the GMO-based bicontinuous cubic nanoparticles viewed along the [100] and [111] directions. Inserted are also high magnifications and their Fourier transformations, which yield reflections consistent with the body centered cubic phase Q229 of the Im3hm space group (or cubic P-type in the minimal surface description of bicontinuous cubic phases).19,20 The calculated lattice parameter of 125.5 Å is in good agreement with the corresponding value of 129 Å obtained from X-ray diffrac-
Figure 1. Representative cryo-TEM micrographs of different nonlamellar lipid nanoparticles. Panels a, b, c and d: Reversed bicontinuous cubic phase particles viewed along [001] (a and b) and [111] (c and d) directions. The dispersion was prepared at the weight ratio GMO/ F127/water ) 1.88/0.12/98.0. Panels e and f: Monodisperse “sponge” phase nanoparticles prepared at the weight ratio DGMO/GDO/P80/ water ) 2.13/2.13/0.74/95.0. Panels g and h: Reversed hexagonal monocrystalline particles made of lipids at the weight ratio DGMO/ GDO/F127/water ) 2.25/2.25/0.5/95.0. Fourier transforms of magnified areas in panels b, d, f, and h show the structural periodicity of the different nanoparticles consistent with the mesophase structures indicated above.
tion (XRD) (Figure 2a). Particle sizes estimated from the images (Figure 1) and measured by laser diffraction (Figure 2b) are also in agreement, showing that the overall dispersion is homogeneous. Note that the mean particle size can be tuned within the range of 100-1000 nm by simply varying the composition prior to the heat-cycling.21 Once prepared, the particle properties do not change noticeably over periods of several months. Until recently only uMGs have been known to possess the aqueous phase behavior suitable for the formation of bicontinous cubic nanoparticles. Our previous study has shown that 3,7,11,15-tetramethyl-1,2,3-hexadecane-triol, which is widely used as active ingredient for the cosmetics industry and commonly known as phytantriol (PtOH), exhibit an aqueous phase behavior similar to uMGs.22 It forms QII phase in excess solution, the main criterion for preparing the corresponding nanoparticle structures. We have discovered that the phytantriol-based cubic phase can be effectively dispersed into stable and reproducible nanoparticles by use of small amounts of D-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (Vitamin E TPGS) (Figure 3). Compared to uMGs, PtOH is more chemically stable, which can be an important advantage in some applications such as cosmetics. To facilitate the preparation of other nonlamellar particle structures the study was extended to systems predicted suitable for forming the other mesophase structures of interest, i.e., “sponge” (L3) and reversed hexagonal (HII) structures. To enable the effective fragmentation and at the same time the tuning of the internal structure, it was necessary to work with multicomponent mixtures. A lipid combination that was found to have particularly suitable 1616
Figure 2. Representative powder XRD patterns and particle-size distributions of cubic (panels a and b), “sponge” (panels c and d) and reversed hexagonal phase (panels e and f) nanoparticles. Polydispersity index (PI) is expressed as the ratio between the standard deviation of the distribution and the mean value. The lipid compositions of the respective samples were as in Figure 1.
properties and aqueous phase behavior was glycerol dioleate (GDO)/diglycerol monooleate (DGMO). At equal weight Nano Lett., Vol. 5, No. 8, 2005
Figure 3. Cryo-TEM micrographs of the phytantriol-based reversed bicontinuous cubic phase particles. The sample was prepared at the weight ratio PtOH/vitamin E TPGS/water ) 1.76/0.24/98.0. Fourier transforms of magnified areas in panel b show the structural periodicity viewed along [100] (left) and [111] (right) directions which are consistent with the Im3hm mesophase structure.
ratio, fortified with small amount of polyoxyethylene(20) sorbitan monooleate (Polysorbate 80, P80), and subsequently mixed with water, the composition forms beautiful nanoparticles with inner “sponge” mesophase structure and spherical shapes (Figures 1e,f). These intricate particle structures form without the need of high energy dispersing methods. The “sponge” particles are essentially self-dispersing, yielding dispersions of extremely high quality in terms of low polydispersity, reproducible mean size in the submicron range, inner morphology, and colloidal stability. Such a system is highly suitable for the formulation of pharmaceutical actives, such as proteins and nucleic acids that are sensitive to high shear forces or harsh preparation conditions and can be prepared using biocompatible excipient combinations. The cryo-TEM images and size distribution (Figure 2d) show that the “sponge”-phase particles consistently have a size of ∼100 nm with an outer layer built of intersecting lamellas and an apparent dense inner core. Due to the highly disordered interior, no Bragg peaks are observed in Fourier transformed cryo-TEM images (Figures 1e,f) and XRD (Figure 2c). A qualitative description of the nature of these “sponge” particles can be obtained if they are looked upon as melted versions of the cubic phase particles shown in Figures 1a-d and Figure 3. The basic driving force forming an L3 rather than lamellar or cubic phase is not an entropy increase due to the disordered structure, but rather optimization of the lipid monolayer curvature.23 Another important feature of this “sponge” phase nanodispersion is that it forms readily also at low hydrations (75 wt % water and below) with nearly identical key properties as dispersions formed at high hydrations (> 90 wt % water). Nano Lett., Vol. 5, No. 8, 2005
The mixture of DGMO/GDO 50/50 (wt/wt) forms a HII phase in water. Contrary to the hexagonal phase formed by phospholipids, such as dioleoylphosphatidylethanolamine (DOPE),24 the HII phase in the DGMO/GDO system is readily dispersed into stable nanoparticles in the presence of poly(ethylene oxide) (PEO) based triblock copolymers, such as the Pluronic F127. The dispersions can be prepared by simple shaking without the need of high-energy treatment. However, the previously mentioned heat treatment step is a prerequisite for forming monocrystalline HII-phase particles of the type shown in Figures 1g,h. From the XRD measurements (Figure 2e), a lattice parameter of 55 Å is calculated for the dispersed sample. This is very close to the value of 55.5 Å determined for the corresponding DGMO/GDO (50/ 50 wt/wt) bulk sample in excess water (50 wt % water) without the F127 stabilizer. Hence, it clearly demonstrates that the F127 polymer is excluded from the particle core “honeycomb” (HII) structure and is therefore necessarily situated at the surface of the particles. Indeed, this is an indispensable condition for forming stable particle dispersions while retaining the internal HII mesophase structure. Notably, fragmentation in the solution with P80, which has a much smaller headgroup (or hydrophilic part) and therefore locates more in the particle interior or core structure, results in a transition from HII to “sponge” mesophase structure, reflecting the drive toward increased monolayer curvature (less negative curvature) induced by P80. The location of the F127 polymer at the surface of the HII particles, with the PEO chains of the triblock copolymer protruding out into the aqueous environment, confers an effective steric (entropic) stabilization of the particles in analogy with so-called “stealth” liposomes.25 The “stealth” functionality facilitates high in vivo stability and long circulation times when injected into the bloodstream of a subject. The above results also demonstrate a strong correlation between the mesophase internal structure and the shape of the nanoparticles. For example, monocrystalline cubic-phase nanoparticles tend to maintain the shape of the cube, which is clearly seen in the [001] projection (Figures 1a,b). For the highly disordered “sponge” phase structures, the spherical particle shape appears most favorable (Figures 1e,f). Interestingly, the most common shape for the HII nanoparticles is the hexagonal prism with an average width of about 200 nm (Figures 1g,h). The fact that these prisms always appear projected along the [01] direction in the cryo-TEM images suggests that they have a high aspect ratio and consequently are aligned in the relatively thin vitrified films of the experiment. To prove this hypothesis, angle-resolved cryoTEM measurements were performed to visualize other facets of the particles and gain further insight into the 3-D structure (Figure 4). The thickness of the particles was evaluated by measuring the projected width as a function of tilting angle and comparing with the corresponding geometric model of a hexagonal prism. Indeed, the high aspect ratio (the width is about five times greater than the thickness) is confirmed by the images shown in Figure 4, providing a final 1617
Figure 4. Angle-resolved cryo-TEM micrographs of the reversed hexagonal nanoparticles together with their theoretical shape models tilted at 0 (a), 10 (b), 20 (c), 30 (d), 40 (e), and 50° (f) angle. The specimen is tilted from right to left. The particle thickness was determined by measuring the width as a function of the tilting angle and relating the results with the geometric hexagonal prism model. The lipid composition of the sample was as in Figure 1.
unambiguous characterization of the structure of the novel monocrystalline HII nanoparticles. uMGs exhibit hemolytic properties at low to moderate concentrations in vivo. Therefore, it was of interest to find alternative, more “biologically friendly” lipid compositions which, in excess water solution, form a bicontinuous cubic phase that can be successfully fragmented into stable nanoparticle dispersions. We have recently discovered that synthetic dioleoyl phosphatidylethanolamine (DOPE) can form a bicontinuous cubic phase when mixed with small amounts of PEGylated GMO under physiologically relevant conditions.26 Here, we introduce a more relevant system based on the naturally occurring HII phase forming soy PE (SPE). As shown in Figure 5, aqueous mixtures of SPE and P80 self-assemble into a QII phase. Although the exact space group is yet to be determined, the angles between reflections obtained from the Fourier transforms (45 and 60° for the [001] and [111] viewing direction, respectively) prove the cubic type lattice. This phospholipid-based system may prove to be very useful for constructing nonlamellar nanoparticles for parenteral drug delivery as well as new matrices for membrane protein crystallization. We conclude that stable and structurally well-defined nanodispersions of nonlamellar mesophases are formed by self-assembly of novel lipid combinations and use of a simple and reproducible preparation method involving heat treatment. Pending applications include utilization of the nanoparticle systems as drug-delivery vehicles. We also envision 1618
Figure 5. Representative phospholipid-based cubic phase fragments (panels a and b) and colloidal particle formation (panel c). The magnified areas and their Fourier transforms in panels a and b show the structural periodicity along the [001] and [111] viewing direction, respectively. The sample was prepared at the weight ratio SPE/P80/water ) 1.65/0.35/98.0.
the use of the soft, nanoporous materials in catalysis and material science. Acknowledgment. We are grateful to Gunnel Karlsson for assistance with the cryo-TEM instrumentation. This work was supported by the Swedish Foundation for Strategic Research, Vinnova, and Camurus Lipid Research Foundation. Supporting Information Available: Detailed descriptions of sample preparation, particle size, XRD and cryoTEM measurements are available. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Luzzati, V. Curr. Opin. Struct. Biol. 1997, 7, 661-668. (2) Landh, T. FEBS Lett. 1995, 13, 13-17. (3) Landau, E. M.; Rosenbusch, J. P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14532-14535. (4) Caffrey, M. Curr. Opin. Struct. Biol. 2000, 10, 486-497. (5) Shah, J.; Sadhale, Y.; Chilikuri, D. M. AdV. Drug DeliVery ReV. 2001, 47, 229-250. (6) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 2000, 4, 449-456. (7) Luzzati, V. in Biological Membranes; Chapman, D., Ed.; Academic Press: New York, 1968; pp 71-123.
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(8) Lindblom, G.; Rilfors, L. Biochim. Biophys. Acta 1989, 988, 221256. (9) Seddon, J. M. Biochim. Biophys. Acta 1990, 1031, 1-69. (10) Bangham, A. D.; Horne, R. W. J. Mol. Biol. 1964, 8, 660-668. (11) Woodle, M. C. AdV. Drug DeliVery ReV. 1995, 16, 249-265. (12) Boswell, G. W.; Buell, D.; Bekersky, I. J. Clin. Pharmacol. 1998, 38, 583-592. (13) Luzzati, V.; Husson, F. J. Cell Biol. 1962, 12, 207-219. (14) Patton, J. S.; Carey, M. C. Science 1979, 204, 145-148. (15) Larsson, K. J. Phys. Chem. 1989, 93, 7304-7314. (16) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M. Larsson, K. Langmuir 1997, 13, 6964-6971. (17) Larsson, K. Nature 1983, 304, 664. (18) Yang, D.; Armitage, B.; Marder, S. R. Angew. Chem., Int. Ed. 2004, 43, 4402-4409. (19) Hyde, S. T.; Andersson, S.; Ericsson, B.; Larsson, K. Z. Kristallogr. 1984, 168, 213-219.
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(20) Hyde, S. T.; Andersson, S.; Blum, Z.; Lidin, S.; Larsson, K.; Landh, T.; Ninham, B. W. The Language of Shape; Elsevier: New York, 1997. (21) Barauskas, J.; Johnsson, M.; Joabsson, F.; Tiberg, F. Langmuir 2005, 21, 2569-2577. (22) Barauskas, J.; Landh, T. Langmuir 2003, 19, 9562-9565. (23) Andersson, D.; Wennerstro¨m, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243-4253. (24) Rand, R. P.; Fuller, N. L. Biophys. J. 1994, 66, 2127-2138. (25) Ceh, B.; Winterhalter, M.; Frederik, P. M.; Vallner, J. J.; Lasic, D. D. AdV. Drug DeliVery ReV. 1997, 24, 165-177. (26) Johnsson, M.; Barauskas, J.; Tiberg, F. J. Am. Chem. Soc. 2005, 127, 1076-1077.
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