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Novel Lipid System Forming Hollow Microtubes at High Yields and Concentration Jean-Paul Douliez,*,† Ce´dric Gaillard,‡ Laurence Navailles,§ and Fre´de´ric Nallet§ INRA/BIA, EÄ quipe Interface et Syste` mes Disperse´ s, rue de la Ge´ raudie` re, F-44316 Nantes, France, INRA/BIA, Plate-Forme Microscopie, rue de la Ge´ raudie` re, F-44316 Nantes, France, and Centre de Recherche Paul Pascal, CNRS, 115 AV. Albert Schweitzer, F-33600 Pessac, France ReceiVed December 2, 2005. In Final Form: February 15, 2006 There is considerable interest in constructing supramolecular hollow tube architectures based on amphiphilic molecules. This can be achieved by using relatively expensive synthetic lipids. Herein, we report on the facile preparation of self-assembled microtubes from a novel low-cost lipid mixture that does not require a previous chemical synthesis step and consists of the ethanolamine salt of 12-hydroxy-stearic acid in water. Tubes of more than 10 µm in length spontaneously form upon cooling from an isotropic solution. They exhibit inner and outer diameters of 400 and 600 nm, respectively, and their walls consist of concentric stacked bilayers of fatty acid salts, each separated by a layer of water.
Introduction The literature reporting on the formation of supramolecular hollow nanotube architectures based on amphiphilic molecules is markedly increasing.1 As in the case of carbon nanotubes,2 there is considerable technological interest. Such self-assembled lipid nanotubes possess a core and an outer surface, both of which are hydrophilic so that they can serve as templates for the fabrication of inorganic nano-objects,3-5 as substrates for the crystallization of macromolecules,6-8 and as low-molecular-mass organogelators.9 There are a limited number of natural and synthetic lipids that can self-assemble into tubular structures, among which are glycolipids1,4,10,11 and their bola-form derivatives12 and phospholipids.13-17 Most often, the tube-forming lipids are chiral molecules, and tubes are obtained in mixtures of alcohol and water following an intermediate step in which helically coiled ribbons form.1 A theoretical description has been proposed with * Corresponding author. E-mail:
[email protected]. Phone: + 33 2 40 67 50 83. † INRA/BIA, E Ä quipe Interface et Syste`mes Disperse´s. ‡ INRA/BIA, Plate-Forme Microscopie. § Centre de Recherche Paul Pascal. (1) Shimizu, T.; Masuda, M. Minamikawa, H. Chem. ReV. 2005, 105, 14011443. (2) Iijima, S. Nature 1991, 354, 56-58. (3) Ipsita, A. B.; Lingtao, Y. Hiroshi, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14678-14682. (4) Zhu, H.; John, G., Wei, B. Chem. Phys. Lett. 2005, 405, 49-52. (5) Gao, P.; Zhan, C. Liu, M. Langmuir 2006, 22, 775-779. (6) Wilson-Kubalek, E. M.; Rhoderick, E. B.; C., H. Milligan, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8040-8045. (7) Matsui, H.; Douberly, G. E. Langmuir 2001, 17, 7918-7922. (8) Lorigan, G. A.; Dave, P. C.; Tiburu, E. K.; Damodaran, K.; Abu-Baker, S.; Karp, E. S.; Gibbons, W. J. Minto, R. E. J. Am. Chem. Soc. 2004, 126, 9504-9505. (9) Zhan, C.; Gao, P. Liu, M. Chem. Commun. 2005, 462, 462-464. (10) Blanzat, M.; Massip, S.; Speziale, V.; Perez, E.; Rico-Lattes, I. Langmuir 2001, 17, 3512-3514. (11) Kamiya, S.; Minamikawa, H.; Jong, H. J.; Bo, Y.; Mitsutoshi, M. Shimizu, T. Langmuir 2005, 21, 743-750. (12) Masuda, M. Shimizu, T. Langmuir 2004, 20, 5969-5977. (13) Spector, M. S.; Easwaran, K. R. K.; Jyothi, G.; Selinger, J. V.; Singh, A. Schnur, J. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12943-12946. (14) Spector, M. S.; Selinger, J. V.; Schnur, J. M. J. Am. Chem. Soc. 1997, 119, 8533-8539. (15) Spector, M. S.; Selinger, J. V.; Singh, A.; Rodriguez, J. M.; Price, R. R.; Schnur, J. M. Langmuir 1998, 14, 3493-3500. (16) Douliez, J.-P.; Lavenant, L.; Renard, D. J. Colloid Interface Sci. 2003, 266, 477-480. (17) Spector, M. S.; Price, C. E.; Schnur, J. M. AdV. Mater. 1999, 11, 337340.
Figure 1. Chemical structure of the 12-hydroxy-stearic salt of ethanolamine.
the aim of determining and understanding the relationship between the size and the shape of the tubes.18 Several models were proposed on the basis of the thermodynamics of the lipid membrane selfassembly including the chiral elastic properties of the membranes. Intuitively, in a membrane, the chiral molecules do not pack parallel to their neighbors, generating a twist that can select a radius for tubes and helical ribbons. Then, in racemic mixtures, only flat membranes should form as in the case of gemini surfactants19 except if phase separation of the racemic mixture occurs.18 Tubes can also form with achiral lipids.20 In such a case, the interpretation with respect to the persistence of tube formation is the chiral symmetry-breaking model.18,21 When the symmetry of both monolayers in the membrane is broken through any mechanism, the membrane will tend to bend in a similar fashion as when using chiral molecules. Lipids with the ability to form tubes are rather different in terms of their molecular shape, and this does not allow the determination of a guideline for tube self-assembly. Then, the formation of tubes involving novel lipid mixtures should help in understanding the mechanism of self-assembly of such supramolecular structures. Here, we report on the observation and characterization of microtubes from a novel low-cost lipid mixture involving the ethanolamine salt of 12-hydroxy-stearic acid (Figure 1).
Results and Discussion Randomly oriented rodlike structures having a size of several tens of micrometers were observed by phase-contrast microscopy (see Supporting Information for the experimental setup) upon investigating the phase behavior of the fatty acid salt, namely, (18) Selinger, J. V.; Spector, M. S.; Schnur, J. M. J. Phys. Chem. B 2001, 105, 7157-7169. (19) Oda, R.; Huc, I.; Schmutz, M.; Candau, S. J.; MacKintosh, F. C. Nature 1999, 399, 566-569. (20) Pakhomov, S.; Hammer, R. P.; Mishra, B. K.; Thomas, B. N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3040-3042. (21) Seifert, U.; Shillcock, J.; Nelson, P. Phys. ReV. Lett. 1996, 77, 52375240.
10.1021/la053262t CCC: $33.50 © 2006 American Chemical Society Published on Web 02/25/2006
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Figure 2. Phase-contrast micrograms of a 1% aqueous dispersion of 12-hydroxy-stearic acid-ethanolamine salt at room temperature. The scale bar represents 10 µm.
at a fatty acid/ethanolamine equimolar ratio (Figure 2). These rods were further assigned to tubes by transmission electron microscopy (TEM). Samples were prepared by weighing the desired amount of fatty acid powder (Aldrich, 98% purity, racemic mixture) in a test tube and adding pure water. Then, the appropriate volume of a freshly prepared 1 M solution of ethanolamine (Sigma, 98% purity) was added. Samples were heated to 75 °C until the solutions become completely clear without any trace of undissolved material and cooled at room temperature. At room temperature, the samples appear as turbid liquid solutions except at high concentration (greater than 50 mg/mL) where a white gel is formed: the sample tube can be turned upside down without any flow. Rods were clearly observed by phase-contrast microscopy up to a concentration of 200 mg/mL. Below a concentration of 5 mg/mL, the lipid rods were curved and aggregated to form stars characteristic of lipid crystals, which might be a consequence of the swelling limit of the lamellar structures.22 Rods were also observed for various fatty acid/ ethanolamine molar ratios, showing that they can be formed in a wide range of compositions in the phase diagram. For instance, rods were obtained in 1% solutions with excess of fatty acid with respect to ethanolamine up to a molar ratio of 2/1. They survive the addition of 10% (by weight with respect to 12-hydroxystearic acid) palmitic acid or stearic acid or in equimolar mixtures of lysine and ethanolamine salts of the fatty acid. Rods were also obtained with derivatives of ethanolamine such as propanolamine, L-alaninol, and 1-amino-2-propanol. However, lamellar phases or vesicles were observed in the case of pure lysine and triethanolamine salts. As in the case of other nanotubes,11 the present lipid mixtures allow for the formation of tubes with a yield higher than 98%. By contrast, our microtubes can also be obtained at high concentrations, which can be relevant for their use as templates. This reveals that our lipid mixture consisting of 12-hydroxy-stearic acid and ethanolamine allows for the facile formation of microtubes under various experimental conditions. The major requirement is that the mixture has to be heated until an isotropic solution occurs and then cooled again. The lipid rods were further characterized by TEM to obtain valuable information on their tubular structure at room temperature (Figure 3). Images were obtained by depositing a lipid drop (previously stained) on carbon-coated TEM grids and recording on a JEM 100S JEOL microscope operating at 80 kV. Two different staining methods were used to characterize either the whole structure or the outer surface of the tubes. With phosphotungstic acid, the dark edges clearly show the tubular wall and the relatively bright center, which is the empty inner (22) Dubois, M.; Zemb, T. Curr. Opin. Colloid Interface Sci. 2000, 5, 27-37.
Figure 3. TEM images of the 1% aqueous dispersion of 12-hydroxystearic acid-ethanolamine salt at room temperature after staining with phosphotungstic acid (top) and uranyl acetate (bottom).
part of the tubes. This unambiguously shows that the lipid rods as observed by phase-contrast microscopy are hollow structures (i.e., microtubes). In the case of the ethanolamine salt, we measured inner and outer diameters of 400 and 600 nm, respectively, averaging over 50 tubes observed in different micrograms. We did not observe a significant effect of composition on the tube diameters, and further details will be given in a forthcoming paper. It must be noted that we never observed any coiled ribbons or vesicles, showing that the tubes formed immediately upon cooling from the transparent, hot solution. However, a coiled ribbonlike structure can be observed when examining the outer surface of the tubes upon staining with uranyl acetate. This is very similar to the structure of lipid tubules with adsorbed Pd-Ni catalyst on the surface23 and allows us to measure a helical pitch angle of about 45° in agreement with theoretical predictions.23,24 Because we used a racemic mixture of 12hydroxy-stearic acid, the microtubes do not form from chiral self-assembly. Instead, the chiral symmetry-breaking model that does not rely on molecular chirality is most probably involved.18 Similar findings were already obtained with achiral diynoic lipids.20 To get structural information on the lipid self-assembly in such tubular structures, we performed small-angle neutron scattering (SANS) experiments on a 1% sample made with the fatty acid salt dissolved in deuterated water. The sample is held in a 2-mm-path, rectangular quartz cell. Data were obtained at room temperature on the PAXY spectrometer (Supporting Information) at Laboratoire Le´on-Brillouin (Laboratoire mixte CEAsCNRS, Saclay, France). The 2D spectrum (Figure 4) is somewhat anisotropic, owing to the wall or flow-induced orientation effects. The pattern anisotropy indicates that, among the microtubes oriented parallel to the cell windows (i.e., perpendicular to the neutron beam), the preferred orientation is along the long axis of the rectangular cell. This shows that the lipid tubes can be rather easily oriented, which may be of practical interest for obtaining structural information on host macromolecules. In addition, fatty acid mixtures are known to self-orient (23) Selinger, J. V.; Schnur, J. M. Phys. ReV. Lett. 1993, 71, 4091-4094. (24) Selinger, J. V.; MacKintoch, F. C.; Schnur, J. M. Phys. ReV. E 1996, 53, 3804-3818.
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thickness of the tubes at room temperature was determined to be about 100 nm by TEM, that is to say, approximately three stacking periods, there are typically four membranes stacked in a concentric fashion to form a microtube. As in the case of glycolipids,1,11,12 our microtubes are thus composed of multiwalled tubes, with a water layer in between. This finding is unique because no one has ever observed a wall structure with such a high repeat distance in microtubes or nanotubes. Preliminary cryo-TEM experiments did not allow us to confirm that wall structure because the samples are too viscous and concentrated and did not freeze properly, but we obtained interesting images from a frozen sample (after drying of the grid) showing a broken tube with a lamellar stacking in agreement with our SANS data (Supporting Information). In the description of the microtube structure, there is thus a hierarchy of characteristic scales, namely, the microtube length L, with typically L ≈ 10 µm, the microtube inner diameter D, with D ≈ 400 nm, the lamellar stacking period d, with d ≈ 35 nm, and ultimately, molecular dimensions as measured, for instance, by the lipid bilayer thickness δ. As far as the SANS experiment is concerned, the first two dimensions L and D can be considered to be practically infinite because the smallest accessible value Qmin for the scattering wave vector, about 6 × 10-3 Å-1, is larger than 2π/D by a safe margin. It then becomes possible to describe the main features of the spectra in terms of a simple model where a finite number N of planar membranes of well-defined thickness δ and large lateral extension are stacked with a period d to build randomly oriented, finite-sized lamellarphase crystallites. On the local scale probed by neutron scattering, there is no difference between randomly oriented large cylinders with thin walls exhibiting a lamellar structure and lamellarphase crystallites. The scattering intensity I(Q) can then be expressed27 as the product of three terms, namely, the form factor P(Q) of a planar membrane of well-defined thickness δ, with the simple expression
Figure 4. Two-dimensional scattering pattern (top) and azimuthally averaged scattering intensity (bottom) of the 1% aqueous dispersion of 12-hydroxy-stearic acid-ethanolamine salt at room temperature. In the 2D picture, the pixel unit corresponds to wave vector increments of 5.8 × 10-4 Å-1 for both the horizontal and vetical scattering directions. The line represents the best fits as obtained with the model described in the text.
field,25
in a magnetic and we will further examine this feature that could be of particular interest for NMR experiments. The azimuthally averaged spectrum, also displayed in Figure 4, exhibits a strong small-angle scattering signal. Three sharp peaks located at Q0 ) 0.018, 0.036, and 0.054 Å-1 (i.e., in an exact ratio of 1:2:3) can be readily identified. Qualitatively similar oscillations have been reported previously for other tubular aggregates26 and described in terms of the scattering signal of randomly oriented, hollow cylinders. Such an analysis is not appropriate in the present case, however, because the expected peak location ratio would be 1:1.88:2.73, in clear disagreement with our SANS data. We therefore describe the peaks as Bragg scattering arising from the 1D periodic staking of lipid membranes, separated by water. According to this interpretation, the microtubes are formed by periodically stacked, concentric layers. Moreover, a lamellar spacing (i.e., the repeat distance corresponding to one lipid layer and a water layer of 2π/Q0 ) 35 nm) can be determined from the experiment. Because the wall (25) Douliez, J.-P. Langmuir 2004, 20, 1543-1550. (26) Terech, P.; Talmon, Y. Langmuir 2002, 18, 7240-7244.
P(Q) )
sin2(Qδ/2) Q2
the structure factor S(Q) of the 1D stack, and a factor of 1/Q2 accounting for the random orientation of the membranes, that is to say,
I(Q) ≈
P(Q) S(Q) Q2
Following ref 27, we use a model for the structure factor S(Q) explicitly taking into account thermally induced elastic distortions in the lamellar stack and the finite resolution of the scattering experiment, which ensures that S(Q) will go quickly to its limiting value of 1 at large wave vectors Q. Note that among the numerous parameters of the model only three are not known a priori and have to be determined by a fitting procedure: an arbitrary intensity scale factor, the Caille´ parameter η controlling the elastic fluctuations in the lamellar stack,27,28 and the thickness δ of the lipid bilayers. Fitting the model to the whole Q range of the SANS data allows an estimation of two relevant quantities: the Caille´ parameter η, which is essentially determined from the Bragg peak region of the spectrum, and from the larger-angle part of the spectrum where S(Q) is close to 1, the membrane thickness δ. The fit, superimposed on the data in Figure 4, is quite (27) Nallet, F.; Laversanne, R.; Roux, D. J. Phys. II 1993, 3, 487-502. (28) Caille´, A. C. R. Hebd. Seances Acad. Sci., Ser. B 1972, 274, 891.
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satisfactory. It yields a value of 4.3 nm for δ and η ) 0.025. Because the stacking period is large, such a small value of the Caille´ parameter suggests very rigid bilayers (curvature modulus on the order of 50kBT), in association with strong interactions in the lamellar stack.28 Besides, as far as the membrane thickness is concerned, it must be remembered that the stearic chain in its extended conformation has a length of about 2.1 nm, that is to say, about half the value that we obtain from the fit. The SANS data are then consistent with lipids organized in bilayers with their alkyl chains in an all-trans conformation (i.e., in their gel state, which is compatible with the above-mentioned high membrane rigidity). Though a membrane repeat distance of 35 nm may appear to be a rather high value for lipids embedded in a gel state, it is a common feature in salt-free catanionic systems.29 Besides, the formation of bilayers from 12-hydroxystearic acid salts indicates that the hydroxyl moiety at position 12 remains within the hydrophobic region of the membrane. Hydrogen bonds between adjacent fatty acids probably stabilize such a structure. In summary, we were able to produce microtubes from a simple, low-cost, novel lipid mixture without using any chemical synthesis. The tubes were obtained in high yields and at high
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concentration without using solvent (i.e., in pure water). They can be doped either within the membrane or at the membrane/ water interface up to 10 and 50%, respectively. This finding should contribute to our understanding of the mechanism by which lipid tubes self-assemble and offers additional perspectives for template synthesis or other chemical and physical properties using such supramolecular objects. Moreover, it shows that hydroxy-derivated fatty acids exhibit interesting self-assembly properties because in addition to supramolecular cones30 they also form tubes. The detailed effects of temperature and composition on the structure of those microtubes are under investigation and will be reported elsewhere. Acknowledgment. We thank Laboratoire Le´on-Brillouin for the allocation of neutron beam time on the PAXY spectrometer, experiment number 7246. We gratefully acknowledge the assistance of our local contact, Laurence Noirez, during the neutron scattering run. Supporting Information Available: Materials and methods and additional TEM images. This material is available free of charge via the Internet at http://pubs.acs.org. LA053262T
(29) Zemb, T.; Dubois, M.; Deme´, B.; Gulik-Krzywicki, T. Science 1999, 283, 816-819.
(30) Douliez, J.-P. J. Am. Chem. Soc. 2005, 127, 15694-15695.
