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Nanocage Aggregates Composed of Bilayer Sheets Keisuke Matsuoka,*,† Tomokazu Yoshimura,‡ Miri Bong,‡ Chikako Honda,† and Kazutoyo Endo† Department of Physical Chemistry, Showa Pharmaceutical UniVersity, Higashi-Tamagawagakuen 3-3165, Machida, Tokyo 194-8543, Japan, and Graduate School of Humanities and Sciences, Nara Women’s UniVersity, Kitauoyanishi-machi, Nara 630-8506, Japan ReceiVed February 26, 2008. ReVised Manuscript ReceiVed April 19, 2008 A partially fluorinated carboxylate-type anionic gemini surfactant, N,N′-di(3-perfluorohexyl-2-hydroxypropyl)N,N′-diacetic acid-ethylenediamine (2C6Fedda), formed aggregates with a cagelike structure in an aqueous alkali solution. These aggregates were composed of several bilayer sheets. The TEM micrograph showed that the bilayer sheets were produced from a condensed self-assembly core. The leaflike bilayer sheets can form folds and link to each other at their edges. The typical size of the spherical cage ranged from ca. 200 to 1500 nm.
The study of self-assembly using simple synthesized molecules helps us to understand the generation mechanisms of complicated biological or natural organisms. Alternatively, biomolecules or natural compounds in such organizations provide several hints for the design of synthetic molecules. A typical example of a self-organizing nanoscale system is a biological membrane, which is mainly composed of phospholipids.1 Amphiphilic molecules possess a unique molecular structure containing two hydrophobic alkyl chains and one hydrophilic group that can self-associate to form liposomes in aqueous solutions. Mimic molecules from preparations that have a particular function can aggregate in a similar manner; these molecules act as drug carriers in the medical field or as solubilizers in nanoscience.2,3 Fluorinated amphiphiles are important in medical and pharmaceutical fields because of their biological inertness and thermal and chemical stability.2,3 A fluorocarbon chain is stiffer than a hydrocarbon chain because of the presence of bulky fluorine atoms. Hence, aggregates of fluorinated amphiphiles tend to form a structure with small surface curvature (i.e., vesicles and lamellar aggregates); this explains why the molecular volume of the aggregates is larger than that of the corresponding hydrocarbons in aqueous solutions.4 Li et al. have reported the formation of a bilayer of polygonal sheets or vesicles with a laterally nanostructured membrane via the self-assembly of a fluorinated terpolymer.5 The report clarifies that the morphology of the aggregate changes when the volume ratios of the hydrophilic group, hydrocarbon chains, and fluorocarbon chains are varied. The authors have synthesized a novel partially fluorinated carboxylate-type anionic gemini surfactant, N,N′-di(3-perfluorohexyl-2-hydroxypropyl)-N,N′-diacetic acid ethylenediamine (2C6Fedda). The synthesis procedure is detailed in the Supporting Information. The molecular structure of 2C6Fedda is shown in Figure 1a. The characteristic molecular structure is referred to as the gemini surfactant, which resembles phospholipid mol* Corresponding author. Tel:+81-42-721-1566. Fax: +81-42-721-1565. E-mail:
[email protected]. † Showa Pharmaceutical University. ‡ Nara Women’s University.
(1) Bangham, A. D.; Horne, R. W. J. Mol. Biol. 1964, 8, 660. (2) Kissa E. In Fluorinated Surfactants: Synthesis, Properties, Applications; Marcel Dekker: New York, 1994. (3) Kissa E. In Fluorinated Surfactants and Repellents, 2nd ed.; Marcel Dekker: New York, 2001. (4) Wang, K.; Karlsson, G.; Almgren, M.; Asakawa, T. J. Phys. Chem. B 1999, 103, 9237. (5) Li, Z.; Hillmyer, M. A.; Lodge, T. P. Nano Lett. 2006, 6, 1245.
Figure 1. Molecular structure of partially fluorinated gemini surfactants. (a) Anionic gemini surfactant (used in the present study) and (b) cationic gemini surfactants (shown for a complementary explanation).8,9
ecules.6,7 To date, the aggregation properties of the analogous surfactant (C6FC3-2-C3C6F), shown in Figure 1b, or those of hybrid gemini surfactants have been reported.8–10 The authors have found that novel 2C6Fedda can form nanosized cagelike aggregates. To the author’s knowledge, the structure of these aggregates has not yet been described. These aggregates consist of several leaflike bilayer sheets, and they resemble a nanocage with a vacant space at its center. The aim of the present study is to elucidate the novel structure of these aggregates by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The crystals of 2C6Fedda are poorly soluble in a neutral aqueous solution because 2C6Fedda has two carboxyl groups, which make it a weak acid, and hydrophobic dimeric fluorocarbon chains (Figure 1a). As a result, sodium hydroxide aqueous solution (0.1 M) was used as the solvent. The solutions were agitated for a day by using a magnetic stirrer and filtered through a membrane filter with a pore size of 0.45 µm (ADVANTEC, Cellulose Acetate Dismic-25cs). The sample solutions were transparent below 0.45 mM at 298.2 K, and their properties have remained unchanged for half a year. The self-association concentration that corresponds to the critical aggregation concentration (CAC) was 2 µM, as shown in Figure 1S. The considerably low threshold concentration (6) Zana, R.; Xia, J. In Gemini Surfactants: Synthesis, Interfacial and SolutionPhase BehaVior, and Applications; Marcel Dekker: New York, 2003. (7) Zana, R. J. Colloid Interface Sci. 2002, 248, 203. (8) Matsuoka, K.; Yoshimura, T.; Shikimoto, T.; Hamada, J.; Yamawaki, M.; Honda, C.; Endo, K. Langmuir 2007, 23, 10990. (9) Yoshimura, T.; Ohno, A.; Esumi, K. Langmuir 2006, 22, 4643. (10) Oda, R.; Huc, I.; Danino, D.; Talmon, Y. Langmuir 2000, 16, 9759.
10.1021/la800618v CCC: $40.75 2008 American Chemical Society Published on Web 05/02/2008
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Figure 2. Change in rh with the concentration of 2C6Fedda at 298.2 K. The arrows indicate the CAC of 2C6Fedda. A-D correspond to those shown in Figure 3.
was determined by a change in the intensity of scattered light at 298.2 K. The CAC of the analogous surfactant (C6FC3-2C3C6F), shown in Figure 1b, in the aqueous solution was 172 µM.9 The difference in the CACs of both the surfactants was significantly large even when different ionic strengths were considered. The decrease in the CAC implies that the molecular structure of 2C6Fedda is quite suitable for aggregation. The change in the average apparent hydrodynamic radii (rh) of the aggregates shown in Figure 2 supported this fact; rh was measured by DLS (ALV-5000, Germany) at 298.2 K. The spontaneous association led to the formation of large aggregates at low concentrations in the system of 2C6Fedda. The rh values showed a maximum of ca. 280 nm near the CAC and then gradually decreased to ca. 80 nm with an increase in concentration. With a few notable exceptions, the rh-concentration curve, Figure 2, of conventional gemini surfactants and fluorinated ones has not been measured. One exceptional case was reported for a type of surfactant, shown in Figure 1b, whose fluorocarbon chain (C4FC3-2-C3C4F) was shorter than C6FC3-2-C3C6F. The aggregates of C4FC3-2-C3C4F that were in the form of vesicles (rh ) 300 nm) became small aggregates (rh ) 25 nm) with an increase in concentration.8 In the present system of 2C6Fedda, the change in the size of the aggregate was also reflected by the change in its morphology. The authors observed the self-association structure by TEM (negative staining (Figure 3a-g) and freeze-fracture techniques (Figure 4a-c)) at several concentrations. The advantage of the former technique is that it is easy to use and it provides relatively highly image contrast as a result of the dispersion of uranyl acetate in the bulk area. The latter technique can cover the former fault (dehydrated observation), and the fracture of replica from frozen sample gives the image of concave-convex for molecular aggregates. In a relatively dilute solution (0.05-0.1 mM), the 2C6Fedda aggregates attained the structure of large aggregates, as shown in the TEM micrograph in Figure 3a. The average rh value at 0.05-0.1 mM was determined to be ca. 280 nm by DLS. As shown in Figure 3a, the core of the aggregate had a vacant space; therefore, it was hollow. The large aggregates were apparently formed by stacked lamellar sheets. In fact, Figure 3b clearly illustrates the generation of a bilayer sheet from a condensed layer. The polydispersity indices (average deviation/average rh) of the aggregates varied from almost 0.7 to 1 within the measurement range, according to the DLS technique. As indicated in Figure 2, the rh value of the aggregates monotonously decreases at concentrations above 0.1 mM; the
Figure 3. Negative-staining TEM images of 2C6Fedda aggregates at different concentrations. Concentrations are indicated in each micrograph, and capital letters correspond to those shown in Figure 2. The generation of a bilayer sheet from a condensed layer (black arrow) is seen in b.The leaflike bilayer sheets fold (white arrow) and link with the edge of another one (black arrow) in d and e.
average rh value is ca. 160 nm at 0.21 mM. The micrographs in Figure 3c-f show the existence of cagelike aggregates at 0.21 mM; these aggregates have vacant spaces at their centers. In Figure 3c, the typical sizes of the spherical cages range from ca. 200 to 1500 nm. The polydisperse state can be easily observed (Figure 3c). The detailed structures of the aggregates are shown in the TEM images in Figure 3d,e. The cagelike aggregates are
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Figure 4. Freeze-fracture TEM micrographs of 2C6Fedda solution at 0.21 mM (a). The leaflike bilayer sheet (black arrow) and the source (white arrow) can be distinctly seen in b. The complicated nanocage aggregate is also seen in c.
composed of several leaf-shaped bilayer sheets. Each bilayer sheet can fold and links with another bilayer leaf at the edge. The freeze-fracture TEM micrograph also shows a cagelike aggregate composed of several bilayer sheets (Figure 4a-c). The outlines of the cagelike aggregates at a relatively low magnification are shown in Figure 4a. The average size and shape of the aggregates can be seen in Figure 4a. The concave-convex surface supports the 3D structure of aggregates. The characteristic leaflike bilayer sheet and the source are clearly observed in Figure 4b. The micrograph in Figure 4b bears some resemblance to Figure 3f. The complicated pattern of the nanocage aggregate is also seen in Figure 4c. The two procedures for TEM observations reached agreement for the cagelike aggregate. In the case of the association of hybrid fluorocarbon/hydrocarbon catanionic sugar-based surfactants, a shape similar to that of coiled membranes on a micrometer scale has been reported by Pasc-Baun et al.11 Finally, the bilayer sheets are separated from the core of the aggregates and are dispersed in relatively small units with increasing concentration. The shape of the small unit makes up bilayer sheets at 0.42 mM (Figure 3g). The following question arises: why are aggregates with a nanocage structure produced by the self-association of 2C6Fedda? This unusual aggregation behavior mainly originates from the molecular structure of 2C6Fedda (Figure 1a). The molecule has a dimeric fluorocarbon chain as the hydrophobic group. In general, (11) Pasc-Banu, A.; Stan, R.; Blanzat, M.; Perez, E.; Rico-Lattes, I.; Lattes, A.; Labrot, T.; Oda, R. Colloids Surf., A 2004, 242, 195.
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the aggregates of fluorinated amphiphiles tend to form a structure with a small surface curvature as a result of the nonflexible fluorocarbon chain. For example, fluorinated monoalkyl surfactants Cn - 1F2n - 1CH2N+(CH3)3Cl- (n ) 8, 10, 12, and 14) and fluorinated gemini surfactants CnFC3-2-C3CnF (n ) 4, 6, and 8), shown in Figure 1b, can form large aggregates by extending their fluorocarbon chains.8,12 Although both 2C6Fedda and C6FC32-C3C6F have the same fluorocarbon chain lengths, the former forms bilayer sheets whereas the latter forms stringlike aggregates.8 When the molecular structure of the former (Figure 1a) is compared with that of the latter (Figure 1b), it is found that the latter gemini surfactant has stereoscopic hindrance due to bulky hydrophilic groups such as quaternary ammonium, which have two methyl groups. In general, the shape of the aggregate depends on the geometrical constraint of each surfactant molecule. Unfortunately, the theory introduced by Israelachvili et al. is not numerically applicable to such a dimeric surfactant; this theory requires a cylindrical molecular shape in the case of a lamellar structure.13–15 Taking an objective view of the matter, the molecular shape of 2C6Fedda may be roughly cylindrical. Moreover, the authors assume that the formation of a nanocage aggregate is related to the thermodynamic stability in a specific concentration range. The large aggregates are suitable for avoiding water molecules at relatively low concentrations. The increase in the concentration of the surfactant monomer leads to the formation of small aggregates because the hydrophilic microenvironments around the aggregates decrease with an increase in concentration. The total free energy for aggregation may consequently decrease with the increase in aggregate concentration (entropy effect). The small units of aggregates separate from large aggregates, and bilayer sheets are produced from the condensed source (Figure 4f). After the supply of molecules from the bilayer source is stopped, the nanocage aggregate is completely formed. Finally, the self-assembly structure attained a small aggregate composed of bilayer sheets at a relatively higher concentration. The nanocage aggregate is formed because the molecular structure has (1) a proper nonflexible fluorocarbon chain length, (2) a roughly cylindrical shape (objective estimation), and (3) a specific concentration range. Acknowledgment. The TEM work was performed at the Hanaichi Ultrastructure Research Institute in Okazaki, Japan. Supporting Information Available: Synthesis of 2C6Fedda, experimental detail, and CAC determination (Figure 1S). This material is available free of charge via the Internet at http://pubs.acs.org. LA800618V (12) Matsuoka, K.; Ishii, M.; Yonekawa, A.; Honda, C.; Endo, K.; Moroi, Y.; Abe, Y.; Tamura, T. Bull. Chem. Soc. Jpn. 2007, 80, 1129. (13) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. Biophys. Acta 1977, 470, 185. (14) Israelachvili, J. N.; Mitchell, D. J. Biochim. Biophys. Acta 1975, 389, 13. (15) Giulieri, F.; Krafft, M. P. Colloids Surf., A 1994, 84, 121.
