A Stimulus-Responsive Shape-Persistent Micelle Bearing a Calix[4

Article pubs.acs.org/Langmuir

A Stimulus-Responsive Shape-Persistent Micelle Bearing a Calix[4]arene Building Block: Reversible pH-Dependent Transition between Spherical and Cylindrical Forms Shota Fujii, Yusuke Sanada, Tomoki Nishimura, Isamu Akiba, and Kazuo Sakurai* Department of Chemistry and Biochemistry, University of Kitakyushu, Hibikino, Kitakyushu 808-0135, Japan

Naoto Yagi and Efstratios Mylonas Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 1-1-1 Kouto, Sayo 679-5198, Japan S Supporting Information *

ABSTRACT: A series of cationic calix[4]arene-based lipids with alkyl chains of varying length were newly synthesized, and the ones with propyl and hexyl tails, denoted by CaL[4]C3 and C6, respectively, were found to form spherical micelles at low pH (protonated state of the amine headgroup). Upon deprotonation with increasing pH, CaL[4]C3 showed a sphere-to-cylinder transition, while CaL[4]C6 changed from sphere, to cylinder, to monolayer vesicle. Synchrotron smallangle X-ray scattering (SAXS) patterns from both spherical and cylindrical CaL[4]C3 micelles exhibited a sharp intensity minimum, indicating shape monodispersity. The monodispersity of the CaL[4]C3 spherical micelles was further confirmed by analytical ultracentrifugation (AUC). SAXS, AUC, and static light scattering agreeingly indicated an aggregation number of 6. In contrast, CaL[4]C6 exhibited polydispersity with an average aggregation number of 12. When the number of carbons of the alkyl chain was increased to 9 (CaL[4]C9), cylinder formed at low pH, while at high pH, no clear morphology could be observed. The present results indicate that a very precise combination of tail length, head volume, and rigidity of the building block is required to produce shape-persistent micelles and that the shape-persistence can be maintained upon a structural transition. An attempt to reconstruct a molecular model for the spherical CaL[4]C3 micelle was made with an ab initio shape determining program.

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INTRODUCTION The potential of supramolecular self-assembly to produce sophisticated structures and functions has attracted significant attention to systems that exhibit these properties. The driving forces for such supramolecular self-assembly consist of multiple anisometric inter- and intramolecular interactions, including hydrophobic, van der Waals, electrostatic, and π−π interactions. Individually, each interaction is subtle and insignificant, but their combination and balance essentially determine the assembled structures. This chemistry can be applied to the production of biocompatible materials that can be used for tissue engineering and drug delivery systems. Because the molecules aggregate through such weak interactions, even a small perturbation of the local environment can trigger rapid and drastic transformation of the equilibrium structures. This behavior can be exploited to introduce stimuli-responsive functions into supramolecular aggregates. Calixarenes are a particularly attractive building block for well-controlled self-assembly. Shinkai et al1 showed that a cone shaped p-sulfonatocalixarene bearing appropriate alkyl groups © 2011 American Chemical Society

forms spherical micelles in aqueous solutions. The nature of the ionic headgroup is a key parameter determining the structure of calixarene amphiphiles; carboxylated calixarenes form vesicles, whereas trimethylammonium headgroups produce spherical micelles.2 Recently, Lee et al.3 synthesized calixarene amphiphiles with several ethylazanediyl-diethanol derivatives and showed that the shape of the micelle changes from spherical to vesicular with increasing bulkiness of the headgroup. The vesicles transform into spherical micelles on dropping the pH from 7 to 5. All of the above-mentioned phenomena can be understood qualitatively in terms of the packing parameter principle.4 Hirsch and his group5 synthesized a calix[4]arene amphiphile with a carboxylic dendritic head that in aqueous solution can assemble into a completely uniform and structurally precise micelle consisting of seven molecules. This shape-persistent micelle is quite unique and Received: September 26, 2011 Revised: December 9, 2011 Published: December 20, 2011 3092

dx.doi.org/10.1021/la2037668 | Langmuir 2012, 28, 3092−3101

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Scheme 1. Synthetic Route of CaL[4]C3 (Compound V) through Click Chemistrya

a

The compounds with alkyl chain lengths 6 and 9 were synthesized in a similar manner.

solutions by use of small-angle X-ray scattering by changing pH for three samples with different alkyl tail lengths.

novel. They also showed that a similarly persistent micelle can be prepared with a fullerene derivative attached to the same dendritic group. Their molecular dynamics simulations show that the metal ion/carboxylic group interaction is the main determinant of the persistent shape and aggregation number, rather than the rigid building blocks of calix[4]arene and fullerene (Supporting Information, S1: chemical structures of other calixarene lipids). pH-responsive structural changes can arise from the attachment of an charged labile headgroup, e.g., an amine group.6 Upon decreasing the environmental pH, so as to protonate the amine, a repulsive force is generated among the headgroups of different molecules in the micelle, inducing structural changes. When the amphiphile has multiple headgroups, e.g., calixarene amphiphiles3 or Gemini type lipids,7,8 intramolecular repulsive forces are generated upon deprotonation and induce conformational transition. This transition can lead to dramatic changes of the micellar structure. Recently, Rodik et al.9 reported that a calix[4]arene amphiphile similar to Lee’s forms a spherical micelle and this system can be used as an effective DNA carrier. The present paper describes the synthesis of a new calix[4]arene cationic amphiphile and the physiochemical characterization of its supramolecular assembly in aqueous

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RESULTS

Synthesis and Molecular Modeling. Scheme 1 presents the synthetic route we followed to prepare the final product V. At each step, we confirmed the product with 1H NMR (the spectra of IV and V are presented in Supporting Information S2). The resulting amphiphilic calixarenes were characterized by elemental analysis and ESI mass spectroscopy (Supporting Information S3) and shown to be in full agreement with the structures presented. Hereinafter, V is denoted by CaL[4]C3. The suffix C3 indicates propyl tails and thus the equivalent hexyl and nonyl compounds are called CaL[4]C6 and CaL[4]C9, respectively. Calix[4]arene can exist in four possible conformations: cone, partial cone, 1,2, and 1,3 alternates.10 Large tail groups such as propyl attached on the lower rim prevent interconversion between conformers. We used a method that yields only the cone conformation. The nucleophilic attack of alkyl iodide was carried out in DMF which locks the conformation of calix[4]arene with intramolecular hydrogen bonding between the lower rim hydroxyl groups. The cone form of the final product CaL[4]C3 was confirmed by 1H NMR. The cone form still has flexibility in the methylene bonds between the aromatic 3093


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Figure 1. Conformational change upon protonation and deprotonation calculated with MOPAC for CaL[4]C3 with a sodium cation coordinated to the four oxygen atoms of the lower rim.

rings, which allows for structures with C 2v and C 4v symmetries.10 The C2v structure has two opposite aromatic rings almost parallel to each other, while the planes of the other two form an almost right angle. In contrast, in a C4v structure the four aromatic rings are equally tilted along the methylene bonds. Although C4v is underrepresented in the crystal state, thus it is less stable than C2v, both structures undergo rapid interconversion in solution.11 On the other hand, when alkaline metals such as sodium and potassium are trapped among the four lower rim oxygen atoms, C4v becomes more favorable due to the shielding of the electrostatic repulsion among the partially negatively charged oxygen atoms.10 Among alkaline metals, the sodium cation has the highest binding affinity toward the calix[4]arene derivatives.12 Figure 1 shows the three-dimensional atomic structure obtained with MOPAC for the protonated and deprotonated CaL[4]C3 with C4v symmetry and sodium coordinated to the four oxygen atoms. At the protonated state, the four amino groups are located as far as possible from each other due to the electrostatic repulsion among the protonated amino groups, while they can come close at the deprotonated state. In terms of the packing parameter theory,4 the head area is drastically altered upon protonation, and thus if they form micelles in aqueous solution, we can expect pH-responsive morphological transition. Micelle Formation and pH Induced Morphological Transition. Pyrene has environmentally sensitive fluorescent peaks: the first peak at 373 nm is intensified in hydrophilic atmosphere, while the third peak at 383 nm is intensified in hydrophobic atmosphere. Therefore, through the relative ratio of these two peaks, the critical micelle concentration (CMC) can be determined by titrating micellar solutions.13 Figure 2a shows the intensity ratio (I 1 /I 3 ) plotted against the concentration of CaL[4]C3 at [NaCl] = 50 mM and pH = 3 and 8. These pH values are below and above the pKa of the amino group, respectively. The CMC was determined from the break point on each plot and summarized in Table 1. As expected, CMC at pH = 8 was smaller than that of pH = 3

Figure 2. CaL[4]C3 concentration dependence of the pyrene fluorescence intensity ratio between 373 and 383 nm (I1/I3) from which the critical micellar concentration (CMC) (a) and the alkyl chain length dependence of CMC (b) are determined.

Table 1. Critical Micelle Concentration, Refractive Index Increment, Partial Specific Volume, and Morphology of the Aggregates at Low and High pH in 5.0 mM NaCl Sample Code

pH

CaL[4]C3

3.0 8.0 3.0 6.3 3.0

CaL[4]C6 CaL[4]C9

CMC/ mM 0.11 0.042 0.0040 0.0029

∂n/∂c g mL−1 v ̅ mL g−1 Morphology 0.202 0.782 Sphere Cylinder 0.195 0.816 Sphere Cylinder Cylinder

because of the increased solubility of the protonated state. Figure 2b shows CMC plotted against the tail length, showing that CMC decreased by more than 1 order of magnitude from C = 3 to C = 6. From these results, it is evident that most molecules are aggregated at concentrations above 0.4 mM. Figure 3 shows the titrimetric curve of a CaL[4]C3 solution at pH = 2.2 adjusted by HCl titrated with a NaOH solution. pH 3094


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Figure 3. Titration curve of a 10 mL CaL[4]C3 solution ([CaL[4]C3] = 1.0 mM in 50 mM NaCl, pH = 2.0) with 0.1 N NaOH and representative AFM images (C, E, G) indicated by red-marked pHs as well as appearances (I and H). For comparison, the AFM images for CaL[4]C6 and CaL [4]C9 are shown at the same pHs.

increased at a titration volume of 1120 μL, which exactly corresponds to the stoichiometric amount of HCl added beforehand and this change is due to the neutralization of HCl. After neutralization, pH was increased in accordance with titration. There is an inflection point observed at a titration volume of 1360 μL, presumably reflecting the deprotonation of the amino head of aggregated CaL[4]C3. The difference between neutralization and this inflection point is 1.5 mM, which is almost half of the total number of the amino groups. Therefore, it can be concluded that at pH < 2−6 the amino groups are fully protonated, at 6 < pH 10 they are completely deprotonated. We carried out microscopic observations at several pHs. At low pH (pH < 6), CaL[4]C3 and CaL[4]C6 showed a spherical shape while CaL[4]C9 showed a rod-like one. For CaL[4]C3, the image changed to rod-like or connected spheres with increasing pH (from C to E) and connected network structures appeared at even larger pH (G). CaL[4]C6 showed a vesicular shape at large pH (F) and a rod-like shape at intermediate pH (D). For CaL[4]C9, we could not observe any ordered structures at large pH. While in images D and E spherical objects coexist with rods, no spherical objects are present in image A. When the CaL[4]C3 solution of pH = 10 was left for a few hours, the solution became partially turbid and gelatinized (H), corresponding to the formation of the network structures at this pH. A more detailed analysis of the AFM images can be seen in Figure 4. The plots show the image height along the indicated lines in the AFM images at pH = 3 and 8 for CaL[4]C3. The image height ranged from 2.3 to 2.5 nm at pH = 3 and from 2.8 to 3.0 nm at pH = 8, indicating that there is a very narrow distribution of the aggregate size. It should be mentioned that, owing to a rather large tip-apex size (ca. 10 nm), the width of the cylinder appears larger than the value obtained by SAXS.

Figure 4. AFM images of the spherical (A) and cylindrical states (B) and their height profiles along the lines indicated at each image.

We believe that the size of the cylinder obtained by SAXS fitting is the more reliable one. Synchrotron Small Angle X-ray Scattering. Figure 5A shows the SAXS profiles of all samples at different pHs in the presence of 50 mM NaCl. For CaL[4]C3 at pH = 3−6, the profiles exhibited typical features of isolated scattering objects; the intensity was satisfying the Guinier law at the low q region and two minima were observed at q = 2 and 5 nm−1 corresponding to features of the internal structure of the particles. With increasing pH, the low q intensity did not follow the Guinier law anymore. Instead, at pH = 7.5 and more, the relation of I(q) ∼ q−α with α = 1 is satisfied, confirming that a rod-like form is present.14,15 In the intermediate region of 6 < pH < 7.5, the scaling factor α is less than 1 and increases with increasing pH. This indicates that at this pH range a mixture of spherical and rod-like aggregates are present in the solution. We 3095


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Figure 5. Absolute SAXS intensities plotted against q for CaL[4]C3 (A), C6 (B), and C9 (C) micelles at different pHs in 50 mM NaCl. For convenience, the profiles are vertically shifted by multiplying with the number shown in parentheses on top of each profile (the number outside the parentheses denotes the pH). The straight lines show the slopes of −1 and −2, representing the expected values for rod- and plate-like scattering objects, respectively. The solid lines overlaid on the data points are calculated from the corresponding core−shell models with the fitting parameters listed in Table 2.

Table 2. SAXS Fitting Parameters for the CaL[4]CX Aggregates at Different pHa Code

pH

Rc /nm

RS /nm

Rs − Rc /nm

σ/Rs

ρC /e nm−1

ρS/ e nm−1

Modelb

CaL[4]C3

4.2 7.5 4.3 6.3 7.8 4.7

0.70 0.55 1.45 1.05 1.15 1.42

2.05 1.68 2.75 2.0 1.91 2.40

1.35 1.0 1.30 0.95 0.76 0.98

0.04 0.03 0.12 0.10 0.18 0.3

270 270 270 270 290 270

391 420 420 440 420 440

sphere cylinder sphere cylinder plate cylinder

CaL[4]C6

CaL[4]C9

Rc: core size, Rs: shell size, ρc: electron density ofthe core, ρS: electron density ofthe shell. bStandard deviation: theoretical expressions for the models are summarized in the Supporting Information S8.

a

= 7.5 also showed a sharp scattering minimum. According to scattering theory,14,15 when the scattering objects are rods (i.e., when the slope of −1 is observed at lower q), the scattering pattern only reflects the cross-section of the rod. Therefore, the presence of the sharp minimum implies that the cross-sectional structure of the CaL[4]C3 is also uniform along the rod and most likely it has a rotational symmetry around the rod axis. For CaL[4]C6 at pH = 4.3 (Figure 5B), the profile exhibited typical behavior of isolated objects similar to that of CaL[4]C3 at pH = 3.0−6.0 and thus was fitted with a core−shell model (solid line). At pH = 6.3, the I(q) ∼ q−1 was held and the data was fitted with a core−shell rod model. These features are common with CaL[4]C3. On the other hand, at pH > 7, it showed different behavior, with a scattering profile corresponding to that of a vesicular object. The scaling factor of α becomes 2, which is consistent with the vesicular objects in AFM. Since the overall size of the vesicle is larger than the q range of SAXS, we fitted the data with a triple layer plate model and the theoretical profile of best fit model is shown as a solid line. The core electron density was 270 e/nm−1 in most cases and this value is normally observed for the alkyl chain domain.17 The exception is the plate model and this could be due to differences among the models or the alkyl chains being more packed or intercalated with each other than in the bilayer state.

used a core−shell sphere or cylinder model to fit the profiles at pH = 3.0 and 7.5, respectively. The best fitted curves are compared with the data and the parameters are listed in Table 2, where RC and RS are the thicknesses of the core and shell of the fitting models and ρC and ρS are the absolute electron densities for the core and shell, respectively. With such a simple core−shell model, the profiles are well reproduced in the range of q < 3 nm−1 (more than 2 nm in the real space). Since the larger q region corresponds to the internal structure of the micelle, in order to fit the SAXS profile at q > 3 nm−1, we need a more elaborate model that can represent the molecular arrangement or the atomic structure of the aggregate. It should be noted that there is a sharp intensity trough at q = 2.0 nm−1. As shown in the Supporting Information S4, this is not an artifact of the oversubtraction of solvent scattering. The presence of such a sharp minimum indicates that the CaL[4]C3 aggregate has a well-defined shape with narrow size distribution and rotational symmetry axes.14−16 This conclusion is consistent with the AFM results in Figure 4. When we examined how other cations change the intensity minimum (Supporting Information S5), Li and K gave less sharp ones than Na. This fact suggests that the cone calix[4]arene conformation with C4v symmetry is essential to provide such a narrow size distribution. Furthermore, the SAXS profile at pH 3096


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Figure 6. I(q)/c vs q plots for different concentrations at [NaCl] = 50 mM, including the extrapolated values at c → 0 in the Guinier region (A). The Guinier plots (i.e., ln I(q)/c vs q2) at c → 0 used to evaluate I(0)/c by extrapolating q → 0(B). The inset C shows the concentration dependence of I(0)/c.

Table 3. Molar Masses Determined with Different Methods and the Aggregation Numbersa sample

SAXS MW/l03 g mol−1

LS MW/ 103 g mol−1

AUC MW/ 103 g mol−1

Mz/MW

Aggregation number

CaL[4]C3 CaL[4]C6

5.80 ± 0.20 14.4 ± 0.95

5.69 ± 0.93 -

6.10 ± 0.20 14.7 ± 0.90

1.07 1.5

6 12

The molar masses of CaL[4]C3 and C6 are 1.0 × 103 and 1.2 × 103g mol−1, respectively. SAXS: synchrotron small-angle X-ray scattering. LS: static light scattering combined with column purification AUC: analytical ultracentrifugation. a

RS − RC, relating to the size of the headgroup, is almost the same for all samples when we use the same model. It also decreases when going from sphere, to rod, to vesicle, consistent with the packing parameter theory where one expects that the headgroup shrinks during this transition. It should be noted that CaL[4]C3 showed a much smaller size distribution of σ/RS than other samples for both sphere and cylinder models (Table 2). This fact is consistent with the uniform peaks in AFM images in Figure 4 and confirms that the aggregates in both forms have a well-defined shape in narrow size distribution. Molar Mass and Its Distribution. We determined the molar mass of the aggregates with three different independent methods: SAXS, light scattering (LS), and analytical ultracentrifugation (AUC). Figure 6A shows the low q regions of both CaL[4]C3 and CaL[4]C6 at pH = 3.0 at different concentrations. The values of I(q) at the limit of c → 0 for each q (i.e., limc→0 I(q)/c) are obtained and plotted in the same figure as colored markers, where c is the CaL[4]C3 concentration (g/mL). Figure 6A indicates that the concentration dependence of the scattering intensities is small enough to allow for the acquisition of accurate values of limc→0 I(q)/c. Figure 6B shows the Guinier plot (i.e., ln I(q) vs q2) for these extrapolated values. From the intercept, the values of limc→0 I(q)/c were obtained, and from these the weight averaged molar mass of the aggregate (MW) was evaluated at 5.8 (±0.2) × 103 g mol−1 for CaL[4]C3 and 14.4 (±1.0) × 103 g mol−1 for CaL[4]C6, respectively. In terms of aggregation numbers (Nagg), Nagg = 6 and 12, for CaL[4]C3 and CaL[4]C6, respectively. The inset C shows I(0)/c values determined by extrapolating at each concentration. The intercept of this plot gave the same values as those in B. The values of MW were

confirmed with LS (Supporting Information S6). The data is summarized in Table 3. Figure 7 presents the results of AUC for CaL[4]C3; the apparent weight-averaged molecular weight (Mw,App) and Q are

Figure 7. Concentration dependence Mw,App and Q (= Mw,App/Mz,App) (inset figure) for CaL[4]C3 solutions containing 50 mM NaCl, determined with analytical ultracentrifugation.

plotted against the concentration, where Q is defined as the ratio of Mz,App/Mw,App, with the z-averaged molecular weight (Mz,App). The values of Mw were evaluated at 6.1 (±0.2) and 14.7 (±0.9) × 103 g mol−1 for CaL[4]C3 and CaL[4]C6, respectively, being consistent with those of SAXS and LS. Since the scattering and the ultracentrifugation mass determination methods lay on different principles and these values agree with 3097


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atomic micellar model was constructed so that the six CaL[4]C3 molecules obtained by MOPAC can assemble in a cubic symmetric way since we know that we have 6 molecules per micelle and the cubic symmetry is the most intuitive way to arrange them. The MOPAC model does not account for thermal fluctuation, and we are in the middle of calculating molecular dynamics of this model to compare with scattering data. It should be noted that the headgroups and tails of the surfactant are very mobile and the overall shape of the micelle is expected to be dynamic. Thus, the dummy atom structures are an average of the multiple conformations adopted.

each other, we can conclude that the aggregation numbers of 6 and 12 are quite reliable. It should be noted that Q is close to 1 for CaL[4]C3, confirming that the sample is monodisperse. On the other hand, Q is about 1.5 for CaL[4]C6, indicating polydispersity. Dummy Atom Models. The scattering pattern of the micelle at pH 1.2 up to resolution q ≅ 5 nm−1 was used to produce the dummy atom model with the program DAMMIN.21 Since we know that the aggregation number of the micelle is six and under the assumption that the monomers are arranged in a symmetric way, we also constructed models with imposed cubic symmetry (each monomer corresponding to one face of a cube). It should be noted that the volumes obtained with DAMMIN are in all cases larger than the dry volume of the particles, and this is due to the larger density of the bound water in the hydration shell compared to the bulk water.16,21 Figure 8 shows a representative model and we

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DISCUSSION pH Dependence of Shape. Figures 3 and 5 show that the micellar shape change is well correlated with the protonation of the headgroup and the transition takes place over a very narrow pH range. For example, the sphere-to-cylinder transition for CaL[4]C3 occurs in the range of pH = 6.5−7.5. Figure 1 shows that the headgroup conformation of CaL[4]C3 is drastically changed upon protonation. This calculation showed that its shape can be represented by a quadrangular pyramid at low pH and by a quadrangular prism at high pH, when sodium cation is coordinated to the four oxygen atoms. Therefore, the micellar shape transition upon pH change is semiquantitatively understood in the framework of packing parameter principle.4 According to the literature, the micellar structure is determined by the packing parameter defined by al/V, i.e.,, the balance of the three geometric factors, where a is the interface area occupied by one headgroup, V is the volume occupied by the hydrophobic moieties such as tails, and l is the length of the hydrophobic moieties. Later, Nagarajan pointed out that, given a headgroup (so a would be constant in the classical manner), al/V is almost constant, because the volumeto-length ratio (l/V) should be independent of the tail length.18 His paper showed that the tail length controls the magnitude of a. For CaL[4]C6, three shapes are observed in the order of sphere, cylinder, and plate (or vesicle) with increasing pH. When the alkyl tails become shorter, there was no plate observed at higher pH. On the other hand, when the alkyl tails become longer, only cylinder was observed at low pH. As pointed out by Nagarajan, this tail length effect cannot be understood in the original principle and its interpretation needs a more sophisticated model. Shape Persistence. The SAXS profiles for CaL[4]C3 at pH = 3−4 and 7.5 showed a sharp intensity minimum and AUC showed monodispersity in the aggregation number at pH = 3. These facts suggest that the molecules assemble in a particular manner in both spherical and cylindrical states. Generally, micelles are in dynamic equilibrium among multiple structures and between the molecules constituting the micelles and the free molecules in the solution. Therefore, it is believed that the shape or aggregation number of micelles is variable. In this sense, the present finding is quite unusual (a similar system was previously reported by Burghardt et al.6). This shape persistence is only observed for CaL[4]C3, suggesting that a suitable combination of head shape and a tail length is necessary to attain the persistence. Furthermore, the presence of the sodium cation is also essential (Supporting Information S5). It is well-known that after certain cations such as sodium coordinate the four oxygen atoms of the lower rim, the conformation of calix[4]arene is fixed at C4v symmetry.10,12 Therefore, the effect of the sodium salt effect to the rigidity of the building block of the lipid is another factor contributing to

Figure 8. Dummy atom model of the CaL[4]C3 micelles at low pH with imposed octahedral symmetry in Panel (A) and its cross section (B). For comparison, the dummy atom model (transparent gray surface representation) is superimposed on a hexameric atomic model (sphere representation) (C). Monomeric models are shown in side (D) and top view (E).

confirmed the fit to the experimental SAXS pattern (Supporting Information S7). The shape is roughly spherical and hollow. This result may seem surprising at first glance, but closer assessment of the properties of the micelle gives a satisfactory interpretation. The electron density of the alkyl tails of the molecule is significantly smaller than that of the headgroups and the water shell surrounding them, while it is similar to the density of the bulk solvent (water). DAMMIN can only represent two phases of uniform density, one for the solvent and one for the particle. Consequently, the ″solvent″ phase in our case represents both the bulk solvent and the alkyl chains while the ″particle″ phase represents the headgroups and the water shell. The higher density of the water shell compared to the bulk solvent also explains why the dummy atom model is larger than the atomic micellar model in Figure 8. Here, the 3098


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cylinder appear. The present results indicate that a precise combination of tail length, head volume, and molecule rigidity is necessary to attain the shape persistence.

the shape persistence of the micelle. One could imagine each molecule as a tetragonal pyramid that can assemble into a cube only if it possesses very specific geometrical dimensions. Hydrophobic interactions between the tail groups and π−π interactions between the aromatic rings would provide stability to the assembly. Hirsch and his group5 were the first to report shape persistence in a calix[4]arene amphiphile with a carboxylic dendritic head and dodecyl tails. They concluded that the micelles consist of seven molecules based on cryo-TEM observations, although they did not determine the aggregation number with a direct method. Their molecular dynamics calculations indicate that the major driving force of the aggregation and the shape persistence is the geometrical packing of the dendritic carboxyl heads and the interaction between the carboxyl heads through cation bridges. In fact, even when they replace calix[4]arene by fullerene and attach the same carboxylic dendritic head, they observe similar shape persistence. Therefore, different features of their molecules are responsible for the shape persistence of their micelles than for the shape persistence of ours. Shape Determination from SAXS. There is a lack of phase information in scattering data, and thus, the inverse Fourier transform cannot be carried out without assumption to reconstruct real space images. If there are enough diffraction peaks, the inverse Fourier transform can be done with less ambiguity;19 however, it is a generally challenging issue to do such reconstruction only from form factor scattering. Recently, several methods for shape reconstruction from SAXS data have emerged.20−22 Although the approaches of each method and the respective programs are different, they all rely on the same principle. Given minimal initial constraints, random shapes are generated, the scattering pattern is calculated and compared to the experimental data, and subsequently the shapes are changed accordingly in small steps in order to improve the fit. Although the fitting parameters to sphere, cylinder, or vesicle models give a general idea of the shape of the molecule or aggregate of interest, they are simplifying and not very informative. In this study, we reconstructed the shape of the CaL[4]C3 low pH micelle and showed that a hexamer arranged in octahedral symmetry is indeed compatible with the experimental SAXS data. Moreover, the shape of the produced dummy atom model is far from the perfect sphere implied by the fitting parameter models. This allows for a better estimation of the relative positions of the monomers that comprise the aggregate and subsequently the interactions that lead to the specific micelle formation. Concluding Remarks. A new calix[4]arene-based cationic lipid with varying alkyl tail length was synthesized and the micellar architecture exhibits strong dependence on pH and tail length. Spherical micelles were formed at low pH (protonated state of the amine head) for both CaL[4]C3 and C6. Upon deprotonation, CaL[4]C3 showed a sphere to cylinder transition, while CaL[4]C6 changed from sphere, to cylinder, to monolayer vesicle. SAXS from the CaL[4]C3 micelle for both spherical and cylinder states exhibited shape monodispersity. The monodispersity of the CaL[4]C3 sphere was confirmed with analytical ultracentrifugation. The aggregation numbers of the protonated states of CaL[4]C3 and CaL[4]C6, as determined by SAXS, static light scattering, and ultracentrifugation, are 6 and 12, respectively. When carbon tail length was increased to 9 (CaL[4]C9), there was no clear morphology observed at high pH, and only at low pH did

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EXPERIMENTAL SECTION

Synthesis and Materials. Synthesis of 5,11,17,23-Tetrakis[tertbutyl((1H-1,2,3-triazol-4-yl)methyl)carbamate-25,26,27,28- tetrapropoxy-calix[4]arene (IV): A solution of compound 3 (0.131 g, 1.60 × 10−4 mol), N-Boc-propargylamine (0.125 g, 8.08 × 10−4 mol), copper sulfate pentahydrate (3.31 mg, 1.32 × 10−5 mol), sodium ascorbate (0.0262 g, 1.32 × 10−4 mol), and anhydrous N,Ndimethylformamide (15 mL) was stirred at 90 °C for 36 h under nitrogen atmosphere, and then water was added and the reactant extracted with EtOAc. The organic layer was washed three times with saturated NaCl solution and dried over MgSO4. The solution was evaporated to dryness, and the residue was purified by flash chromatography over silica gel using CH2Cl2:Methanol = 15:1 as eluent, IV was obtained after evaporation of the solvent (0.170 g, 1.19 × 10−4 mol, 73%). 1 H NMR (500 MHz, CDCl3): δ = 7.46 (s, 4H), 6.49 (s, 8H), 5.43 (s, 4H), 5.21 (s, 8H), 4.40 (s, 8H), 4.38 (d, J = 13.5 Hz, 4H), 3.80 (t, J = 7.5 Hz, 8H), 3.06 (d, J = 13.5 Hz, 4H), 1.88 (m, J = 7.5 Hz, 8H), 1.41 (s, 36H), 0.97 (t, J = 7.5 Hz, 12H). Synthesis of 5,11,17,23-Tetrakis[(1H-1,2,3-triazol-4-yl)methanaminehydrochloride-25,26,27,28- tetrapropoxy-calix[4]arene (V): A solution of the compound IV (0.17 g, 1.19 × 10−4 mol) was treated with 4 M HCl/EtOAc for 2 h, and then washed with CH2Cl2. The residue was recovered with methanol and V was obtained after evaporation of the solvent (0.157 g, 98%) 1HNMR (500 MHz, methanol-d4): δ = 8.12 (s, 4H), 6.69 (s, 8H), 5.36 (s, 8H), 4.42 (d, J = 13.5 Hz, 4H), 4.29 (s, 8H), 3.81 (t, J = 7.5 Hz, 8H), 3.13 (d, J = 13.5 Hz, 4H), 1.90 (m, J = 7.5 Hz, 8H), 0.99 (t, J = 7.5 Hz, 12H). ESI-MS (m/z): [M−Na]+ calcd for C56H72N16NaO4 1056.27; found 1055.6. The compounds I, II, and III were synthesized with the reported method.23 Calix[4]arene and synthesis chemicals and solvents were purchased from Tokyo Chemical Industry Co., Sigma-Aldrich Co., Wako Chemical Industries and used without further purification. Water was purified with a Millipore Milli-Q water purification system. Light Scattering. Light scattering (LS) was carried out with a Dawn-Heleos-II (Wyatt) coupled with a Shodex GPC-101 system. A 400 μL sample of unfiltered solution was injected into a system consisting of a Waters 590 programmable HPLC pump (Waters, Milford, MA) and a Shodex degassing unit (ERS-3000). The chromatogram was measured with an Optilab DSP interferometric differential refractive index detector (Wyatt) and a UV absorbance detector SPD-10A (Shimazu). The column was a Shodex IC SI-90 4E (polyvinyl alcohol quaternary ammonium, particle size; 9 μm) which is generally used for chromatographic analysis or separation of anions. We used this column to optically purify the sample solution. The elution of sufficient amount of intact micelles required the injection of an untypically large amount of CaL[4]C3 sample in order to saturate the hydrophobic groups of the column resin (that otherwise destroy the micelles). The mobile phase was a 50 mM NaCl solution with a pH of 2.8. Data acquisition and analysis was performed using Wyatt’s ASTRA for Windows software (v 4.73.04). Scattered light intensities at scattering angles 14−163° were measured and the weight averaged molecular mass (MW) was determined. The specific refractive index increment (∂n/∂c) of CaL[4]C3 and CaL[4]C6 in a 50 mM NaCl solution (pH = 3.0) were 0.202 and 0.198 cm3 g−1, respectively, determined with a DRM-1021 differential refractometer (Otsuka Electronics) at 633 nm and 25 °C (Supporting Information S8) . Synchrotron SAXS Measurements. SAXS measurements were performed at BL-40B2 of SPring-8, Japan. A 30 cm × 30 cm imaging plate (Rigaku R-AXIS VII) detector was placed at 0.7 or 1.8 m away from the sample. The wavelength of the incident beam (λ) was either 0.071 or 0.10 nm. The 0.7 and 1.8 m set-ups provided a q range of 0.2−10 nm−1 and 0.07−4.0 nm−1, respectively, where q is the magnitude of the scattering vector defined by q = 4π sin θ/λ with the 3099


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configuration X that minimizes the function f(X) = χ2 + αP(X). Here, χ2 is the discrepancy

scattering angle of 2θ. A bespoke SAXS vacuum sample chamber was used and the X-ray transmittance of the samples was determined with an ion chamber located in front of the sample and a Si photodiode for X-ray (Hamamatsu Photonics S8193) after the sample. The detailed experimental procedures are reported elsewhere.17,24 Fluorescence Measurements. The fluorescence probe pyrene was used without further purification. All solutions were prepared by diluting a stock solution (5 mM of CaL[4]Cn solution) with distilled water containing 50 mM NaCl (pH = 3.0). The concentration of pyrene was fixed at 1.0 × 10−5 M. The fluorescence measurements were carried out with a fluorescence spectrophotometer (Hitachi F4500), by exciting at 335 nm and recording the emission spectrum in the range 350−650 nm. The scan speed and the slit widths were 260 nm min−1 and 5.0 nm, respectively. Atomic Force Microscopy Observations. 50 uL of the solution was drop-cast on the mica surface and dried in air. AFM experiments were performed immediately following sample.25,26 The films were imaged by AFM (SII NanoTechnology Inc.) operating in tapping mode at room temperature using a silicon tip (SI-DF20(AL)). The frequency of the tapping mode was 118 kHz and the radius of the tip apex is around 10 nm. Analysis of SAXS Data. Form Factor of Multilayer Models. The SAXS intensity from a dilute solution containing identical, randomly oriented particles may be expressed by27

I(q) = NVM 2( ρ̅ − ρ0)2 P(q)

χ2 =

j

(3)

where N is the number of experimental points, and I(q), Iexp(q), and σ(q) denote the calculated intensity of the model, the experimental intensity, and the experimental error, respectively. The penalty term P(X) taken with a positive weight α > 0 ensures that the model has low resolution with respect to the packing radius r0 and ″loose″ or very detailed shapes are discouraged. It is also possible to impose point symmetry conditions on the models. Molar Mass Determination. Light and X-ray Scattering. The forward scattering intensity I(0) can be written as M 2 I(0) = NVM 2( ρ̅ − ρ0)2 = cv ( ρ̅ − ρ0)2 NA ̅

(4)

where NA is the Avogadro number, v ̅ is the partial specific volume of a micelle, M is the molar mass, and c is the weight concentration. For static light scattering experiments, the magnitude of v2̅ (ρ̅ − ρ0)2can be directly determined by the concentration dependence of the refractive index. In the case of SAXS, however, the terms of v ̅ and ρ̅ − ρ0 have to be given separately, where ρ̅ − ρ0 can be calculated from the atomic scattering factor.14 In this paper, we determined v ̅ by measuring the density increment upon addition of the solute molecules (Supporting Information S10). The values used in this paper are listed in Table 1. Analytical Ultracentrifugation. Sedimentation equilibriums of CaL[4]C3 and CaL[4]C6 in 50 mM NaCl at pH = 3.0 was studied in a Beckman Optima XL-1 ultracentrifuge at 25 °C. A 12 mm doublesector cell was used and the liquid column was adjusted to 2.0 mm. The rotor speeds were set at 2.0 × and 1.5 × 104 rpm for C3 and C6, respectively. From analyzing the Rayleigh fringe, the apparent weight average molecular weight Mapp,W−1 and Q (= Mapp,W/Mapp,Z) were determined according to the established method.29

(1)

where N is the number of the particle in the unit volume, VM is the volume of the particle, and ρ0 and ρ̅ are the scatteringlength density (or electron density) of the solvent and the particle, respectively. When the particle is inhomogeneous, ρ̅ is given by taking average all over the scattering object. P(q) is the form factor of the particle, reflecting the inner structure of the particle (i.e., internal electron density distribution) and the overall shape. The form factor for multilayer models is generally written by k ⎡ A (ρ − ρ )B(qr ) ⎤ 1 1 P(q) = a ∑ ⎢ i i i+1 i ⎥ 2 q ( ρ̅ − ρ ) qri ⎣ ⎦ 0 i=1

⎡ I(q ) − I (q ) ⎤2 exp j j 1 ⎥ ∑ ⎢⎢ ⎥ N−1 ( q ) σ j ⎦ ⎣

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2

ASSOCIATED CONTENT

S Supporting Information *

Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

(2)

■ ■

Here, A is the relative volume (Vi) for sphere, the crosssectional area (Si) for cylinder, the radius (ri) for plate, and B is spherical Bessel function, the first-order Bessel function, or sine function, when it applied to sphere, cylinder, or plate, respectively. ρi is the electron density of the i the layer, except for ρk+1 being the electron density of the solvent (i.e., ρk+1 = ρsolv). ri is the relative the radius of the i the layer. When we apply eq 2 to core−shell spheres, we chose a = 0, k = 2, A is Vi, and B(x) as the first-order spherical Bessel function, and the relative volume is defined so as to P(q) = 1 at the limit of q = 0. For core−shell cylinders, a = 1, k = 2, A is Si, B(x) is the firstorder Bessel function, and qP(q) = 1 at the limit of q = 0. For the plate, a = 2, k = 2, A is ri, B(x) is the sine function, and q2P(q) = 1 at the limit of q = 0. When applied to the data, we assumed a Gaussian distribution for the radius. More details are presented in the Supporting Information (S9). Ab Initio Modeling. The maximum dimension (Dmax) and the distance distribution function of the micelle was determined using the indirect Fourier transform program package GNOM.28 The program DAMMIN21 was used for ab initio shape determination. A sphere of diameter Dmax is filled with densely packed small spheres (dummy atoms) with diameter r0 ≪ Dmax. At the initial step of the minimization each bead is assigned randomly either to the solvent or to the particle phase. A simulated annealing procedure is employed to find a bead

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS This work is financially supported by JST CREST program and all SAXS measurements were carried out at SPring-8 40B2 (2009A0012, 2009B1397, 2010A1089, 2010B1726). We acknowledge the help of Profs. T. Sato and K. Terao at Osaka University with analytical ultracentrifugation, and Prof. K. Yamaguchi and Dr. I. Ohara with mass analysis.

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REFERENCES

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A Stimulus-Responsive Shape-Persistent Micelle Bearing a Calix[4

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