Glutamic Acids Bearing Calix[4]arene Micelle: pH-Controllable

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Glutamic Acids Bearing Calix[4]arene Micelle: pH-Controllable Aggregation Number Corresponding to Regular Polyhedra Shota Fujii, Rintaro Takahashi, and Kazuo Sakurai Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b00603 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on March 31, 2017

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Glutamic Acids Bearing Calix[4]arene Micelle: pHControllable Aggregation Number Corresponding to Regular Polyhedra Shota Fujii, Rintaro Takahashi, and Kazuo Sakurai*

Department of Chemistry and Biochemistry, University of Kitakyushu, 1-1 Hibikino, Kitakyushu, Fukuoka 808-0135, Japan

KEYWORDS: monodisperse micelles, pH-responsiveness, amino acid-based zwitterions

ACS Paragon Plus Environment

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ABSTRACT

We have prepared a new calix[4]arene-based lipid containing glutamic acid as the hydrophilic group. The α-amine and the γ-carboxylic acid groups of the glutamic acid moiety allowed a continuous change of the state of the head-group from cationic to zwitterionic and then to anionic with increasing pH. Accompanying this head group change, micelles of the lipid underwent a morphological transformation from spherical to cylindrical, and again to spherical. The morphological transition was ascribed to the change in the lipid conformation corresponding to the pH conditions. Interestingly, at acidic and basic pH, the spherical micelles demonstrated monodispersity in terms of the aggregation number, which agreed with the vertex numbers of Platonic solids, indicating the formation of Platonic micelles. At acidic and basic pH, the lipid conformations were almost identical, but there was a slight difference in the hydrophilic volume, which might affect the packing behavior of the lipid into micelles and account for the difference in the aggregation number. This study clearly demonstrates the precisely pH-controllable aggregation number of micelles, which belong to the Platonic micelle systems.


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INTRODUCTION Dispersion of amphiphilic compounds, including lipids and block copolymers, in water leads to self-assembly of these molecules to form micelles.1-4 The driving force for this selfassembly is determined by the balance between two opposite forces; the hydrophobic tails tend to avoid unfavorable interactions with polar water, while the head groups favorably interact with water. Israelachvili applied a simple thermodynamic model called the “Parking Parameter Principle” to micelle formation and introduced the parameter, ae , which is related to the equilibrium area per molecule at the aggregate interface.5 A geometrical criterion for micelles to adopt a spherical shape was also proposed as follows: V / ae h < 1/ 3 , where V and h are the volume and length of the surfactant tails, respectively. The factor of 1/3 is related to the geometrical relations for the volume and surface area of a sphere made up of N surfactants, subject to the constraint that the radius of the sphere R cannot exceed the extended tail length h. Based on the Parking Parameter Principle, the morphological changes of the micelles are rationalized by the variation of V / ae h . In the case where V / ae h > 1/ 3 , the micelle morphology is expected to change to cylindrical, and a further increase of V / ae h leads to plate formation. Furthermore, the aggregation number ( Nagg ) of the micelles is mainly determined by the following relation:

N agg ≈ (4πh 2 ) / ae

(1)

Many previous studies have shown that this principle is quite successful and useful for designing and controlling the morphology of micelles.6-10


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Calix[4]arene is a macrocyclic molecule consisting of four phenol units. It is an attractive building block for precise construction of designed structures because of its rigid backbone.11-15 For example, we synthesized a series of calix[4]arene lipids containing alkyl chains in the lower rim and hydrophilic groups in the upper rim using triazole units as a spacer.16-18 This design imparts cone-shaped conformations to the lipids when the charged hydrophilic groups are separated as far as possible by the electrostatic repulsion. One of the interesting findings is that the lipids form monodisperse micelles with Nagg values of 6, 8, 12, or 20 (Figure 1). Furthermore, these values agree with the vertex numbers of Platonic solids. Thus, we termed these micelles Platonic micelles and proposed a hypothesis to explain why the Nagg corresponds to the Platonic solid numbers.19 In a series of studies, we found that primary amino groups bearing calix[4]arene-based lipids self-assemble into cubic-like hexamer micelles. When either choline phosphate or cysteine is employed as the hydrophilic group instead of the primary amine, the micelle structure changes to octahedral or dodecahedral. We attributed this phenomenon to the difference in the head-group volume, which contributes to the interfacial area, ae .

Figure 1. Schematic illustration of Platonic micelles composed of calix[4]arene-based lipids.


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According to the Packing Parameter theory, another important factor for controlling the micellar aggregation nubmer is the length of the hydrophobic moiety (h), in addition to ae . In this study, a new system comprising glutamic acids bearing a calix[4]arene-based lipid composed of four propyl chains in the lower rim and four glutamic acids in the upper rim (denoted as ECaL3, shown in Figure 2) is synthesized. In the attached glutamic acid head, the αamine and the γ-carboxylic acid are separated by a three-carbon spacer. Based on previous work, these two functional groups can act in a zwitterionic manner depending on the pH. This means that changing the pH leads to three states: the positively charged amino group state, inter- and intra-molecularly interacting ion-pairs (i.e., zwitterions), and the negatively charged carboxylic state.20-24 The difference in the position of the charge may change the tail length, h as well as ae . This is because when only the amines are charged, a hydrophilic/hydrophobic interface is created between the amine and calix[4]arene moieties, which produces a shorter tail length than that of the carboxylic acid charged state, while the magnitude of the interfacial area is almost identical for both charged states in terms of the chemical structure. Variation of the parameters including h and ae by the pH-responsiveness may lead to a change in Nagg as suggested by Eq. 1. This external-stimulus induced morphological change is the main subject of this report.


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EXPERIMENTAL SECTION Materials and Synthesis. All chemical reagents were purchased from Tokyo Chemical Industry Co., Sigma-Aldrich Co., and Watanabe Chemical Co., and were used without further purification. All reactions were carried out under nitrogen atmosphere, and all solvents were dehydrated by standard methods. The progress of the reactions was monitored using thin layer chromatography (TLC), and detected using ultraviolet (UV; 254 nm) irradiation and staining with a basic solution of potassium permanganate. All products were purified by column chromatography with silica gel 60 (240–400 mesh). Nuclear magnetic resonance spectra were recorded with a 500 MHz Bruker spectrometer using chloroform-d and methanol-d4 as solvents. All chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilane using the solvent resonance as the internal standard. Synthesis of N-(2-propynyl)-Boc-L-Glu(OtBu) (I). A solution of Boc-Glu(OtBu)-OH (3.90 g, 12.9 mmol), propargyl amine (0.850 g, 15.4 mmol), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium chloride (DMT-MM) (4.26 g, 15.4 mmol), and anhydrous methanol (15 mL) was stirred at room temperature for 2 h under nitrogen atmosphere. Water was then added, and the reactant was 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 (1:1 EtOAc/Hexane), which afforded I as a white solid (3.62 g, 10.6 mmol, 82%). 1H NMR (400 MHz, CDCl3): δ = 6.73 (s, 1H), 5.36 (d, J = 7.88 Hz, 1H), 4.14 (s, 1H), 4.04 (m, 2H), 2.43–2.26 (m, 2H), 2.21 (t, J = 2.54 Hz, 1H), 2.11–1.85 (m, 2H), 1.44–1.43 (m, 18H).


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Synthesis of O-n-propoxytetraazidomethylenecalix[4]arene (II). Compound II was synthesized according to the reported method.25 Synthesis of calix[4]arene lipid bearing protected glutamic acids (III). Compound I (0.810 g, 2.38 mmol), copper(II) sulfate pentahydrate (11.9 mg, 47.6 µmol), and sodium ascorbate (28.3 mg, 0.143 mmol) were dissolved in dry DMF (10 mL); a solution of calixarene II (0.391 g, 0.476 mmol) in dry DMF (5 mL) was then added to the mixture. The mixture was stirred at 80 °C for 24 h and then cooled to room temperature. Water was then added, and the reactant was 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 (10:1 EtOAc/methanol), which afforded a brown solid (0.798 g, 0.367 mol, 77%). 1H NMR (400 MHz, CDCl3): δ (ppm) = 7.68 (br, 4H), 7.54 (s, 4H), 6.45 (s, 8H), 5.62 (br, 4H), 5.16 (s, 8H), 4.59–4.44 (m, 8H), 4.36 (d, J = 13.2 Hz, 4H), 4.18 (br, 4H), 3.78 (t, J = 7.44 Hz 8H), 3.06 (d, J = 13.4 Hz, 4H), 2.28 (br, 8H), 2.08–1.84 (m, 8H), 1.87 (m, 8H), 1.41–1.38 (m, 18H), 0.96 (t, J = 7.36 Hz, 12H). Synthesis of calix[4]arene lipid bearing glutamic acids (IV: ECaL3). A solution of compound III (0.362 g, 0.167 mol) was treated with 4 N HCl/EtOAc for 1 h. The solvent was evaporated, and the residue was washed with CH2Cl2 and EtOAc. IV was obtained as a white solid (0.253 mg, 0.163 mmol, 98%). 1H NMR (400 MHz, methanol-d4): δ = 8.02 (s, 4H), 6.67 (s, 8H), 5.33 (m, 8H), 4.55 (m, 8H), 4.43 (d, J = 13.2 Hz, 4H), 3.99 (t, J = 6.32 Hz, 4H), 3.83 (t, J = 7.28 Hz, 8H), 3.15 (d, J = 13.5 Hz, 4H), 2.47 (m, 8H), 2.14 (m, 8H), 1.92 (m, 8H), 1.00 (t, J = 7.48 Hz, 12H). 13

C NMR (100 MHz, methanol-d4): δ (ppm) = 172.7, 168.5, 156.9, 135.4, 128.3, 128.2, 124.2,

76.8, 54.3, 52.4, 51.1, 33.9, 30.3, 28.7, 26.1, 23.0, 9.33. ESI–MS (M–): calcd for C76H101N20O16 1548.75, found 1548.50.


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Critical micelle concentration (CMC) measurements. The concentration of an aqueous ECaL3 solution was adjusted to 10 mM, and the solution was diluted with 50 mM aqueous NaCl. The pH of the solution was controlled with 1 M HCl, Tris-HCl buffer, and 1 M NaOH aqueous solutions. Sodium 8-anilino-1-naphthalenesulfonic acid (ANS) was used as a fluorescence probe. The stock solution of ANS was prepared at a concentration of 0.1 mM in water, and was then diluted to 10 µM in each solution. Before the fluorescence measurements, all samples were incubated for at least 30 min in the dark at room temperature. The fluorescence measurements were carried out with a fluorescence spectrometer (JASCO FP-6600). ANS was excited at 350 nm and the emission spectra were recorded at 400–700 nm. The scan speed was 240 nm min−1. The fluorescence intensity of ANS sensitively reflects the polarity of its environment; therefore, the CMC can be determined from a plot of the fluorescence intensity vs. the ECaL3 concentration.26

pH titration and zeta potential measurements. The concentration of the ECaL3 solution was adjusted to 10 mM using 50 mM NaCl at pH = 2 using aqueous HCl. Aqueous 0.1 M NaOH was titrated into the micellar solution and the pH values were recorded after each titration. Zeta potential measurements of the 2.0 mM ECaL3 solutions in 50 mM NaCl aqueous solutions at different pH were performed using a Zeta-sizer 3000 (Malvern, UK) instrument.

Small angle X-ray scattering (SAXS) measurements. The concentration of the ECaL3 solutions was adjusted to 5.0 mg mL-1 with ultrapure water and then diluted to the required concentration with 50 mM aqueous NaCl. The pH of the solution was adjusted with aqueous 1 M


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HCl and 1 M NaOH. The prepared samples were left for at least one day to equilibrate at room temperature. Small angle X-ray scattering (SAXS) measurements were carried out at the BL40B2 beamline of the SPring-8 facility, Hyōgo Prefecture, Japan. A 30 × 30 cm imaging plate (Rigaku R-AXIS VII) detector was placed 1 m from the sample. The wavelength of the incident beam (λ) was adjusted to 0.10 nm. This setup provided a q range of 0.20−4 nm−1, where q is the magnitude of the scattering vector, defined as q = 4π sin θ/λ, with a scattering angle of 2θ. The X-ray transmittance of the samples was determined by using ion chambers located in front of and behind the sample. The detailed experimental procedures are reported elsewhere.16 The absolute SAXS intensities were recorded using the absolute scattering intensities of water.27, 28

The micellar SAXS profiles were fitted by using the following equations: j ( qRC ) j ( qRS )   I ( q ) = 3VC ( ρC − ρS ) 1 + 3VS ρS 1  qRC qRS  

2

(3)

2

Lπ  J 1(qRC ) J 1(qRS )  I (q) = + SS ( ρS－ρSol ) SC ( ρC－ρS )  q  qRC qRS 

I (q ) =

f (q,α) = 2( ρC－ρS )VC j0 (qHcosα )

N VS

∫

(4)

π/2

f 2 ( q,α )sinαdα

0

(5)

J 1(qrsinα) J 1[q (r + t )sinα ] + 2( ρS－ρSol )VS j0 [q( H + t )cosα ] (qrsinα ) [q(r + t )sinα ] (6)

Here, Eq. (3), (4), and (5)-(6) express core-shell spheres and infinite core-shell cylinders, and finite core-shell cylinders, respectively. RC and RS are the outer radii of the core and micelle


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(core + shell), and ρC, ρS, and ρSol are the scattering length (or electron density) of the core, the shell, and the solvent, respectively. J1, j0, and j1 are the first Bessel function and first and second spherical Bessel function, respectively. VC and VS are the particle volume of the core and micelle (core + shell), respectively. SC and SS are the cross-sectional areas of the core and micelle (core + shell), respectively.

The SAXS profiles in the low q region follow the Guinier relation given by the following equation:

I (q) = I (0)exp(−q 2 Rg 2 / 3)

(7)

where I(0) is the forward scattering intensity at q = 0. I(0) and the gyration radius (Rg) are determined from the intercept and the slope of the ln(I(q)) vs. q2 plot (Guinier plot). Due to interparticle interference, the I(0) and Rg values depend on the sample concentration. In order to remove the concentration effects, the SAXS intensities recorded at different concentrations were extrapolated to zero concentration.

Determination of micellar molar mass by SAXS. The molar mass of the micelles can be given by the following equation:

M w = I (0){ N A c (∆ρυ ) 2 }

(8)

Where Mw is the weight-average molar mass, c is the concentration of lipids, NA is Avogadro's number, and ∆ρ is the scattering length difference, which can be calculated from the electron


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number and the molecular weight of the lipid and the solvent. The term

υ indicates the specific

volume of micelles in the solution, which can be determined by the density of the micellar solutions and the solvent (Figure S1).

Multi-angle light scattering coupled with field flow fractionation (FFF-MALS) measurements. ECaL3 (10 mg mL-1) was prepared in 50 mM aqueous NaCl at pH = 3.0 or 10. The solution was optically purified by using an ultracentrifuge to remove large dust particles and other impurities, the signals of which often overlap with those of the micelles. The 60 µL sample solutions were immediately injected into an Eclipse 3+ separation system (Wyatt Technology Europe GmbH, Dernbach, Germany) for field-flow fractionation (FFF) at 22–28 oC. The output from FFF was then passed sequentially through a Dawn Heleos II multiangle light-scattering (MALS) detector (Wyatt Technology), UV detector, and an Optilab rEX DSP differential refractive index (RI) detector (Wyatt Technology), operating at a wavelength of 658 nm. A Wyatt channel (Eclipse 3 channel LC) attached to a membrane (polyether sulfone membrane; 1 kDa LC) at the bottom of the channel was used for the measurements. The cross-flow and channel-flow rates were fixed at 4.0 and 1.0 mL min-1, respectively. Detailed experimental procedures are reported elsewhere.17 The specific refractive index increments ( ∂n / ∂c ) and the extinction coefficients (ε at 270 nm) of the micelles in aqueous solution were determined using a DRM-1021 differential refractometer (Otsuka Electronics, Osaka) and a Jasco V-630 spectrometer, respectively (see Figure S1).


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RESULTS AND DISCUSSION Synthesis and characterization of micelles composed of ECaL3 ECaL3 was synthesized as shown in Figure 2. The propynyl group was attached to the αcarboxylic acid of glutamic acid via dehydration condensation using 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride. Glutamic acid was introduced on the upper rim of the calix[4]arene via the copper(I)-catalyzed azide-alkyne click reaction; the protection groups (Boc of the α-amine and t-Bu of the γ-carboxylic acid) were removed by deprotection with trifluoroacetic acid. The chemical structures generated at each step were confirmed by 1H and 13

C NMR as well as mass spectroscopy (see Supporting Information). The cone conformation of

the calix[4]arene-based lipids was stabilized in C4v symmetry by the Na ion trapped by the four oxygen atoms at the lower rim; this ion might be necessary to form polyhedral micelles.16 Therefore, all experiments were carried out in the presence of excess Na ions. The critical micelle concentration (CMC) of the ECaL3 micelles under acidic, neutral, and basic conditions was determined with 8-anilinonaphthalene-1-sulfonate as a fluorescence probe (Figure S2); the data are listed in the first column of Table 1. The CMC at neutral pH (7.0) was lower than that obtained under other pH conditions since the intermolecular electrostatic repulsions are cancelled due to the zwitterionic nature.


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I

II III

IV: ECaL3

Figure 2. Synthesis of glutamic acid-containing calix[4]arene-based lipid (compound IV) via copper(I)-catalyzed azide-alkyne click reaction. The amino groups are protonated at acidic pH +

COOH (pH < pKa ), and the carboxylic acids are deprotonated at basic pH (pH > pK a NH3 ). The tail

length, h, differs under each pH condition, presumably hNH3+ < hCOOH.

Figure 3a shows a titration curve for the ECaL3 micelles. It is clear that there are two buffering regions, i.e., at pH= 3–5 and 6.5–10; the former is related to the equilibrium between


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the carboxylic acids and the carboxylate ions, and the latter is ascribed to the equilibrium between the amines and the ammonium ions. In both regions, the titration curve changes continuously. This may be interpreted based on the fact that adjacent charged groups interact closely, thus, the first ionization process changes the secondary processes, leading to broad titration changes. This titration curve indicates that at pH > 3–5 the carboxylic group is negatively charged and at pH < 6.5–10, the amino group is positively charged. Therefore, in the range of 3 < pH < 10, the amino and carboxylic groups are oppositely charged. When oppositely charged groups are present in the same molecule or aggregates, they should undergo ion-paring. Figure 3b shows a plot of the zeta-potential versus pH. The data indicate that the micelles are positively charged at pH < 5 and negatively charged at pH > 7, which is consistent with the titration curves.

10 8 6 4

ECaL3 ( ) ECaL3 ( )

2 0

400

800

1200

Zeta potential / mV

(b)

(a)

pH

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30 20 10 0 -10 -20 -30 2

Titration volume / µL

4

6

8

10

pH

Figure 3. (a) Titration curve of 10 mL ECaL3 solution ([ECaL3] = 10 mM in 50 mM NaCl, pH = 2.0) with 0.1 N NaOH. Unfilled circles and triangles denote the titration result with and without ECaL3 in the solutions, respectively. (b) pH dependence of zeta potential of ECaL3 micelles in 50 mM aqueous NaCl.


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Morphological transition of ECaL3 micelles in response to pH

10 10

9

8

(b)

pH 10.1 (a=106)

6.2 (a=102)

Slope at low q region

(a)

5.4 (a=101)

(c)

3

2

3.2 (a=100)

9.2 (a=105) 10 10

7

8.4 (a=104) 6

7.3 (a=103) 10 10 10 10 10 10 10

5

4

0.0 -0.2 -0.4 -0.6 -0.8 -1.0

L for Cylindrical micelles / nm

[I(q) / cm-1] ✕ a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1

0

14 12 10 8

-1 3

4

5 6 7 8 9

0

2

10 q / nm-1

3

4

6

8

10

pH

Figure 4. (a) SAXS profiles for ECaL3 micelles in 50 mM aqueous NaCl with variation of the pH. The solid lines were calculated using the core-shell sphere and cylinder models; the fitting parameters are listed in Table 1. pH dependence of the slope at low q region for each SAXS profile and fitting parameters of L for cylindrical models are shown in (b) and (c), respectively. Figure 4a shows the SAXS profiles of the ECaL3 micelles with variation of the pH. Under acidic and basic conditions, the sailing factor was α = 0 from the SAXS intensity in the α low q region, where the scattering intensity, I(q), is expressed by: I (q) ∝ q . This indicates that


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these solutions contain isolated scattering objects without secondary aggregation; presumably, these objects are spherical micelles. When the pH is changed to neutral, α takes a value of −1 (Figure 4b), which indicates transition to a cylindrical morphology. The transition regions are almost identical to the two buffering zones in the titration curves. Therefore, we can conclude that the pH dependence of α clearly demonstrates that the presence and absence of intramolecular electrostatic repulsions among the head-groups induces morphological transition of the micelles. This feature is quite similar to our previous observations, where we attached four primary amino groups as the headgroup of calix[4]arene and observed spherical micelles at low pH (protonated amino) and cylindrical micelles at higher pH (deprotonated).

In the neutral pH region, we found 0 < α < 1. The intermediate value can be interpreted in two ways: these solutions may contain a mixture of spherical and cylindrical micelles, or the cylinder is not long enough to give α = −1 at very low q. The first interpretation can be eliminated as follows: if there is a mixture of spherical and cylindrical micelles, the charge on the lipids consisting of the cylinders may be neutralized owing to ion-pair formation, and the lipids comprising the spheres are fully charged as cations or anions. Such an unbalanced charge distribution is energetically unfavorable. Therefore, we can presume that 0 < α < 1 is related to the short cylinders.

The SAXS profiles at all pH values were fitted to the core-shell spherical or cylindrical model with or without a finite contour length; the fitting parameters are listed in Table 1, and the average cylinder length (L) is plotted against pH in Figure 4c. The size of the core was in the


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range of 0.4–0.7 nm, and the ρC was 270 e nm-1, both of which are reasonable in terms of the length of the n-propyl chain and the electron density of the alkyl chain in this system.16 The size of the hydrophilic shell: RS−RC becomes smaller at pH = 8.0, which can be explained by the synergistic interaction between the α-amine and the γ-carboxylic acid, i.e., the folded back structure of the glutamic acid moieties. RS−RC at pH = 3.0 was smaller than that at pH = 10, which can be explained by the difference in the position of the charge in the head-group.

Table 1. SAXS Fitting Parameters and Critical Micelle Concentration (CMC) for ECaL3 Micelles at Different pH CMC

Rg

pH

RC

RS

L

RS−RC

Model / µM

/ nm

3.0

4.4±0.2

1.64

5.4

－

6.2

σ / RS

ρC

ρS

/ e nm-1

/ e nm-1

/nm

/nm

/nm

/nm

sphere

0.700

2.15

－

1.45

0.06

270

400

1.77

sphere

0.600

2.35

－

1.75

0.04

270

385

－

－

finite cylinder

0.550

1.90

9.35

1.35

－

270

385

7.4

－

－

infinite cylinder

0.420

1.80

－

1.38

0.05

270

365

8.3

1.0±0.1

－

infinite cylinder

0.420

1.80

－

1.38

0.05

270

365

9.2

－

－

finite cylinder

0.530

1.98

9.45

1.45

－

270

385

10

1.8±0.2

1.82

sphere

0.700

2.38

－

1.68

0.05

270

400

RC: core size, RS: shell size, ρC: electron density of the core, ρS: electron density of the shell, σ: standard deviation

When we compared L (or α) and the titration behavior, there was some discrepancy, i.e., the protonated amino groups have a greater tendency to form spheres than the deprotonated carboxylic groups. This may be related to the difference in the position of the charge in the headgroup, which leads to a difference in h, as mentioned in the Introduction, i.e., when the amino groups are charged, a hydrophilic/hydrophobic interface would be created between the amino


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and calixarene moieties and thus the tail length, h, would become shorter than that formed when the carboxylic groups are charged. A smaller h would be more favorable for formation of spherical micelles than a larger h.

Determination of Nagg at acidic and basic pH At pH = 3.0 or 10, either the carboxylic or the amino group is charged and the other group is not. For simplicity, we first examined these pH ranges. The molar mass of the micelles and its dispersity were evaluated using FFF-MALS (Figure 5). Only one sharp peak corresponding to the micelles was observed at each condition. At pH = 10, the maximum peak was obtained at 6.8 min, while at pH = 3, the maximum was observed at 6.1 min. Since larger particles take longer to elute in FFF,29 this difference indicates that at pH = 3.0, smaller particles are formed than at pH = 10. Notably, the LS and UV peaks overlapped with each other under both pH conditions, indicating that the molecular weight distribution over all the peaks is quite small. The inset shows the weight-averaged molar mass (Mw) plotted against time, where the Mw values determined from the Zimm plot were 9.30 × 103 and 19.0 × 103 g mol-1 at pH = 10 and 3.0, respectively (Figure S3). The Nagg values were calculated from the respective Mws, giving values of Nagg = 6.0 ±0.15 at pH = 3.0 and Nagg =12±0.20 at pH = 10. The molecular weight distribution (Mw/Mn) calculated from the micellar peaks was almost 1.0 under both pH conditions. The obtained values are summarized in Table 2.


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10

10

3

5 0

2

1

15

5

5.6

10 6.8

6.0 6.4 time / min

1

15 10 5

0

0 4 -1 2

0.5

10 10 10 6.4

10

6.8 7.2 time / min

5 4 3 2

3 2 1

-1

10 Conc. / mg mL

1.0

4 3 2 1 0

Molar mass / g mol

pH 10

1.5

Conc. / 102 mg mL-1

10 Conc. / mg mL

2

10

-1

Rayleigh ratio / 107 cm-1

-1

pH 3.0

3

Molar mass / g mol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.0

0 0

5

10

15

20

25

time / min

Figure 5. FFF-MALS fractogram of ECaL3 micelles at pH = 3.0 and 10, measured with LS at 90° (circles) and UV at 270 nm (solid lines). Inset shows time dependence of the molecular concentration and molar mass of ECaL3 micelles.

According to the SAXS profiles, the micellar molar masses determined from the intercept values (I(0)) obtained by extrapolating the scattering intensities to infinite dilution were 9.10 × 103 g mol-1 at pH = 3.0 and 18.6 × 103 g mol-1 at pH = 10 (Figure 6a and 6b). Although the excess electron number used for determination of the molar mass was calculated from the chemical composition of the lipid and solvent without any consideration of the ion aggregates on


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the micellar shell, the values of the micellar molar mass determined by SAXS are consistent with the FFF-MALS results, as shown in Table 2. This indicates that the sodium and chloride ions in the sample solutions do not aggregate on the micelles very much, presumably due to the weakly basic and acidic nature of the primary amine and carboxylic acid of the glutamic acid. These results led us to conclude that the aggregation numbers of the ECaL3 micelles are 6.0 and 12 at acidic and basic pH, respectively. As mentioned in the Introduction, some calix[4]arene-based lipids form Platonic micelles that show monodispersity in terms of the aggregation numbers, which are consistent with the vertex numbers of Platonic solids (6, 8, 12, 20). Under acidic and basic pH conditions, the ECaL3 micelles also demonstrate similar properties, implying the formation of Platonic micelles.

Table 2. Molar masses determined with different methods and the aggregation numbers. pH

Mw (SAXS)

Mw (FFF-MALS)

Nagg

/ 103 g mol-1

/ 103 g mol-1

Mw/Mn

3.0

9.10 ± 0.10

9.30 ±0.20

1.04

6.0

10

18.6 ± 0.10

18.9 ± 0.20

1.00

12

Figure 6b presents a comparison of the Guinier plot for the pH range between 10 and 3.2. It is clear that at pH 10, the Rg (c.a., 1.82 nm) is larger than that at pH = 3.2 (1.64 nm). For the core-shell sphere model, Rg is related to RC and RS by the following equation30:

Rg 2 =

3[VC RC 2 (ρC − ρS ) + VS RS 2 ρS ] 5[VC (ρC − ρS ) + VS ρS ]

(9)

From the values of RC and RS obtained by fitting (Table 1), the calculated values of Rg were 1.67 and 1.85 nm at pH = 3.0 and pH = 10, respectively; these values are quite consistent with the


20

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experimentally determined values. The q value associated with the first minimum (q1st min) is related to the micelle size. The q1st min at pH = 10 was 0.17 nm-1 smaller than that at pH = 3.2. This is also consistent with the size change observed in the Guinier region.

(b)

(a) 2

0 6 4 2

-1 2

3

4

5

q/

6

nm-1

1.5 1.0 0.5

Rg / nm

ln(I/c)

4.0 mg/ml 3.0 mg/ml 2.0 mg/ml 1.0 mg/ml Conc. = 0 (pH3.0) Conc. = 0 (pH10)

16 12 8 4 0.000

0.002

7 8 9

10

0

pH = 10

0.05

0.10 0.15 q2 / nm-2

1.75 1.70 1.65

0.004

Conc. / g mL-1

0.0 0.00

pH = 10

1.80

2.0

2

10

1.85

2.5

1 6 4

10

(c)

I(0)/c / cm-1 mL g-1

10 I(q) / cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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pH = 3.0

1.60 0.20

0

1

pH = 3.0

2 3 Cycles

4

5

Figure 6. (a) I(q)/c as a function of q for different ECaL3 concentrations in 50 mM NaCl solution at pH = 3.0 and 10. The extrapolated values at infinite dilution for each q are shown by the red markers. (b) Guinier plot (i.e., ln I(q)/c versus q2) constructed from the extrapolated intensities. Inset shows concentration dependence of the I(0)/c values. Unfilled red and blue circles denote the results of ECaL3 micelles at pH = 3.0 and 10, respectively. The micellar molar mass determined from the intercept values at q = 0 and concentration = 0 are consistent. (c) Reversible switching of micellar gyration radius with variation of pH.

To evaluate the reversibility of the morphological transition of the micelles under acidic versus basic conditions, the gyration radius was measured several times for one sample with variation of the pH. As seen in Figure 6c, the gyration radius increased and decreased reversibly


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with variation of the pH. This result clearly demonstrates a reversible pH-responsive morphological transition of the ECaL3 micelles. These SAXS result demonstrate that the aggregation number changes with variation of the pH. Notably, the sharp intensity minimum at q1st min indicates that the distribution of the shape and the aggregation number is quite small under both pH conditions. Although electron microscopy may not provide sufficient resolution for such small particles, we confirmed the uniform size of the micelles via AFM (Figure S4).

Molecular modeling of ECaL3 with the charged head Figure 7 shows the energy-minimized structures of ECaL3 with either protonated amino groups or deprotonated carboxylic acid groups, corresponding to acidic and basic conditions, respectively. For the energy-minimized structure with deprotonated carboxylic acids, the glutamic acid moieties exhibited folded back structures, resulting in a smaller hydrophilic volume than that with the protonated amino groups. The aggregation number of the ECaL3 micelles was different under each condition, presumably due to the difference in the volume of the head-group, as well as the length of the hydrophobic moiety under acidic versus basic conditions. In general, lipids with larger head groups tend to self-assemble into micelles with smaller aggregation number. This idea is applied to Platonic micelles as well. Based on the FTIR spectra of the freeze-dried micellar samples prepared at pH = 3.0 or 10, the absorbance peak of the amide I band at basic pH, which is mainly associated with the C=O stretching vibration,31, 32

shifts to lower wavenumber compared to that at acidic pH (Figure S5). This is presumably due

to the different packing behavior of the micelles between at pH = 3.0 and 10. The folded back structure of the glutamic acid moieties at basic pH might allow for intermolecular interactions


22

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via the hydrogen bonding interactions among the amide groups in the micelle. The pHresponsive morphological transitions of the Platonic micelles composed of ECaL3 are summarized in Figure 8.

Figure 7. Energy minimized molecular models of ECaL3 calculated using the SCIGRESS program (Fujitsu Ltd., Japan) with MOPAC in water. Before starting the calculation for ECaL3, the model was pre-optimized via the molecular mechanics (MM) method using MM3. The molecular models are shown in side view (a, c) and top view (b, d). The α-amines of the upper models are protonated, and the γ-carboxylic acids of the lower models are deprotonated.


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Figure 8. Schematic representation of the morphological transitions between monodisperse spherical micelles with pH-controllable aggregation numbers.

Concluding Remarks A new calix[4]arene-based lipid containing four glutamic acids in the upper rim was synthesized, and micelles based on this lipid undergo morphological transitions from spherical to cylindrical and back to spherical with increasing pH. The morphological transition is attributed to the lipid conformation: the glutamic acid moieties are positioned away from each other at acidic and basic pH due to the intramolecular electrostatic repulsions, while the zwitterionic nature at neutral pH does not permit the 'head-group spread'. The spherical micelles generated at acidic and basic pH are monodisperse in terms of the aggregation number, which agrees with the vertex numbers of Platonic solids: 6 and 12 at acidic and basic pH, respectively. The difference in the tail length influences the interfacial area created between the hydrophilic and hydrophobic regions, which affects the aggregation behavior of the Platonic micelles. This is the first demonstration of a reversible pH-responsive morphological transition in Platonic micellar structures. The concept of controlling Platonic micelle structures by tuning the packing


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parameters, including ae and h, provides a new strategy for designing a new class of smart materials. ASSOCIATED CONTENT Supporting Information. 1H and 13C NMR spectra of compound I, II, III, and IV. UV spectrum, concentration dependence of refractive index increment, and density increment for ECaL3 micelles in 50 mM aqueous NaCl. The CMC values of ECaL3 micelles at different pH conditions detemined by Fluorescent measurements. The Zimm plots for ECaL3 micelles at different pH conditions. AFM and TEM images of the ECaL3 micelles at different pH conditions. FT-IR spectrum of the micelles 50 mM aqueous NaCl at pH = 3.0 and 10. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We appreciate JST/CREST program for financial support and all SAXS measurements were carried out at SPring-8 40B2 [2014A1268, 2014A1268, 2013B1203, 2013A1564]. This work


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was supported by the Photon and Quantum Basic Research Coordinated Development Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Table of Contents


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Glutamic Acids Bearing Calix[4]arene Micelle: pH-Controllable

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