Self-Assembly of Calix[4]arene-Based Amphiphiles Bearing

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Self-assembly of Calix[4]arene-Based Amphiphiles Bearing Polyethylene Glycols: Another Example of “Platonic Micelles” Kenta Yoshida, Shota Fujii, Rintaro Takahashi, Sakiko Matsumoto, and Kazuo Sakurai Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02196 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 10, 2017

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Self-assembly of Calix[4]arene-Based Amphiphiles Bearing Polyethylene Glycols: Another Example of “Platonic Micelles”

Kenta Yoshida†, Shota Fujii†, Rintaro Takahashi, Sakiko Matsumoto and Kazuo Sakurai*

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

†: These authors contributed equally to this work.

KEYWORDS: monodisperse micelles, poly(ethylene glycol), platonic micelles

ACS Paragon Plus Environment

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ABSTRACT The aggregation number of classical micelles exhibits a certain distribution, which is a recognizable feature of conventional micelles. However, we recently identified perfectly monodisperse calix[4]arene-based micelles whose aggregation numbers agree with the vertex numbers of regular polyhedra, that is, Platonic solids, and thus they are named “Platonic micelles.” Regarding our hypothesis of the formation mechanism of Platonic micelles, both repulsive interactions including steric hindrance and electrostatic repulsions among the headgroups are important for determining their aggregation number; however, neither of these is necessarily needed to consider. In this study, we employed polyethylene glycols (PEGs) as the non-ionic headgroup of calix[4]arene-based amphiphiles to study the effects of only repulsive interactions caused by steric hindrance on the formation of Platonic micelles. The amphiphiles containing relatively low-molecular-weight PEGs (550 or 1000 g mol−1) form dodecamer or octamer micelles, respectively, with no variation in the aggregation number. However, relatively high-molecular-weight PEGs (2000 g mol−1) produce polydispersed micelles with a range of aggregation number. PEG 2000 exhibits greater affinity for water than PEG 550 and 1000, resulting in fewer hydrophobic interactions in micelle formation, as indicated by the drastic increase of critical micelle concentration (CMC) value in the PEG 2000 system. The instability of the structure of PEG2kCaL5 micelles might contribute to the higher mobility of PEG in the micellar shell, resulting in a non-Platonic aggregation number with polydispersity.


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INTRODUCTION Micelles have been widely used for many applications in daily life, including cosmetics, soups, detergents, and drug delivery, as well as them being key materials in nanotechnology.1-5 Spherical micelles are one of the various types of micelle that have been extensively studied over the past hundred years.6-7 When surfactants, and more generally amphiphilic molecules, satisfy a certain geometric balance between their hydrophobic and hydrophilic moieties, they selfassemble to form a spherical micelle in aqueous solutions.6, 8 The aggregation number (Nagg) of micelles is not typically fixed but instead varies across a certain distribution, which depends not only on the molecular structure and its concentration but also on the environment, including solvent conditions and temperature. For instance, the Nagg of micelles composed of sodium dodecyl sulfate has experimentally been determined9 to be around 50 to 64, and it approaches 100 with increasing concentration of the surfactant and ionic strength due to the transition in micellar shape from spheres to ellipsoids.8, 10 In contrast to this conventional micelle system, we recently identified no variation of Nagg of spherical micelles composed of calix[4]arene-based amphiphiles, in which electrolyte functional groups are attached to the upper rim and alkyl chains are added to the lower rim.11-15 Owing to the presence of a triazole ring between the calix[4]arene moiety and the electrolyte group as a spacer, the conformation of the surfactants would be cone-shaped due to the electrostatic intramolecular repulsions in the headgroup, which satisfies the geometric balance needed to form spherical micelles. In addition, Nagg varies depending on the functional group in the hydrophilic part and the length of the alkyl chains, being one of a range of numbers including 2, 6, 8, 12, 20, and 32.14 Since some of these numbers coincide with the vertex numbers of


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Platonic solids (regular polyhedrons), we named these micelles “Platonic micelles.”14 Calix[4]arene-based micelles have been studied for decades16-17 and some of them also shows the monodipersity in the aggregation number which was determined by the reconstructed 3D structure of the micelle using cryo-electron microscopy.18 Although it does not provide absolute micellar molar mass, the 3D image suggested that seven molecules of calix[4]arene-based amphiphiles form into one micelle. Furthermore, employing calix[4]arene backbone into amphiphiles led the formation of uniform micelles which was used in gene delivery19 and bioimaging20. To explain this Platonic nature (i.e., monodispersity and a preference for adopting certain discontinuous Nagg values), we considered the best packing of the unit spherical surface by equally sized cap objects such that the distance between the objects can be maximized21-22 because the thermodynamic stability of micelles relies on how efficiently the micelle hydrophobic core is covered with the hydrophilic part of the amphiphile. Considering the repulsive interactions between the spherical caps, the arrangement where the total energy is minimized is the optimal one.23 The total energy is described as follows:

=

Here, the position of N caps is represented by XN = {x1, x2, …, xN}. The repulsive interaction potential between xi and xj is described by φ(rij) ( ≡ − is the spherical distance between xi and xj) and represented by the following equation: =

1


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The case of m = 1 corresponds to the unscreened Coulomb potential when charges are placed at each cap, while the caps can be regarded as a rigid body with hard core potential when m = ∞. The coverage density [D(N)], defined by the ratio of the summation of the cap area to the total surface area of the unit sphere, is determined by the optimal arrangement: XN* = {x1*, x2*, …, xN*}, considering the interactions among the caps. In the case of m = 1 or ∞, the interactions are related to the Thomson22 or Tammes21 problem, whose general analytical solutions have yet to be obtained for several numbers of caps. Both cases have a maximum at certain cap numbers corresponding to 2, 4, 6, 8, 12, 20, and 32 caps. These numbers agree with the Nagg experimentally determined for various calix[4]arene-based micelles, and therefore we speculate that the discrete and monodispersed nature of aggregates is controlled by the packing considerations. It is necessary to consider interactions relating to both the Thomson and Tammes problems as the calix[4]arene-based amphiphiles always contain an electrolyte functional group with the flexible spacer of a triazole ring. The question here is whether the electrostatic repulsive interactions are necessary for the formation of Platonic micelles. Based on the above-mentioned consideration, Platonic micelles should be formed when considering only the effect of steric hindrance without charge, which is only related to the Tammes problem. In this study, we attached polyethylene glycols (PEGs) as non-ionic hydrophilic groups to the upper rim of calix[4]arene-based amphiphiles (denoted by PEGmCaL5, as shown in Figure 1: m indicates the molecular weight of PEG and the suffix number is the number of carbons in each alkyl chain) in order to demonstrate Platonic micelles without electrostatic repulsive interactions, which is relating to only Tammes problem. The amphiphile conformation would be cone-shaped, which is necessary for the formation of spherical micelles, owing to the high flexibility of PEG


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and the large exclusion volume in water.24-26 We also characterized the structures of the micelles bearing PEGs and investigated the effects of the molecular weight of PEG on the aggregation behavior of the micelles.

PEG550CaL5

PEG1kCaL5

PEG2kCaL5

Figure 1. Chemical structures of calix[4]arene-based amphiphiles bearing PEGs.


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RESULTS and DISCUSSION We synthesized calix[4]arene-based amphiphiles bearing PEGs by a copper(I)-catalyzed azide-alkyne cycloaddition reaction (Scheme S1). The PEGs used in this study are relatively monodisperse, with molecular weights of 550, 1000, and 2000 g mol−1. The chemical structure of the products was confirmed by 1H-NMR at each step. The four oxygen atoms at the lower rim of the calix[4]arene moiety can trap one sodium ion, which allows the formation of C4v symmetrical structures.27 Since a highly symmetrical structure might be necessary for formation of the Platonic micelles,11 we performed all experiments under conditions of excess sodium ions in this study. Figure S2 shows the critical micelle concentration of PEGmCaL5 determined with 8anilinonaphthalene-1-sulfonate as a fluorescence probe. The CMC values were 0.14, 0.27, and 4.5 µM for PEG550CaL5, PEG1kCaL5, and PEG2kCaL5, respectively. The drastic increase of the CMC value of PEG2kCaL5 micelles was ascribed to the reduction in the number of hydrophobic interactions due to the higher affinity to water of the relatively high-molecular-weight PEG.

Figure 2a shows the small angle X-ray scattering (SAXS) profiles of PEGmCaL5 micelles in 50 mM aqueous NaCl solutions. At the low-q region of all SAXS profiles, I(q) follows Guinier law without appreciable interparticle interactions, indicating that the micelles are isolated without the formation of secondary aggregation. The first minimum position of the scattering profiles is shifted to the low-q region with increasing molecular weight of PEG, suggesting an increase of micellar size. In fact, the gyration radius (Rg) of the micelles determined using a Guinier plot (Figure 2c) increases with increasing PEG molecular weight (summarized in Table 1). Figure 2b shows the extrapolated scattering profiles of PEGmCaL5 micelles to zero concentration, and the weight-averaged molar mass (Mw) of the micelles determined from the intercept values [I(0)] extrapolating the scattering intensity to zero angle


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were 3.90 × 104, 4.18 × 104, and 2.99 × 104 g mol−1 for PEG550CaL5, PEG1kCaL5, and PEG2kCaL5, respectively (Figure 2b). The Nagg values were calculated from the respective Mw values, giving Nagg = 12, 8.0, and 3.5 for the micelles of PEG550CaL5, PEG1kCaL5, and PEG2kCaL5, respectively. The chemical structure of PEG550CaL5 differs slightly from those of the others in terms of the linker between the calix[4]arene moiety and the PEG group. Since the PEG as a hydrophilic group would be dominant in determining the micellar structure due to its high molecular weight, the small difference in the chemical structure would presumably have a negligible impact on the micellar structure. As evidence of that, we performed SAXS measurements of micelles composed of PEG550-bearing calix[4]arene derivatives whose chemical structure of the linker region is exactly the same as the other derivatives (Figure S3). The SAXS profile is almost consistent with that of PEG550CaL5 micelles, with the determined molar mass also being the same.


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(a)

(b) 10

PEG550CaL5

10

I (q)/c / cm-1mLg-1

10

6

PEG1kCaL5

10

10

0

PEG550CaL5 4.20mg/mL 3.18mg/mL 1.90mg/mL 1.12mg/mL Conc. = 0

1

PEG1kCaL5 0

-1

4.02mg/mL 2.92mg/mL 1.98mg/mL 1.07mg/mL Conc. = 0

10 1 10

PEG2kCaL5 10

1

-1

10

10

5

10

I(q) / (a.u.)

10

0

-1 -2

PEG2kCaL5 4.05mg/mL 2.98mg/mL 1.88mg/mL 1.18mg/mL Conc. = 0

2

3

10

4

-4

4

5

6

7

8 9

q / nm-1

(c)

1

PEG550CaL5

-5

10

In I (q) /c

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3

PEG1kCaL5

-6

PEG2kCaL5

-7

10

2 2

3

4

5

6

7 8 9

2

1

q / nm

3

4

5

-8 0.00

-1

0.05

0.10 2

q / nm

0.15

0.20

-2

Figure 2. (a) SAXS profiles of PEG-bearing calix[4]arene-based micelles in 50 mM aqueous NaCl (gray points) and fitting lines calculated from the core-corona model (shown in Figure S4). (b) I(q)/c as a function of q for different PEGmCaL5 concentrations in 50 mM aqueous NaCl. The extrapolated values at an infinite dilution for each q are shown by the black markers. (c) The Guinier plot [i.e., ln I(q)/c versus q2] constructed from the extrapolated intensities.


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Table 1. Fitting Parameters for PEGmCaL5 Micelles, Gyration Radius (Rg), Molar Mass of the Micelles, and PEG Density of the Micellar Shell a b f g c d e RC RS Rg Mw RS−RC h Nagg sample i PEG -3 -3 -3 4 -1 / nm

/nm

/nm

/ e nm

/ e nm

/ e nm

/ nm

/ 10 g mol

PEG550CaL5

0.65

2.90

2.25

270

358

334

2.26 ± 0.14

3.90 ± 0.15

12

3.35 ± 0.24

PEG1kCaL5

0.60

3.60

3.00

270

358

334

2.63 ± 0.12

4.18 ± 0.17

8.0

3.32 ± 0.15

PEG2kCaL5

0.59

4.10

3.51

270

358

334

3.75 ± 0.34

2.99 ± 0.29

3~4

1.61 ± 0.45

a

Rc: core size, bRs: shell size, cρc: electron density of the core, dρs: electron density of the shell, eρsol: electorn density of the solvent. fRg: gyration radius, gMw: weight-averaged molar mass, hNagg: aggregation number, iPEG: PEG density in the miecllar shell.

All SAXS profiles were fitted with the core-corona spherical model, the fitting parameters for which are listed in Table 1. The size of the core was in the range of 0.57–0.65 nm, which is reasonable in terms of the length of the n-pentyl chain. was in the range of 350–360 e

nm−3 and ! was fixed at 270 e nm−3, both of which are almost consistent with previously reported values.11 The hydrophilic shell size (Rs − Rc) increased in accordance with the molecular

weight of PEG. The increment of shell size when changing the PEG molecular weight from 550 to 1000 was 0.75 nm, which is larger than that when changing from 1000 to 2000, namely, 0.23 nm. This implies that the PEG crowding state at the core-shell interface of the micelles differs from the system of PEG550CaL5 and PEG1kCaL5 to PEG2kCaL5. The chain density at the interface can be calculated by the following equation:28-29

σ PEG =

2 N agg π Rg,PEG

4π( RC + Rg,PEG ) 2

(4)

where Rg,PEG is the gyration radius of a single chain of PEG. When σPEG is less than 1, the tethered chains do not interact with each other, which is called the mushroom region. For σPEG over 1, the chains overlap with each other (brush region). The calculated values of σPEG are summarized in Table 1. The values of σPEG for PEG550CaL5 and PEG1kCaL5 are over 3.3,


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indicating a highly stretched chain conformation compared with that of the micelles composed of PEG2kCaL5 whose σPEG is 1.61. The relatively low density of the PEG chain indicates the higher mobility of PEG chains in the hydrophilic shell than in the other systems. This is presumably because the excessively high affinity for water of PEG 2000 contributes to instability of the micelle, in agreement with the relatively high CMC value of PEG2kCaL5. Scattering objects with a high symmetrical structure and monodispersity exhibit a sharper first minimum in the SAXS profile. The first minimum around 1.3 nm−1 in the SAXS profiles of PEG550CaL5 and PEG1kCaL5 micelles is relatively sharp compared with that of PEG2kCaL5 micelles, indicating the formation of highly symmetrical micelles with a narrow size distribution. The molar mass of the PEGmCaL5 micelles and its dispersity were evaluated via multiangle light scattering coupled with field flow fractionation (FFF-MALS), the results of which are shown in Figure 3a. The elution took longer for the micelles bearing higher-molecular-weight PEG. Since larger particles take longer to elute in FFF30, this difference shows the formation of larger micelles when PEG molecular weight is increased. It is worth noting that the LS and UV peaks overlapped with each other for PEG550CaL5 and PEG1kCaL5 micelles, indicating a quite small distribution of micellar molar mass (Figure S4). The LS and UV peaks of PEG2kCaL5 micelles do not overlap, thus exhibiting the formation of polydispersed micelles. The weightaveraged molar masses (Mw) determined from the Zimm plot were 3.82 × 104, 4.09 × 104, and 2.96 × 104 g mol−1 for PEG550CaL5, PEG1kCaL5, and PEG2kCaL5, respectively (Figure S5). The calculated values of Nagg from the corresponding values of Mw were 12, 8.0, and 3 for PEG550CaL5, PEG1kCaL5, and PEG2kCaL5, respectively. The molecular weight distribution (Mw/Mn) calculated from the micellar peaks was almost 1.0 for PEG550CaL5 and PEG1kCaL5


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micelles, while PEG2kCaL5 micelles showed polydispersity with Mw/Mn = 1.35. The obtained values are summarized in Table 2. We also performed analytical ultracentrifugation (AUC) measurements for PEG550CaL5 and PEG1kCaL5 micelles. Figures 3b and 3c present the concentration dependence of the apparent weight-averaged molecular weight (Mw,App) and the ratio of Mw,App/Mz,App (Q), where Mz,App is the apparent z-averaged molecular weight. The values of the molar mass were 3.76 × 104 for PEG550CaL5 and 4.09 × 104 for PEG1kCaL5, being consistent with those determined by SAXS and FFF-MALS measurements. The plots of lnC(r) vs. r2 for AUC show linearity, indicating monodispersity (Figure S6). In fact, the calculated molecular weight distribution (Mz/Mw) was almost 1.0 for both micelles. The agreement of the molar mass values in the three independent measurements led us to conclude that the aggregation numbers of PEG550CaL5 and PEG1kCaL5 micelles are 12 and 8.0, respectively.


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(a)

UV absorbance at 270 nm

1.2

PEG550CaL5

0.8

10

Absorbance Molar Mass

1.0

PEG1kCaL5

10

6

5

0.6 0.4

10

PEG2kCaL5

4

Molar Mass / g mol-1

0.2 10 0.0

8

10

12

14

3

16

time / min (b)

(c) 0.030

0.04

PEG550CaL5

PEG1kCaL5 0.025

1.4 1.2

Q

1.0 0.8

0.020 1.4

0.4

0.010

0.2 0.0 0.0000

0.0010

0.0020

1.0 0.8 0.6

0.6

0.01

1.2

0.015 Q

0.02

Mapp-1/2/(mol g-1)1/2

0.03

Mapp-1/2/(mol g-1)1/2

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0.4 0.2 0.0 0.0000

0.0030

0.0010

0.0020

0.0030

-1

-1

Conc. / g mL

0.00 0.0000

0.0010

0.0020

Conc. / g mL

Conc. / g mL

0.005 0.0030

0.0000

0.0010

-1

0.0020

Conc. / g mL

0.0030

-1

Figure 3. (a) FFF-MALS fractograms of PEGmCaL5 micelles in 50 mM aqueous NaCl measured with UV at 270 nm (lines); the points indicate the calculated molar mass of the micelles at each retention time. (b) and (c) show the concentration dependence of Mw,APP and Q (= Mw.APP/Mz.APP) (inserted figures) determined by analytical ultracentrifugation measurements for PEG550CaL5 and PEG1kCaL5 micelles, respectively.


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Table 2. Micellar Molar Mass Determined by Field Flow Fractionation coupled with Multi-Angle Light Scattering and Analytical Ultracentrifugation Measurements a b Mw Mw a b sample Mw/Mn Mz/Mw Nagg 4 -1 4 -1 / 10 g mol / 10 g mol

a b

PEG550CaL5

3.82 ± 0.11

1.00

3.76 ± 0.12

1.00

12

PEG1kCaL5

4.09 ± 0.17

1.00

4.09 ± 0.15

1.01

8.0

PEG2kCaL5

2.96 ± 0.35

1.35

―

―

3~4

Weight averaged micellar molar mass and its distribution determined by FFF-MALS Weight averaged micellar molar mass and its distribution determined by AUC

The characteristics of the monodispersity and the aggregation numbers corresponding to the vertex numbers of Platonic solids indicate the formation of Platonic micelles. As described in the introduction, considering the coverage density D(N), we can find local maxima at N = 2, 4, 6, 12, and 20 in both Tammes and Thomson problems, which are consistent with the Platonic numbers. For the system of PEG-bearing calix[4]arene-based amphiphiles, the created interfaces (ae) between hydrophilic domain and hydrophobic domain can be regarded as being equal in size and having the potential to provide a hard core. Exhibiting a proportionate relationship to ae, the molecular weight of PEG seems to be the dominant factor determining the aggregation number. For the PEG 550 and 1000 systems, the micelles demonstrated monodispersity in the aggregation number because the densely crowded state of PEG, which is indicated by the high σPEG value, determined a certain ae of the amphiphiles comprising the micelle. However, for the following two reasons, PEG 2000-bearing calix[4]arene-based amphiphiles (PEG2kCaL5) showed neither monodispersity nor Platonic numbers in the aggregation numbers. First, the self-assembly force was dependent on the balance of hydrophobicity and the affinity for water of the hydrophilic part. The relatively high molecular weight of PEG in PEG2kCaL5 conferred excessive affinity for water and large ae, leading to instability of the micelle. Second, owing to the instability arising


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from the high affinity for water of PEG 2000, the PEG chain could be mobile in the micellar shell, which presumably induces deformed ae, resulting in a non-Platonic number of the aggregation number with polydispersity. The effect of molecular weight of PEG on the aggregation behavior of the micelles composed of calix[4]arene-based amphiphiles was investigated in previous work14, but the critical factor for determining the aggregation number and the polydispersity of the micelle was still unclear. In this paper, we characterized the micellar physical properties including the CMC and the σPEG as well as the morphology. The comprehensive understanding of the properties led us to conclude that the ae might be significant factor to determine the aggregation behavior in the system of Platonic micelles. Figure 4 shows a summary of the effects of PEG molecular weight on the aggregation behavior of PEGmCaL5 micelles.


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Figure 4. Schematic illustration of the effects of molecular weight of PEGs in PEGmCaL5 on the micellar aggregation behaviors including Nagg and its polydispersity.

CONCLUSIONS We synthesized calix[4]arene-based amphiphiles bearing PEGs, non-ionic hydrophilic polymers, and demonstrated the formation of Platonic micelles composed of the amphiphiles. The interfacial area (ae) between hydrophilic and hydrophobic domains of the amphiphiles can


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be controlled by changing the molecular weight of PEG, which then makes it possible to control the aggregation number of the micelles. PEG 550- or 1000-bearing calix[4]arene-based amphiphiles self-assemble into monodisperse micelles whose aggregation number is 12 or 8, respectively. A relatively high molecular weight of PEG (2000 g mol−1) reduces the hydrophobic interactions, leading to instability of the micellar structure. The unstable structure endows the PEG chain with mobility in the micellar shell, which induces distinct ae and results in a nonPlatonic number for the aggregation number with polydispersity. This study demonstrates that non-ionic hydrophilic polymers can act as a hydrophilic shell for the formation of Platonic micelles, which would be helpful for the design of such completely monodisperse micelles for certain applications, including drug delivery systems and nano/micro-reactors.


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EXPERIMENTAL SECTION Materials and Synthesis. All chemical reagents were purchased from Tokyo Chemical Industry Co., and Sigma-Aldrich Co., and were used without further purification. All reactions were carried out under nitrogen atmosphere, and all solvents were dehydrated by standard methods. We monitored the progress of the reactions using the thin layer chromatography (TLC) in which the reactions were detected using ultraviolet (UV; 254 nm) irradiation and staining with a basic solution of potassium permanganate. Products were purified by column chromatography with silica gel 60 (240–400 mesh) or dialysis against water for 5 days. Nuclear magnetic resonance spectra were recorded with a 500 MHz JEOL spectrometer using chloroform-d as solvents. All chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilane using the solvent resonance as the internal standard. The compounds I, II, III, V was synthesized according to the reported method.11, 31 Synthesis of polyethylene glycol (PEG) (MW = 550 g/mol) bearing calix[4]arene amphiphile with pentyl tails(PEG550CaL5). A PEG derivative (0.163 g, 0.295 mmol), copper (II) sulfate pentahydrate (2.20 mg, 8.81 µmol), and sodium ascorbate (3.70 mg, 18.7 µmol) were dissolved in dry DMF (10 mL), and then a solution of azide bearing calix[4]arene derivative (54.6 mg, 0.0590 mmol) in dry DMF (5 mL) was added to the mixture. The mixture was stirred at 90 °C for 24 h, and then cooled to room temperature. The crude product was purified with Spectra/Por Float-A-Lyzer (cellulose membrane; cut off 1 kDa) for 5 days. The compound was purified by reversed-phase high performance liquid chromatography (RP-HPLC) using CH3CN as the eluent. (137 mg, 0.0437 mmol, 74%). 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.46 (s, 4H), 6.55 (s, 8H), 5.22 (s, 8H), 4.65 (s, 8H), 4.38 (d, J = 10.0 Hz, 4H), 3.84 (t, J = 7.50 Hz, 8H), 3.70–3.51


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(m, 176H), 3.38 (s, 12H), 3.08 (d, J = 15.0 Hz 4H), 1.88–1.85 (m, 8H), 1.38–1.36 (m, 16H), 0.93 (t, J = 7.50 Hz, 12H).

Synthesis of polyethylene glycol (PEG) (MW = 1000 or 2000 g/mol) bearing calix[4]arene amphiphile with pentyl tails(PEG1kCaL5 or PEG2kCaL5). The other PEGs bearing calix[4]arene-based amphiphiles (PEG1kCaL5 and PEG2kCaL5) were synthesized by a similar process to that used for PEG550CaL5; yield: 206 mg (80 %) and 101 mg (70 %) for PEG1kCaL5 and PEG2kCaL5, respectively. 1H NMR for PEG1kCaL5 (500 MHz, CDCl3): δ (ppm) = 7.69 (s, 4H), 6.61 (s, 8H), 4.53 (m, 16H), 4.40 (d, J = 10.0 Hz, 4H), 4.22 (s, 8H), 3.86 (m, 16H), 3.68– 3.50 (m, 336H), 3.11 (d, J = 10.0 Hz 4H), 1.90–1.87 (m, 8H), 1.38–1.37 (m, 16H), 0.94 (t, J = 7.50 Hz, 12H). 1H NMR for PEG2kCaL5 (500 MHz, CDCl3): δ (ppm) = 7.69 (s, 4H), 6.60 (s, 8H), 4.53 (m, 16H), 4.40 (d, J = 10.0 Hz, 4H), 4.22 (s, 8H), 3.87 (m, 16H), 3.68–3.49 (m, 672H), 3.11 (d, J = 15.0 Hz 4H), 1.90–1.87 (m, 8H), 1.37–1.34 (m, 16H), 0.93 (t, J = 7.50 Hz, 12H).

Critical micelle concentration (CMC) measurements. The powder of PEGmCaL5 was dissolved in 50 mM aqueous NaCl to be 10 mM as a stock solution, and then the solution was diluted. 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 50 mM aqueous NaCl, and was then diluted to 10 µM in each solution. All samples were incubated for at least 1 hour in the dark at room temperature before the fluorescence measurements. The fluorescence measurements were performed 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 is sensitive to the polarity of its environment;


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therefore, the CMC can be determined from a plot of the fluorescence intensity vs. the sample concentration.32 Small angle X-ray scattering (SAXS) measurements. The powder of PEGmCaL5 was dissolved in 50 mM aqueous NaCl to be required concentration 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 BL-40B2 beamline of the SPring-8 facility, Hyōgo Prefecture, Japan. A 30 × 30 cm imaging plate (Rigaku R-AXIS VII) detector was placed 2 m from the sample. The wavelength of the incident beam (λ) was adjusted to 0.10 nm. This setup provided a q range of 0.10−5 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.11 The absolute SAXS intensities were recorded using the absolute scattering intensities of water.33 The micellar SAXS profiles were fitted by using the following equations: 7

:; 3*sin#.& − #.& cos #.& 1 ' − ' 7 sin# /# 9 = "#~ %& − ' (& + 45 6 #.& 2 :
). Due to inter-particle interference, the I(0) and .> values depend on the sample concentration. In order to remove the effects of sample concentration, the SAXS intensities measured 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 = I (0){ N A c( ∆ρυ ) 2 }

(4)

where Mw is the weight average molecular weight, c is the concentration of the samples, NA is Avogadro's number, and ∆ is the scattering length difference, which can be calculated from the

electron number and the molecular weight of the molecules and the solvent. The @̅ is the specific


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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. PEGmCaL5 (10 mg mL-1) was prepared in 50 mM aqueous NaCl. Aliquots (60 µL) of the sample solution were injected into an Eclipse 3+ separation system (Wyatt Technology Europe GmbH, Dernbach, Germany) for field-flow fractionation (FFF) at 22–28°C. The output from FFF was then passed sequentially through a Dawn Heleos II multiangle lightscattering (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 crossflow and channel-flow rates were fixed at 4.0 and 1.0 mL min-1, respectively. Detailed experimental procedures are reported elsewhere.32 The specific refractive index increments (BC/BD) and the extinction coefficients (E 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). Analytical ultracentrifugation (AUC). Sedimentation equilibriums of PEGmCaL5 in 50 mM NaCl 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 × 104 rpm. The apparent weight average molecular weight Mapp,W−1 and Q (= Mapp,W/Mapp,Z) were determined by analyzing the Rayleigh fringe according to the established method.35


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Supporting Information.

1

H NMR spectra of PEGmCaL5. UV specta, concentration

dependence of refractive index increment, and density increment for PEGmCaL5 micelles in 50 mM aqueous NaCl. The CMC values of PEGmCaL5 micelles detemined by Fluorescent measurements. The SAXS data of control sample for PEG550CaL5 micelle. FFF-MALS fractogram for PEGmCaL5 micelles. The Zimm plots for PEGmCaL5 micelles. The evidence of the monodispersity of PEGmCaL5 micelles indicated from the linearity of the relationship in lnC(r) vs. r2 for AUC. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions †Kenta Yoshida and Shota Fujii contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT


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We appreciate JST/CREST program for financial support and all SAXS measurements were carried out at SPring-8 40B2 [2016A1242, 2016B1481, 2017A1414].


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Graphical abstract


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Self-Assembly of Calix[4]arene-Based Amphiphiles Bearing

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