CoreâShellâCorona Micelles from a Polyether-Based Triblock

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Core-Shell-Corona Micelles from a Polyether-Based Triblock Terpolymer: Investigation of the pH-Dependent Micellar Structure Shotaro Miwa, Rintaro Takahashi, Carsten Rossel, Sakiko Matsumoto, Shota Fujii, Ji Ha Lee, Felix H Schacher, and Kazuo Sakurai Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01168 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

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Core−Shell−Corona Micelles from a Polyether-Based Triblock

Terpolymer:

Investigation

of

the

pH-Dependent Micellar Structure Shotaro Miwa,†§ Rintaro Takahashi,†§ Carsten Rössel,‡ Sakiko Matsumoto,† Shota Fujii,† Ji Ha Lee,† Felix H. Schacher,‡ Kazuo Sakurai*† †

Department of Chemistry and Biochemistry, University of Kitakyushu, 1-1, Hibikino,

Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan ‡

Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller

University Jena, Lessingstraße 8, D-07743 Jena, Germany, and Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany §

These authors equally contributed to this work.

*(K.S.) E-mail: [email protected].

Abstract: Core-shell-corona micelles featuring a pH-responsive shell have been characterized in dilute aqueous solution at different pH values (= 4−8) by using field-flow fractionation coupled with multi-angle light scattering detector (FFF-MALS), steady-state fluorescence, and small-angle X-ray scattering (SAXS). The micelles are formed by self-assembly of a polyether-based triblock terpolymer consisting of a hydrophobic poly(tert-butyl glycidyl ether) block (PtBGE), a pH-responsive modified poly(allyl glycidyl ether) segment (PAGECOOH), and a neutral hydrophilic poly(ethylene oxide) block (PEO). Due to the side-chain carboxylic acids in the middle block, the

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micellar structure and size depends on the solution pH. Hereby, we show that an increase in pH induces a decrease in the aggregation number ( N agg ). In addition, the combination of the above measurements revealed an unexpected morphological change from spherical to ellipsoidal micelles by increasing pH.

INTRODUCTION Micellization of block copolymers in solution has been extensively studied over the last 40 years.1−3 In particular, stimuli-responsive block copolymers attract considerable attention, for example to achieve the controlled release of drugs toward the application to drug delivery systems.4−6 Among the many stimuli, pH is one of the most important ones for prospective drug delivery systems because the pH value inside cells and around tumor tissues is distinctly lower than the physiological pH (~ 7.4).7,8 To impart pH-sensitivity, a usual strategy is to use block copolymers containing ionic segments, since the ionic block chain can ionize/unionize depending on the pH, which leads to changes in the electrostatic interaction between the ionic segments as well as between the ionic segments and potential ionic guest molecules (drugs). Hydrophobic−ionic hydrophilic−nonionic hydrophilic block copolymers can simultaneously encapsulate hydrophobic drugs and hydrophilic oppositely-charged ionic materials. This is because such micelles feature both a hydrophobic core, an ionic shell, and a hydrophilic but non-ionic corona as, e.g., shown for polyether-based core-shell-corona micelles of varying composition.9,10 Moreover, the terminal non-ionic hydrophilic segments stabilize such micelles even if oppositely charged materials are 2 ACS Paragon Plus Environment

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contained. Such systems might be very promising in terms of co-delivery approaches, and recently the advantage and potential therapeutic application have been shown.11 Nevertheless, few studies have been performed so far where the pH-dependent aggregation number ( N agg ) or changes in micellar structure have been in focus for such systems. In the present study, we investigate the solution structure of amphiphilic poly(ethylene oxide)-block-poly(allyl glycidyl etherCOOH)-block-poly(tert-butyl glycidyl ether) (PEO-b-PAGECOOH-b-PtBGE; Chart 1) triblock terpolymers in aqueous solution at different pH. The middle block, PAGECOOH, features carboxylic acids in the side chain and, hence, the repulsive interactions between adjacent PAGECOOH segments can be changed with pH. We have investigated the pH-dependent micellar characteristics of PEO-b-PAGECOOH-b-PtBGE by using small-angle X-ray scattering (SAXS), field flow fractionation coupled with multi-angle light scattering detector (FFF-MALS), dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and steady-state fluorescence.


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Chart 1. Chemical Structure of PEO-b-PAGECOOH-b-PtBGEa

k = NPEO = 42 l = NPAGECOOH = 12 m = NPtBGE = 22 a

Triblock terpolymer consisting of poly(ethylene oxide) (PEO), poly(allyl glycidyl

ether) functionalized by COOH (PAGECOOH), and poly(tert-butyl glycidyl ether) (PtBGE).

EXPERIMENTAL SECTION Materials. Polyethylenoxid monomethyl ether (Molecular weight: 2000, PEO), NaH (95%), CaH2 (95%), 3-mercaptopropionic acid (99%), 2,2-dimethoxy-2-phenylacetophenone (99%, DMPA), allyl glycidyl ether (99%, AGE), tert-butyl glycidyl ether (99%, tBGE), and 1,6-Diphenyl-1,3,5-hexatriene (DPH) were purchased from Sigma Aldrich. PEO was purified by precipitation in cold diethyl ether and dried by azeotropic distillation with dry toluol in vacuum. The glycidyl ethers were dried over CaH2 and distilled under reduced pressure. Deuterated chloroform was obtained from Deutero GmbH (Kastellaun, Germany). For dialysis, a regenerated cellulose membrane (Spectrum, Inc., Spectra/Por® 6 pre-wetted dialysis tubing) with a nominal molecular 4 ACS Paragon Plus Environment

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weight cut-off of 1 kDa was used. The buffer and salts were purchased from Wako Pure Chemical Industries and used without any purification. Water was purified by a Millipore Milli-Q system. Synthesis of PEO-b-PAGE-b-PtBGE. In a glove box under Ar atmosphere PEO (2.024 g, 1.08 mmol) was melted at 110 °C and NaH (0.034 g, 1.35 mmol) was added. After 2 hours, allyl glycidyl ether (1.7 mL, 14.33 mmol) was added. After further 23 hours, tert-butyl glycidyl ether (3.6 mL, 25.36 mmol) was added and the mixture was stirred for 24 hours. The polymerization was terminated with MeOH (1.0 mL), cooled to room temperature and dried under vacuum at 100 °C to afford 6.996 g of the triblock terpolymer (PEO42-b-PAGE12-b-PtBGE22) as orange solid. 1

H-NMR (400 MHz, CDCl3, 24 °C): δ [ppm] = 0.90-1.15 (PtBGE); 3.15-3.65 (polymer

backbone); 3.75-3.95 (PAGE); 4.90-5.25 (PAGE); 5.60-5.90 (PAGE). Synthesis

of

PEO-b-PAGECOOH-b-PtBGE.9

In

a

flask

PEO-b-PAGE-b-PtBGE (1.998 g, 0.32 mmol), 3-mercaptopropionic acid (1.2 mL, 13.66 mmol) and DMPA (0.285 g, 1.11 mmol) were dissolved in 16 mL THF. The reaction mixture was flushed with Ar to degas the solution and irradiated with UV-light in a UV-cube (UVACUBE 100 (100 W) from Hoehnle UV technology, Germany) for 2 hours. The crude product was purified by dialysis against water, a water:THF-mixture (1:1) and pure THF. The solvent was removed and the polymer was dried under vacuum to afford 2.401 g polymer with a degree of functionalization over 99%. 1H-NMR shown in Figure S1 (400 MHz, CDCl3, 24 °C): δ [ppm] = 1.10-1.20 (PtBGE); 1.75-1.90 (PAGECOOH); 2.50-2.90 (PAGECOOH); 3.30-3.70 (polymer backbone); 6.00-7.20 5 ACS Paragon Plus Environment

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(PAGECOOH). Micelle preparation. The triblock terpolymer sample was dissolved in tetrahydrofuran (THF) in a graduated cylinder. Milli-Q water was added into the solution, and THF was vaporized by gentle heating (~ 50 °C) and reduced pressure with stirring in the cylinder for at least 10 hours. During this process, water was further added to adjust the solution volume. After that, the solution was mixed with each aqueous salt solution. The salt solutions (solvents) were composed of NaCl (50 mM) and HCl (small amount) at pH = 4 and 5; NaCl (50 mM) and sodium citric buffer (50 mM) at pH = 6; NaCl (50 mM) and sodium borate buffer (50 mM) at pH = 8. The triblock terpolymer concentration ( c ) of the test solution used in the following measurements was fixed to 0.003 g/cm 3 . It cannot be excluded that the final solutions may contain traces of THF. Dynamic Light Scattering (DLS). Dynamic light scattering (DLS) was performed at a scattering angle of θ = 90° on an ALV CGS-3 instrument and a He−Ne laser operating at a wavelength of λ0 = 633 nm at 25 °C. The CONTIN algorithm was applied to analyze the obtained correlation functions and get the relaxation time spectra [A(τ)]. The first cumulant ( Γ ) was calculated by the summation of A(τ)/τ to obtain the apparent hydrodynamic radii (RH) according to the Stokes−Einstein equation:

RH =

kBT q 2 6πη Γ

(1)

where kBT is the Boltzmann constant multiplied by the absolute temperature, η is the viscosity coefficient of the solvent, and q is the magnitude of the scattering vector


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defined by

with the refractive index ( ) of the solvent.

FFF-MALS. FFF-MALS measurements were performed using an Eclipse 3+ separation system (Wyatt Technology Europe GmbH, Dernbach, Germany) coupled with a Dawn Heleos II MALS detector (Wyatt Technology, CA) and an Optilab rEX DSP differential refractive index (RI) detector at a wavelength of 658 nm (Wyatt Technology). The weight-average molar mass at each fraction was also determined by a Guinier plot:

1 R  2 4 ln  θ  = ln M w − 〈S 2 〉1/2 z q + O(q )  Kc  3

(2)

where Rθ is the Rayleigh ratio, K is the optical constant in light scattering, and is the z-average radius of gyration. The weight-average aggregation number 〈S 2 〉1/2 z ( N agg ) was calculated by

N agg = M w / M 1,w

(3)

with the weight-average molecular weight ( M 1,w ) of the single triblock terpolymer chain. Steady-state fluorescence. DPH was used as the fluorophore, which is suitable to determine the critical micellar concentration (CMC).12 A THF solution of DPH (0.5 mM) was added into the aqueous triblock terpolymer solution (prepared as explained above). The final DPH concentration was fixed to 0.5 µM. Steady-state fluorescence spectra of the micellar solution containing DPH, excited by the incident light at the wavelength of 350 nm, were recorded by a F-4500 fluorescence spectrometer (Hitachi Ltd., Tokyo) at 25 °C.


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SAXS. Synchrotron SAXS measurements were conducted at the beamline BL40B2, SPring-8, Hyogo, Japan. The sample solution was poured into a quartz capillary cell and maintained at 25 °C. The scattering intensity was detected by an imaging

plate

detector

R-AXIS

VII

(Rigaku

Corporation,

Tokyo)

at

a

sample-to-detector distance of 1 or 4 m. The excess scattering intensity was transformed to the differential scattering cross section [denoted by I(q) for the simplicity] by the standard method.13 The SAXS intensity was also analyzed by the equation:

 I (q)  1 = ln M w − 〈S 2 〉1/2 ln  q 2 + O(q 4 ) z  3  Kec 

(2´)

= ln  M w Pz (q)  where Ke is the optical constant in SAXS (see ref. 14), and Pz(q) is the z-average particle scattering function. Cryo-TEM. Cryo-transmission electron microscopy (TEM) measurements were performed on a FEI Tecnai G2 20 cryo-transmission electron microscope. The acceleration voltage was set to 200 kV and samples were prepared on Quantifoil grids (3.5/1) after cleaning by argon plasma treatment for 120 s. 8.5 µL of the solutions were blotted by using a Vitrobot Mark IV. The aqueous samples (typical concentrations are 1 mg mL-1) were plunge-frozen in liquid ethane and stored under nitrogen before being transferred to the microscope utilizing a Gatan transfer stage. TEM images were acquired with a 200 kV FEI Tecnai G2 20 equipped with a 4k x 4k Eagle HS CCD and a 1k x 1k Olympus MegaView camera. Micrographs were adapted in terms of brightness and contrast using the software ImageJ 1.47v.


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RESULTS AND DISCUSSION Synthesis and Micelle formation. The corresponding triblock terpolymer, PEO42-b-PAGE12,COOH-b-PtBGE22,

was

synthesized

using

sequential

anionic

ring-opening polymerization as reported earlier.9 Afterwards, the PAGE block was modified using thiol-ene chemistry to attach –COOH functionalities in the side chain. The success of the modification was confirmed by 1H-NMR spectroscopy, where the signals of the allyl group completely disappeared (Figure S1, Signals e, f, g) and new signals corresponding to the introduced 3-mercaptopropionic acid are visible. The molecular weight characteristics are listed in Table 1.

Table 1. Molecular Characteristics of the Synthesized Triblock Terpolymer Entry

Mn,1 a

Mw,1 b

M1,w / M1,n c

PEO

1800e

1900 e

1.06

PEO-b-PAGE

2500 e

2700 e

1.10

PEO-b-PAGE-b-PtBGE

7400f

8000g

1.08

a

N PEG,n d

N PAGECOOH,n d

N PtBGE,n d

42

12

22

Number-average molecular weight. bWeight-average molecular weight. cDispersity

index determined by SEC (Figure S2). dNumber-average degree of polymerization of i block chain (i = PEO, PAGECOOH, PtBGE) determined by 1H NMR. eDetermined by SEC (chloroform; PEO calibration). fDetermined by 1H NMR. gCalculated from M1,n and M1,w / M1,n


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Micelle formation was induced by self-assembly in aqueous solution into core-shell-corona structures featuring a hydrophobic PtBGE core, a PAGECOOH shell and a PEO corona. Therefore, an aqueous solution was added to a terpolymer solution in THF and the organic co-solvent was evaporated. The micellar solutions at different pH values were subsequently analyzed by DLS. The hydrodynamic radii of the micelles increased from 4 to 7 nm by increasing the pH from 4 to 6 (Figure 1). The COOH groups were partially deprotonated from pH 4 to 6, resulting in an increased electrostatic repulsion, a stretched shell, and larger hydrodynamic radii. Furthermore, at pH 8 the COOH groups are almost completely deprotonated, which leads to a drastic shift of the hydrophilic-to-hydrophobic balance of the entire triblock terpolymer and presumably also a lower aggregation number.

A(τ)

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1 0.8 0.6 0.4 0.2 0 0 10

-●-●-●-●-

101

pH = 4 pH = 5 pH = 6 pH = 8

102

k BT 2 q τ nm 6πη Figure 1. Relaxation time spectra of PEO-b-PAGECOOH-b-PtBGE at pH = 4 (blue, Rh = 4 nm), pH = 5 (red, Rh = 6 nm), pH = 6 (green, Rh = 7 nm) and pH = 8 (purple, Rh = 10 ACS Paragon Plus Environment

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6 nm).

FFF-fractograms. Figure 2 shows the FFF fractograms at different pH. At pH = 4 and 6, only one aggregate population was detected, and the N agg value was determined to 140. However, at pH = 8 a bimodal fractogram was observed. Please note that the smaller component flows faster and the larger component flows slower in FFF measurements. The obtained N agg values of the main micelles decreases with increasing pH which we attribute to an increased hydrophilicity of the PAGECOOH block with an increasing degree of ionization. The M w / M n values of at pH = 4−8 are quite narrow (< 1.2) and the obtained values of N agg and dispersity index ( M w / M n ) are listed in Table 2. The N agg and M w / M n values of the smaller distribution at pH = 8 are 6.6 and 5.0, respectively, indicating that these are aggregates formed by only a few terpolymer chains.


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107

(a)

1

105

0.5

104 0

103 0

10

20

107 106

1

(b)

105

0.5

104 0

103 0

10

20

107 106

1

(c)

105

0.5

Scattering intensity at 90° / arb. units

106

Mw / g mol−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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104 0

103 0

10

20

Elusion time / min Figure 2. FFF-MALS fractograms of PEG-b-PAGECOOH-b-PtBGE in aqueous solutions at pH = 4 (a), 6 (b), and 8 (c).


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1 2 3 4 5 6 7 8 9 Table 2. Micelle Characteristics 10 11 −1 3 2 1/2 N agg (by N agg (by / Mw / M n pH 〈S 2 〉1/2 CMC / z Rcore,M / nm a 〈S 〉corona / nm (Wcore / ccore ) / g cm 12 13 SAXS) FFF-MALS) nm 10 −5 g cm −3 14 15 16 17 4 230 ± 5 140 9.0 ± 0.2 < 1.03 0.5 ± 0.5 6.0 2.0 ± 0.2 0.32 ± 0.02 18 19 20 21 5 230 ± 5 8.8 ± 0.2 1 ± 0.5 6.0 2.0 ± 0.2 0.32 ± 0.02 22 23 24 25 26 6 40 ± 2 74 6.9 ± 0.1 1.2 ± 0.1 1 ± 0.5 2.8 2.0 ± 0.2 0.32 ± 0.02 27 28 29 30 31 8 38 ± 2 40 6.7 ± 0.1 1.2 ± 0.1 1 ± 0.5 2.5 2.0 ± 0.2 0.32 ± 0.02 32 33 34 35 a 36 Core radius calculated from eq 8 using the M w value obtained by SAXS. 37 38 39 40 41 13 42 43 44 ACS Paragon Plus Environment 45 46 47

fcore

ε

σ

−0.08±0.

1

3.6

1

3.6

02 −0.08±0. 02 −0.08±0. 2.4±0. 02

2

−0.08±0. 3.0±0. 02

1.1

2

1.0

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CMC. The CMC was directly determined by steady-state fluorescence using DPH as a fluorophore, since the small distribution observed in FFF-fractograms at higher pH could be indicative of dissociation of the micelles under these conditions. It is known that DPH is hydrophobic and fluoresces if encapsulated inside a hydrophobic micellar core. The wavelength at the maximum fluorescence intensity was 432 nm for all solutions measured. Figure 3 displays the fluorescence intensity at the wavelength of 432 nm as a function of c for PEO-b-PAGECOOH-b-PtBGE solutions containing DPH. Although almost no fluorescence was detected at lower c , with the increase in c the fluorescence intensity starts increasing at a certain c value. This means that PEO-b-PAGECOOH-b-PtBGE behaves as unimer at low c values, and the micelle was gradually formed by increasing c . The c values at the folding point in the figure were defined as CMC, although the determined CMC values are only rough estimates.


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Fluorescence intensity / arb. units

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106 105 104

pH = 8

103

pH = 6

102 pH = 5 101

pH = 4

100 -7 10 10-6 10-5 10-4 10-3

c / g cm−3 Figure 3. Fluorescence intensity at the wavelength of 432 nm as a function of the polymer concentration c in aqueous solution of PEG-b-PAGECOOH-b-PtBGE containing DPH (0.5 µM).

The CMC values are listed in Table 1 and are much lower (~ 10−5 g/cm3) than the actually injected polymer concentration (2 × 10−3 g/cm3) in the FFF measurements, even if the pH value was 6 or 8. However, as mentioned above, FFF-MALS results suggests a partial disruption of the micelles in the solution at pH = 6 and 8. This inconsistency may be interpreted as follows: The actual CMC value is low even at pH = 6 and 8. However, the flows (focusing flow, cross-flow, or channel flow) in the FFF 15 ACS Paragon Plus Environment

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measurement may induce micellar disruption due to shear forces, as already observed for a different kind of block copolymer micelles.15 Therefore, we believe the small distribution to only occur during the FFF measurements. Please note that the disrupted amount is quite small and may scarcely influence the N agg value of the micellar distribution as determined by FFF-MALS. SAXS profiles. Figure 4 shows the SAXS profiles at different pH. A clear oscillation was observed at pH = 4 and 5, indicating that the size dispersity of the micelles is quite narrow. The profile at pH = 4 is not significantly different from that at pH = 5, as can be seen from the upper right panel of the absolute intensity plots. By further increase in pH to 6 and 8, the oscillation diminishes. The slope at the low q region is not steep and a so-called Guinier region is observed, which allows us to obtain both the N agg and 〈S 2 〉1/2 values from the Guinier plots (Figure 5). Both of the z obtained N agg and 〈S 2 〉1/2 values decrease by increasing pH as listed in Table 2. The z N agg values obtained by SAXS (= 230 at pH = 4) are not completely equal to those by

FFF-MALS (= 140 at pH = 4). This may be due to the difference of the conditions such as concentration during the measurements and flows. However, the tendency that pH induces the decrease in N agg observed by SAXS is similar to that observed earlier by FFF-MALS.


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107 106

 I (q ) 1  K c g mol  × A  e 

10

9

10

8

10

7

105 104 103 102

10-1

100

106

pH = 8 (A = 103)

105

pH = 6 (A = 102)

104

pH = 5 (A = 10)

103

pH = 4 (A = 1)

10-1

100 q / nm

1

Figure 4. SAXS profiles of PEG-b-PAGECOOH-b-PtBGE in aqueous solution at pH = 4 (blue circle), 5 (red circle), 6 (green circle), and 8 (purple circle). The data are shifted vertically (multiplied by A) for clarity. Upper right panel: Plots without the shift. The black solid and red solid curves represent the fitted curves by spherical (eq 3) and ellipsoidal (eq 3´) core−corona models, respectively.

15  I (q)  ln  g mol −1   Kec 

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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14

● pH = 4, ● pH = 5 ● pH = 6, ● pH = 8

13 12 11 0

0.01 0.02 0.03 0.04 0.05 q / nm−1

Figure 5. Guinier plots of PEG-b-PAGECOOH-b-PtBGE in aqueous solution at pH = 4 (blue circle), 5 (red circle), 6 (green circle), and 8 (purple circle). 17 ACS Paragon Plus Environment

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Analysis of the SAXS profiles. The micellar morphology was inferred to be spherical due to earlier experience9 and because the size of the micelles was rather small ( 〈S 2 〉1/2 < 10 nm). Further, it was expected that the micelle is composed of a z hydrophobic core, a (partially) charged shell, and a neutral hydrophilic corona. Therefore, to fit the SAXS profiles, we chose the spherical micelle model which is composed of spherical rigid core and attached coronal Gaussian chains. Such a model can be formulated as follows:16

P(q) = [ fcore Esph,core (q) + (1− fcore )Esph,corona ]2 +

1− fcore 2 2 [Echain (q) − Esph,corona (q)] N agg

(4)

where fcore is the contrast of the core region, and Esph,core (q) , Esph,corona (q) , and

Echain (q) are the time-averaged amplitudes of the scattering electric field from the core, the corona, and each coronal chain, respectively, given by

Esph,core (q) =

3[sin(qRcore ) − qRcore cos(qRcore )] (qRcore )3

Esph,corona (q) = 2 Echain (q) =

1− exp(−q 2 〈S 2 〉 corona ) sin[q(Rcore + 〈S 2 〉1/2 corona )] 2 2 2 1/2 q 〈S 〉corona q(Rcore + 〈S 〉 corona )

2[exp(−q 2 〈S 2 〉corona ) − 1+ q 2 〈S 2 〉corona ] (q 2 〈S 2 〉corona )2

(5) (6) (7)

Here, Rcore is the radius of the micelle core, which is related to the molar mass ( M mic ) and the concentration of the core region ( ccore ) by

Rcore

 3Wcore M  =   4π N A ccore 

1/3

(8)


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with the weight fraction of the core region in the micelle ( Wcore ) and Avogadro constant ( N A ). The dispersity of the micellar component was taken into account by assuming the log-normal distribution given by17

M w Pz (q) =

1 2π ln( M w / M n )

 ln 2 ( M / M M / M )  w n w  dM × ∫ P(q) exp  − 2 ln( M / M )   w n

(9)

The scattering profile was well described by the model as represented by the black solid curves in Figure 4. Because the shape of the oscillation (or the minima positions) in the SAXS profile is quite sensitive to the radii and electron densities [i.e., , Wcore , and fcore ], almost all the parameters can be Rcore ∝ (Wcore M / ccore )1/3 , 〈S 2 〉1/2 corona uniquely determined as listed in Table 2. The fitted curves by the spherical core−corona model indicate the black solid curves in Figure 4. Note that M w was determined by the intensity at the low q limit, and Nagg was calculated by eq 3 from M w determined by the Guinier plots (eq 2´), which was substituted to the second term of the right hand in eq 4. The Wcore and ccore could not separately determined, but the ratio ( Wcore / ccore ) is 0.32 g −1cm 3 at the pH of 4 and 5. If Wcore / ccore is 1.1 g/cm 3 (near the value of PEO density; PtBGE main chain is the same as PEO), the Wcore is 0.36 which is similar to the weight fraction WPtBGE (= 0.39 in the unionized state; 0.38 in the sodium salt state) of the PtBGE block in the copolymer chain, calculated by

WPtBGE =

M PtBGE,0 N PtBGE,n M PEO,0 N PEO,n + M PAGECOOH,0 N PAGECOOH,n + M PtBGE,0 N PtBGE,n

(10)

Here, M i,0 is the molecular weight of the monomer, and the subscript notation of PEO,


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PAGECOOH, and PtBGE represent the quantities of each block chain. That indicates that the micellar core is consisting of PtBGE chains only. The obtained 〈S 2 〉1/2 value is corona 2±0.2 nm in pH = 4−8, which is equal to the radius of gyration in the coil limit of the PEO and PAGECOOH block chain (calculated from the contour length of ca. 17 nm), although the electrostatic repulsive interaction between adjacent PAGECOOH blocks drastically increases at higher pH. Here, X-ray scattering from PAGECOOH is stronger than that from PEO because the electron density of the PAGECOOH block is higher. The value of 〈S 2 〉1/2 may be influenced by such an effect, namely, the coronal chains corona actually might be stretched. In the fit of the profiles at the pH of 6 and 8, although we initially used the spherical core−corona model (eqs 4−7) described above, the quite high M mic,w / M mic,n values (> 5) were necessary to fit the SAXS profile particularly on the diminished oscillation. The fitted model curves by the spherical core−corona model represent the black solid curves. The high M mic,w / M mic,n values disagree with the FFF-MALS results. Hence, we used an ellipsoidal core−corona model, since the SAXS profile of the monodisperse ellipsoid resembles that of the disperse sphere. The particle scattering function and the time-averaged amplitudes of the scattering electric field from the core and corona of the ellipsoidal core−corona micelle are given by18 Pmic (q) = ∫

π /2

0

{

 fcore Eell,core (q) + (1− fcore )Eell,corona   

2

 1− fcore 2 2  Echain (q) − Eell,corona   sin(Θ)dΘ + (q)  N agg  


(4´)

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Eell,core (q) =

3{sin[qr( Rcore , ε )] − qr( Rcore , ε )cos[qr( Rcore , ε )]} [qr( Rcore , ε )]3

2 1/2 1− exp(−q 2 〈S 2 〉corona ) sin { q[r(Rcore , ε ) + 〈S 〉corona ]} Eell,corona (q) = q 2 〈S 2 〉 corona q[r(Rcore , ε ) + 〈S 2 〉1/2 corona ]

(5´) (6´)

Here, r(Rcore , ε ) is the orient dependent radius given by r( Rcore , ε ) = Rcore sin 2 Θ + ε 2 cos 2 Θ

(11)

with the aspect ratio ( ε ). We chose ε = 2.4 at pH = 6 and 3.0 at pH 8 for the best fit ( ε > 1 means prolate). Even if the M mic,w / M mic,n is equal to the values obtained by FFF-MALS, the model nicely describes the experimental data, shown as the red solid curves in Figure 4. The parameters in the fit are summarized in Table 2. The 〈S 2 〉1/2 z values of the core−corona model can be calculated,19 which are almost the same as in the values determined by the Guinier plots. It should be noted that the fitted model curves of the disperse spherical core−corona model are not significantly different from the monodisperse ellipsoidal core−corona model as can be seen from that the black and red solid curves at pH = 6 and 8 in Figure 4. However, by taking into account the FFF-MALS result (narrow dispersity index), we can speculate that prolate spheroidal micelles are formed at pH = 6 and 8 from the point of the rather narrow dispersity. The fit also figured out that ε became large by increasing pH, and the Wcore / ccore and 〈S 2 〉1/2 remained 0.32 g/cm 3 corona and 2 nm, respectively. Concerning the coronal chain density of the spherical or prolate spheroid micelle, this can be discussed by using σ as defined by20


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σ=

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π 〈S 2 〉corona,0  (Rcore + 〈S 2 〉1/2 )2 corona,0  2π  arcsin 1− (1/ ε 2 )  +( R ε + 〈S 2 〉1/2 )( R + 〈S 2 〉1/2 ) core corona,0 core corona,0  1− (1/ ε 2 ) 

(

)

     

(12)

N agg

where 〈S 2 〉1/2 is the unperturbed radius of gyration of the coronal chain. In eq 12, corona,0 the denominator represents the cross-sectional area of a coronal chain, and the numerator is the surface area per chain. When σ is lower than 4, the coronal chains are not crossed over and, instead, higher σ indicates that the chains are more crowded. Here, 〈S 2 〉1/2 is assumed to be 2 nm (coil limit), and Rcore,M values listed in Table 2 corona,0 are used as Rc in eq 13. The calculated σ values decreased from 3.6 to 1 by increasing pH as shown in Table 2, which demonstrates that the coronal density decreases with pH. We explain this by increasing electrostatic repulsion between the PAGECOOH segments due to increasing ionization. Cryo-TEM observation. Additional cryo-TEM experiments have been carried out at pH 4 and pH 8 (exemplarily) in order to compare the results with DLS and SAXS data as discussed above (Figure 6). At pH = 4 (panel a), clearly spherical objects are observed, and the core diameter with 6.3 ± 1.2 nm agrees well with the scattering data shown in Table 2 (obtained by analyzing approximately 70 micelles within 5 different micrographs). However, at pH = 8 (panel b), the size of the micellar cores with 4.1 ± 0.8 nm is distinctly smaller and it is difficult to judge the morphology from the obtained micrographs. Also, the inherent contrast of the images was lower if compared to the measurements performed at pH 4. 22 ACS Paragon Plus Environment

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Langmuir

(a)

(b)

Figure 6. Cryo-TEM images of PEG-b-PAGECOOH-b-PtBGE at pH = 4 (a) and 8 (b).

Origin of the morphological change. Our first explanation for the sphere−prolate spheroid morphological change was based on the packing parameter as defined by21

λp =

v a0lc

(13)

where v is the effective volume of the hydrophobic chain, a0 is the area per chain of the core−corona domains interface, and lc is the length of the hydrophobic chain in the core domain. In the case of pH = 4 and 5, the micellar morphology was spherical, indicating that the λp value was within the range of 1/3 < λp < 1/2 (if the morphology would be ellipsoidal, λp would be larger). By increasing the pH value, the ionization degree of PAGECOOH increases, and with that a0 increases as well, as confirmed above with σ . Consequently, λp decreases and the morphological change


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should not take place in view of the λ p value. Nevertheless, a morphological change from spherical micelle to ellipsoidal micelle can be observed by increasing pH from 4 to 8 and, hence, in our opinion the morphological change cannot be explained by the packing parameter. Here, let us consider the interfacial energy. The total interfacial area ( Acore ) of the core domain is related to the total core volume ( Vcore ) of the micelles in the solution by22 Acore =

2(3 − d )Vcore D

(14)

where D (= 2 Rcore ) denotes the thickness of the core domain. In general. d is a parameter related to the micellar morphology and dimension, with d = 0 and 1 for spherical and cylindrical micelles, respectively, and 0 < d < 1 for prolate spheroidal micelles or cap-ended cylinders. In eq 14, Vcore is constant as the overall mass stays constant. The relation of Vcore = Nυ is true where N and υ denote the number micelles in the solution and the volume of one micelle. Similarly, Acore = N α where

α is the interfacial area of the core domain for one single micelle. When D decreases due to the decrease in N agg , Acore (and thus the interfacial energy) increases under the condition of a fixed Vcore . In order to reduce the penalty of the interfacial energy, d has to increase by the morphological change towards a prolate spheroidal micelle. It should be mentioned that it has been reported that D for cylindrical micelles is lower than D for spherical micelle in the morphological change of some kinds

of

block

copolymer

micelles

such

as

PEO-b-poly(ethyl


ethylene),23

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Langmuir

PEO-b-poly(butadiene),24

PEO-b-polystyrene,25

polystyrene-b-polyisoprene,26

polystyrene-b-poly(acrylic acid),27 and a polyelectrolyte complex micelle.17,22 Thus, in our opinion the described morphological change can be explained by the penalty of the interfacial energy, i.e, the increment of N due to the enhanced polymer solubility increases Acore , thus the morphology has to change from sphere to ellipsoid in order to reduce the interfacial free energy.

CONCLUSION We herein investigated the pH-dependent micellar characteristics of core-shell-corona micelles formed from polyether-based triblock terpolymers. Our results first demonstrate a quite low CMC values and decreasing N agg aggregation numbers by increasing pH – mainly due to increasing hydrophilicity of the PAGECOOH block. The SAXS profiles at pH = 4 and 5 exhibited clear oscillations and the analysis revealed that narrowly dispersed spherical micelle are formed. However, SAXS profiles at pH = 6 and 8 were quite different and could be fitted either by the disperse spherical core−corona or the monodisperse prolate spheroid core−corona model. By considering additional FFF-MALS results (low dispersity micelles were formed at pH = 6 and 8), our current interpretation points towards the prolate spheroid model. These results were confirmed by additional DLS and cryo-TEM measurements, where at least in the latter case slightly distorted spherical micelles were observed. In other words, the herein used combination of methods represents a powerful toolbox for determining micellar morphologies, also at sizes below 50 nm. In our opinion, the morphological change can 25 ACS Paragon Plus Environment

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be interpreted in terms of the interfacial energy.

ACKNOWLEDGEMENTS The SAXS experiments were carried out at SPring-8 under the approval by JASRI (Proposal number: 2017B1481, 2017A1414, and 2017A1238). This study was supported by the Photon and Quantum Basic Research Coordinated Development Program from MEXT, and a Grant-in-Aid for CREST-JST (JPMJCR1521). F. H. S. gratefully acknowledges funding within the collaborative research center PolyTarget (SFB 1278, Project C03) by the Deutsche Forschungsgemeinschaft (DFG). The authors thank Philip Biehl for performing the cryo-TEM measurements.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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in

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Solution.

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for Table of Contents use only

Core−Shell−Corona Micelles from a Polyether-Based Triblock Terpolymer: Investigation of the pH-Dependent Micellar Structure Shotaro Miwa, Rintaro Takahashi, Carsten Rössel, Sakiko Matsumoto, Shota Fujii, Ji Ha Lee, Felix H. Schacher, Kazuo Sakurai


CoreâShellâCorona Micelles from a Polyether-Based Triblock

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