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Complexes Covered with Phosphorylcholine Groups Prepared by Mixing Anionic Diblock Copolymers and Cationic Surfactants Keita Nakai, Kazuhiko Ishihara, and Shin-ichi Yusa Langmuir, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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Original Article for Langmuir

Complexes Covered with Phosphorylcholine Groups Prepared by Mixing Anionic Diblock Copolymers and Cationic Surfactants

Keita Nakai†, Kazuhiko Ishihara§, Shin-ichi Yusa*,†

†Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan §

Department of Materials Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-8656, Japan

ABSTRACT: Anionic diblock copolymers (PmAn) composed of biocompatible polybetaine, poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC), and anionic poly(sodium 2-(acrylamido)-2-methylpropane sulfonate) (PAMPS) were synthesized via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Two types of diblock copolymers (P24A217 and P100A99) were prepared with different compositions. The PmAn/CTAB complexes were formed by a stoichiometrically charge-neutralized mixture of anionic PmAn and cationic cetyltrimethylammonium bromide (CTAB) micelles in water. The complexes prepared using P24A217 and P100A99 were vesicles and micelles, respectively, and were covered with hydrophilic PMPC shells. The complexes dissociated upon addition of NaCl because the complex was maintained through electrostatic interactions. The

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P24A217/CTAB vesicles could encapsulate uncharged hydrophilic guest molecules into the interior of the aqueous phase.

Graphical Abstract

INTRODUCTION Complexes and supramolecular assemblies formed by interactions between polymers and low-molecular-weight surfactants have been investigated for several decades.1-5 The interactions between polymers and surfactants are of interest for theoretical as well as practical purposes for application in fields such as cosmetics, coatings, paints, detergents, food science, and drug delivery systems.6,7 In particular, the electrostatic interactions involved in complex formation between polyelectrolytes and oppositely charged surfactants have been extensively studied. The shape, size, and properties of the complexes are influenced by factors such as polymer and surfactant concentrations, molecular weight of the polymer,8 charge density of the polymer,9 the head group polarity, and the length of the alkyl chain in the surfactant.10 Fundin et al.11 reported the formation of a complex involving a polyelectrolyte, sodium

poly(styrene

sulfonate)

(PSSNa),

and

oppositely

2


charged

surfactant,

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cetyltrimethylammonium bromide (CTAB), in water. A dilute solution of the complex in water could be formed. The size and shape of the PSSNa/CTAB complexes could be adjusted by changing the molecular weight of the PSSNa, the mixing ratio, and the salt concentration. Mezei et al.12 reported the formation of complexes between cationic hyper-branched poly(ethylene imine) and the anionic surfactant sodium dodecyl sulfate (SDS). Formation of the complexes depended on pH and ionic strength. These complexes, composed of a polyelectrolyte and oppositely charged surfactant, were soluble in water when the concentration was very dilute or the charge balance was not equalized. In general, however, mixing a polyelectrolyte and oppositely charged surfactant produced phase separation in water at high concentrations with a stoichiometric charge balance. Water-soluble complexes can be prepared by mixing the surfactant and an oppositely charged diblock copolymer composed of nonionic hydrophilic and polyelectrolyte blocks.13-17 Berret et al.18 prepared a water-soluble complex composed of a cationic n-dodecyltrimethyl ammonium bromide and a diblock copolymer composed of nonionic polyacrylamide with anionic poly(sodium acrylate) blocks. The core-shell microphase-separated structure of the complex was confirmed using light scattering and small-angle X-ray scattering techniques. The shape and structure of the water-soluble complexes formed from surfactants and oppositely charged diblock copolymers composed of nonionic water-soluble blocks with polyelectrolyte blocks depended on the composition and molecular weight of the diblock copolymer.

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Figure 1. (a) Chemical structure of anionic diblock copolymers (PmAn) and (b) conceptual illustration of PmAn/CTAB complexes in 0.1 M NaCl aqueous solution.

Cationic diblock copolymers (PmMn) composed of a water-soluble biocompatible polybetaine, poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) with cationic poly(((3-(methacrylamino)propyl)trimethylammonium

chloride)

copolymers

PMPC

(PmAn)

composed

of

2-(acrylamido)-2-methylpropanesulfonate),

were

prepared

and

anionic

with

diblock

poly(sodium via

reversible

addition-fragmentation chain transfer (RAFT) controlled radical polymerization (Figure 1).19 The subscripts m and n indicate the degree of polymerization (DP) for the PMPC and polyelectrolyte blocks, respectively. When PmMn and PmAn were mixed in aqueous solution to neutralize the charges in the ionic blocks, water-soluble polyion complex (PIC) aggregates formed. When m and n were about 100, water-soluble spherical PIC micelles with a core-shell structure were formed. The PIC micelles were colloidally stabilized in water by nonionic betaine PMPC shells. Water-soluble PIC vesicles formed when m and n were about 20 and 200, respectively. The interior of the aqueous phase of the PIC vesicles could encapsulate hydrophilic nonionic guest macromolecules. 4


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The water-soluble PIC aggregates produced upon mixing the oppositely charged diblock copolymers could form micelles or vesicles depending on the composition of the block copolymers. The current study describes the preparation of water-soluble complexes (PmAn/CTAB) due to electrostatic interactions resulting from mixing anionic PmAn block copolymers and the cationic surfactant CTAB micelles. Two types of PmAn with different compositions (P24A217 and P100A99) were prepared via RAFT radical polymerization. The effect of anionic diblock copolymer composition on the shape of the PmAn/CTAB complexes was also investigated. If the PmAn/CTAB system exhibits association behavior similar to that of the water-soluble PIC aggregates formed from PmMn/PmAn, then P24A217/CTAB and P100A99/CTAB could be expected to form vesicle and micelle structures, respectively. The association behavior of PmAn/CTAB was studied with 1H NMR relaxation time, dynamic and static light scattering, transmission electron microscope (TEM), and fluorescence probe techniques. The effect of adding NaCl to aqueous PmAn/CTAB solutions was also investigated because of the electrostatic interactions involved with the complexes. Furthermore, encapsulation of hydrophilic guest molecules into the interior of the aqueous phase of P24A217/CTAB vesicles was evaluated using a fluorescence method.

EXPERIMENTS Materials. 2-(Methacryloyloxy)ethyl phosphorylcholine (MPC) was purchased from NOF Corp., and was purified using a previously reported method.20 4-Cyanopentanoic acid dithiobenzoate (CPD), used as a chain transfer agent (CTA), was synthesized according to a previously reported method.21 2-(Acrylamido)-2-methylpropanesulfonic acid (AMPS, 95%), 4,4’-azobis(4-cyanopentanoic acid) (V-501, 98%), cetyltrimethylammonium bromide (CTAB, 98%), and pyrene (97%) were purchased from Wako Pure Chemicals, and Texas red-labeled dextran (TD70, Mw = 70,000) was purchased from Life Technologies; all were used as 5


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received without further purification. Phosphate-buffered saline (PBS) tablets were obtained from Sigma Aldrich and used by dissolving one tablet in 200 mL of pure water. Methanol was dried over 4 Å molecular sieves and distilled. Water was purified with an ion-exchange column system. Other reagents were used as received. Preparation of PMPCm. PMPC24 was prepared by modifying previously reported methods.22 MPC (5.02 g, 17.0 mmol), CPD (237 mg, 0.847 mmol), and V-501 (94.9 mg, 0.339 mmol) were dissolved in 16.9 mL of water. The aqueous solution was degassed by purging with Ar gas for 30 min. Polymerization was conducted at 70°C for 2 h. The reaction mixture was dialyzed against pure water for two days. PMPC24 was recovered by freeze-drying (4.72 g, 94.0%). The number-average molecular weight (Mn(NMR)) and degree of polymerization (DP) estimated from 1H NMR, and the molecular weight distribution (Mw/Mn) estimated from gel-permeation chromatography (GPC), were 7.50 × 103, 24, and 1.04, respectively. PMPC100 was prepared by a method similar to that described above (7.28 g, 98.8%). The Mn(NMR) and DP estimated from 1H NMR data, and Mw/Mn estimated from GPC were 2.98 × 104, 100, and 1.14 respectively. Preparation of Anionic Diblock Copolymers (PmAn). A predetermined amount of AMPS (4.22 g, 20.4 mmol) was neutralized with 1 M NaOH in 20.5 mL water. To this solution was added PMPC24 (0.751 g, 0.100 mmol, Mn(NMR) = 7.50 × 103, Mw/Mn = 1.04) and V-501 (11.4 mg, 0.0407 mmol). The solution was deoxygenated by purging with Ar gas for 30 min. Block copolymerization was conducted at 70°C for 4 h. The diblock copolymer was purified by dialysis against pure water for two days. The anionic diblock copolymer (P24A217) was recovered by freeze-drying (5.19 g, 95.4%, Mn(NMR) = 5.73 × 104, Mw/Mn = 1.19). The P100A99 was prepared by a similar method (1.17 g, 91.2 %, Mn(NMR) = 5.25 × 104, Mw/Mn = 1.23). Preparation of PmAn/CTAB Complexes. PmAn (0.5 g/L) and CTAB (1.0 g/L) in 0.1

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M NaCl aqueous solutions were prepared separately. To prepare the PmAn/CTAB complex, the CTAB solution was added to the PmAn solution, followed by stirring for 5 min. The mixing ratio of PmAn and CTAB was represented by the mole fraction of the positively charged unit (f+ = [CTAB]/([AMPS] + [CTAB])). Complete charge neutralization was achieved at f+ = 0.5. Concentrations of P24A217 and CTAB in the complex at f+ = 0.5 were 0.296 g/L (1.12 mmol/L of AMPS unit) and 0.408 g/L (1.12 mmol/L), respectively. Final concentrations of P100A99 and CTAB in P100A99/CTAB complex were 0.372 g/L (0.702 mmol/L of AMPS unit) and 0.256 g/L (0.702 mmol/L), respectively. Measurements. GPC measurements were performed using a Tosoh RI-8020 refractive index detector equipped with a Tosoh 13.0-µm particle size TSKgel α-M column operating at 40°C under a flow rate of 0.6 mL/min. Phosphate buffer (50 mmol/L, pH 8) containing 10 vol% acetonitrile was used as the eluent. Values of Mn(GPC) and Mw/Mn were calibrated using standard poly(sodium styrenesulfonate) samples. 1H NMR spectra were obtained with a Bruker DRX-500 spectrometer. 1H NMR spin-spin relaxation time (T2) was determined using a Carr-Purcell-Meiboom-Gill (CPMG) method.23 A 90° pulse of 13.85 µs duration was calibrated and used for the measurements. Echo peak intensities at 12 different numbers of the 180° pulse were measured. Light scattering measurements were obtained using an Otsuka Electronics Photal DLS-7000HL equipped with a multi-τ, digital time correlator (ALV-5000E) at 25°C. A He-Ne laser (10.0 mW at 632.8 nm) was used as the light source. Sample solutions were filtered through a 0.2-µm membrane filter. For dynamic light scattering (DLS) measurements, the data obtained were analyzed using ALV-correlator software version 3.0. For static light scattering (SLS) measurements, weight-average molecular weight (Mw), radius of gyration (Rg), and the second virial coefficient (A2) values for PmAn and the complexes were estimated from Zimm plots. Values of dn/dc at 633 nm were determined with an Otsuka Electronics Photal DRM-3000 differential refractometer at 25°C.

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The ζ-potential measurements were performed using a Malvern Zetasizer Nano-ZS ZEN3600 instrument equipped with a He-Ne laser light source (4 mW at 632.8 nm). The ζ-potential was calculated from electrophoretic mobility (µ) using the Smoluchowski relationship, ζ = ηµ/ε (κa >> 1), where ε is the dielectric constant of the medium, κ is the Debye-Hückel parameter, and a is the particle radius. The TEM observations were performed with a Jeol JEM-2100 electron microscope at an accelerating voltage of 200 kV. Samples were prepared by placing one drop of the aqueous solution on a copper grid coated with thin films of Formvar. Excess water was blotted using filter paper. The samples were stained by sodium phosphotungstate and dried under vacuum overnight. Fluorescence emission and excitation spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer. Pyrene was used as a fluorescence probe. Excitation spectra were monitored at 390 nm. The slit widths for the excitation and emission were both maintained at 2.5 nm. Sample solutions of P24A217/CTAB complexes were prepared in pyrene-saturated (6.0 × 10-7 M) 0.1 M NaCl aqueous solutions. Fluorescence emission spectra of TD70 were obtained by excitation at 550 nm. The excitation and emission slit widths were both maintained at 5 nm. Encapsulation of TD70. The TD70 (0.0237 mg, 3.39 × 10-10 mol, Mw = 70,000) was dissolved in PBS buffer (2.37 mL), while the P24A217 (1.18 mg, 4.49 µmol of AMSP units) was dissolved in PBS buffer solution containing TD70. The CTAB (1.63 mg, 4.47 µmol) was dissolved in PBS buffer (1.63 mL). The CTAB solution was added to the P24A217 solution with stirring for 5 min to incorporate TD70 into inside of the P24A217/CTAB complex. The mixing ratio was kept constant at f+ = 0.5. The TD70 concentration in the P24A217/CTAB complex solution was 5.93 mg/L (0.0847 µmol/L); 4 mL of the complex solution was transferred to the Fast Dialyzer (Harvard Apparatus, 5000 µL chamber) equipped with 50-nm pore size polycarbonate membrane (Harvard Apparatus) and the solution was dialyzed against 400 mL of PBS buffer for 120 h to remove free TD70 molecules not encapsulated inside the

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complex. The outer PBS buffer was exchanged with fresh buffer 5 times. After dialysis, 1.5 M NaCl was added to dissociate the complex and avoid fluorescence self-quenching. The TD70 fluorescence intensity was measured. The loading efficiency (LE) and loading capacity (LC) of TD70 into P24A217/CTAB complex were calculated using the following equations:

LE (%) =

Weight of encapsulated TD 70 × 100 Weight of total TD 70

(1)

LC (%) =

Weight of encapsulated TD 70 × 100 Weight of complex

(2)

RESULTS AND DISCUSSION Anionic diblock copolymers, P24A217 and A100A99, were prepared via RAFT controlled radical polymerization. No interaction occurred between CTAB and the PMPC block. In contrast, CTAB and the anionic PAMPS block became associated due to electrostatic interactions, forming water-soluble PmAn/CTAB complexes with hydrophilic outer PMPC shells. Values for m and n of PMPCm and PmAn, which indicated DP, were estimated from 1H NMR data. Figure S1 compares GPC elution curves for PMPCm and PmAn. The GPC retention times for P24A217 and P100A99 were shorter than those of parent PMPCm, indicating that the molecular weights of the block copolymers were larger than those of PMPCm. The theoretical number-average molecular weight (Mn(theo)) was calculated from the following equation:

M n (theo ) =

[M ]0 xm M + M [CTA ]0 100 m CTA

(3)

where [M]0 is initial monomer concentration, [CTA]0 is initial CTA concentration, xm is 9


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percent conversion of the monomer, Mm is molecular weight of the monomer, and MCTA is the molecular weight of CTA. The molecular characteristics of the polymers are summarized in Table 1. The Mn(NMR) values for PMPCm were calculated by determining the integral area intensity ratio of pendant methylene protons at 3.7 ppm to the terminal dithiobenzoate protons at 7.4-8.0 ppm. The Mn(NMR) values for PMPCm were close to the theoretical Mn(theo) values, and the Mw/Mn values were within a narrow range (1.04–1.14), indicating that polymerization proceeded through a controlled mechanism. Therefore, PMPCm can be used as macro-CTA to prepare diblock copolymers.

Table 1. Number-average molecular weight (Mn), degree of polymerization (DP), and molecular weight distribution (Mw/Mn) of PMPCm and PmAn Sample

a

Mn(theo)

DP

Mn(NMR)

Mn(GPC)

× 10-4

(NMR)

× 10-4

× 10-4

PMPC24

0.621

24

0.750

0.496

1.04

PMPC100

2.98

100

2.98

1.58

1.14

P24A217

5.17

217a

5.73

5.29

1.19

P100A99

5.25

99a

5.25

3.15

1.23

DP of the PAMPS block estimated from 1H NMR data.

10


Mw/Mn

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Figure 2. 1H NMR spectra for (a) CTAB, (b) P24A217, (c) P100A99, (d) P24A217/CTAB, and (e) P100A99/CTAB complexes with f+ = 0.5 in 0.1 M NaCl containing D2O. Assignments for resonance peaks are indicated.

Figure 2a shows the 1H NMR spectrum of CTAB in D2O. Figure 2b and c shows 1H NMR spectra of P24A217 and P100A99, respectively, in D2O. The DP (= n) of the PAMPS block was calculated from the integral intensity ratio of the pendant methylene proton in the PAMPS block at 3.3-3.5 ppm to the pendant methylene protons in the PMPC block at 3.7 ppm. The Mn(NMR) values estimated from 1H NMR measurements for P24A217 and P100A99 were 5.73 × 104 and 5.25 × 104, respectively. The Mn(NMR) values for PmAn were similar to the Mn(theo) values, and the Mw/Mn values were within a narrow range (1.19–1.23), indicating controlled polymerization. The Mn(GPC) values for P100A99 were smaller than the theoretical values and

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the Mn(NMR) values. The Mn(GPC) value is an apparent value because of the inherent error involved in the use of sodium poly(styrenesulfonate)s as calibration standards. The concentrations of PmAn and CTAB in aqueous solution before mixing were fixed at 0.5 and 1.0 g/L, respectively. The critical micelle concentration (cmc) for CTAB in water has been reported to be 0.328 g/L.24 The CTAB concentration (1.0 g/L) was larger than the cmc, which indicates that CTAB formed micelles. The aqueous CTAB micelle solution was added to the aqueous PmAn solution to prepare the PmAn/CTAB complex. The mixing ratio (f+) of PmAn and CTAB was adjusted by changing the relative volumes of these aqueous solutions. The anionic PAMPS block and cationic CTAB micelles were neutralized to obtain f+ = 0.5. The ζ-potential values for P24A217/CTAB and P100A99/CTAB complexes at f+ = 0.5 were -5.32 and -2.12 mV, respectively, which indicates that the charges were nearly neutralized. The ζ-potential values for CTAB micelles, P24A217, and P100A99 were +49.7, -32.2, and -17.6 mV, respectively. The PmAn/CTAB complexes were prepared at f+ = 0.5 unless otherwise noted. Figure 2d and e shows 1H NMR spectra for PmAn/CTAB complexes in D2O. All NMR signals for P24A217/CTAB were broadened compared to those before mixing. After mixing with CTAB, the AMPS pendant methylene protons at 3.3-3.5 ppm in P24A217 disappeared. The mobility of the PAMPS block in P24A217/CTAB was restricted, because the anionic PAMPS block and cationic CTAB micelles interacted electrostatically. In contrast, a trace of pendant methylene protons at 3.7 and 4.0-4.3 ppm for the PMPC block were observed for the P24A217/CTAB complex. The DP of the PMPC block in P24A217 was 24, which is approximately 10 times shorter than that of the anionic PAMPS block. The motion of the PMPC chains surrounding the complex may be significantly restricted in P24A217/CTAB complex, because the mobility of the PMPC chains was affected by the low-mobility PAMPS chains. The motion of CTAB was not suppressed as much as that of P24A217, because broad NMR signals for CTAB can be observed for the P24A217/CTAB complex. For the

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P100A99/CTAB complex, relative intensities of the pendant methylene protons in the PAMPS block at 3.3-3.5 ppm and in the PMPC block at 3.7 and 4.0-4.3 ppm were similar before and after mixing with CTAB. The motion of the PMPC blocks in P100A99/CTAB complex was not restricted, because the PMPC chains formed shells. All NMR signals for CTAB in the P100A99/CTAB complex broadened slightly after mixing, suggesting that cationic CTAB molecules interacted with the anionic PAMPS block.

Table 2. 1H NMR spin-spin relaxation time (T2) for protons in the PMPC blocks and CTAB in D2O containing 0.1 M NaCl 3.3a ppm

3.1b ppm

1.3c ppm

CTABd

-

309

148

P24A217e

317

-

-

P100A99e

370

-

-

P24A217/CTABf

2.55

2.49

1.06

P100A99/CTABf

266

27.0

9.19

Sample

a

PMPC pendent methyl protons. bCTAB methyl protons next to a nitrogen atom. cCTAB

methylene protons. dCTAB micelles. eFree unimer state. fPmAn/CTAB complex.

To obtain further information about the motional restriction of PmAn and CTAB upon the formation of PmAn/CTAB complexes, 1H NMR spin-spin relaxation times (T2) were determined in D2O containing 0.1 M NaCl. NMR signal broadening suggests a decrease in T2 and molecular motions.25-27 In general, T2 decreased with the motion of the molecules. The T2 values of the methyl and methylene protons at 3.1 and 1.3 ppm for CTAB micelles were 309

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and 148 ms, respectively. The T2 values of the pendant methyl protons in the PMPC block for P24A217 and P100A99 under unimer states were 317 and 370 ms, respectively. The T2 values for the methyl protons in the PMPC block at 3.3 ppm and for CTAB at 3.1 ppm in the P24A217/CTAB complex decreased to 2.55 and 2.49 ms, respectively. This observation suggests that the mobility of the PMPC shells in P24A217/CTAB complex was reduced as much as that of the CTAB core of the complex. The T2 values for the methyl protons of the PMPC block at 3.3 ppm and of CTAB at 3.1 ppm in the P100A99/CTAB complex decreased to 266 and 27.0 ms, respectively, which indicates that the mobility of the PMPC shells in the P100A99/CTAB complex was not suppressed compared to that of P24A217/CTAB. The mobility of CTAB in P24A217/CTAB was less than that in P100A99/CTAB. The T2 value for the PAMPS block in the PmAn/CTAB complexes could not be obtained, because the T2 value was too low, indicating that motion of the PAMPS blocks was more restricted than that of the CTAB molecules. These observations suggest that CTAB micelles interacted with the anionic PAMPS block to form aggregates covered with PMPC shells through electrostatic interactions.

Figure 3. Hydrodynamic radius (Rh) distributions for (a) CTAB micelles at 10 g/L, (b) P24A217, (c) P100A99 at 5 g/L, (d) P24A217/CTAB, and (e) P100A99/CTAB complexes in 0.1 M NaCl aqueous solutions. 14


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Figure 3 shows Rh distributions for CTAB micelles, PmAn at unimer states, and PmAn/CTAB complexes in 0.1 M NaCl aqueous solutions. All Rh distributions were unimodal. The Rh value for CTAB at 10 g/L was 2.8 nm, which indicates size of micelles, because this concentration (10 g/L) was greater than the cmc (0.328 g/L).24 The Rh values for P24A217 and P100A99 were 4.3 and 4.4 nm, respectively. These small Rh values were reasonable for unimer states. The Rh for the P24A217/CTAB complex was 49.6 nm, which was larger than that (Rh = 22.7 nm) of the P100A99/CTAB complex. Two explanations are possible for this observation. First, the P24A217/CTAB complex formed by electrostatic interactions between the PAMPS block and CTAB micelles was larger than the P100A99/CTAB complex, because the DP of the PAMPS block in P24A217 was about two times larger than that of P100A99. Second, the DP of the hydrophilic PMPC block of the P24A217/CTAB complex was about four times less than that of the P100A99/CTAB complex. Shorter PMPC shells may make dissolution of the water-insoluble PAMPS/CTAB core in water difficult. Therefore, the aggregation number (number of polymer chains and CTAB molecules to form one complex) for P24A217/CTAB may be larger than that for P100A99/CTAB.

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Figure 4. TEM images for (a) P24A217/CTAB and (b) P100A99/CTAB complexes.

To confirm the shape and size of the PmAn/CTAB complexes, TEM measurements were obtained (Figure 4). TEM images showed that the P100A99/CTAB and P24A217/CTAB complexes were spherical. The P100A99/CTAB complex produced spherical objects with uniform contrast, suggesting that it was composed of micelles with a PAMPS/CTAB core and PMPC shells. The P24A217/CTAB complex may form vesicles, because the contrast of the spherical objects in the center was lower than that of the outer side. However unfortunately, we cannot obtain clear TEM data of the vesicle structure. The vesicle structure may break for drying process to prepare the TEM sample, because the P24A217/CTAB vesicles are formed by weak interaction between polymers and small surfactant molecules. To confirm the vesicle structure, we performed fluorescence studies later. Average radii estimated from the TEM images for P24A217/CTAB and P100A99/CTAB complexes were 46 and 22 nm, respectively. These radii were similar to the Rh values estimated from DLS. The structure of the PmAn/CTAB complexes could be directed to be micelles or vesicles by adjusting the DP of the

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water-soluble PMPC block and anionic PAMPS block. Han et al.28 reported that spherical and rod-like aggregates are formed in the poly(ethylene glycol)-b-poly(sodium glutamate) (PEG113-PGlu50)/dodecyltrimetylammonium bromide (DTAB) mixture, while the vesicular aggregates are formed in the PEG113-PGlu100/DTAB mixture in solution. These results are consistent with our data. The time dependence of the Rh values of PmAn/CTAB in 0.1 M NaCl aqueous solution was monitored (Figure S2). Although the value of Rh of P24A217/CTAB just after preparation was 48.7 nm, it increased slightly to 54.1 nm after 48 h. Although the Rh of P100A99/CTAB just after preparation was 21.8 nm, it also increased slightly to 22.5 nm after 48 h. These Rh changes with time were negligibly small. After 48 h, the Rh values were constant for two months. Further aggregation of PmAn/CTAB complexes with time could not be observed because of good dispersibility of the complexes covered with PMPC shells in aqueous solution.

Figure 5. Typical Zimm plots for (a) P24A217/CTAB and (b) P100A99/CTAB in 0.1 M NaCl

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aqueous solutions at scattering angles from 30 to 130° with a 20° increment.

Table 3. Dynamic and static light scattering data for PmAn and PmAn/CTAB Mw(SLS)a Sample × 10

-4

NPaggb

Rge

Rhf

dn/dCpg Rg/Rh

3 -2

(cm g mol) (nm) (nm)

P24A217

9.55

1

P100A99

6.54

1

P24A217/CTAB

5080

344

P100A99/CTAB

435

43

a

A2d × 104

NSaggc

-

dh

(mg/L) (g/cm3)

13.1

9.7

4.3

2.26

0.134

0.147

6.45

9.7

4.4

2.21

0.137

0.304

74600

-0.443

33.4

49.6 0.673

0.126

0.165

4280

0.495

20.1

22.7 0.888

0.149

0.476

-

Apparent weight-average molecular weight estimated from SLS. bAggregation number of

PmAn calculated using Eq. 4. cAggregation number of CTAB calculated using Eq. 5. dSecond virial coefficient estimated from SLS.

e

Radius of gyration estimated from SLS.

f

Hydrodynamic radius estimated from DLS. gRefractive index increment against concentration.

h

Density calculated using Eq. 6.

To further characterize the PmAn/CTAB complexes, SLS measurements were performed in 0.1 M NaCl aqueous solutions. Figures 5 and S3 show examples of Zimm plots for PmAn/CTAB and PmAn. The differential refractive index increment (dn/dCp) was measured using an Otsuka Electronics Photal DRM-3000 differential refractometer. Table 3 summarizes apparent weight-average molecular weight (Mw(SLS)), aggregation numbers, A2, Rg, Rh, Rg/Rh, dn/dCp, and density (d) for PmAn and PmAn/CTAB complexes estimated from SLS and DLS measurements. The Mw(SLS) values for P24A217 and P100A99 were 9.55 × 104 and 6.54 × 104, respectively, which were similar to the Mn(theo) and Mn(NMR) values shown in Table 1. The Mw(SLS) values for P24A217/CTAB and P100A99/CTAB were 5.08 × 107 and 4.35 × 106, respectively. The numbers of PmAn chains (NPagg) and CTAB molecules (NSagg) used to form 18


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one PmAn/CTAB complex were calculated using the following equations:

N Pagg =

M wC M wP + m × MWS

N Sagg = N Pagg × m

(4)

(5)

where MwP is Mw(SLS) of PmAn in the complex, m is DP of the PAMPS block, and MWS is molecular weight of CTAB (= 364.45). The values of NPagg and NSagg for P24A217/CTAB were 334 and 74,600, respectively. The values for NPagg and NSagg for P100A99/CTAB were 43 and 4,280, respectively. The NPagg and NSagg values for P24A217/CTAB were larger than those for P100A99/CTAB. The number of CTAB molecules (NSagg) needed to neutralize the anionic charges in PAMPS in P24A217/CTAB was larger than that for P100A99/CTAB, because the DP of the PAMPS block in P24A217 was approximately two times larger than that for P100A99. The value of NPagg for P24A217/CTAB was about eight times larger than that for P100A99/CTAB. The water-insoluble PAMPS/CTAB portion of P24A217/CTAB was larger than that in P100A99/CTAB, because the DP of the water-soluble PMPC block in P24A217, which acts as a dispersion stabilizer of the complex, was about four times lower than that in P100A99. The structures of PmAn/CTAB were characterized further by combining DLS and SLS to calculate the Rg/Rh ratio. The Rg/Rh ratio is a structure-sensitive parameter that provides information about the density distribution of the particles and, therefore, also about particle morphology.29,30 The Rg/Rh ratio equals 1.0 for a thin hard spherical shell (e.g., micelle or vesicle). A Rg/Rh ratio greater than 2 indicates a rod-shape structure. The Rg/Rh ratios for P24A217 and P100A99 were 2.26 and 2.21, respectively, suggesting that these unimers were rod-shaped in aqueous solution due to electrostatic repulsion among the pendant sulfonate groups. The Rg/Rh ratios for P24A217/CTAB and P100A99/CTAB complexes were 19


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0.673 and 0.888, respectively, which suggests that the complexes were spherical, such as spherical micelles or vesicles. In general, A2 is related to solubility and dispersibility in a solvent. The solubility of a polymer increases with the value of A2.31-33 The solubility of P24A217 in 0.1 M NaCl aqueous solution was greater than that of P100A99, because the A2 value for P24A217 was larger than that for P100A99. The A2 value for P100A99/CTAB was smaller than that for P100A99, suggesting that solubility of the complex in 0.1 M NaCl aqueous solution decreased relative to the unimers. In addition, the A2 value for P24A217/CTAB became -4.43 × 10-5, indicating that the solubility of P24A217/CTAB was decreased significantly compared to P100A99/CTAB, likely because of the short water-soluble PMPC shells in P24A217/CTAB. The d values for PmAn and PmAn/CTAB can be calculated using the following equation:

d=

M w (SLS) N A ×V

(6)

where V is the volume of anionic diblock copolymers or PmAn/CTAB calculated from 4/3πRh3. The d values for P24A217, P100A99, P24A217/CTAB, and P100A99/CTAB were 0.147, 0.304, 0.165, and 0.476 g/cm3, respectively. The d values for complexes were slightly larger than the anionic diblock copolymer under unimer states, suggesting that the polymer chains in the complex were more densely packed than were the unimers since aggregates composed of PAMPS/CTAB formed by electrostatic and hydrophobic interactions. The d value for P24A217/CTAB was smaller than that for P100A99/CTAB, which is consistent with P24A217/CTAB forming vesicles with a hollow core and with P100A99/CTAB forming solid micelles.

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Figure 6. (a) Rh and (b) %T at 800 nm for P24A217/CTAB (〇) and P100A99/CTAB (◇) as a function of NaCl concentration ([NaCl]) in water. Insert in (a) is a digital photograph of the aqueous P24A217/CTAB complex at [NaCl] = 0.9 M.

Sodium chloride concentration ([NaCl]) in the aqueous solution was expected to affect the association behavior of the complex, because the PmAn/CTAB complexes were formed by electrostatic interactions between anionic PmAn polymers and cationic CTAB micelles. Therefore, the dependence of Rh and percent transmittance (%T) at 800 nm on [NaCl] was determined for PmAn/CTAB in aqueous solution (Figure 6). The structure of P24A217/CTAB was not affected by [NaCl] ≤ 0.75 M, because the Rh (ca. 50 nm) and %T (ca. 95%) were nearly independent of [NaCl]. At 0.75 M < [NaCl] ≤ 1.3 M, the Rh of P24A217/CTAB could not be measured because the solution was turbid. In aqueous solution, the P24A217/CTAB complex aqueous solution may undergo liquid-liquid phase separation due to the salting out effect and formation of coacervate in this [NaCl] region.34,35 However, at [NaCl] > 1.3 M, the aqueous solution became transparent again (i.e., %T = 100%). This 21


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observation suggests that P24A217/CTAB was dissociated to P24A217 unimers and CTAB micelles, because the electrostatic interactions between the anionic PAMPS block and cationic CTAB micelle were completely screened at [NaCl] > 1.3 M. The %T values for P100A99/CTAB were constant (100%) independent of [NaCl] at 0.1 M ≤ [NaCl] ≤ 1.5 M. At [NaCl] ≤ 0.6 M, the Rh values for P100A99/CTAB remained nearly constant at ca. 23 nm. At 0.6 M < [NaCl] ≤ 1.0 M, the Rh values for P100A99/CTAB slightly increased as the PAMPS/CTAB micelle core was hydrated due prevention of electrostatic interactions between anionic PAMPS and cationic CTAB micelles. At [NaCl] > 1.0 M, Rh for P100A99/CTAB decreased to the same value as that of the P100A99 unimer state (Rh = 4.4 nm), suggesting that P100A99/CTAB was completely dissociated to P100A99 unimers and CTAB micelles.

Figure 7. (a) Rh for PmAn/CTAB and (b) pyrene emission intensity ratio (I338/I335) in the presence of CTAB and PmAn/CTAB as a function of CTAB concentrations in 0.1 M NaCl aqueous solutions: CTAB (○), P24A217/CTAB (◇), and P100A99/CTAB (□).

22


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The PmAn/CTAB complex was formed by electrostatic interactions between PmAn polymers and CTAB micelles. When the concentration of PmAn/CTAB was less than the cmc of CTAB micelles, the shape of PmAn/CTAB changed due to dissociation of CTAB micelles in the complex. Therefore, the dependence of Rh for PmAn/CTAB on concentration was determined through DLS measurements. Figure 7a plots Rh values for PmAn/CTAB against CTAB concentration. When CTAB concentration for P24A217/CTAB and P100A99/CTAB were less than 8.76 × 10-4 and 1.80 × 10-3 g/L, respectively, the light scattering intensities were too low to measure DLS. At CTAB concentrations larger than these, the Rh values for P24A217/CTAB and P100A99/CTAB were ca. 54 and 24 nm, respectively, independent of CTAB concentration. The DLS measurements could not determine shape changes of the complexes at low CTAB concentrations. At low concentrations, the cmc can be determined with greater sensitivity using a fluorescent probe than using DLS. The maximum wavelength (λmax) of the 0-0 band in the pyrene excitation spectrum depends strongly on the microenvironmental polarity of the pyrene molecules.36 When hydrophobic pyrene molecules are in the aqueous phase, the λmax is 335 nm. In contrast, when pyrene molecules are in a hydrophobic environment, such as the interior of the CTAB micelle core, λmax shifts to 338 nm.37,38 Therefore, the intensity ratio (I338/I335) from the pyrene excitation spectra at 338 and 335 nm becomes small for pyrene in an aqueous phase. When the pyrene molecules are in a hydrophobic environment, I338/I335 becomes larger. Figure 7b plots I338/I335 for pyrene in the presence of CTAB or PmAn/CTAB against CTAB concentration. The I338/I335 ratio was 0.58 for the aqueous CTAB solution in a low CTAB concentration region. As CTAB concentration increased, I338/I335 also increased beginning at 2.01 × 10-2 g/L. Because the pyrene molecules begin to incorporate into the hydrophobic core of CTAB micelles above this concentration, it was defined as the cmc. The cmc values for CTAB micelles in PmAn/CTAB were evaluated using the same pyrene

23


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fluorescence probe method. The cmc values for P24A217/CTAB and P100A99/CTAB were 8.76 × 10-4 and 1.80 × 10-3 g/L, respectively. The 1H NMR spin-spin relaxation times (Table 2) indicated that CTAB in P24A217/CTAB was more restricted than that in P100A99/CTAB. This observation suggests that the interactions of P24A217 with CTAB micelles were stronger than those of P100A99. The composition and DP of the water-soluble PMPC block and anionic PAMPS blocks may determine the degree of interactions between PmAn and CTAB micelles. The cmc for P24A217/CTAB was less than that for P100A99/CTAB, because P24A217 interacts electrostatically with CTAB more strongly than P100A99 does.

Figure 8. Fluorescence emission spectra from TD70 after dialysis against PBS buffer for 120 h excited at 550 nm in the presence (──) and absence (----) of P24A217/CTAB complex in PBS buffer.

When P24A217/CTAB formed vesicles, hydrophilic guest molecules could be incorporated into the interior aqueous phase of the vesicles. The vesicle structure of P24A217/CTAB was confirmed using TEM observations (Figure 4). Additional evidence for vesicle formation by P24A217/CTAB was provided by fluorescence experiments using Texas red-labeled nonionic hydrophilic dextran (TD70) as a guest molecule. The P24A217/CTAB complex was prepared using PBS buffer containing TD70. When the PBS solution was dialyzed using a membrane with a 50 nm pore size, the vesicles (Rh = ca. 50 nm) remained in 24


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the dialysis bag after dialysis. However, TD70, which cannot be incorporated into the interior aqueous phase of the vesicle, was completely removed. To avoid self-quenching of TD70 fluorescence, 1.5 M NaCl was added to the PBS buffer solution after dialysis to dissociate the P24A217/CTAB complex (Figure 6). As a reference experiment, TD70 without P24A217/CTAB was dialyzed under the same conditions and fluorescence spectra for these PBS buffer solutions were obtained (Figure 8). No fluorescence was observed from TD70 without P24A217/CTAB because TD70 was completely removed from the dialysis bag. In the presence of P24A217/CTAB, TD70 could be incorporated into the interior aqueous phase of the vesicle, indicated by the observation of TD70 fluorescence. The amount of TD70 incorporated into P24A217/CTAB vesicle was obtained from the calibration curve. Furthermore, the loading efficiency (LE) and loading capacity (LC) of TD70 were calculated from the measured amount and Eqs. (1) and (2) as 9.45 and 0.189 %, respectively, which were lower than those for other polymer vesicles.39-42 These low LE and LC values suggest that no interactions occurred between P24A217/CTAB vesicles and TD70.

CONCLUSIONS Colloidally stabilized complexes (PmAn/CTAB) were prepared by mixing anionic diblock copolymers (P24A217 and P100A99) and cationic CTAB micelles in aqueous solution. The mixture of P24A217 and CTAB formed P24A217/CTAB vesicles, which was confirmed by TEM observations and the encapsulation of hydrophilic guest molecules. The mixture of P100A99 and CTAB formed P100A99/CTAB micelles. The T2 results revealed that CTAB micelle motions in P24A217/CTAB were more restricted than those in P100A99/CTAB. The Rh values for P24A217/CTAB vesicles and P100A99/CTAB micelles were ca. 50 and 23 nm, respectively. The aggregation numbers (NPagg and NSagg) of P24A217/CTAB vesicles were considerably larger than those of P100A99/CTAB micelles. The P24A217/CTAB vesicles and P100A99/CTAB micelles

25


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could be dissociated by adding NaCl at concentrations greater than 1.3 M. The cmc values of CTAB micelles in P24A217/CTAB vesicles and P100A99/CTAB micelles were more than one order of magnitude lower than that of the CTAB micelles alone. The P24A217/CTAB vesicles could encapsulate nonionic water-soluble guest macromolecules into the interior aqueous phase.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.xxxxxxx. Additional figures as described in the text (PDF).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

ORCID Shin-ichi Yusa: 0000-0002-2838-5200

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by a Grant-in-Aid for Scientific Research (25288101, 17H03071, and 16K14008) from the Japan Society for the Promotion of Science (JSPS), JSPS Bilateral Joint Research Projects, and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices (20164026).”

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