Formation of Polyion Complex (PIC) Micelles and Vesicles with

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Formation of Polyion Complex (PIC) Micelles and Vesicles with Anionic pHResponsive Unimer Micelles and Cationic Diblock Copolymers in Water Sayaka Ohno, Kazuhiko Ishihara, and Shin-ichi Yusa Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00637 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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Original Article for Langmuir Formation of Polyion Complex (PIC) Micelles and Vesicles with Anionic pH-Responsive Unimer Micelles and Cationic Diblock Copolymers in Water

Sayaka Ohno†, Kazuhiko Ishihara§, and 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:

A

random

copolymer

2-(acrylamido)-2-methylpropanesulfonate 11-methacrylamidoundecanate

(MaU)

(p(A/MaU)) (AMPS)

was

prepared

of and

via

sodium sodium

conventional

radical

polymerization, which formed a unimer micelle under acidic conditions due to intramolecular hydrophobic interactions between the pendant undecanoic acid groups. Under basic conditions, unimer micelles were opened up to an expanded chain conformation by electrostatic repulsion between the pendant sulfonate and undecanoate anions.

A

cationic

diblock

copolymer

poly(3-(methacrylamido)propyl)trimethylammonium

(P163M99) chloride

consisting

of

(PMAPTAC)

and

hydrophilic polybetaine, 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) blocks was prepared via controlled radical polymerization. Mixing of p(A/MaU) and P163M99 in 0.1 M aq. NaCl under acidic conditions resulted in the formation of spherical polyion complex (PIC) micelles and vesicles, depending on polymer concentration before mixing. Shapes of the PIC micelles and vesicles changed under basic conditions due to

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collapse of the charge balance between p(A/MaU) and P163M99. The PIC vesicles can incorporate nonionic hydrophilic guest molecules, and the PIC micelles and vesicles can accept hydrophobic guest molecules in the hydrophobic core formed from p(A/MaU).

Graphical Abstract

INTRODUCTION

Amphiphilic diblock copolymers form polymer micelles in water due to hydrophobic interactions between the hydrophobic blocks. Conventional polymer micelles are composed of a core and shell formed from hydrophobic and hydrophilic blocks, respectively. Aggregation behavior of these amphiphilic diblock copolymers has been widely studied.1 Investigations of the self-assembly of amphiphilic polymers containing nanoparticles have been reported.2-4 Li et al.5 reported synthesis of the amphiphilic diblock copolymer (PEO-b-P(MMA/TMSPMA)) containing a linear poly(ethylene oxide) (PEO) block as the hydrophilic portion and a nanoparticle as the hydrophobic portion.

The

nanoparticle

(P(MMA/TMSPMA))

was

composed

a

hydrophobic

random

methyl

methacrylate

of

copolymer (MMA)

block and

3-(trimethoxysilyl)propyl methacrylate (TMSPMA). Intramolecular hydrolysis and polycondensation of silane moieties in the pendant chain of PEO-b-P(MMA/TMSPMA)

2


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under dilute conditions led to formation of a hybrid nanoparticle-containing polymer with a silica-like head and a PEO tail. The polymers were self-assembled by hydrophobic interactions of the silica-like heads in water. At low polymer concentrations, the polymers with a silica-like head formed micelles. At high polymer concentrations, the polymers formed vesicles. The shape of the aggregates can be controlled by adjusting polymer concentration. Morishima et al. reported that random copolymers of anionic sodium 2-(acrylamido)-2-methylpropanesulfonate (AMPS) and hydrophobic methacrylamides N-substituted with bulky hydrophobes, such as cyclododecyl,6 n-dodecyl,7 and adamantyl groups8, formed spherical unimer micelles due to hydrophobic intra-polymer interactions in water independent of polymer concentration. Therefore, the unimer micelle did not reach critical micelle concentration. The unimer micelle allows the loading of hydrophobic low-molecular-weight compounds into the hydrophobic domain. Recently, Akashi et al.9 reported that biodegradable random copolymers composed of hydrophilic γ-glutamic acid with a hydrophobic L-phenylalanine (L-Phe) main chain formed unimer micelles in water. The unimer micelle could load hydrophobic fluorescence molecules, such as pyrene, into the hydrophobic L-Phe core. Releasing guest molecules loaded by the unimer micelle is difficult due to the stable micelle structure. Random copolymers (p(A/AaU)) composed of AMPS and 11-acrylamido undecanoic acid (AaU), which has a pH-responsive pendant fatty acid group, can control the formation and collapse of the unimer micelles.10 A large difference in pKa exists between the pendant sulfonate and carboxylate groups. The AMPS unit was not affected by acidic conditions, but the pendant carboxylate group in the AaU unit becomes hydrophobic by selective protonation. Therefore, p(A/AaU)

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forms a unimer micelle due to intramolecular hydrophobic interactions under acidic conditions in water. Under basic conditions, the unimer micelle opens up to an extended chain conformation due to electrostatic repulsion between the pendant sulfonate and carboxylate anions. Formation and collapse of the unimer micelle can be controlled by adjusting the pH. A

stoichiometrically

charge-neutral

mixture

of

oppositely

charged

polyelectrolytes in water led to spontaneous formation of a polyion complex (PIC) that was insoluble in water.11 Mixing of oppositely charged diblock copolymers containing a nonionic water-soluble block resulted in the formation of water-soluble PIC micelles in water.12-14 These PIC micelles are promising candidates for various applications, because they can contain substances such as metal ions,15 enzymes,16 and DNA.17 2-(Methacryloyloxy)ethyl phosphorylcholine (MPC) has a pendant hydrophilic phosphorylcholine group which possesses the same chemical structure as the hydrophilic surface of a cell membrane.18 MPC can be copolymerized with functional vinyl comonomers, and the properties of the copolymers obtained can be controlled by choosing an appropriate comonomer.19 MPC-containing copolymers can inhibit both blood coagulation and immune responses.20 Therefore, PIC micelles covered with poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) shells are good candidates for carriers in drug delivery systems (DDS).21,22 In the present study, a random copolymer (p(A/MaU)) composed of equimolar amounts of AMPS and sodium 11-methylacrylamidoundecanate (MaU) was prepared via conventional radical polymerization (Figure 1a). Reversible formation and collapse of p(A/MaU) unimer micelles could be controlled by adjusting the solution pH. Under acidic conditions, p(A/MaU) formed unimer micelles by intra-polymer association of

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the pendant undecanoic acid groups. Under basic conditions, electrostatic repulsion between the pendent sulfonate and undecanoate anions led to an open-chain conformation (Figure 1b). Cationic diblock copolymer (P163M99) consisting of poly(3-(methacrylamido)propyl)trimethylammonium chloride (PMAPTAC) and PMPC blocks was prepared via reversible addition-fragmentation chain transfer (RAFT) radical polymerization. Mixing aqueous solutions of p(A/MaU) and P163M99 under acidic conditions resulted in the formation of PIC aggregates (p(A/MaU)/P163M99) due to electrostatic interactions (Figure 1c). The polymers formed spherical PIC micelles and vesicles depending on the polymer concentration before mixing. The PIC micelles and vesicles were characterized by dynamic light scattering (DLS), static light scattering (SLS), transmittance electron microscopy (TEM), and fluorescence techniques.

Figure 1. (a) Chemical structures of p(A/MaU) and P163M99. (b) Illustration of pH-responsive unimer micelle. (c) Illustration of PIC micelle and vesicle composed of anionic p(A/MaU) and cationic P163M99.

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EXPERIMENTAL SECTION Materials. 2-(Methacryloyloxy)ethylphosphorylcholine (MPC) was purchased from NOF Corp., and purified by a previously reported method.18 4-Cyanopentanoic acid dithiobenzoate (CPD) was synthesized according to the method reported by McCormick and co-workers.23 11-Aminoundecanoic acid (97%) was purchased from Aldrich, methacryloyl chloride (>80%) from Tokyo Chemical Industry, Texas red-labeled dextran (Dex, Mw = 70000, neutral) from Life Technologies, and 2-(acrylamido)-2-methylpropanesulfonic

acid

(AMPS,

>97%),

(3-(methacryloylamino)propyl) trimethylammonium chloride (MAPTAC, >96%), and 4,4’-azobis-(4-cyanopentanoic acid) (V-501, > 98%) from Wako Pure Chemical; all were used as received without further purification. Pyrene (>98.0%) from Kanto Chemical was recrystallized from methanol. Methanol was dried over 4Å molecular sieves and purified by distillation. Water was purified with a Millipore Milli-Q system. Other regents were used as received. Synthesis

of

11-methacrylamidoundecanoic

acid

(MaU).

11-Aminoundecanoic acid (21.6 g, 0.08 mol) and NaOH (14.4 g, 0.36 mol) were dissolved in water (600 mL). The solution was cooled in an ice bath. Methacryloyl chloride (26.1 g, 0.25 mol) was added to the solution over 10 min at 0°C and stirred for 3 h at room temperature. After reaction, the pH was adjusted to 3 by adding 12 N HCl. The precipitate was filtered, and then dissolved in chloroform. The chloroform solution was washed with 1 N HCl and sat. NaCl solution, followed by drying over anhydrous Na2SO4. The organic solvent was evaporated, and the crude product was recrystallized from acetone/n-hexane (1/1, v/v) twice. The 11-methacrylamidoundecanoic acid (MaU)

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obtained was dried under vacuum for 24 h: yield 19.9 g (92.4%); mp 68-69°C. 1H NMR (500 MHz, CDCl3, δ): 5.83 (br, 1H, -NH-), 5.66 (br, 1H, HCH=C(CH3)-), 5.32-5.31 (br, 1H,

HCH=C(CH3)-),

3.32-3.28

(t,

2H,

-NH-CH2-CH2-),

2.36-2.33

(t,

2H,

-CH2-CH2-COOH), 1.96 (s, 3H, CH2=C(CH3)-), 1.65-1.60 (m, 2H, -CH2-CH2-COOH), 1.56-1.50 (m, 2H, -NH-CH2-CH2-), 1.30-1.28 (m,12H ,-CH2-(CH2)6-CH2-). Preparation of p(A/MaU). AMPS (4.00 g, 0.0193 mol), MaU (5.20 g, 0.0193 mol), NaOH (1.54 g, 0.0386 mol), and V-501 (74.3 mg, 0.265 mmol) were dissolved in water (19.3 mL). The solution was deoxygenated by purging with Ar gas for 30 min. Polymerization was conducted at 70°C for 3 h. The reaction mixture was dialyzed against pure water for 7 days. The polymer (p(A/MaU)) was recovered by freeze-drying: yield 6.20 g (67.4%). The composition of MaU in p(A/MaU) was 50 mol%, determined using 1H NMR in D2O at pH 9. (Figure S1). Number-average molecular weight (Mn(GPC)) and molecular weight distribution (Mw/Mn) estimated from gel-permeation chromatography (GPC) were 1.55 × 104 and 4.67, respectively (Figure S2). Preparation of cationic diblock copolymer (P163M99). MAPTAC (20.0 g, 90.7 mmol) was dissolved in water (27.5 mL). CPD (90.3 mg, 0.323 mmol) and V-501 (18.0 mg, 0.0643 mmol) were dissolved in methanol (3.03 mL), and added to the aqueous MAPTAC solution. This solution was deoxygenated by purging with Ar gas for 30 min. Polymerization was conducted at 70°C for 4 h. After reaction, 1H NMR analysis indicated that the conversion was 51.3%. The reaction mixture was purified by precipitating twice from methanol into an excess of acetone. The polymer obtained (PMAPTAC163) was dried under vacuum oven at 40°C for 24 h: yield 11.0 g (54.7%). Mn(GPC) and Mw/Mn estimated from GPC were 2.61 × 104 and 1.10, respectively

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(Figure S3). Mn(NMR) and degree of polymerization (DP) determined from 1H NMR were 3.63 × 104 and 163, respectively (Figure S4a). MPC (1.01 g, 3.42 mmol), V-501 (3.78 mg, 0.0135 mmol), and PMAPTAC163 (1.23 g, 0.0339 mmol, Mn(NMR) = 3.63 × 104, Mw/Mn = 1.10) were dissolved in water (3.38 mL). The solution was deoxygenated by purging with Ar gas for 30 min. Polymerization was conducted at 70°C for 1 h. After reaction, 1H NMR analysis indicated that conversion was 99.0%. The reaction mixture was dialyzed against pure water for 3 days. The diblock copolymer (P163M99) was recovered by freeze-drying: yield 1.77 g (79.1%). Mn(GPC) and Mw/Mn were estimated to be 3.49 × 104 and 1.11, respectively (Figure S3). Mn(NMR) for P163M99 and DP for the PMPC block were 6.55 × 104 and 99, respectively (Figure S4b). Preparation of unimer micelles. p(A/MaU) was dissolved in 0.1 M NaCl at a polymer concentration (Cp) of 2 g/L and pH 3. The solution was stirred overnight at 40°C to achieve complete dissolution. Sample solutions were filtered with a 0.2 µm pore size membrane filter prior to measurement. Solution pH was adjusted by adding the proper amount of aqueous NaOH or HCl. Preparation of PIC micelles and vesicles. p(A/MaU) and P163M99 were separately dissolved in 0.1 M NaCl at pH 3. To prepare PIC micelles and vesicles, the P163M99 solution was added dropwise to the p(A/MaU) solution at Cp = 0.1 or 1 g/L, respectively. Sample solutions were filtered using a 0.2 µm membrane filter prior to measurement. The mixing ratio of the two polymers was represented by the mole fraction of positively charged units (f+ = [cationic charge]/([anionic charge] + [cationic charge])); hence, complete charge neutralization was attained at f+ = 0.5. PIC micelles and vesicles were prepared at f+ = 0.5 in 0.1 M NaCl unless otherwise noted.

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Measurements. 1H NMR spectra were obtained with a Bruker BioSpin DRX-500 spectrometer. GPC spectra of p(A/MaU) were obtained using a chromatograph equipped with a Shodex Asahipak GF-1G guard column and 7.0 µm bead size GF-7 M HQ column at 40°C under a flow rate of 0.6 mL/min using a refractive index (RI) detector. Phosphate buffer (pH 9) containing 10 vol% acetonitrile was used as an eluent. The values of Mn(GPC) and Mw/Mn for p(A/MaU) were obtained using standard sodium poly(styrenesulfonate) samples. GPC measurements of P163M99 were obtained using a chromatograph equipped with a Shodex Ohpak SB-G guard column and 10 µm bead size SB-804 HQ column at 40°C under a flow rate of 0.6 mL/min and an RI detector. A 0.3 M Na2SO4 solution containing 0.5 M acetic acid was used as an eluent. The values of Mn(GPC) and Mw/Mn for P163M99 were obtained using standard poly(2-vinylpyridine) samples. Static light scattering (SLS) measurements were performed at 25°C with an Otsuka Electronics Photal DLS-7000 light scattering spectrometer. A He-Ne laser (10 mW at 632.8 nm) was used as a light source. The weight-average molecular weight [Mw(SLS)], z-average radius of gyration (Rg), and second virial coefficient (A2) values were estimated from the relation:

KCP 1  1  = 1 + Rg 2 q 2  + 2 A2C p Rθ Mw  3 

(1)

where Rθ is the Rayleigh ratio; K = 4π2n2(dn/dCp)2/NAλ4, with n the refractive index of the solvent, dn/dCp the refractive index increment against Cp, NA Avogadro’s number, and λ the wavelength (= 632.8 nm); q = (4πn/λ)sin(θ/2), with θ as the scattering angle. Toluene was used for calibrating the instrument. Values of dn/dCp were determined

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using an Otsuka Electronics Photal DRM-3000 differential refractometer at a wavelength of 633 nm. Dynamic light scattering (DLS) measurements were obtained using a Malvern Instruments Zetasizer Nano ZS instrument equipped with a He-Ne laser (4 mW at 633 nm). Measurements were taken at a scattering angle of 173°. The hydrodynamic radius (Rh) was determined using the Stokes-Einstein equation.24 The ζ potential was measured using a Malvern Zetasizer Nano-ZS ZEN3600 instrument equipped with a He-Ne laser light source (4 mW at 632.8 nm) at 25°C. 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 and

κ and a are the Debye-Huckel parameter and particle radius, respectively. Transmission electron microscopy (TEM) images were obtained using a JEOL TEM-2100 electron microscope operated at an accelerating voltage of 200 kV. Samples for TEM 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 for one day. Fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer. Pyrene was used as the fluorescence probe. The pyrene-saturated aqueous stock solution was prepared according to a previously reported method.25 The p(A/MaU) was dissolved in an aqueous 0.1 M NaCl solution saturated with pyrene at pH 3. The P163M99 was dissolved in 0.1 M NaCl at pH 3. To prepare pyrene-loaded PIC micelles and vesicles, the P163M99 solution was added dropwise to the aqueous p(A/MaU) solution. Loading amounts of Texas red-labeled dextran (Dex). Dex (0.24 mg, 3.43 × 10-10 mol) was dissolved in 0.1 M NaCl at pH 3 (24 mL), followed by addition of

10


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P163M99 (1 g/L) and then p(A/MaU) (1 g/L). This solution (4 mL) was dialyzed against fresh 0.1 M NaCl at pH 3 (400 mL) using a polycarbonate membrane with 50 nm pore size (Harvard Apparatus) for 48 h. The 0.1 M NaCl solution was replaced 8 times to remove the free Dex which was not incorporated into the hollow cores of the PIC vesicles. After dialysis, fluorescence emission of 0.1 M NaCl solution in the dialyzer was measured. As a reference, the fluorescence of a 0.1 M NaCl solution at pH 3 of Dex without PIC vesicles was measured using a similar procedure. The weight of Dex incorporated into PIC vesicles was calculated using a calibration curve. Loading efficiency (LE) and loading capacity (LC) of Dex were calculated according to the following equations:

LE (%) =

Weight of encapsulated Dex × 100 Weight of total Dex

(2)

LC (%) =

Weight of encapsulated Dex × 100 Weight of polymer

(3)

RESULTS AND DISCUSSION Characterization of p(A/MaU). Figure S1 shows a comparison of the 1H NMR spectra of p(A/MaU) at pH 3 and 9 in D2O containing 0.1 M NaCl. The composition of MaU was estimated to be 50 mol% by comparing the integral area intensity ratio of the pendant methylene protons in ionized MaU at 2.1 ppm and the overlapped peak with the pendant methylene protons in AMPS and MaU at 2.9-3.6 ppm at pH 9. The Mn(GPC) and Mw/Mn of p(A/MaU) estimated from GPC were 1.55 × 104 and 4.67, respectively (Figure S2).

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Figure 2. (a) Typical examples of Rh distributions for p(A/MaU) at Cp = 2 g/L and pH 3 (―) or pH 9 (---) in 0.1 M NaCl. (b) Hydrodynamic radius (Rh, ○) and light scattering intensity (LSI,

) for p(A/MaU) at Cp = 2 g/L as a function of pH in 0.1 M NaCl.

Table 1. Dynamic and static light scattering data for p(A/MaU) at pH 3 and 9 in 0.1 M NaCl Mw(SLS) ×10-6

Rg

Rh

pH

A2 Rg/Rh

(cm3·mol/g2)

(g/mol)

(nm)

(nm)

3

1.12

27.5

17.4

1.58

4.78×10-5

9

1.14

52.8

19.6

2.69

1.19×10-3

Figure 2a shows unimodal Rh distributions for p(A/MaU) at pH 3 and 9 in 0.1 M NaCl. The Rh values for p(A/MaU) at pH 3 and 9 were 17.4 and 19.6 nm,

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respectively. Light scattering intensity (LSI) values for p(A/MaU) at pH 3 and 9 were 3.49 and 1.43 Mcps, respectively. The Rh value at pH 3 was smaller than that at pH 9. However, the LSI value at pH 3 was larger than that at pH 9 because the density of p(A/MaU) at pH 3 is higher than that at pH 9. Figure S5 shows Zimm plots for p(A/MaU) at pH 3 and 9 in 0.1 M NaCl. Table 1 summarizes the light scattering data. The Mw(SLS) value at pH 3 was similar to that at pH 9, suggesting that p(A/MaU) could be dissolved in aqueous solution as an unimer independent of pH. The Rg/Rh ratio is a structure-sensitive parameter that provides information about the density of the particles and their morphology.26 The Rg/Rh ratio is 0.775 for a homogeneous hard sphere and 1.0 for a thin hard spherical shell, e.g., vesicle, and increases substantially for a less dense structure, e.g., rod, and in a polydisperse situation because large molecules of a broad distribution will contribute more to Rg than Rh provided that internal modes of motion are absent.27 In the present study, the Rg/Rh ratio for p(A/MaU) at pH 3 was calculated to be 1.58, indicating that the shape of p(A/MaU) is close to spherical. In contrast, the Rg/Rh ratio at pH 9 was 2.69, indicating that p(A/MaU) may be an expanded open chain conformation. These results indicate that p(A/MaU) forms spherical unimer micelles by intra-polymer hydrophobic interactions at pH 3. However, at pH 9, p(A/MaU) forms an open-chain conformation due to electrostatic repulsion between the pendant sulfonate and undecanoate anions. The A2 value for p(A/MaU) at pH 3 was smaller than that at pH 9, suggesting that the solubility of p(A/MaU) in 0.1 M NaCl at pH 3 was less than that at pH 9.28 The average radius for unimer micelles formed from p(A/MaU) at pH 3 was 14 nm, as estimated from TEM (Figure S6). The radius estimated from TEM was smaller than the corresponding radii estimated from light scattering measurements, because the unimer micelles may contract during the drying process of the TEM sample

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preparation method. Figure 2b shows the Rh and LSI values for p(A/MaU) at Cp = 2 g/L in 0.1 M NaCl as a function of pH. As pH increased from 3 to 12, the Rh values increased slightly from pH 6 to 8, while LSI decreased from pH 6 to 8. The pH value where the Rh and LSI values began to change was close to the pKa for the low-molecular-weight fatty acid decanoic acid, which is about 7.2.29 If particles have a similar size with the same quantity, LSI is proportional to particle density. These observations suggest that, under acidic conditions, p(A/MaU) forms intra-polymer aggregates, i.e., unimer micelles due to the hydrophobic interactions between the pendant protonated undecanoic acid groups. In contrast, under basic conditions, electrostatic repulsions between the pendant deprotonated undecanoate anions resulted in an open-chain conformation of p(A/MaU).

Characterization of P163M99. Figure S4 compares the

1

H NMR spectra of

PMAPTAC163 and P163M99 in D2O. The DP and Mn(NMR) values for PMAPTAC163 were 163 and 3.63 × 104, respectively, estimated by comparing the integral area intensity ratio of the terminal phenyl protons at 7.4-7.5 ppm and the pendant methylene proton at 2.0 ppm. The DP, Mn(NMR), Mn(GPC), and Mw/Mn values for PMAPTAC163 are summarized in Table 2. When polymerization is assumed to be an ideal controlled/living process, theoretical number-average molecular weight (Mn(theo)) can be calculated from the following equation:

M n (theo) =

[M ]0 χ m M + M [CTA ]0 100 m CTA

(4)

where [M]0 is initial monomer concentration, [CTA]0 is initial chain transfer agent

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(CTA) concentration, χm is percent conversion of the monomer, Mm is the molecular weight of monomer, and MCTA is the molecular weight of CTA. The Mn(theo) for PMAPTAC163 was close to that of Mn(NMR) of PMAPTAC163. The Mw/Mn for PMAPTAC163 (= 1.10) was narrow. These observations indicate that PMAPTAC163 has well-controlled structure. The DP values of the PMPC block and the Mn(NMR) value for P163M99 were estimated by comparing integral area intensities of the pendant methylene protons in the PMPC block at 3.6 ppm and the pendant methylene protons in the PMAPTAC block at 3.3 ppm (Table 2).

Table 2. DP, Mn, and Mw/Mn values for PMAPTAC163 and P163M99 DP of

DP of Mn(theo) Mn(NMR) Mn(GPC)

Polymer PMAPTAC PMPC

×10-4

×10-4

×10-4

Mw/Mn

PMAPTAC163

163

―

3.74

3.63

2.61

1.10

P163M99

163

99

6.55

6.55

3.49

1.11

Characterization of PIC micelles and vesicles

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Figure 3. (a) Typical examples of hydrodynamic radius (Rh) distributions for p(A/MaU)/P163M99 PIC aggregates at Cp = 0.1 (―) and 1 g/L (---) in 0.1 M NaCl at pH 3. (b) Hydrodynamic radius (Rh) for p(A/MaU)/P163M99 PIC aggregates at pH 3 as a function of Cp after mixing p(A/MaU) and P163M99. The p(A/MaU)/P163M99 PIC aqueous aggregate solutions at Cp = 0.1 (○) and 1 g/L ( ) were diluted with 0.1 M NaCl.

The effect of the Cp value before mixing was examined on the size and structure of PIC aggregates formed from p(A/MaU) and P163M99 at pH 3 (Figure 3). When polymer concentrations were 0.1 and 1 g/L before mixing, Rh values for PIC aggregates were 33.1 and 62.7 nm, respectively. The size of PIC aggregates increased with p(A/MaU) and P163M99 polymer concentration before mixing. When solutions of PIC aggregates were diluted, the sizes remained constant, independent of polymer concentrations up to 0.01 g/L (Figure 3b). These observations suggest that the size and structure of PIC aggregates formed from p(A/MaU) and P163M99 may not change upon

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dilution.

Table 3. Dynamic and static light scattering data for p(A/MaU)/P163M99 PIC micelles at Cp = 0.1 g/L and vesicles at Cp = 1 g/L at pH 3 and 9 in 0.1 M NaCl Cp

Mw(SLS)×10-6

(g/L)

(g/mol)

3

0.1

30.4

29.5

3

1

71.2

9

0.1

9

1

Rg

A2×106

Rh Rg/Rh

Nagg

33.1

0.89

88

16.5

68.0

62.7

1.08

208

2.02

1.37

18.4

19.4

0.95

4

2640

1.43

22.0

21.8

1.01

4

1470

pH

(nm) (nm)

(cm3·mol/g2)

To further investigate the structures of PIC aggregates prepared from different Cp before adding p(A/MaU) and P163M99, SLS measurements were obtained for PIC aggregates prepared at Cp = 0.1 and 1 g/L (Figure S7). Table 3 summarizes light scattering data for PIC aggregates prepared at Cp = 0.1 and 1 g/L at pH 3. At pH 3, the Mw(SLS) and Rg values prepared at Cp = 1 g/L were larger than those prepared at 0.1 g/L. Theoretically, an Rg/Rh ≈ 1 indicates spherical molecular assemblies.30 The Rg/Rh ratios for PIC aggregates prepared at Cp = 0.1 and 1 g/L were close to 1, suggesting that both PIC aggregates may be spherical with narrow particle size distribution. The aggregation number (Nagg) was defined as the total polymer chain number required to form one PIC aggregate. The Nagg value can be calculated from the molar ratio of p(A/MaU) with P163M99, molecular weight of each polymer, and Mw(SLS) values of PIC aggregates. The Nagg value of PIC aggregates prepared at Cp = 0.1 g/L, which were composed of 22 anionic unimer micelles formed from p(A/MaU) at pH 3

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and 66 cationic P163M99 chains, was 88. The Nagg value of PIC aggregates prepared at Cp = 1 g/L, which were composed of 52 anionic unimer micelles and 156 cationic P163M99 chains, was 208. The value for A2 for PIC aggregates prepared at Cp = 1 g/L was less than that of aggregates prepared at 0.1 g/L, which indicates that the PIC aggregates prepared at 0.1 g/L were more soluble than those prepared at 1 g/L.28

Figure 4. TEM images of p(A/MaU)/P163M99 PIC aggregates: (a) Cp = 0.1 g/L, pH 3 (PIC micelle); (b) Cp = 1 g/L, pH 3 (PIC vesicle); (c) Cp = 0.1 g/L, pH 9; and (d) Cp = 1 g/L, pH 9.

The influence of Cp before mixing on the structure of PIC aggregates was examined using TEM observations (Figure 4). For PIC aggregates prepared at Cp = 0.1 g/L at pH 3, TEM data suggest PIC micelle formation, because the uniform spherical objects were observed. The TEM data at pH 3 showed that PIC aggregates prepared at

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Cp = 1 g/L formed PIC vesicles with a spherical shape and a contrasting center. The average radii for PIC micelles and vesicles estimated from TEM were 22 and 30 nm, respectively. The radii estimated from TEM were smaller than those estimated from light scattering measurements, because the TEM samples may have contracted during the drying process of the sample preparation method. In particular, the constrictive ratio for PIC vesicles during the drying process may be large, because of a hollow structure. If the pH of the PIC aggregate solution was adjusted to 9, the pendant undecanoic acid groups in the PIC aggregates were ionized. Therefore, at pH 9, PIC micelles and vesicles may collapse due to disruption of the charge balance between anionic p(A/MaU) and cationic P163M99. Figure S8 shows Rh distributions for PIC micelles and vesicles at pH 3 and 9. At pH 9, the Rh values for PIC micelles and vesicles were 19.4 and 21.8 nm, respectively. Measurements obtained from TEM observations at pH 9 gave average radii for PIC micelles and vesicles of 18 and 15 nm, respectively (Figure 4). At pH 9, p(A/MaU) adopted an open-chain conformation due to electrostatic repulsion between the pendant sulfonate and undecanoate anions. Therefore, small PIC aggregates with low Nagg can be formed from expanded p(A/MaU) and P163M99 chains due to unbalanced electrostatic interactions. SLS measurements were obtained for PIC aggregates in 0.1 M NaCl at pH 9 (Figure S9). Table 3 summarizes all light scattering data at pH 9. The PIC aggregates at pH 9 are spherical, because the Rg/Rh ratios for PIC micelles and vesicles at pH 9 were close to 1. The Nagg values for PIC micelles and vesicles at pH 9 were both approximately 4. The Mw(SLS), Nagg, and Rg values for PIC micelles and vesicles at pH 9 were less than those at pH 3. The Nagg value for PIC aggregates at pH 9 indicated that the aggregates were composed of one expanded p(A/MaU) chain and three P163M99

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chains. The charge balance between the anions and cations was disrupted due to the increase in the number of anionic charges at pH 9. The small PIC aggregates with small Nagg may be formed from expanded p(A/MaU) with P163M99 at pH 9. The A2 values for both PIC micelles and vesicles at pH 9 were greater than those at pH 3, suggesting that PIC aggregates are more soluble at pH 9 than at pH 3. The dependence of Rh and LSI on time for PIC micelles and vesicles at pH 3 and 9 was monitored by light scattering measurements (Figure S10). The Rh and LSI values were nearly constant and independent of time until 119 h from sample preparation. The Rh and LSI values for PIC micelles and vesicles at Cp = 0.01 g/L immediately after sample preparation were almost same as those after one day. The diluted PIC micelles and vesicles were stable at least one day.

Figure 5. Light scattering intensity (LSI) for p(A/MaU)/P163M99 PIC micelles at Cp = 0.1 g/L (○) and for PIC vesicle at Cp = 1 g/L ( ) as a function of pH in 0.1 M NaCl.

Figure 5 shows the dependence of LSI on pH for PIC micelles and vesicles prepared in 0.1 M NaCl at pH 3. The LSI values for both PIC aggregates under basic conditions were less than those at pH 3. Under basic conditions, the unimer micelles convert to an open-chain conformation due to electrostatic repulsion of the pendant

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anionic groups, and the anionic charge numbers in p(A/MaU) increases two-fold. The PIC micelles and vesicles collapsed under basic conditions due to disruption of the charge balance of cationic P163M99 and anionic p(A/MaU). With increasing pH values from 3 to 12, the LSI values for PIC micelles and vesicles remained nearly constant at pH ≤ 6. The LSI values for PIC micelles and vesicles started to decrease at pH > 6, suggesting that the density of PIC micelles may decrease due to hydration at pH values between 6 and 8. PIC micelles and vesicles collapsed at pH > 8.

Figure 6. (a) Hydrodynamic radius (Rh, ○), light scattering intensity (LSI,

), and (b)

ζ-potential for p(A/MaU)/P163M99 PIC micelles at pH 3 as a function of f+ (= [MAPTAC]/([MAPTAC] + [AMPS])) in 0.1 M NaCl. Total polymer concentration was fixed at 0.1 g/L.

The Rh, LSI, and ζ-potential values for PIC micelles prepared at Cp = 0.1 g/L were plotted as a function of f+ at pH 3 (Figure 6). At f+ = 0.5, i.e., a stoichiometrically

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charge-neutral mixture of anionic unimer p(A/MaU) micelles and cationic P163M99, Rh (= 33.1 nm) and LSI (= 5.37 Mcps) reached a maximum, while the ζ-potential was close to 0 mV, suggesting formation of PIC micelles. When the charge balance of anionic unimer micelles and cationic P163M99 was not neutralized, i.e., f+ was not 0.5, the PIC micelles became smaller. The Rh, LSI, and ζ-potential values for PIC vesicles prepared at Cp = 1 g/L and pH 3 were plotted as a function of f+ (Figure S11). Results showed that, at f+ = 0.5, Rh (= 62.7 nm) and LSI (= 119 Mcps) reached maxima, and the ζ-potential was nearly 0 mV, suggesting formation of PIC vesicles. Electrostatic interactions can be examined by adding a salt such as NaCl. The stability of PIC aggregates depends on the NaCl concentration.31 The Rh distributions and the LSI for PIC micelles and vesicles were determined at varying NaCl concentrations (Figure S12). At pH 3, the Rh and LSI values for PIC micelle at 0.1 M NaCl were 33.1 nm and 5.37 Mcps, respectively, but were 51.0 nm and 1.01 Mcps, respectively, in 2 M NaCl. These results indicate that PIC micelles cannot dissociate in 2 M NaCl, because the Rh values for the unimer state of p(A/MaU) and P163M99 at pH 3 were 17.4 and 6.3 nm, respectively. The Rh and LSI values for PIC vesicles in 0.1 M NaCl were 59.7 nm and 119 Mcps, respectively, at pH 3. The Rh and LSI values decreased to 43.3 nm and 9.32 Mcps, respectively, in 2 M NaCl. However, the Rh value for PIC vesicles in 2 M NaCl was greater than those for unimer states of p(A/MaU) and P163M99. Therefore, 2 M NaCl cannot dissociate PIC vesicles to unimers. The electrostatic interactions between anionic p(A/MaU) and cationic P163M99 can be examined at 2 M NaCl. However, 2 M NaCl cannot dissociate PIC micelles and vesicles, because p(A/MaU) aggregates due to salting out at pH 3. To confirm this, the dependence of Rh on salt concentration for unimer micelles formed from p(A/MaU) at

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pH 3 and Cp = 1 g/L was determined. The Rh and LSI values for p(A/MaU) in 0.1 M NaCl were 15.0 and 1.79 Mcps, respectively (Figure S12e). At 2M NaCl, Rh and LSI for p(A/MaU) increased to 23.7 and 5.26 Mcps, respectively (Figure S12f). Increasing NaCl concentrations promoted intermicellar aggregation of the unimer micelles, presumably because of the salting out effect of the pendant hydrophobic undecanoic acid groups.

Figure 7. Fluorescence emission spectra for Texas red-labeled dextran after dialysis against 0.1 M NaCl at pH 3 for 48 h, excited at 530 nm in the presence (─) and absence (- - -) of p(A/MaU)/P163M99 PIC vesicles.

To confirm the vesicle structure of the PIC aggregate formed from oppositely charged polymers at Cp = 1 g/L and pH 3, Texas red-labeled nonionic water soluble dextran (Dex, MW = 70,000) was incorporated as a guest molecule into the interior hydrophilic core of the vesicle. If oppositely charged polymers form PIC vesicle structures, hydrophilic guest molecules can be incorporated into the internal water phase. If the polymers form PIC micelles, hydrophilic guest molecules cannot be incorporated into the hydrophobic core of the micelle. PIC vesicles were prepared by mixing p(A/MaU) and P163M99 at Cp = 1 g/L and pH 3 in the presence of Dex. This aqueous

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solution was transferred to a dialysis bag with 50 nm pores to remove unloaded Dex. PIC vesicles cannot permeate through the dialysis membrane, whereas Dex can permeate the membrane. The reference solution was prepared to dissolve Dex without p(A/MaU) and P163M99 in water at pH 3. The PIC vesicle and reference solutions were dialyzed against 0.1 M NaCl at pH 3 for 48 h. Fluorescence spectra for the PIC vesicle and reference aqueous solutions were obtained after dialysis (Figure 7). In the presence of PIC vesicles, fluorescence emission at 605 nm from Texas red was observed clearly. Fluorescence emissions were not observed from the reference solution without PIC vesicles. The PIC aggregates formed from mixing oppositely charged polymers at 1 g/L possessed a hollow structure, which allows the loading of Dex into the internal water phase. Loading efficiency (LE) and loading capacity (LC) of Dex were 0.0572 and 5.73%, respectively, estimated from the calibration curve and equations 2 and 3. The low LE value suggests that there is no interaction between the PIC vesicle and guest molecule.32 At pH 3, PIC micelles were prepared by mixing p(A/MaU) and P163M99 at Cp = 0.1 g/L in the presence of Dex. The aqueous solution was dialyzed against 0.1 M NaCl at pH 3 using a dialysis bag with 50 nm pores. Fluorescence from Dex could not be observed from the aqueous PIC micelle solution after dialysis. PIC micelles could not load hydrophilic Dex, because the micelles do not have an internal hydrophilic hollow structure.

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Figure 8. I338/I335 for Py excitation spectra monitored at 390 nm in the presence p(A/MaU)/P163M99 PIC micelles at Cp = 0.1 g/L (○) and PIC vesicles at Cp = 1 g/L ( ) as a function of pH in 0.1 M NaCl.

Pyrene excitation spectra in the presence of PIC micelles and vesicles were obtained in solutions of varying pH to confirm that the micelles and vesicles could incorporate and release hydrophobic pyrene molecules (Figure S13). The maximum wavelength of the 0-0 band of pyrene excitation spectra in water was observed at 335 nm. If pyrene exists in a hydrophobic environment, the maximum wavelength is red-shifted.33 The intensity ratio (I338/I335) of pyrene emission intensities of the 0-0 band at 335 and 338 nm in a hydrophobic environment is greater than that in water. Figure 8 shows I338/I335 in the presence of PIC micelles and vesicles as a function of pH. At pH 3, the I338/I335 ratios for PIC micelles and vesicles were similar (approximately 1.25) because the pyrene molecules are incorporated into the hydrophobic microdomain formed from the pendant undecanoic acid groups in p(A/MaU) of PIC micelles and vesicles. The I338/I335 ratios for PIC micelles and vesicles decreased under basic conditions. At pH 9, the pyrene molecules moved from the hydrophobic microdomain to the aqueous bulk phase due to ionization of the pendant undecanoate groups that collapsed the hydrophobic microdomain formed from p(A/MaU).

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At pH 3 and 9, I338/I335 ratios for pyrene without polymers were identical (0.57). Under basic conditions (pH > 9), the I338/I335 ratio for PIC vesicles was greater than that for PIC micelles, suggesting that pyrene molecules may not be completely released from the hydrophobic domain of PIC vesicles. The I338/I335 ratios at pH 3 and 9 in the presence of p(A/MaU) at Cp = 0.05 g/L were 1.20 and 0.62, respectively. The I338/I335 ratios at pH 3 and 9 in the presence of p(A/MaU) at Cp = 0.5 g/L were 1.25 and 0.70, respectively. At pH 9, the I338/I335 ratio (0.70) for p(A/MaU) at Cp = 0.5 g/L was greater than that (0.62) at Cp = 0.05 g/L. Unimer micelles may collapse at pH 9. However, when the p(A/MaU) concentration is high (Cp ≥ 0.5 g/L), pyrene molecules may get trapped into the hydrophobic portion of the MaU side chain and backbone. When PIC vesicles were prepared at Cp = 1 g/L, the concentration of p(A/MaU) was 0.5 g/L in the aqueous PIC vesicle solution. Therefore, pyrene molecules may exist in the hydrophobic environment around the MaU units after collapse of the PIC vesicle at Cp = 1 g/L under basic conditions.

CONCLUSIONS The random copolymer p(A/MaU), composed of equimolar AMPS and MaU units, was prepared via conventional radical polymerization. The p(A/MaU) formed unimer micelles under acidic conditions. In contrast, the unimer micelle was opened into a chain conformation under basic conditions. P163M99 with a well-controlled structure was prepared via RAFT-controlled radical polymerization. To prepare PIC micelles and vesicles, unimer micelles of p(A/MaU) and liner P163M99 were mixed in a stoichiometrically charge-neutral solution under acidic conditions. When the polymer concentrations of p(A/MaU) and P163M99 before mixing were 0.1 g/L, these polymers

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formed spherical PIC micelles. When both polymer concentrations before mixing were 1 g/L, these polymers formed PIC vesicles. When the pH of aqueous PIC micelle and vesicle solutions were > 9, the negative charges in p(A/MaU) increased two-fold due to ionization of the pendant undecanoic acid groups in MaU, causing a shift in the balance of charges. At pH > 9, PIC micelles and vesicles collapsed to form low-aggregation-number aggregates consisting of p(A/MaU) and P163M99 with expanded open chain structures. PIC vesicles can load hydrophilic guest molecules into the interior water phase. Furthermore, PIC micelles and vesicles can incorporate hydrophobic guest molecules into the hydrophobic microdomain formed from the pendant undecanoic acid groups. Thus, pH-responsive PIC micelles and vesicles are promising candidates for carriers of DDS, because the surface of these micelles and vesicles is covered with biocompatible PMPC shells.

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]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

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This work was financially supported by a Grant-in-Aid for Scientific Research (No. 25288101) from the Japan Society for the Promotion of Science (JSPS), and the Cooperative Research Program “Network Joint Research Center for Materials and Devices” (No. 2015467).

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