Preparation and Characterization of Polyion Complex Micelles with

Article pubs.acs.org/Langmuir

Preparation and Characterization of Polyion Complex Micelles with Phosphobetaine Shells Keita Nakai,† Midori Nishiuchi,† Masamichi Inoue,† Kazuhiko Ishihara,‡ Yusuke Sanada,§ Kazuo Sakurai,§ and Shin-ichi Yusa*,† †

Department of Materials Science and 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 § Department of Chemistry and Biochemistry, University of Kitakyushu, 1-1 Hibikino, 808-0135 Kitakyushu, Japan S Supporting Information *

ABSTRACT: A pair of oppositely charged diblock copolymers, poly(2-(methacryloyloxy)ethyl phosphorylcholine)block-poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMPC-b-PMAPTAC) and poly(2(methacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2-methylpropanesulfonate) (PMPC-bPAMPS), was prepared via reversible addition−fragmentation chain transfer radical polymerization using a PMPC-based macro chain transfer agent. The pendant phosphorylcholine group in the hydrophilic PMPC block has anionic phosphate and cationic quaternary amino groups, which are neutralized within the pendant group. Therefore, the mixing of aqueous solutions of PMPC-b-PMAPTAC and PMPC-b-PAMPS leads to the spontaneous formation of simple core−shell spherical polyion complex (PIC) micelles comprising of a segregated PIC core and PMPC shells. The PIC micelles were characterized using 1H NMR spin−spin (T2) and spin−lattice relaxation times (T1), diffusion-ordered NMR spectroscopy, static light scattering, dynamic light scattering (DLS), and transmission electron microscopy techniques. The hydrodynamic size of the PIC micelle depended on the mixing ratio of PMPC-b-PMAPTAC and PMPC-b-PAMPS; the maximum size occurred at the mixing ratio yielding stoichiometric charge neutralization. The PIC micelles disintegrated to become unimers with the addition of salts.

■

INTRODUCTION Self-assembly of amphiphilic block copolymers in water has been widely used to prepare polymer-based nanostructured materials such as spherical, worm-shaped, cylindrical, toroidal, lamellar, and vesicular objects.1−3 Self-assembly of block copolymers is usually induced by noncovalent interactions including hydrophobic, hydrogen bonding, van der Waals, and electrostatic interactions. In particular, spherical core−shell polymer micelles formed from amphiphilic diblock copolymers by hydrophobic interactions in water are widely studied because they are thought to be potentially important in many applications including separation4 and delivery systems.5 Stoichiometrically mixing a pair of oppositely charged polyanions and polycations in water leads to spontaneous formation of polyion complexes (PICs), which are not ordinarily soluble in any solvent.6 Stoichiometric PICs are electronically neutral because the charges of components are mutually neutralized. Therefore, they lose their colloidal stability and precipitate from the solution.7 The precipitation of PIC can be prevented if a hydrophilic nonionic block such as poly(ethylene glycol) (PEG) is attached to at least one of the polyelectrolytes.8−10 In these cases, the aggregates have a PIC micelle with a well-defined core−shell structure comprising of a PIC core and hydrophilic shell constructed from the nonionic © 2013 American Chemical Society

blocks. Kataoka and Harada reported on this type of PIC micelle prepared from a pair of oppositely charged poly(amino acid) block copolymers of PEG.11−13 The PIC micelles were formed from a stoichiometric mixture of PEG-block-poly(Llysine) and PEG-block-poly(α,β-aspartic acid). PIC micelles are promising candidates for a variety of applications such as those involving metal ions,14 enzymes,15 RNA,16 and DNA.17 Each of these cases corresponds to a type of core−shell PIC micelle. In particular, these micelles may be useful as carriers of oligonucleotides and plasmid DNA for human gene therapy because of their sizes and characteristic core−shell structures.18 Most PIC micelles have a PEG shell as a nonionic hydrophilic block. On the other hand, few studies have reported on a PIC micelle comprising of shells other than PEG chains. Liu et al.19 reported on the preparation and characterization of PIC micelles with thermoresponsive poly(N-isopropylacrylamide) graft chains. In addition, hydrophilic nonionic blocks such as poly(N,N-dimethylacrylamide),20 poly(poly(ethylene oxide) methyl ether methacrylate),21 and polyacrylamide22 are used as PIC shells. Cohen-Stuart et al. Received: March 21, 2013 Revised: June 2, 2013 Published: July 11, 2013 9651

dx.doi.org/10.1021/la401063b | Langmuir 2013, 29, 9651−9661

Langmuir

Article

reported on Janus PIC micelles prepared with incompatible water-soluble neutral polymer chains in the shells, such as polyacrylamide and polyethyleneoxide, giving rise to a symmetry breaking in the shells.23 The phosphorylcholine group of the 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) monomer is a component of cell membranes.24,25 Water-soluble MPC can be polymerized with other vinyl compounds, and the properties and functions of such polymers can be controlled by choosing an appropriate comonomer. Polymers with MPC units show extremely high biocompatibility and antithrombogenicity. MPC polymer (PMPC) is a polyampholyte bearing both positive and negative charge in its phosphorylcholine group. To the best of our knowledge, there is no report on PIC micelles with polyampholyte PMPC shells. However, PIC micelles covered with biocompatible PMPC shells may be an excellent candidate for gene and drug delivery systems. Therefore, we investigated the influence of polyampholyte PMPC shells on the formation of PIC micelles. The stability of the micelles and micellization behavior depend on various factors such as the concentration and chemical structure of the polymer. PIC micelles, as well as ordinary micelles comprising of amphiphilic block copolymers, have a critical association concentration,26 which suggests that the PIC micelles may be in an equilibrium state in which the block copolymers are exchanged between the micelle and bulk water phases. In addition, the salt concentration is a key parameter for the stability of the PIC micelles because the electrostatic interactions between charged blocks are screened by added salt. In the present work, we prepared a pair of oppositely charged diblock copolymers comprising a polyampholyte PMPC block and charged block (Figure 1), that is, poly(2(methacryloyloxy)ethyl phosphorylcholine)-block-poly((3-

(methacryloylamino)propyl)trimethylammonium chloride) (PMPC100-b-PMAPTACm) and poly(2-(methacryloyloxy)ethyl phosphorylcholine)-block-poly(sodium 2-(acrylamido)-2methylpropanesulfonate) (PMPC100-b-PAMPSn), via reversible addition−fragmentation chain transfer (RAFT) polymerization using a PMPC-based macro chain transfer agent (CTA) with a number-average degree of polymerization (DP) of 100. We obtained diblock copolymers with different, well-controlled PMAPTAC [DP (m) = 27, 48, and 96] and PAMPS [DP (n) = 27, 45, and 99] block lengths. When aqueous solutions of the oppositely charged diblock copolymers were mixed, spherical PIC micelles comprising a PIC segregated core and PMPC shells formed spontaneously. The PIC micelles were characterized by the 1H NMR relaxation time, diffusion-ordered spectroscopy (DOSY), static light scattering (SLS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) techniques. The size of the micelles depended on the mixing ratio of PMPC100-b-PMAPTACm and PMPC100-bPAMPSn; the maximum size occurred at the mixing ratio yielding stoichiometric charge neutralization. To investigate the dynamic properties of the PIC micelles, the effect of additional salt was investigated using a light scattering technique.

■

EXPERIMENTAL SECTION

Materials. MPC was synthesized as previously reported and recrystallized from acetonitrile.25 (3-(Methacryloylamino)propyl)trimethylammonium chloride (MAPTAC, 96%), 2-(acrylamido)-2methylpropanesulfonic acid (AMPS, 95%), and 4,4′-azobis(4-cyanopentanoic acid) (V-501, 98%) from Wako Pure Chemical Industries, Ltd. were used as received without further purification. 4Cyanopentanoic acid dithiobenzoate (CPD) was synthesized according to the method reported by McCormick and co-workers.27 Water was purified with a Millipore Milli-Q system. Other reagents were used as received. Homopolymerization of MPC by RAFT. MPC homopolymer was synthesized according to a modified version of previously reported methods.28 MPC (15.0 g, 50.8 mmol) was dissolved in 45.7 mL of water, and then CPD (0.142 g, 0.508 mmol) and V-501 (71.2 mg, 0.254 mmol) were added to the solution. The aqueous solution was degassed by purging with Ar gas for 30 min. Polymerization was performed at 70 °C for 4 h. The reaction mixture was dialyzed against pure water for five days. The homopolymer (PMPC100-CTA) was recovered by freeze-drying (12.2 g, 80.5%). Gel-permeation chromatography (GPC) was performed to estimate the numberaverage molecular weight [Mn (GPC)] and molecular weight distribution (Mw/Mn) values as 1.91 × 104 and 1.05, respectively. Mn(NMR) and DP for PMPC100-CTA were 2.98 × 104 and 100, respectively, as estimated by 1H NMR. The obtained MPC homopolymer could be used as a CTA to prepare block copolymers. Preparation of Cationic Diblock Copolymers (PMPC100-bPMAPTACm). A representative example of the preparation of the diblock copolymer of PMPC and PMAPTAC is as follows: MAPTAC (1.09 g, 4.96 mmol), V-501 (7.00 mg, 0.0250 mmol), and PMPC100CTA (1.00 g, 0.0496 mmol, Mn(NMR) = 2.98 × 104, Mw/Mn = 1.05) were dissolved in water (10.0 mL). The mixture was degassed by purging with Ar gas for 30 min. Polymerization was performed at 70 °C for 6 h. The reaction mixture was dialyzed against pure water for two days. The diblock copolymer (PMPC100-b-PMAPTAC96) was recovered by freeze-drying (1.79 g, 85.6%). Mn(GPC) and Mw/Mn were estimated to be 9.91 × 104 and 1.11, respectively, by GPC. Mn(NMR) and DP for the PMAPTAC block were 4.99 × 104 and 96, respectively, as estimated by 1H NMR. Preparation of Anionic Diblock Copolymers (PMPC100-bPAMPSn). A representative example of the preparation of the diblock copolymer of PMPC and PAMPS is as follows: A predetermined amount of AMPS (0.487 g, 2.35 mmol) was neutralized with NaOH aqueous solution. To this solution was added predetermined amounts

Figure 1. (a) Chemical structures of diblock copolymers, PMPC100-bPMAPTACm (P100Mm) and PMPC100-b-PAMPSn (P100An). (b) Schematic representation of PIC micelle comprising P100Mm and P100An. 9652


Langmuir

Article

of V-501 (3.27 mg, 0.0117 mmol) and PMPC100-CTA [0.700 g, 0.0231 mmol, Mn(NMR) = 2.98 × 104, and Mw/Mn = 1.05]. The solution was degassed by purging with Ar gas for 30 min. Polymerization was performed at 70 °C for 2 h. The reaction mixture was dialyzed against pure water for two days. The polymer (PMPC100-b-PAMPS99) was recovered by freeze-drying (0.794 g, 66.9%). Mn(GPC) and Mw/Mn were estimated to be 3.07 × 104 and 1.11, respectively, by GPC. Mn(NMR) and DP for the PAMPS block were 5.03 × 104 and 99, respectively, as estimated by 1H NMR. Block Copolymerization Using PMPC100 -CTA in D2 O. Predetermined amounts of MAPTAC (0.557 g, 2.52 mmol), PMPC100-CTA (0.752 g, 0.0248 mmol), and V-501 (3.54 mg, 0.0126 mmol) were dissolved in 5.0 mL of D2O. The solution was transferred to several NMR tubes and degassed by purging with Ar gas for 30 min. The cap was sealed, and the solution was heated at 70 °C in an oil bath for varying reaction times. Polymerization was terminated by rapid cooling in an ice bath. The monomer conversion estimated by 1H NMR was monitored as a function of reaction time. GPC of the reaction mixture was used to estimate Mn and Mw/Mn. AMPS was polymerized using PMPC100-CTA in D2O in a similar manner. Preparation Methods of PIC Micelles. PMPC100-b-PMAPTACm and PMPC100-b-PAMPSn were separately dissolved in an aqueous solution, and the solutions were allowed to stand overnight at room temperature to achieve complete dissolution. The solutions contained 0.1 M NaCl unless otherwise noted. To prepare PIC micelles, a PMPC100-b-PMAPTACm solution was added dropwise to a PMPC100b-PAMPSn solution over 5 min with stirring, and the mixture was allowed to equilibrate for at least one day prior to measurement. The mixing ratio of the two block copolymers in the solution was represented by the mole fraction of positively charged units ( f + = [MAPTAC]/([AMPS] + [MAPTAC])); hence, complete charge neutralization was attained at f + = 0.5. The PIC micelles were prepared at f+ = 0.5 unless otherwise noted. Measurements. 1H NMR. Proton NMR spectra were obtained with a Bruker DRX-500 spectrometer operating at 500.13 MHz using a deuterium lock during the entire run at 20 °C. The sample solutions of the polymer for 1H NMR measurements were prepared in D2O containing 0.1 M NaCl. The proton NMR spin−spin relaxation time (T2) was determined using the Carr−Purcell−Meiboom−Gill pulse sequence.29 For T2 measurements, NMR tubes containing the D2O solution were deaerated by purging with Ar gas for 30 min. A 90° pulse of 13.85 μs was calibrated and used for measurements. The echo peak intensity was measured at 12 different values of the 180° pulse. The proton spin−lattice relaxation times (T1) were measured using the inversion−recovery sequence (180°−tr−90°), where the recovery time (tr) was varied from 50 ms to 10 s, recording 12 experimental points. The T1 values were calculated by single-exponential fitting of the inversion recovery curves.30 Diffusion-Ordered NMR Spectroscopy (DOSY). Proton NMR selfdiffusion experiments were performed using a Bruker DRX500 at 20 °C. The sample solution was prepared in D2O containing 0.1 M NaCl and was transferred to a 5 mm NMR tube. A 2D sequence for diffusion measurements using the standard pulse program, stebpgp1s19 (Bruker TopSpin 1.3), employing a stimulated echo, bipolar gradient pulses, and one spoil gradient was used with 3−9−19 pulse sequence (WATERGATE) solvent suppression to suppress the H2O signal.31 The variation in the intensity of one selected resonance peak in the 1H NMR spectrum (I) is related to the strength of the gradient (g) by the following equation: ⎡ ⎛ I δ ⎞⎤ = exp⎢− DNMR (γδg )2 ⎜Δ − ⎟⎥ ⎝ ⎣ I0 3 ⎠⎦

with the peaks of the solutes. Prior to recording the 2D DOSY experiment, the δ and Δ values were optimized for each sample using the 1D sequence (stebpgp1s191d) for diffusion measurements.32 For all of the experiments, δ = 3.5−4.5 ms and Δ = 250−400 ms were used. The data were analyzed with TopSpin 1.3, which directly provided DNMR. The hydrodynamic radius (Rh,NMR) estimated from DOSY is given by the Stokes−Einstein equation, Rh,NMR = kBT/ (6πηDNMR), where kB is the Boltzmann constant, T is the absolute temperature, and η is the solvent viscosity. GPC. GPC measurements of cationic polymer samples were performed using a refractive index (RI) detector equipped with a Shodex Ohpak SB-G guard column and 10 μm bead size SB-804 HQ column (exclusion limit, ∼107) working at 40 °C under a flow rate of 0.6 mL/min. A 0.3 M Na2SO4 aqueous solution containing 0.5 M acetic acid was used as an eluent. The values of Mn and Mw/Mn for cationic polymer samples were calibrated with standard poly(2vinylpyridine) samples of six different molecular weights from 5.70 × 103 to 3.16 × 105. GPC measurements of PMPC homopolymer and anionic polymer samples were performed using an RI detector equipped with a Shodex Asahipak GF-1G guard column and 7.0 μm bead size GF-7 M HQ column (exclusion limit, ∼107) working at 40 °C under a flow rate of 0.6 mL/min. A phosphate buffer (pH 9) containing 10 vol % acetonitrile was used as an eluent. The values of Mn and Mw/Mn for anionic polymers were calibrated with standard sodium poly(styrenesulfonate) samples of 11 different molecular weights from 1.37 × 103 to 2.16 × 106. Light Scattering Measurements. Light scattering measurements were performed using an Otsuka Electronics Photal DLS-7000HL light scattering spectrometer 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 a light source. Sample solutions for light scattering measurements were filtered using a membrane filter with 0.2 μm pores. In the SLS measurements, the weight-average molecular weight (Mw), z-average radius of gyration (Rg), and second virial coefficient (A2) values were estimated from the following relationship:

KC p Rθ

=

⎞ 1 ⎛⎜ 1 1 + R g 2q2⎟ + 2A 2 C p ⎝ ⎠ Mw 3

(2)

where Rθ is the difference between the Rayleigh ratio of the solution and that of the solvent, q is the magnitude of the scattering vector, and K = 4π2n2(dn/dCp)2/NAλ4, with dn/dCp being the RI increment against the polymer concentration (Cp) and NA being Avogadro’s number. The q value was calculated using q = (4π/λ)sin(θ/2), where n is RI of the solvent, λ is the wavelength of the light source (= 632.8 nm), and θ is the scattering angle. By measuring Rθ for a set of Cp and θ, the values of Mw, Rg, and A2 were estimated from Zimm plots. The known Rayleigh ratio of toluene was used to calibrate the instrument. The values of dn/dCp at 633 nm were determined with an Otsuka Electronics Photal DRM-3000 differential refractometer at 25 °C. In the DLS measurements, to obtain the relaxation time distribution, τA(τ), inverse Laplace transform analysis was performed using the algorithm REPES.33 g(1)(t ) =

∫ τA(τ)exp(−t /τ)d ln τ

(3)

where τ is the relaxation time and g(1)(t) is the normalized autocorrelation function. The relaxation rate (Γ = τ−1) is a function of θ.34 The diffusion coefficient (D) is calculated from D = (Γ/q2)q→0. The hydrodynamic radius (Rh) is given by the Stokes−Einstein equation, Rh = kBT/(6πηD). The details of the DLS instrumentation and theory are described in the literature.35 ζ Potential. The ζ potential was measured using a Malvern Zetasizer Nano-ZS ZEN3600 equipped with a He−Ne laser light source (4 mW at 632.8 nm) at 25 °C. The ζ potential was calculated from the 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−Hückel parameter and particle radius, respectively.36

(1)

where γ is the gyromagnetic ratio of the proton, δ is the length of the gradient pulse (small delta), g is the gradient strength, Δ is the delay time between the midpoints of the gradients (big delta), I0 is the initial intensity at Δ = 0, and DNMR is the self-diffusion coefficient estimated from the DOSY experiment. It was necessary to accumulate 128 scans per spectrum to increase the signal-to-noise (S/N) ratio associated 9653


Langmuir

Article

TEM. TEM observations were performed with a JEOL JEM-2100 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.

CTA. A significant increase in the molecular weight occurred upon polymerization of MAPTAC, indicating formation of the block copolymer, PMPC100-b-PMAPTACm. Neither a new peak nor a shoulder due to homopolymers of MAPTAC was recognized. In Figure 2c, the values of Mn and Mw/Mn estimated by GPC for the polymers are plotted as a function of the conversion of MAPTAC determined by 1H NMR. Mn increased with conversion, whereas Mw/Mn remained fairly constant (Mw/Mn < 1.05) independent of conversion. If the polymerization was assumed to be ideally living process, the theoretical number-average molecular weight (Mn(theo)) can be calculated as

■

RESULTS AND DISCUSSION Synthesis of Diblock Copolymers. PMPC100-b-PMAPTACm diblock copolymers were synthesized using PMPC100CTA (Mn(NMR) = 2.98 × 104 and Mw/Mn = 1.05). Figure 2a

M n(theo) =

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

(4)

where [monomer]0 is the initial monomer concentration, [CTA]0 is the initial PMPC100-CTA concentration, xm is the percent conversion of the monomer, Mm is the molecular weight of the monomer, and MCTA is the molecular weight of PMPC100-CTA. The observed values of Mn(GPC) estimated from GPC deviated from the theoretical values of Mn(theo) estimated from eq 4 (Table 1). The reason for the considerable deviation is currently an open question, but it may be due to the use of poly(2-vinylpyridine) as a standard polymer to calibrate the Mn values, the volume-to-mass ratio of which may differ greatly from that of PMPC100-b-PMAPTACm.38 The number-average molecular weights (Mn(NMR)) of the PMPC100-b-PMAPTACm samples were calculated from 1H NMR data. As illustrated in Table 1, the Mn(NMR) values for PMPC100-b-PMAPTACm were in fair agreement with the Mn(theo) values. The polymerization of AMPS in the presence of PMPC100CTA also proceeded in a controlled manner, as illustrated in Figure 3. Figure 3a shows the time−conversion and first-order kinetic plots. Polymerization started after an induction period of ca. 8 min, reaching a monomer conversion of 95.8% within 60 min. The GPC elution curves in Figure 3b demonstrate that the molecular weight of the resulting block polymer, PMPC100b-PAMPSn, increases with monomer conversion, and the molecular weight distribution is unimodal. The values of Mn and Mw/Mn estimated from GPC are plotted in Figure 3c as a function of conversion. The Mn value increases almost linearly with the conversion, whereas Mw/Mn lies in a narrow range of 1.05−1.10 independent of conversion. For PMPC100-bPAMPSn, Mn(GPC) deviated from Mn(theo) (Table 1). However, for PMPC100-b-PAMPSn, Mn(NMR) was close to Mn(theo). In this work, we prepared a set of PMPC100-b-PMAPTACm and PMPC100-b-PAMPSn samples having different lengths of the cationic and anionic blocks, while the length of the polyampholyte PMPC block did not vary (Mn(NMR) = 2.98 × 104, DP = 100). The values of Mn and Mw/Mn for all the block copolymers are listed in Table 1. The block copolymers, PMPC100-b-PMAPTACm and PMPC100-b-PAMPSn, are further abbreviated as P100Mm and P100An, where P, M, and A represent PMPC, PMAPTAC, and PAMPS blocks, respectively, and the subscript indicates DP of the block. 1 H NMR. Proton NMR spectra for P100M96 and P100A99 are compared in Figure 4a and b. As shown in Figure 4a, the resonance bands observed at 0.8−1.1 ppm and at 1.8 ppm were attributed to α-methyl protons and main chain methylene protons, respectively. DP (= m) and Mn(NMR) of the

Figure 2. (a) Time−conversion (○) and first-order kinetic plots (△) for the polymerization of MAPTAC in the presence of PMPC100-CTA in D2O at 70 °C. [M]0 and [M] are the concentrations of the monomer at polymerization time 0 and corresponding time, respectively. (b) GPC elution curves demonstrating evolution of molecular weight during synthesis of PMPC100-b-PMAPTACm. Conversions are shown for each peak. (c) Dependence of Mn (○) and Mw/Mn (△) on monomer conversion during polymerization of MAPTAC.

shows the time−conversion relationship and first-order kinetic plots for the polymerization of MAPTAC. The monomer consumption was monitored by 1H NMR spectroscopy as a function of the polymerization time. There is an induction period of ca. 10 min, which may be due to a slow rate of formation of the 4-cyanopentanoic acid radical fragment, as reported by McCormick and co-workers.37 Monomer conversion of 96.0% was reached within 300 min. The first-order kinetic plot suggests that the concentration of propagating radicals remained constant during polymerization. Figure 2b shows the GPC elution curves (RI response) for the polymerization of MAPTAC in the presence of PMPC1009654


Langmuir

Article

Table 1. Number-Average Molecular Weight (Mn), Molecular Weight Distribution (Mw/Mn), and Hydrodynamic Radius (Rh) of the Block Copolymers samples PMPC100-b-PMAPTACm

PMPC100-b-PAMPSn

P100M27 P100M48 P100M96 P100A27 P100A45 P100A99

Mn(theo)a × 10−4

Mn(GPC)b × 10−4

Mn(NMR)c × 10−4

Mw/Mnb (GPC)

Rhd (nm)

3.63 4.07 5.04 3.59 3.81 5.03

7.48 8.20 9.91 2.29 2.47 3.07

3.58 4.09 4.99 3.59 3.91 5.03

1.08 1.08 1.11 1.06 1.06 1.11

2.3 2.6 3.6 1.5 2.9 3.1

a Calculated from eq 4. bEstimated from GPC. cEstimated from 1H NMR for the purified diblock copolymers. dDetermined by DLS at 25 °C in 0.1 M NaCl aqueous solution at Cp = 1.0 g/L.

Figure 4. 1H NMR spectra measured for (a) PMPC100-b-PMAPTAC96 (P100M96), (b) PMPC100-b-PAMPS99 (P100A99), and (c) PIC micelle of P100M96/P100A99 with f+ = 0.5 at Cp = 1.0 g/L in D2O containing 0.1 M NaCl. Assignments are indicated for the resonance peaks.

Figure 3. (a) Time−conversion (○) and first-order kinetic plots (△) for the polymerization of AMPS in the presence of PMPC100-CTA in D2O at 70 °C. [M]0 and [M] are the concentrations of the monomer at polymerization time 0 and corresponding time, respectively. (b) GPC elution curves demonstrating evolution of molecular weight during synthesis of PMPC100-b-PAMPSn. Conversions are shown for each peak. (c) Dependence of Mn (○) and Mw/Mn (△) on monomer conversion during polymerization of AMPS.

Figure 4c shows a 1H NMR spectrum for a mixed solution of P100M96 and P100A99 with a 0.5 mole fraction of positively charged units (f+ = 0.5) prepared in D2O containing 0.1 M NaCl by adding a solution of P100M96 to that of P100A99 at Cp = 1.0 g/L. A stoichiometric mixture of P100Mm and P100An was expected to form a core−shell micelle. As shown in Figure 4c, the intensity of resonance bands associated with the PMAPTAC and PAMPS blocks was extremely weak compared with those associated with the PMPC block. Broadening of the signal indicates a decrease in the spin−spin relaxation time (T2) and molecular motion.39 Therefore, the motions of the PMAPTAC and PAMPS blocks were highly restricted because these oppositely charged block chains were confined in a PIC micelle core. To obtain further information about the motional restriction of the PMAPTAC, PAMPS, and PMPC blocks when PIC micelles were formed, the T2 values of P100Mm, P100An, and P100Mm/P100An PIC micelles were measured in D2O containing

PMAPTAC block in P100Mm were determined from the integral intensity ratio of the resonance bands at approximately 3.1 and 3.2 ppm attributed to pendant methyl protons in the PMAPTAC and PMPC blocks, respectively. As shown in Figure 4b, the resonance bands observed at 0.8−2.2 ppm for P100An were attributed to the sum of the main chain and pendant methyl groups in the PAMPS block. DP (= n) of the PAMPS block in P100An and Mn(NMR) were calculated from the integral intensity ratio of the resonance bands at 3.4 and 3.2 ppm attributed to the pendant methylene protons in the PAMPS and PMPC blocks, respectively. 9655


Langmuir

Article

Table 2. Spin−Spin (T2) and Spin−Lattice Relaxation Times (T1) for Protons in the PMPC, PMAPTAC, and PAMPS Blocks in D2O Containing 0.1 M NaCl T2 (ms) sample

3.2 ppma

d

267 273 262 259 262 234 237 238 189

P100M27 P100A27d P100M27/P100A27e P100M48d P100A45d P100M48/P100A45e P100M96d P100A99d P100M96/P100A99e a

3.1 ppmb

T1 (ms) 1.5 ppmc

123 40.4 8.6

36.6 101

37.3 5.5

13.9 98.3

32.5 3.5

5.3

3.2 ppma

3.1 ppmb

481 477 483 487 482 481 486 484 493

416

1.5 ppmc 516 670

482 436

566 923

575 439

570 1059

558

PMPC pendant methyl protons. bPMAPTAC pendant methyl protons. cPAMPS pendant methyl protons. dFree unimer state. ePIC micelles.

Table 3. Diffusion-Ordered NMR Spectroscopy (DOSY), Dynamic Light Scattering (DLS), and Static Light Scattering (SLS) Data for PIC Micelles in 0.1 M NaCl sample

Rh,NMRa (nm)

Rhb (nm)

Mwc × 10−5

Rgc (nm)

A2c × 104 (mol mL/g2)

Rg/Rh

Naggd

dn/dCp (mL/g)

P100M27/P100A27 P100M48/P100A45 P100M96/P100A99

7.0 11.0 17.8

8.6 16.2 18.5

2.01 5.40 18.1

10.2 19.5 17.5

2.01 0.30 0.065

1.23 1.11 0.97

6 14 37

0.141 0.139 0.150

a Estimated by 1H NMR DOSY using a PMPC pendant methyl proton signal at 3.2 ppm. bEstimated by DLS. cEstimated by SLS. dAggregation number of PIC micelles calculated from Mw of the micelles determined by SLS and Mw of the corresponding unimers.

0.1 M NaCl.40,41 Matched pairs (in terms of the lengths of the charged blocks) of the oppositely charged diblock copolymers were chosen to prepare PIC micelles, that is, P100M27/P100A27, P100M48/P100A45, and P100M96/P100A99. To monitor the motional restriction of the blocks, the relaxation times for the peaks attributed to PMAPTAC, PAMPS, and PMPC pendant methyl protons at 3.1, 1.5, and 3.2 ppm, respectively, were analyzed. Table 2 compares the T2 relaxation times of the PMAPTAC, PAMPS, and PMPC blocks in the free unimer and PIC micellar states. In the unimer state, the values of T2 for PMAPTAC in P100M27 and PAMPS in P100A27 were found to be 123 and 40.4 ms, respectively. When P100M27 and P100A27 formed PIC micelles, the values of T2 for PMAPTAC and PAMPS decreased significantly to 36.6 and 8.6 ms, respectively. This decrease in T2 was indicative of the restricted motions of the charged blocks in the PIC micelles. It should be noted that the T2 values for the longer charged blocks decreased much smaller values as they formed a PIC micelle. In the unimer state, the T2 values for PMAPTAC in P100M96 and PAMPS in P100A99 were found to be 98.3 and 32.5 ms, respectively. When P100M96/P100A99 PIC micelles were formed, the T2 values for PMAPTAC and PAMPS became 5.3 and 3.5 ms, respectively. This indicates that the motions of the charged blocks were more restricted in the PIC micelle formed from a pair of longer charged blocks. In contrast to the T2 values for the charged blocks, those for the PMPC block decreased slightly when the PIC micelle was formed; that is, the T2 values of 267 and 273 ms for free P100M27 and P100A27, respectively, decreased to 262 ms as they formed a PIC micelle. However, for a pair of longer charged blocks, that is, for the PMPC block, T2 decreased to a smaller value of 189 ms as they formed a PIC micelle from 237 and 238 ms for the unimer states of P100M96 and P100A99, respectively. The PMPC blocks may form the shell of the PIC micelles because the pendant charged phosphorylcholine groups were neutralized within a single polymer chain. However, from the T2 data, the motion of the PMPC shells in the PIC micelles

formed from longer PMAPTAC and PAMPS blocks (i.e., P100M96 and P100A99) was slightly restricted. The mobility of the PMPC shells, especially at the interface between the core and shell, may be influenced by the highly restricted PIC cores comprising of long PMAPTAC and PAMPS blocks. Furthermore, the PMPC chains were crowded into the limited space in the PIC shells. To obtain more insight into the motion of associated molecules, the spin−lattice relaxation time (T1) was studied quantitatively. Spin−lattice relaxation occurs most efficiently through molecular motion at frequencies comparable to the NMR frequency. Therefore, T1 decreases concurrently with T2 as the molecular motion decreases. After reaching a minimum value, T1 then increases with a further decrease in the molecular motion, whereas T2 remains at the minimum value. The T1 values for P100M96 and P100A99 in the unimer state were 439 and 570 ms, respectively. When these oppositely charged diblock copolymers formed a PIC micelle, the T1 values for the PMAPTAC and PAMPS blocks increased to 558 and 1059 ms, respectively, which were larger than those in the unimer state. An increase in T1 and a decrease in T2 for the PMAPTAC and PAMPS blocks in PIC micelles, respectively, suggest that the motion of the charged segments was restricted in the PIC core. The T1 values for the PMPC blocks in the unimer state were almost the same as those in PIC micelles. However, the T2 values for the PMPC blocks in PIC micelles were slightly shorter than those in the unimer state, suggesting that the motion of PMPC blocks was partially restricted in the PIC shells. We performed 1H NMR DOSY experiments to obtain the hydrodynamic radius (Rh,NMR). The water signal was suppressed using a WATERGATE pulse sequence. The resonance band observed at 3.2 ppm and attributed to PMPC pendant methylene protons was used to estimate Rh,NMR because it exhibited a good S/N ratio and did not overlap other peaks. The DNMR values for the P100M27/P100A27, P100M48/P100A45, and P100M96/P100A99 PIC micelles in 0.1 M NaCl were 2.43 × 10−11, 9656


Langmuir

Article

1.56 × 10−11, and 9.61 × 10−12 m2/s, respectively, which can be converted to Rh,NMR using the Stokes−Einstein equation. The Rh,NMR values for the PIC micelles are summarized in Table 3. SLS. The apparent values of the weight-average molecular weight (Mw(SLS)), radius of gyration (Rg), and second virial coefficient (A2) for the PIC micelles determined by SLS measurements are listed in Table 3. The dn/dCp values at 633 nm for the PIC micelles determined using a differential refractometer are also listed in Table 3. Figure 5 shows a typical

Figure 5. Typical example of Zimm plots for P100M96/P100A99 PIC micelles with f+ = 0.5 in 0.1 M NaCl aqueous solution at angles from 30 to 130° with an increment of 20°. The polymer concentrations (Cp) were 2, 5, and 10 g/L, respectively.

example of Zimm plots for P100M96/P100A99 PIC micelles in a Cp range of 2−10 g/L at 25 °C. As Table 3 demonstrates, Rg and Mw(SLS) depend strongly on the length of the charged blocks (PMAPTAC and PAMPS); the size and molecular weight are larger for longer charged blocks. The Mw(SLS) value for the P 100 M 96 /P 100 A 99 PIC micelle was more than approximately nine times larger than that for the P100M27/ P100A27 PIC micelle. The end-to-end distance (LPMPC) of a fully extended PMPC block with a DP of 100 can be calculated to be 25.0 nm. The value of Rg = 17.5 nm found for the P100M96/P100A99 PIC micelle (Table 3) was smaller than that of the fully extended chain lengths of P100M96 and P100A99 (i.e., ca. 50.0 nm). The value of Rg = 10.2 nm found for the P100M27/P100A27 PIC micelle was also smaller than that of the fully extended chain lengths of P100M27 and P100A27 (i.e., ca. 31.8 nm). These observations suggest that the PIC micelles may be simple core− shell structures without intermicellar aggregation. The aggregation number (Nagg) for the PIC micelles, defined as the total number of PMPC polymer chains forming one PIC micelle, can be calculated from the ratio of Mw(SLS) for the PIC micelle estimated from SLS and Mw for a single polymer chain (unimer) calculated from 1H NMR (Mn(NMR)) and GPC (Mw/Mn) data. The Nagg value for the P100M96/P100A99 PIC micelle was found to be 37 which is much larger than that for the P100M27/P100A27 PIC micelle (Nagg = 6). The small A2 values shown in Table 3 for the polymer micelles indicated their low solubility in the solvent.42,43 Therefore, the solubility of P100M96/P100A99 in 0.1 M NaCl aqueous solution was lower than that of P100M27/P100A27 because the volume of the water-insoluble segregated PIC core (Vc) for the former may be larger than that for the latter. We will discuss this point in more detail in the following subsection. DLS. The values of the hydrodynamic radius (Rh) for the diblock copolymers were determined by DLS at Cp = 1.0 g/L in 0.1 M NaCl at 25 °C and are listed in Table 1. Figure 6a shows the Rh values for P100M27/P100A27, P100M48/P100A45, and

Figure 6. (a) Hydrodynamic radius (Rh), (b) light scattering intensity, and (c) ζ potential for PIC micelles as a function of f+ (= [MAPTAC]/ ([MAPTAC] + [AMPS])) in 0.1 M NaCl aqueous solutions at 25 °C: P100M27/P100A27 (◇), P100M48/P100A45 (△), and P100M96/P100A99 (○). The total concentration of block copolymers was fixed at 1.0 g/L.

P100M96/P100A99 PIC micelles in 0.1 M NaCl as a function of f+. In this experiment, the total polymer concentration was maintained constant at 1.0 g/L. An increase in Rh indicates an increase in the size of the PIC micelles. Maximum Rh values were observed at f+ = 0.5. Figure 6b shows the light scattering intensity for the three types of micelles in 0.1 M NaCl as a function of f+. The light scattering intensity depends on the molecular weight of the particles. An increase in the scattering intensity indicates an increase in Nagg for the PIC micelles. These results indicate that stoichiometric charge neutralization in the mixture of two oppositely charged diblock copolymers produces PIC micelles with the maximum size and aggregation number. To confirm neutralization of the PIC micelles at f+ = 0.5, the ζ potential was measured as a function of f+ (Figure 6c). At f+ = 0, the aqueous solutions of P100A27, P100A45, and P100A99 showed negative ζ potential values because the PAMPS block has pendant anionic sulfonate groups. At f+ = 1, the aqueous solution of P100M27, P100M48, and P100M96 showed positive ζ potential values because the PMAPTAC block has pendant cationic quaternary amino groups. At f+ = 0.5, the ζ potential was zero because the PAMPS and PMAPTAC blocks were stoichiometrically neutralized. The PIC micelles were made up of a PIC core and PMPC shell. The pendant phosphorylcholine groups in the PMPC shells have anionic phosphate and cationic quaternary amino groups. However, the ζ potential of the PMPC homopolymer was zero (data not shown) because a pair of pendant anion and cation groups was neutralized within the single-polymer PMPC chain. 9657


Langmuir

Article

The Rh values from 1.5 to 3.6 nm appear to be reasonable for unimers of these block copolymers (Table 1). Figure 7a shows

(Γ) measured at different scattering angles are plotted as a function of the square of the scattering vector (q2) in Figure 7c. A linear relation passing through the origin indicates that all the relaxation modes were virtually diffusive.44 The diffusion coefficients (D) estimated from the slope of the Γ−q2 plots were in good agreement with those calculated from Γ at the peak of the relaxation time (τ) distribution obtained at θ = 90°. Because the angular dependence was negligible, the Rh values were estimated at a fixed θ of 90° and are listed in Table 3. The Rg/Rh ratio is useful for characterizing the shape of molecular assemblies. The theoretical value of Rg/Rh for a homogeneous hard sphere is 0.778, and it increases substantially for a less dense structure and polydisperse mixture. For example, Rg/Rh = 1.5−1.7 for flexible linear chains in a good solvent, whereas Rg/Rh ≥ 2 for a rigid rod.45−47 The Rg/Rh ratios listed in Table 3 for the P100M27/ P100A27, P100M48/P100A45, and P100M96/P100A99 micelles (1.23, 1.11, and 0.97, respectively) suggest that their shape was close to a monodisperse sphere. The core radius (Rc) of the PIC micelles formed at f+ = 0.5 with a simple core−shell structure can be calculated from the volume of each charged block as follows: Rc =

⎛ 3Vc ⎞1/3 ⎜ ⎟ ⎝ 4π ⎠

1/3 ⎛ ⎛ M n,PMAPTAC M n,PAMPS ⎞ Nagg ⎞ 3 ⎟ ⎜⎜ ⎟⎟ × = ⎜⎜ + ρPAMPS ⎠ 2 ⎟⎠ ⎝ 4πNA ⎝ ρPMAPTAC

(5)

where Vc is core volume of a PIC micelle, Mn,PMAPTAC and Mn,PAMPS are number-average molecular weights of the PMAPTAC and PAMPS blocks, and ρPMAPTAC and ρPAMPS are densities of the PMAPTAC and PAMPS blocks, respectively. To calculate Rc, 1.05 and 1.21 g/cm3 were used as bulk densities of MAPTAC (ρPMAPTAC) and AMPS (ρPAMPS) monomers, respectively. From Rh and Rc, the shell thickness (Ls) can be calculated as Ls = Rh − Rc. The calculated Rc, Ls, dPIC, and ΦPIC values for the three types of micelles are listed in Table 4. The density of the PIC micelle (dPIC) can be calculated by

Figure 7. (a) Typical examples of hydrodynamic radius (Rh) distributions for PIC micelles with f+ = 0.5 at Cp = 1.0 g/L in 0.1 M NaCl aqueous solutions, (b) relationship between Rh and Cp for PIC micelles, and (c) relationship between the relaxation rate (Γ) and square of the magnitude of the scattering vector (q2) for PIC micelles at Cp = 1.0 g/L: P100M27/P100A27 (◇), P100M48/P100A45 (△), and P100M96/P100A99 (○).

the Rh distributions for the three types of PIC micelles with f+ = 0.5 at θ = 90°. The Rh distributions were unimodal. The Rh values for the P100M27/P100A27, P100M48/P100A45, and P100M96/ P100A99 micelles calculated from the distribution were 8.6, 16.2, and 18.5 nm, respectively (Table 3), which were close to the values of Rh,NMR. These Rh values for the PIC micelles were smaller than the lengths of the fully extended block copolymer chains, supporting the suggestion that these PIC aggregates may be simple core−shell structures (Table 4). In Figure 7b, the Rh values of the PC micelles are plotted against Cp. Rh was practically constant in the polymer concentration range 0.5 ≤ Cp ≤ 10 g/L. The relaxation rates

dPIC =

3M w,PIC 4πNAR h 3

(6)

where Mw,PIC is weight-average molecular weight of a PIC micelle (= Mw(SLS)). Values of dPIC for the PIC micelles of P100M27/P100A27, P100M48/P100A45, and P100M96/P100A99 were calculated to be 0.125, 0.050, and 0.113 g/cm3, respectively. The smaller dPIC for the P100M48/P100A45 PIC micelle implies that this micelle is more hydrated; that is, more water molecules are present in its PMPC shells than those in the other two. The polymer density at the core−shell interface (ΦPIC) for the PIC micelles can be calculated by

Table 4. Core Radius (Rc), Shell Thickness (Ls), Counter Length of PMAPTAC (LPMAPTAC) and PAMPS (LPAMPS), Density (dPIC), and Polymer Density at Core−Shell Interface (ΦPIC) for PIC Micelles in 0.1 M NaCl sample

Rca (nm)

Lsb (nm)

LPMAPTACc (nm)

LPAMPSc (nm)

dPICd (g/cm3)

ΦPICe (chains/nm2)

P100M27/P100A27 P100M48/P100A45 P100M96/P100A99

2.3 3.7 6.6

6.3 12.5 11.9

6.8 12.0 24.0

6.8 11.3 24.8

0.125 0.050 0.113

0.090 0.081 0.068

Estimated from eq 5. bEstimated from Ls = Rh − Rc. cCounter length of the repeating unit is 0.25 nm for the polymer chains. dEstimated from eq 6. Estimated from eq 7.

a e

9658


Langmuir ΦPIC =

Article

Nagg Sc

=

amount of NaCl to adjust [NaCl]. At [NaCl] ≤ 0.3 M, the Rh values for the P100M96/P100A99 PIC micelle were constant at approximately 18 nm. At [NaCl] ≤ 0.2 M, the Rh values for the P100M48/P100A45 PIC micelle were constant at approximately 15 nm. At [NaCl] ≤ 0.1 M, the Rh values for the P100M27/P100A27 PIC micelle were constant at approximately 9 nm. These observations suggest that the structure of the PIC micelles remained constant at low NaCl concentrations. On the other hand, the Rh values decreased when [NaCl] increased above the critical concentrations. At [NaCl] ≥ 0.8 M, the Rh values were approximately 3 nm for all three types of micelles, suggesting that they completely dissociated into unimers. These observations indicate that screening of electrostatic interactions increases with [NaCl], which causes the PIC to dissociate into sets of oppositely charged unimers.49,50

Nagg 4πR c

2

(7)

where Sc is the surface area of the core. The ΦPIC values decreased with increasing Rh of the PIC micelles. TEM Observations. The structure of the PIC micelle was further confirmed by TEM observations, which are illustrated in Figure 8 and Figure S1 of the Supporting Information. The

■

CONCLUSIONS The diblock copolymers, PMPC 100 -b-PMAPTAC m and PMPC100-b-PAMPSn, with well-defined block lengths were prepared via RAFT-controlled radical polymerization using a PMPC macro CTA. Pairs of the oppositely charged diblock copolymers formed PIC micelles in 0.1 M NaCl aqueous solutions. When the MAPTAC/AMPS unit mole ratio was unity in the PIC micelles, that is, f+ = 0.5, the size and scattering intensity exhibited maximum values, indicating stoichiometric PIC formation from PMPC100-b-PMAPTACm and PMPC100-bPAMPSn. Small T2 values in 1H NMR were observed for the core of the PIC micelles, suggesting that the motion of the PIC core was highly restricted. Light scattering and TEM data suggest that the PIC micelles were spherical particles. The Nagg and Rh values for the P100M96/P100A99 PIC micelle were considerably larger than those for the P100M27/P100A27 PIC micelle. The PIC micelles disintegrated to become unimers with the addition of NaCl.

Figure 8. Typical examples of TEM images of (a) P100M27/P100A27, (b) P100M48/P100A45, and (c) P100M96/P100A99 PIC micelles.

micelles can be observed to have spherical shapes. The mean diameters for the P100M27/P100A27, P100M48/P100A45, and P100M96/P100A99 PIC micelles observed in the TEM images were 18, 36, and 39 nm, respectively, which were close to those determined by DLS measurements. The 2Rh values for P100M27/P100A27, P100M48/P100A45, and P100M96/P100A99 were 17, 32, and 37, respectively. Effect of Salt Addition. The presence of salts can screen electrostatic interactions between the oppositely charged blocks, which can cause the PIC micelles to decompose.48 Figure 9 shows Rh for the PIC micelles with f+ = 0.5 as a function of NaCl concentration ([NaCl]). A pair of the cationic and anionic diblock copolymers was mixed in water to prepare PIC micelles with f+ = 0.5, to which was added a predetermined

■

ASSOCIATED CONTENT

S Supporting Information *

Example TEM of P100M96/P100A99 micelles. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid (No. 21106518) for Scientific Research on Innovative Areas, “Molecular SoftInterface Science,” from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

■

REFERENCES

(1) Cui, H.; Chen, Z.; Zhong, S.; Wooley, K. L.; Pochan, D. J. Block copolymer assembly via kinetic control. Science 2007, 317, 647−650. (2) Zhang, L.; Eisenberg, A. Multiple morphologies of “crew-cut” aggregates of polystyrene-b-poly(acrylic acid) block copolymers. Science 1995, 268, 1728−1731. (3) Zhang, L.; Eisenberg, A. Multiple morphologies and characteristics of “crew-cut” micelle-like aggregates of polystyrene-b-poly(acrylic acid) diblock copolymers in aqueous solutions. J. Am. Chem. Soc. 1996, 118, 3168−3181.

Figure 9. Hydrodynamic radius (Rh) for PIC micelles with f+ = 0.5 at Cp = 1.0 g/L in 0.1 M NaCl aqueous solutions as a function of sodium chloride concentration ([NaCl]): P100M27/P100A27 (◇), P100M48/ P100A45 (△), and P100M96/P100A99 (○). 9659


Langmuir

Article

(22) Lindhoud, S.; Norde, W.; Stuart, M. A. C. Reversibility and relaxation behavior of polyelectrolyte complex micelle formation. J. Phys. Chem. B 2009, 113, 5431−5439. (23) Voets, I. K.; Fokkink, R.; Hellweg, T.; King, S. M.; de Waard, P.; de Keizer, A.; Cohen-Stuart, M. A. Spontaneous symmetry breaking: formation of Janus micelles. Soft Matter 2009, 5, 999−1005. (24) Iwasaki, Y.; Ishihara, K. Cell membrane-inspired phospholipid polymers for developing medical devices with excellent biointerfaces. Sci. Technol. Adv. Mater. 2012, 13, 064101. (25) Ishihara, K.; Ueda, T.; Nakabayasi, N. Preparation of phospholipid polymers and their properties as hydrogel sheet. Polym. J. 1990, 22, 355−360. (26) Kakizawa, Y.; Harada, A.; Kataoka, K. Environment-sensitive stabilization of core−shell structured polyion complex micelle by reversible cross-linking of the core through disulfide bond. J. Am. Chem. Soc. 1999, 121, 11247−11248. (27) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Water-soluble polymers. 81. Direct synthesis of hydrophilic styrenicbased homopolymers and block copolymers in aqueous solution via RAFT. Macromolecules 2001, 34, 2248−2256. (28) Yusa, S.; Fukuda, K.; Yamamoto, T.; Ishihara, K.; Morishima, Y. Synthesis of well-defined amphiphilic block copolymers having phospholipid polymer sequences as a novel biocompatible polymer micelle reagent. Biomacromolecules 2005, 6, 663−670. (29) Meiboom, S.; Gill, D. Modified spin-echo method for measuring nuclear relaxation times. Rev. Sci. Instrum. 1958, 29, 688−691. (30) Seki, M.; Morishima, Y.; Kamachi, M. Characterization of the complexes of amphiphilic polyanions and double-chain cationic surfactants. Macromolecules 1992, 25, 6540−6546. (31) Piotto, M.; Saudek, V.; Sklenár,̌ V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 1992, 2, 661−665. (32) Söderman, O.; Stilbs, P.; Price, W. S. NMR studies of surfactants. Concepts Magn. Res. Part A 2004, 23A, 121−135. (33) Jakes, J. Regularized positive exponential sum (REPES) programa way of inverting Laplace transform data obtained by dynamic light scattering. Collect. Czech. Chem. Commun. 1995, 60, 1781−1797. (34) Stockmayer, W. H.; Schmidt, M. Effects of polydispersity, branching and chain stiffness on quasielastic light scattering. Pure Appl. Chem. 1982, 54, 407−414. (35) Phillies, G. D. J. Quasielastic light scattering. Anal. Chem. 1990, 62, 1049A−1057A. (36) Ali, S. I.; Heuts, J. P. A.; van Herk, A. M. Controlled synthesis of polymeric nanocapsules by RAFT-based vesicle templating. Langmuir 2010, 26, 7848−7858. (37) Donovan, M. S.; Lowe, A. B.; Sumerlin, B. S.; McCormick, C. L. Raft polymerization of N,N-dimethylacrylamide utilizing novel chain transfer agents tailored for high reinitiation efficiency and structural control. Macromolecules 2002, 35, 4123−4132. (38) Yusa, S.; Konishi, Y.; Mitsukami, Y.; Yamamoto, T.; Morishima, Y. pH-Responsive micellization of amine-containing cationic diblock copolymers prepared by reversible addition−fragmentation chain transfer (RAFT) radical polymerization. Polym. J. 2005, 37, 480−488. (39) Wu, T.; Wu, Q.; Guan, S.; Su, H.; Cai, Z. Binding of the environmental pollutant naphthol to bovine serum albumin. Biomacromolecules 2007, 8, 1899−1906. (40) Yusa, S.; Shimada, Y.; Mitsukami, Y.; Yamamoto, T.; Morishima, Y. pH-Responsive micellization of amphiphilic diblock copolymers synthesized via reversible addition−fragmentation chain transfer polymerization. Macromolecules 2003, 36, 4208−4215. (41) Yusa, S.; Yokoyama, Y.; Morishima, Y. Synthesis of oppositely charged block copolymers of poly(ethylene glycol) via reversible addition−fragmentation chain transfer radical polymerization and characterization of their polyion complex micelles in water. Macromolecules 2009, 42, 376−383. (42) Quintana, J. R.; Jánez, M. D.; Villacampa, M.; Katime, I. Diblock copolymer micelles in solvent binary mixtures. 1. Selective solvent/ precipitant. Macromolecules 1995, 28, 4139−4143.

(4) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. Block copolymer micelles as vehicles for drug delivery. J. Controlled Release 1993, 24, 119−132. (5) Hurter, P. N.; Hatton, T. A. Solubilization of polycyclic aromatic hydrocarbons by poly(ethylene oxide-propylene oxide) block copolymer micelles: effects of polymer structure. Langmuir 1992, 8, 1291− 1299. (6) Michaels, A. S.; Miekka, R. G. Polycation-polyanion complexes: preparation and properties of poly-(vinylbenzyltrimethylammonium) poly-(styrenesulfonate). J. Phys. Chem. 1961, 65, 1765−1773. (7) Kudlay, A.; de la Cruz, M. O. Precipitation of oppositely charged polyelectrolytes in salt solutions. J. Chem. Phys. 2004, 120, 404−412. (8) Cohen Stuart, M. A.; Besseling, N. A. M.; Fokkink, R. G. Formation of micelles with complex coacervate cores. Langmuir 1998, 14, 6846−6849. (9) Kabanov, A. V.; Bronich, T. K.; Kabanov, V. A.; Yu, K.; Eisenberg, A. Soluble stoichiometric complexes from poly(N-ethyl-4-vinylpyridinium) cations and poly(ethylene oxide)-block-polymethacrylate anions. Macromolecules 1996, 29, 6797−6802. (10) Gohy, J. F.; Varshney, S. K.; Jérôme, R. Morphology of watersoluble interpolyelectrolyte complexes formed by poly(2-vinylpyridinium)-block-poly(ethylene oxide) diblocks and poly(4-styrenesulfonate) polyanions. Macromolecules 2001, 34, 2745−2747. (11) Kataoka, K.; Togawa, H.; Harada, A.; Yagusi, K.; Matsumoto, T.; Katayose, S. Spontaneous formation of polyion complex micelles with narrow distribution from antisense oligonucleotide and cationic block copolymer in physiological saline. Macromolecules 1996, 29, 8556− 8557. (12) Harada, A.; Kataoka, K. Novel polyion complex micelles entrapping enzyme molecules in the core: preparation of narrowlydistributed micelles from lysozyme and poly(ethylene glycol)− poly(aspartic acid) block copolymer in aqueous medium. Macromolecules 1998, 31, 288−294. (13) Harada, A.; Kataoka, K. Chain length recognition: core-shell supramolecular assembly from oppositely charged block copolymers. Science 1999, 283, 65−67. (14) Sanson, N.; Bouyer, F.; Destarac, M.; In, M.; Gérardin, C. Hybrid polyion complex micelles formed from double hydrophilic block copolymers and multivalent metal ions: size control and nanostructure. Langmuir 2012, 28, 3773−3782. (15) Lindhoud, S.; Norde, W.; Cohen Stuart, M. A. Effects of polyelectrolyte complex micelles and their components on the enzymatic activity of lipase. Langmuir 2010, 26, 9802−9808. (16) Oishi, M.; Nagasaki, Y.; Itaka, K.; Nishiyama, N.; Kataoka, K. Lactosylated poly(ethylene glycol)-siRNA conjugate through acidlabile β-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J. Am. Chem. Soc. 2005, 127, 1624−1625. (17) Kim, W.; Yamasaki, Y.; Jang, W.-D.; Kataoka, K. Thermodynamics of DNA condensation induced by poly(ethylene glycol)-blockpolylysine through polyion complex micelle formation. Biomacromolecules 2010, 11, 1180−1186. (18) Katayose, S.; Kataoka, K. Water-soluble polyion complex associates of DNA and poly(ethylene glycol)−poly(L-lysine) block copolymer. Bioconjugate Chem. 1997, 8, 702−707. (19) Zhang, J.; Zhou, Y.; Zhu, Z.; Ge, Z.; Liu, S. Polyion complex micelles possessing thermoresponsive coronas and their covalent core stabilization via “click” chemistry. Macromolecules 2008, 41, 1444− 1454. (20) Sotiropoulou, M.; Cincu, C.; Bokias, G.; Staikos, G. Watersoluble polyelectrolyte complexes formed by poly(diallyldimethylammonium chloride) and poly(sodium acrylate-cosodium 2-acrylamido-2-methyl-1-propanesulphonate)-graf t-poly(N,Ndimethylacrylamide) copolymers. Polymer 2004, 45, 1563−1568. (21) Shovsky, A.; Varga, I.; Makuška, R.; Claesson, P. M. Formation and stability of water-soluble, molecular polyelectrolyte complexes: effects of charge density, mixing ratio, and polyelectrolyte concentration. Langmuir 2009, 25, 6113−6121. 9660


Langmuir

Article

(43) Villacampa, M.; Apodaca, E. D.; Quintana, J. R.; Katime, I. Diblock copolymer micelles in solvent binary mixtures. 2. Selective solvent/good solvent. Macromolecules 1995, 28, 4144−4149. (44) Xu, R.; Winnik, M. A.; Hallett, F. R.; Riess, G.; Croucher, M. D. Light-scattering study of the association behavior of styrene-ethylene oxide block copolymers in aqueous solution. Macromolecules 1991, 24, 87−93. (45) Huber, K.; Bantle, S.; Lutz, P.; Burchard, W. Hydrodynamic and thermodynamic behavior of short-chain polystyrene in toluene and cyclohexane at 34.5.degree.C. Macromolecules 1985, 18, 1461−1467. (46) Akcasu, A. Z.; Han, C. C. Molecular weight and temperature dependence of polymer dimensions in solution. Macromolecules 1979, 12, 276−280. (47) Konishi, T.; Yoshizaki, T.; Yamakawa, H. On the “universal constants” ρ and Φ of flexible polymers. Macromolecules 1991, 24, 5614−5622. (48) De Santis, S.; Ladogana, R. D.; Diociaiuti, M.; Masci, G. Pegylated and thermosensitive polyion complex micelles by selfassembly of two oppositely and permanently charged diblock copolymers. Macromolecules 2010, 43, 1992−2001. (49) Solomatin, S. V.; Bronich, T. K.; Eisenberg, A.; Kabanov, V. A.; Kabanov, A. V. Colloidal stability of aqueous dispersions of block ionomer complexes: effects of temperature and salt. Langmuir 2004, 20, 2066−2068. (50) Park, J. S.; Akiyama, Y.; Yamasaki, Y.; Kataoka, K. Preparation and characterization of polyion complex micelles with a novel thermosensitive poly(2-isopropyl-2-oxazoline) shell via the complexation of oppositely charged block ionomers. Langmuir 2007, 23, 138− 146.

9661


Preparation and Characterization of Polyion Complex Micelles with

Recommend Documents