Thermoresponsive Copolymer Brushes Possessing Quaternary Amine

Article pubs.acs.org/Biomac

Thermoresponsive Copolymer Brushes Possessing Quaternary Amine Groups for Strong Anion-Exchange Chromatographic Matrices Kenichi Nagase,† Mike Geven,‡ Saori Kimura,§ Jun Kobayashi,† Akihiko Kikuchi,∥ Yoshikatsu Akiyama,† Dirk W. Grijpma,‡,⊥ Hideko Kanazawa,§ and Teruo Okano*,† †

Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWIns, 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan ‡ MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Biomaterials Science and Technology, University of Twente, P.O. Box 217 7500 AE Enschede The Netherlands § Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan ∥ Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika, Tokyo 125-8585, Japan ⊥ University Medical Center Groningen, University of Groningen, W.J. Kolff Institute, Department of Biomedical Engineering P.O. Box 196, 7600 AD Groningen, The Netherlands S Supporting Information *

ABSTRACT: A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices.

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have been developed.2−5 PIPAAm has been used as a controllable drug carrier,6−8 while the functions of PIPAAm-modified peptides or enzymes are able to be modulated by external temperature change.9,10 The phase transition properties of PIPAAm have been applied to biosensors11 and a successful application of PIPAAm is in cell culture dishes used to fabricate “cell-sheets”. The surface wettability of PIPAAm-modified cell

INTRODUCTION

Stimuli-responsive polymers and the polymer-modified substrates have attracted attention as functional materials because these polymers alter their properties through external stimuli. Several polymers have been investigated for the purpose of biomedical applications, with one of the well-known polymers being poly(N-isopropylacrylamide) (PIPAAm). This polymer shows temperature-dependent hydrophilic and hydrophobic alteration across its lower critical solution temperature (LCST) of 32 °C.1 Because the LCST of PIPAAm is similar to body temperature, several biomedical applications of this polymer © 2014 American Chemical Society

Received: December 27, 2013 Revised: January 25, 2014 Published: January 27, 2014 1031

dx.doi.org/10.1021/bm401918a | Biomacromolecules 2014, 15, 1031−1043

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Figure 1. Scheme for preparing P(IPAAm-co-APTAC-co- tBAAm) brush-grafted silica beads through surface-initiated ATRP as thermoresponsive protein adsorption materials with a strong basic moiety (A), and illustration of thermally modulated protein adsorption on the cationic copolymer brush (B).

adducts and the analyte can be obtained, compared with other types of PIPAAm-modified beads. Furthermore, thermoresponsive ion-exchange chromatography has been developed by preparing thermoresponsive ioniccopolymer-modified stationary phases.29,30 The ionic properties of thermoresponsive anionic or cationic copolymers having weak acid or base groups can be modulated by changing temperature, attributed to an environmental change in hydrophobicity.31,32 Thus, a copolymer-modified stationary phase can control electrostatic interaction between ionic analytes and the copolymer simply by changing the external temperature.33,34 Using ionic copolymer brush-modified beads, thermally modulated adsorption/desorption of several proteins was achieved, and the properties were applied to purification of pharmaceutical proteins by temperature change.35,36 The performance of thermoresponsive ion exchange chromatographic matrices will be affected by the properties of the ionic group. For preparation of the thermoresponsive cationic copolymer above, a comonomer possessing tertiary amine groups was used.30 However, in ordinary ion-exchange chromatographic matrices, quaternary amine groups are widely used as a stationary phase modifier, because of their strong basic properties.37,38 From this viewpoint, a thermoresponsive copolymer brush having quaternary amine groups would be an effective stationary phase modifier for thermoresponsive ionexchange chromatography.

culture dishes can be controlled simply by temperature change, leading to controllable cell adhesion and detachment.12−14 Some types of cell-sheets have already been used as effective therapies in clinical practice.15−17 Additionally, PIPAAm-modified surface have been investigated as a chromatographic stationary phase, named “thermoresponsive” or “green” chromatography.18,19 In this system, PIPAAm-modified stationary phases, mainly silica beads, are used, and the PIPAAm adduct alters their hydrophobicity upon temperature change. Thus, the chromatography system can control the interaction between the analyte and stationary phase simply by temperature change. This thermally modulated chromatography system does not require organic solvents for modulating the interaction between the stationary phase and analytes, avoiding the deactivation of biological analytes and reducing pollution from effluents. The separation efficiency of the PIPAAm-modified stationary phase depends on the configuration of the PIPAAm modification. To improve separation efficiency, several PIPAAm modification methods have been investigated.18−21 Among these, a PIPAAm brush-modified stationary-phase prepared though surface-initiated atom transfer radical polymerization (ATRP) has proven to be an effective stationary phase. Surface-initiated ATRP allows for formation of densely packed polymer-brush structures on the substrate,22−27 leading to larger amounts of PIPAAm per surface area of beads.21,28 Thus, strong interaction between the PIPAAm 1032


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Table 1. Characterization of Thermoresponsive Strong Cationic Copolymer P(IPAAm-co-APTAC-co-tBAAm) IPAAm/APTAC/tBAAm (molar ratio) codeb

in feed

in copolymerc

IPAtB-0 IPAtB-1 IPAtB-3 IPAtB-5 IPAtB-7 IPAtB-10 IPDtB-5

80.0/0/20.0 79.0/1.0/20.0 77.0/3.0/20.0 75.0/5.0/20.0 73.0/7.0/20.0 70.0/10.0/20.0 75.0/5.0/20.0

80.9/0.0/19.1 77.9/1.60/20.5 77.4/3.74/18.9 75.6/4.79/19.6 71.4/8.71/20.0 69.7/10.5/19.8 76.0/3.26/20.7

LCSTa Mnc by H NMR

Mnd by GPC

Mw/Mnd

in 66.7 mmol/L PB

in 33.3 mmol/L PB

in 16.7 mmol/L PB

7600 8800 6600 5000 5700 7800 4600

6200 6200 5500 5100 4000 4800 8000

1.16 1.24 1.26 1.27 1.31 1.47 1.19

16.9 17.9 20.2 22.9 27.6 n.d. 21.9

18.0 19.3 21.9 23.8 32.0 n.d. 24.1

18.3 20.2 21.4 23.5 32.0 n.d. 25.0

1

a

Defined as the temperature where the sample solution had a transmittance of 90%. bAll samples were named using the monomer abbreviation and feed molar composition of APTAC or DMAPAAm. “IP”, “A”, “D”, and “tB” represent IPAAm, APTAC, DMAPAAm, and tBAAm, respectively. c Determined by 1H NMR measurement using deuterium oxide as an NMR solvent. dMeasured by GPC using DMF containing 50 mmol/L LiCl with poly(ethylene glycol) standards.

Figure 2. Phase transition profile of poly(IPAAm-co-APTAC-co-tBAAm) in (A) 66.7 mM, (B) 33.3 mM, and (C) 16.7 mM phosphate buffer solutions (pH 7.0). A cellulose dialysis membrane (molecular weight cut off, MWCO, 1000 Da) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). ((Chloromethyl)phenylethyl)trimethoxysilane (mixed m, p isomers) was obtained from Gelest (Morrisville, PA, USA). Silica beads with an average diameter of 5 μm, a pore size of 300 Å, and a specific surface area of 100 m2/g were obtained from Chemco Scientific (Osaka, Japan). A stainless steel column (50 × 4.6 mm i.d.) was purchased from GL Science (Tokyo, Japan). ATRP Initiator Modification of Silica Beads. Modification of ATRP initiator on silica beads surface was performed through a silane coupling reaction as shown in Figure 1. Silica beads were washed with hydrochloric acid at 90 °C for 3 h. Subsequently, the beads were rinsed with water until the pH of supernatant was neutral, and dried at 150 °C overnight in a high-vacuum drying oven (DP33, Yamato, Tokyo, Japan). Washed beads (21.0 g) were placed into a 500 mL flask and stirred for 4 h at a relative humidity of 60%. ATRP initiator solution was prepared by dissolving ((chloromethyl)phenylethyl)trimethoxysilane in toluene (53.4 mmol/L), and the solution was poured onto the beads in a flask. The reaction mixture was stirred for 16 h at room temperature. After the reaction, the ATRP initiator-modified beads were rinsed with toluene, methanol, dichloromethane, and acetone, and dried at 110 °C for 2 h in a high-vacuum drying oven. Copolymer Grafting on Silica Beads by ATRP. Silica beads with thermoresponsive cationic copolymer brushes were prepared through surface-initiated-ATRP as shown in Figure 1. The typical procedure was as follows. APTAC (3.95 g, 14.7 mmol, 5 mol %) was dissolved in 294 mL of 2-propanol. The feed composition of APTAC was varied to be 0, 1, 3, 5, 7, and 10 mol %. The solution was passed though the inhibitor removal column. IPAAm (3.66 g, 32.3 mmol, 75 mol %) and tBAAm (1.09 g, 8.6 mmol, 20 mol %) were then dissolved in 42.8 mL

In this work, silica-beads modified with a thermoresponsive copolymer brush possessing quaternary amine groups were prepared through surface-initiated ATRP using 3-acrylamidopropyl trimethylammonium chloride (APTAC) as a cationic comonomer having quaternary amine groups. The copolymer and the copolymer-modified beads were characterized, and performance of the beads as chromatographic matrices was investigated by the elution behavior of acidic biomolecules and proteins.

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EXPERIMENTAL SECTION

Materials. IPAAm and APTAC containing 25 wt % water in the product were kindly provided by Kohjin (Tokyo, Japan). IPAAm was purified by recrystallization from n-hexane. Polymerization inhibitor in APTAC was removed by passing it through an inhibitor-remover column (Sigma, St. Louis, MO, USA). tert-Butylacrylamide (tBAAm) was purchased from Tokyo Chemical Industry (Tokyo, Japan) and recrystallized from acetone. N,N-dimethylaminopropylacrylamide (DMAPAAm) was purchased from Wako Pure Chemicals (Osaka, Japan) and purified by distillation at 113 °C and 1 mmHg. CuCl, CuCl2 α-chloro-p-xylene, formic acid, formaldehyde, tris(2-aminoethyl)amine (TREN), toluene (dehydrate), 2-propanol (HPLC grade), dichloromethane, methanol, acetone, and ethylenediamine-N, N, N′, N′-tetraacetic acid disodium salt dehydrate (EDTA·2Na), phosphate buffer powder (66.7 mM, pH7.0) and steroids were purchased from Wako Pure Chemicals. Adenosine nucleotides were purchased from Tokyo Chemical Industry. Proteins were obtained from Sigma. Tris(2-N,N-dimethylaminoethyl)amine (Me6TREN) was synthesized from TREN following the methods described in previous report.39 1033


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Table 2. Elemental Analyses of P(IPAAm-co-APTAC-co-tBAAm)-Brush Grafted Silica Beads by an XPS Take-off Angle of 90° atom (%)a code

b

initiator modified IPAtB-0 IPAtB-1 IPAtB-3 IPAtB-5 IPAtB-7 IPDtB-5 calcd of IPAAmc calcd of APTACc calcd of tBAAmc calcd of DMAPAAmc

C

N

O

22.37 ± 6.53 58.9 ± 4.11 63.3 ± 0.95 63.6 ± 1.47 63.1 ± 0.94 59.1 ± 4.84 54.2 ± 0.48 75.0 75.0 77.8 72.7

0.38 ± 0.66 7.10 ± 1.12 8.41 ± 1.07 8.64 ± 1.08 9.09 ± 0.43 9.97 ± 2.19 4.99 ± 1.35 12.5 16.7 11.1 18.2

47.9 ± 2.35 31.6 ± 9.47 20.9 ± 0.95 20.7 ± 1.06 19.5 ± 0.50 26.0 ± 3.50 28.5 ± 1.52 12.5 8.33 11.1 0.09

Si 27.4 8.93 6.99 6.71 7.25 4.63 11.0

± ± ± ± ± ± ± -

Cl 5.67 2.01 0.55 0.82 0.22 0.29 0.33

2.00 1.52 0.50 0.40 1.07 0.31 1.28

± ± ± ± ± ± ± -

N/C ratio 0.96 1.57 0.44 0.36 0.65 0.37 0.56

0.126 0.133 0.136 0.144 0.169 0.092 0.167 0.222 0.143 0.250

Data from three separate experiments are shown as mean ± SD. bAll samples were named using the monomer abbreviation and feed molar composition of APTAC or DMAPAAm. “IP”, “A”, “D”, and “tB” represent IPAAm, APTAC, DMAPAAm, and tBAAm, respectively. cEstimated atomic composition of each monomer.

a

Table 3. Characterization of P(IPAAm-co-APTAC-co-tBAAm) Brush-Modified Beads elemental composition (%) codea initiator modified IPAtB-0 IPAtB-1 IPAtB-3 IPAtB-5 IPAtB-7 IPDtB-5

Cb 4.66 16.8 16.9 17.1 16.3 17.6 16.0

± ± ± ± ± ± ±

Hb 0.05 0.07 0.08 0.93 0.07 0.22 0.06

0.28 2.40 2.41 2.58 2.45 2.59 2.24

± ± ± ± ± ± ±

immobilized initiatorc (μmol/m2)

Nb 0.08 0.05 0.04 0.11 0.07 0.02 0.02

0.04 2.65 2.62 2.76 2.71 2.91 2.53

± ± ± ± ± ± ±

0.02 0.06 0.01 0.08 0.04 0.04 0.04

grafted copolymer (mg/m2)

Mnd

Mw/Mnd

graft density (chains/nm2)

2.59 2.62 2.68 2.47 2.84 2.37

10500 11910 12200 13800 8400 15800

1.22 1.31 1.38 3.48 1.53 1.67

0.149 0.132 0.133 0.108 0.204 0.090

4.52

All samples were named using the monomer abbreviation and feed molar composition of APTAC or DMAPAAm. “IP”, “A”, “D”, and “tB” represent IPAAm, APTAC, DMAPAAm, and tBAAm, respectively. bDetermined by CHNS elemental analysis (n = 3). cEstimated from carbon composition. d Determined by GPC using DMF containing 50 mmol/L LiCl as a mobile phase; calibration curves were obtained using a PIPAAm standard. a

Figure 3. Chromatograms of adenosine nucleotides separated on HPLC, for which the packing materials were P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads at various temperatures (A) P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads (IPAtB-5 in Table 2), (B) P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads (IPAtB-3 in Table 2), and (C) P(IPAAm-co-DMAPAAm-co-tBAAm) brush-grafted silica beads (IPDtB-5 in Table 2) for comparison. Mobile phase is 66.7 mmol/L phosphate buffer (pH 7.0). Peak No. 1 represents AMP; No. 2, ADP; No. 3, ATP. of APTAC solution. The feed composition of IPAAm was varied at 70, 73, 75, 77, 79, and 80 mol % and the total monomer concentration was set at 1 mol/L. The monomer solutions were deoxygenated by argon gas bubbling for 1 h. CuCl (84.7 mg, 0.86 mmol) and CuCl2 (11.5 mg, 0.086 mmol) were added to the monomer solutions and stirred for 10 min using magnetic stirrer under an argon atmosphere. Subsequently,

Me6TREN (0.22 g, 0.959 mmol) was added to the solution and stirred for 10 min under an argon atmosphere. The monomer solution was reacted with the premodified ATRP initiator beads for 16 h at 25 °C with continuous shaking of the reaction mixture. After the ATRP reaction, the copolymer grafted beads were rinsed with acetone, methanol, EDTA solution, and Milli-Q water, and dried in a vacuum oven at 50 °C for 5 h. 1034


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Figure 4. Temperature-dependent retention time changes of adenosine nucleotides on (A) a P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads packed column (IPAtB-5 in Table 2), (B) a P(IPAAm-co-APTAC-co-tBAAm)-brush grafted silica-beads packed column (IPAtB-3 in Table 2), and (C) a P(IPAAm-co-DMAPAAm-co-tBAAm) brush-grafted silica beads packed column (IPDtB-5 in Table 2) for comparison. The closed diamonds represent AMP; the closed triangles, ADP; the open circles, ATP. Numerical numbers −1, −2, and −3 represent data using 66.7, 33.3, and 16.7 mmol/L phosphate buffer (pH 7.0) as mobile phase. Characterization of Prepared Beads. Silica beads were analyzed by a CHNS elemental analyzer (PE 2400 series, PerkinElmer, Waltham, MA, USA) to obtain the amount of initiator and copolymer on the bead surface. The amount of initiator on silica bead surfaces (g/m2) was estimated using the following equation.

where %CP is the increase in carbon percent over that of the initiator beads, determined by elemental analysis and %CP(calcd) is the calculated weight percent of carbon in the copolymer. The grafted copolymers were retrieved from the silica bead surface to obtain the molecular weight and polydispersity index. Copolymer grafted beads were immersed in 10 mol/L of concentrated sodium hydroxide solution overnight under continuous stirring. Subsequently, the solution was neutralized with hydrochloric acid, filtered and dialyzed against water using a dialysis membrane with MWCO 1000 Da for seven days by changing the water daily. After purifying, the solution was freeze-dried, and the copolymer was obtained. Molecular weight and PDI value were obtained using gel permeation chromatography (GPC-8020 model II, Tosoh, Tokyo, Japan). N,N-dimethylformamide (DMF) containing 50 mmol/L LiCl was used as a mobile phase with a flow rate of 1.0 mL/min. The temperature of the GPC columns (TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000) (Tosoh, Tokyo) was maintained at 45 °C. Calibration curves were established to determine the molecular weight of copolymer using polyethylene glycol standards and poly(N-isopropylacrylamide) standards. The elution profile of copolymer was observed using an in-line

Immobilized initiator =

%CI %C I(calcd) × (1 − %CI /%CI(calcd)) × S

(1)

where %CI is the carbon percentage obtained from elemental analysis, %CI(calcd) is the calculated carbon percentage in the initiator molecules, and S is the surface area of the beads (100m2/g). The amount of the grafted copolymer on silica beads (g/m2) was estimated by following equation: Grafted copolymer =

%Cp %CP(calcd) × (1 − %Cp /%Cp(calcd) − %CI /%CI(calcd)) × S

(2) 1035


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refractometer. The copolymer graft density was estimated using the following equation: Graft density =

mc · NA Mn

(3)

where mc is the amount of grafted copolymer on the silica bead surface (g/m2), NA is Avogadro’s number, and Mn is the number average molecular weight of the grafted copolymer. The surface elemental composition was measured by X-ray photoelectron spectroscopy (XPS) (K-Alpha, Thermo Fisher Scientific, Waltham, MA, USA). Wide scans were performed to analyze all existing elements on silica bead surfaces. Peak deconvolution of carbon C1s signals were performed to obtain information about the chemical bonds. Synthesis of Copolymer by ATRP. To characterize the thermoresponsive copolymer with quaternary amine groups, solution phase ATRP was performed using similar ATRP procedure, except that the ATRP-initiator α-chloro-p-xylene (53.4 mg, 380 μmol) was used instead of initiator-immobilized beads. After ATRP, the solution was dialyzed using a dialysis membrane with MWCO 1000 Da against EDTA solution for 3 d to remove unreacted monomers and ATRP catalyst. Subsequently, the dialysis was continued for 2 d against pure water to remove residual EDTA. The purified copolymer was obtained by lyophilization. Characterization of Copolymer. Molecular weight and PDI of the prepared copolymer was measured by GPC using the GPC system described above. The temperature-dependent phase transition profile of the prepared copolymers was observed by monitoring the change in transmittance of the copolymer solutions. Copolymer solutions (10 mg/mL) were prepared with 66.7, 33.3, and 16.7 mM phosphate buffer solutions (pH 7.0). Temperature-dependent optical transmittance change was monitored at 600 nm using a UV/visible spectrometer (V-530, JASCO, Tokyo, Japan) by heating the sample cuvette at 0.20 °C/min. The LCST of the copolymer was defined as the temperature at which there was 90% transmittance. The composition of each monomer in the prepared copolymer was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer, Varian, Palo Alto, CA, USA) using deuterium oxide as a solvent. Temperature-Modulated Elusion of Bioactive Compounds and Proteins. The copolymer-modified beads were packed into a stainless steel column (50 × 4.6 mm i.d., GL Science, Tokyo, Japan) as previously reported.40 The column was connected to an HPLC system controlled with Borwin software (JASCO). Various concentrations of phosphate buffers (PBs, pH 7.0), 66.7, 33.3, and 16.7 mM, were used as mobile phases. Adenosine nucleotides (AMP, ADP, and ATP) were used to investigate the surface electrostatic properties of the copolymer-grafted beads. The properties of analytes are shown in Tables S1 and S2 (Supporting Information). AMP (0.56 mg), ADP (3.72 mg) and ATP (40.0 mg) were dissolved in 12 mL of PB, and an adenosine nucleotides mixture sample was prepared. The mobile phases were flowed at 1.0 mL/min using a pump (PU-980, JASCO). The elution behavior was observed at 254 nm with a UV-970 UV/ visible detector. Column temperature was controlled with a thermostatted water circulator (CTA400, Yamato, Tokyo, Japan). Hydrophobic steroids were used to investigate the hydrophobic properties of the bead surfaces. Hydrocortisone (3.83 mg) and dexamethasone (8.46 mg) were dissolved in 12 mL of ethanol, and a steroids mixture sample was prepared. Elution behavior of the steroids was also observed at 254 nm at the same flow rate of mobile phases (1.0 mL/min). To investigate the protein adsorption properties, proteins (3.00 mg) (aprotinin, β-galactosidase, conalbumin fibrinogen, human serum albumin, γ-globulin, ovalbumin, transferrin, trypsin inhibitor from soybean) were dissolved in 6 mL of phosphate buffer. Elution behavior of each protein was observed at 280 nm at a mobile phase flow rate of 1.0 mL/min.

Figure 5. Chromatograms of hydrophobic steroids separated on HPLC, for which packing materials were P(IPAAm-co-APTAC-cotBAAm) brush-grafted silica beads at various temperatures (A) P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads (IPAtB-5 in Table 2), (B) P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads (IPAtB-3 in Table 2), and (C) P(IPAAm-co-DMAPAAm-cotBAAm) brush-grafted silica beads (IPDtB-5 in Table 2) for comparison. Mobile phase is 66.7 mmol/L phosphate buffer (pH 7.0). Peak No. 1 represents hydrocortisone; No. 2, dexamethasone.

copolymers possessing various contents of quaternary amine groups P(IPAAm-co-APTAC-co-tBAAm) were characterized by 1 H NMR, GPC and observation of temperature-dependent changes in optical transmittance. The data are summarized in Table 1. The copolymers are named using the monomer abbreviation and cationic monomer APTAC or DMAPAAm feed composition. “IP”, “tB”, “A”, and “D” denote IPAAm, tBAAm, APTAC, and DMAPAAm, respectively. 1H NMR measurement of the prepared copolymer revealed that the mole fraction observed was similar to the feed composition, because all monomers are similar acrylamide derivatives and have similar reactivity under these ATRP conditions using CuCl/CuCl2/Me6TREN as catalyst and 2-propanol as a solvent. GPC measurement revealed that the molecular weight and PDI decreased and increased, respectively, with increasing APTAC composition. On the contrary, molecular weight determined by 1H NMR exhibited similar values regardless of APTAC composition. These results indicate that APTAC interacted with the GPC column, leading to relatively lower molecular weight and larger PDI at higher APTAC contents. Actually, the gel permeation chromatogram exhibits peak tailing in Figure S1. Thus, although a relatively larger PDI was observed by GPC measurement at higher APTAC content, polymerization would be controlled under the ATRP. Phase transition behavior of the thermoresponsive copolymer possessing quaternary amine groups was observed with various concentrations of PB (Figure 2), with those of P(IPAAm-co-DMAPAAm-co-tBAAm) shown in Figure S2. Phase transition temperature increased with increasing APTAC composition. Above 5% APTAC feed composition, the optical transmittance of the copolymer solution did not decrease with increasing temperature, indicating that the copolymers do not induce hydrophobic aggregation, because the strong basic property of the quaternary amine groups provided hydrophilicity to the copolymer. Also, shrinkage and aggregation of the copolymer was suppressed by the repulsive forces of the quaternary amine groups in the copolymers. Our previous report indicated that a similar thermoresponsive cationic copolymer with tertiary amine groups P(IPAAm-co-DMAPAAmco-tBAAm) exhibited phase transition profiles even at a 10 mol % DMAPAAm composition.35 Therefore, P(IPAAm-co-APTAC-cotBAAm) has quite a strongly basic nature compared with that of

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RESULTS AND DISCUSSION Characterization of Thermoresponsive Copolymer Possessing Quaternary Amines. Prepared thermoresponsive 1036


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Figure 6. Temperature-dependent retention time changes of hydrophobic steroids on (A) a P(IPAAm-co-APTAC-co-tBAAm) brush-grafted silica beads packed column (IPAtB-5 in Table 2), (B) a P(IPAAm-co-APTAC-co-tBAAm)-brush-grafted silica-beads packed column (IPAtB-3), and (C) a P(IPAAm-co-DMAPAAm-co-tBAAm) brush-grafted silica beads packed column (IPDtB-5 in Table 2) for comparison. The open circles represent hydrocortisone; the closed triangles, dexamethasone. Numerical numbers −1, −2, and −3 represent data using 66.7, 33.3, and 16.7 mmol/L phosphate buffer (pH 7.0) as mobile phase.

P(IPAAm-co-DMAPAAm-co-tBAAm). These results indicate that 5 mol % APTAC is a suitable composition to achieve thermal modulation of the copolymer. Characterization of Copolymer Brush-Grafted Silica Beads. XPS measurement of prepared beads was performed to obtain the elemental composition of bead surfaces. Table 2 summarizes the elemental composition of the prepared beads. All samples are named using the initial of the monomers and the APTAC or DMAPAAm feed composition. C1s peak deconvolution was performed and is shown in Figure S3. Larger carbon and nitrogen contents and smaller silicone content were observed on copolymer brush-grafted beads compared with those of initiator-immobilized beads. Peak deconvolution of C1s peaks was performed and is shown in Figure S3. An additional peak at 288 eV, which is attributed to the CO bond, was observed on copolymer-grafted beads, while there were no peaks on initiator-modified beads. These results indicate that

copolymer was successfully grafted onto the silica bead surfaces through surface-initiated ATRP. CHN elemental analysis of the prepared beads was performed to obtain the amount of modified initiator and copolymer on the silica bead surfaces. Table 3 summarizes the elemental composition of the prepared beads, the amounts of initiator and copolymer on the bead surfaces and the graft density of copolymer. Larger amounts of grafted copolymer on silica bead surfaces were observed compared with that prepared by conventional free radical polymerization, previously reported.28 This is because the grafted copolymer formed a densely packed structure on silica bead surfaces, attributed to the high initiating efficiency of the ATRP-initiator and controlled polymerization. Grafted copolymer was retrieved from silica beads with concentrated sodium hydroxide solution, and their molecular weight and PDI were measured by GPC. Gel permeation chromatograms are shown in Figure S4. Molecular weights of the retrieved copolymers were slightly 1037


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Figure 7. Temperature-dependent peak area changes of proteins eluted from IPAtB-5: (A) aprotinin, (B) β-galactosidase, (C) conalbumin, (D) fibrinogen, (E) human serum albumin, (F) γ-globulin, (G) ovalbumin (H) transferrin, (I) trypsin inhibitor from soybean. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mmol/L phosphate buffer (pH 7.0) as mobile phase.

lower than those of the PIPAAm homopolymer, as previously reported.41 Also, larger PDIs were observed. These can be attributed to interaction between APTAC and the GPC column. The copolymer graft density exceeded 0.1 chains/ nm2, indicating that a dense polymer brush structure with quaternary amine groups was successfully formed on the silica bead surfaces. Elution Behavior of Bioactive Compounds. Copolymer modified beads were packed into a column, and temperaturedependent surface properties of the beads were investigated by observing the elution profiles of adenosine nucleotides and hydrophobic steroids. Figure 3 shows the elution behavior of an adenosine nucleotides mixture containing AMP, ADP, and ATP using 66.7 mM PB. Elution behaviors using 33.3 and 16.7 mM PB are shown in Figures S5 and S6, respectively. Figure 4 shows the retention time of each adenosine nucleotide at various temperatures. IPAtB-5 and IPAtB-3 columns exhibited relatively longer retention times and higher resolution separation than the IPDtB-5 column. This is because of the strong

electrostatic properties of the quaternary amines on the IPAtB-5 and IPAtB-3 columns. The strong basic properties of the quaternary amine group led to strong electrostatic interactions between the grafted copolymer and analytes, leading to longer retention times and high-resolution separation of adenosine nucleotides. The retention times for adenosine nucleotides were prolonged with decreasing concentrations of PB, because the electrostatic interaction between copolymer and analytes strengthens at lower ionic concentrations. Different retention time change-profiles were observed on IPAtB-5 and IPAtB-3 columns, compared with that on the IPDtB-5 column (Figure 4). This can be attributed to the difference in basic properties between quaternary amine and tertiary amine groups. The basic properties of the tertiary amine groups on the IPDtB-5 column decreased with increasing temperature, because an increasing environmental hydrophobicity in the vicinity of the tertiary amine group and shrinkage of the copolymer induces deprotonation of the amine group.30,35 Therefore, retention time decreased with increased deprotonation of the grafted copolymer 1038


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Figure 8. Temperature-dependent peak area changes of proteins eluted from IPAtB-3: (A) aprotinin, (B) β-galactosidase, (C) conalbumin, (D) fibrinogen, (E) human serum albumin, (F) γ-globulin, (G) ovalbumin (H) transferrin, (I) trypsin inhibitor from soybean. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mmol/L phosphate buffer (pH 7.0) as mobile phase.

on IPDtB-5, and the IPDtB-5 column exhibited a rectilinear retention time change, with an inflection point at the LCST. On the contrary, the quaternary amine groups on IPAtB-5 or IPAtB-3 columns are always protonated, indicating that the charge density of the grafted copolymer is always retained. Thus, the retention time did not decrease linearly. The decrease in retention time at lower and higher temperature regions was probably attributed to the following factors. The first is the collapse of modified brushes. Grafted copolymer brushes would collapse with increasing temperature, and adenosine nucleotides molecules would not tend to diffuse into the collapsed copolymer brush. Thus, retention time would decrease with increasing temperature. Second, the solubility of analytes in the mobile phase would be increased and electrostatic interactions would decrease with increasing temperature. These are also the possible reason for decreased retention time of analytes with increasing temperature. Hydrophobicity of the copolymer brush was investigated by the elution profiles of hydrophobic steroids. Figure 5 shows the

elution behavior of a steroids mixture containing hydrocortisone and dexamethasone, using 66.7 mM PB. Elution behaviors using 33.3 and 16.7 mM PB are shown in Figures S7 and S8, respectively. Figure 6 shows the retention time of each hydrophobic steroid at various temperatures. Retention time increased with increasing temperature, indicating that dehydration of the copolymer brush on the bead surfaces proceeded and hydrophobic interactions between copolymer and steroids were enhanced at increased temperature. Relatively high retention times were observed on IPDtB-5 compared with those on the IPAtB-5 and IPAtB-3 columns, because of the high ionic strength of the quaternary amine. The quaternary amine incorporated in the copolymer provides hydrophilicity to the copolymer brush, leading to weakening of the hydrophobic interactions between the copolymer and hydrophobic steroids. The retention times of steroids increased slightly with increasing concentration of the mobile phase. Previous reports indicated that dehydration of PIPAAm and its copolymer was enhanced by increasing salt concentration.42,43 1039


Biomacromolecules

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Figure 9. Temperature-dependent peak area changes of proteins eluted from IPDtB-5: (A) aprotinin, (B) β-galactosidase, (C) conalbumin, (D) fibrinogen, (E) human serum albumin, (F) γ-globulin, (G) ovalbumin (H) transferrin, (I) trypsin inhibitor from soybean. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mmol/L phosphate buffer (pH 7.0) as mobile phase.

Figure 10. Temperature-dependent elution of proteins from IPAtB-5: (A) fibrinogen, (B) human serum albumin, (C) ovalbumin, (D) trypsin inhibitor from soybean, and (E) transferrin. (A), (B) and (C) were obtained using 33.3 mM PB, and (D) and (E) were obtained using 66.7 mM PB as mobile phase. 1040


Biomacromolecules

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Figure 11. Temperature-dependent elution of proteins from IPAtB-3: (A) fibrinogen, (B) human serum albumin, (C) ovalbumin, (D) trypsin inhibitor from soybean, and (E) transferrin. (A), (B) and (C) were obtained using 33.3 mM PB, and (D) and (E) were obtained using 66.7 mM PB as mobile phase.

Figure 12. Temperature-dependent elution of proteins from IPDtB-5: (A) fibrinogen, (B) human serum albumin, (C) ovalbumin, (D) trypsin inhibitor from soybean, and (E) transferrin. (A), (B) and (C) were obtained using 33.3 mM PB, and (D) and (E) were obtained using 66.7 mM PB as mobile phase.

interaction also contributes to protein adsorption on the copolymer brush. Comparing the protein adsorption capacity of the beads, IPAtB-5 exhibited protein adsorption even at moderate PB concentrations and low temperature, while the IPDtB-5 column required a reduced PB concentration to enhance electrostatic interactions and the column temperature to be relatively high. This behavior is also attributed to the strong basic properties of the quaternary amine groups on IPAtB-5. Strong basic properties of quaternary amine groups induced strong electrostatic interactions between the copolymer and proteins. Thus, these proteins tend to be adsorbed more strongly on IPAtB-5 beads, compared with IPDtB-5 beads. Additionally, the thermoresponsive copolymer on IPAtB-5 retains its charge density, even at elevated temperature, because the quaternary amine is always protonated. On the contrary, the charge of the copolymer brush on IPDtB-5 decreased with increasing temperature, attributed to deprotonation of the tertiary amine groups, induced by hydrophobicity increasing in the vicinity of the amino groups and shrinkage of the copolymer.26,30 Thus, the thermoresponsive copolymer with quaternary amine groups is effective for protein adsorption at elevated temperature, compared with that having tertiary amine groups. These results demonstrate that a thermoresponsive copolymer with quaternary amine groups is effective for acidic protein

Thus, dehydration of the grafted copolymer was enhanced with increasing PB concentration. Proteins Adsorption on Copolymer Brush. Our previous reports indicated that thermoresponsive ionic copolymer modified beads functioned as thermoresponsive protein adsorption materials.35,36 Thus, a thermoresponsive copolymer brush having strong basic groups could be used as an effective protein adsorption material. Nine types of protein were used as model proteins and thermoresponsive protein adsorption properties were investigated. Figures 7−9 show the temperaturedependent peak area changes for the proteins, and Figures 10−12 show chromatograms of the proteins that adsorbed on the copolymer brush. Chromatograms of transferrin are also shown as a nonadsorbed protein for comparison. The peak areas of fibrinogen, human serum albumin, ovalbumin, and trypsin inhibitor from soybean decreased with increasing temperature, indicating that protein adsorption on the copolymer brush was enhanced with increasing temperature. Also, adsorbed proteins were successfully recovered simply by reducing the temperature (Figure 13). Adsorption of these proteins was enhanced with decreasing PB concentration in the mobile phase, because these adsorbed proteins have strong acidic properties and their electrostatic interactions with the copolymer were strengthened at lower PB concentrations. Protein adsorption was enhanced with increasing temperature, indicating that hydrophobic 1041


Biomacromolecules

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Figure 13. Protein recoveries from the IPAtB-5 column by reducing column temperature. (A) fibrinogen, (B) human serum albumin, (C) ovalbumin, (D) trypsin inhibitor from soybean. (A), (B) and (C) were obtained using 33.3 mM PB, and (D) was obtained using 66.7 mM PB as mobile phase.

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adsorption, even at relatively low temperature and moderate concentrations of PB, compared with tertiary amine groups. Thus, these copolymer brush-modified beads are expected to be useful as effective protein adsorption materials that can purify proteins simply by changing temperature.

Corresponding Author

*Phone: +81-3-5367-9945 Ext. 6201; Fax: +81-3-3359-6046; E-mail: [email protected].

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Notes

The authors declare no competing financial interest.

CONCLUSIONS A thermoresponsive copolymer brush with quaternary amine groups, P(IPAAm-co-APTAC-co-tBAAm), was grafted onto silica beads through surface-initiated ATRP to synthesize strong cationic, thermoresponsive chromatographic matrices. The characterization of the prepared beads indicates that densely packed P(IPAAm-co-APTAC-co-tBAAm) brushes were grafted onto the silica bead surfaces. Phase transition profiles of copolymers exhibited that a content of APTAC above 5 mol % is not suitable for thermal modulation of properties in the copolymer, which can be attributed to the strongly basic properties of the quaternary amine groups. Chromatographic analysis using the prepared beads indicates that P(IPAAm-coAPTAC-co-tBAAm) beads show high retention of adenosine nucleotides compared with P(IPAAm-co-DMAPAAm-cotBAAm) beads, because of the strong electrostatic interactions between adenosine nucleotides and the quaternary amine groups. Also, intrinsic temperature-dependent retention time changes were observed on a P(IPAAm-co-APTAC-co-tBAAm) beads column, probably because of the persistently protonated quaternary amine groups in the copolymer brush. Temperaturedependent protein adsorption was performed using the prepared beads. Fibrinogen, human serum albumin, ovalbumin, and trypsin inhibitor from soybean were adsorbed on the copolymer brushes with increasing temperature. Using P(IPAAm-co-APTAC-cotBAAm) beads, these proteins could be adsorbed, even at relatively low temperature and at moderate phosphate buffer concentrations. These results indicate that a thermoresponsive copolymer brush with quaternary amine groups could be an effective thermally modulated cationic chromatographic matrix for separation of acidic biomolecules or purification of proteins.

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AUTHOR INFORMATION

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ACKNOWLEDGMENTS This research was partly financially supported by the Development of New Environmental Technology Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned by the Ministry of Environment, Japan; a Grants-in-Aid for Young Scientists (B) No. 24760580 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; and subsidies from the Kumagai Foundation for Science and Technology. We thank Dr. Atsushi Tamura for valuable discussion.

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ASSOCIATED CONTENT

S Supporting Information *

Properties of the analyte, gel permeation chromatograms of the copolymer, phase transition profiles of IPDtB-5, XPS peak deconvolution of the C1s peaks, and chromatograms of adenosine nucleotides and hydrophobic steroids. This information is available free-of-charge via the Internet at http://pubs.acs.org/. 1042


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Thermoresponsive Copolymer Brushes Possessing Quaternary Amine

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