Thermoresponsive Anionic Copolymer Brushes Containing Strong

Article pubs.acs.org/Biomac

Thermoresponsive Anionic Copolymer Brushes Containing Strong Acid Moieties for Effective Separation of Basic Biomolecules and Proteins Kenichi Nagase,*,† Jun Kobayashi,† Akihiko Kikuchi,‡ Yoshikatsu Akiyama,† 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 ‡ Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika, Tokyo 125-8585, Japan § Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan S Supporting Information *

ABSTRACT: A thermoresponsive copolymer brush possessing the sulfonic acid group, poly(N-isopropylacrylamide (IPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-tert-butylacrylamide (tBAAm)), was grafted onto the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer and copolymer brushes on silica beads were characterized by observing the phase transition profile, CHNS elemental analysis, X-ray photoelectron spectroscopy, and gel permeation chromatography. The phase transition profile indicated that an appropriate AMPS composition for enabling thermally modulated property changes is 5 mol %, while excessive amounts of sulfonic acid groups prevented copolymer phase transition. Chromatographic elutions of catecholamine derivatives and basic proteins were observed, using the prepared copolymer brushmodified beads as chromatographic matrices, and the results suggest that the beads interact with these analytes through relatively strong electrostatic interactions. Thus, poly(IPAAm-co-AMPS-co-tBAAm) brush-modified beads will be useful for effective thermoresponsive chromatography matrices that separate basic biomolecules through strong electrostatic interactions.

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INTRODUCTION Functional polymers that respond to external stimuli have been investigated over the past few decades. Among these polymers, the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) has been used in various biomedical applications,1−3 because it exhibits thermoresponsive hydrophobicity changes across the lower critical solution temperature (LCST) of 32 °C,4 which is near body temperature. Drug and gene carriers using PIPAAm and its copolymers have been investigated as thermally modulated drug delivery systems.5,6 PIPAAmconjugated enzyme or peptides have been investigated to control activity through external temperature change7,8 and biosensor or diagnostic systems using the phase transition behavior of PIPAAm have also been investigated.9,10 In addition, PIPAAm-modified cell culture dishes that control cell adhesion and detachment have been developed.11,12 Such thermoresponsive cell culture dishes can fabricate “cell-sheets” and some types of cell sheets have been used in clinical practice as an effective therapy for patients.13−15 Additionally, PIPAAm has been used as a chromatography stationary phase whose surface properties can be modulated by changing column temperature,16 eliminating the need for organic solvents in the mobile phase during elution, because the © 2014 American Chemical Society

interaction between stationary phase and analytes can be modulated simply by changing column temperature. This thermoresponsive chromatography system preserves the activity of biological analytes, such as proteins, while conventional chromatography systems that use organic solvents may deactivate the analytes. The separation efficiency of the chromatography system depends on the PIPAAm configuration on the stationary phase surfaces, mainly silica bead surfaces.17 Thus, several PIPAAm modification methods have been investigated to develop an effective PIPAAm-modified stationary phase. In one example, a PIPAAm brush-modified stationary phase prepared through surface-initiated atom transfer radical polymerization (ATRP) exhibited a high separation efficiency.18 ATRP has been widely used for surface modification of substrates by polymers, because the densely packed polymer chains can be grafted on the substrate, while chain length and density can be precisely modulated.19−25 Previous reports have suggested that protein adsorption behavior can be effectively modulated by changing polymer Received: August 18, 2014 Revised: September 11, 2014 Published: September 13, 2014 3846

dx.doi.org/10.1021/bm5012163 | Biomacromolecules 2014, 15, 3846−3858

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Figure 1. Scheme for preparing P(IPAAm-co-AMPS-co-tBAAm) brush-grafted silica beads through surface-initiated ATRP as thermoresponsive protein adsorption materials containing a strong acid group (A) and illustration of thermally modulated interactions between protein, biomolecules, and copolymer brushes (B).

brush length and density.24−29 Using ATRP to prepare PIPAAm-modified silica beads as a thermoresponsive chromatographic stationary phase enables very large amounts of PIPAAm to be grafted onto silica bead surfaces, which is attributed to the densely packed PIPAAm structure, leading to strong interactions with analytes.18 Furthermore, by introducing ionic groups into the PIPAAm brush, thermoresponsive ion exchange chromatography has been developed, which can modulate electrostatic interactions with ionic biomolecules. Cationic or anionic copolymer brushes were prepared by copolymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA),30,31 N,N-dimethylaminopropylacrylamide (DMAPAAm),32 or acrylic acid (AAc)33,34 into a PIPAAm copolymer brush. These acidic or basic properties can be modulated by changing the external temperature, because protonation or dissociation of the ionic groups can be modulated by changing vicinal hydrophobicity35−37 induced dehydration of the copolymer. In addition, we have developed temperature-modulated protein adsorption materials using similar thermoresponsive copolymer brush-modified beads.32,38 In this application, proteins were adsorbed on a copolymer brush through both electrostatic and hydrophobic interactions. In contrast, in ordinary ion exchange chromatography matrices, strong ion exchange group-modified beads are widely used because of their strong electrostatic interaction with analytes. Thus, we have prepared a thermoresponsive copolymer brush possessing quaternary amine groups by copolymerization of 3-acrylamidopropyl trimethylammonium chloride (APTAC) with the thermoresponsive copolymer brush.39 As a result, the copolymer brush exhibited strong electrostatic interactions with acidic biomolecules compared with a brush possessing tertiary amine groups. From this viewpoint, incorporation of strongly acidic groups such as

sulfonic acid into a copolymer brush would be an effective approach for preparing effective thermoresponsive cationexchange matrices. However, polymerization of acidic monomers by ATRP is difficult, because acidic monomers deactivate the ATRP catalyst, leading to poor polymerization.40 In the present study, we have prepared poly(IPAAm-co-2acrylamido-2-methylpropanesulfonic acid (AMPS)-co-tert-butylacrylamide (tBAAm) brush-modified silica beads through surface-initiated ATRP. To prevent deactivation of the ATRP catalyst, the pH of the ATRP reaction solution was adjusted to 7.0. Characterization of these prepared copolymers and beads was performed by observing the phase transition profiles, CHNS elemental analysis, X-ray photoelectron spectroscopy (XPS), and gel permeation chromatography (GPC). Chromatographic analysis was also performed using the beads as chromatographic packing materials.

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EXPERIMENTAL SECTION

Materials. IPAAm was kindly provided by Kohjin (Tokyo, Japan) and purified by recrystallization from n-hexane. AMPS, tertbutylacrylamide (tBAAm), and acrylic acid were purchased from Tokyo Chemical Industry (Tokyo, Japan). AMPS was used as received. tBAAm was purified by recrystallization from acetone. Polymerization inhibitor in AAc was removed by passing it through an inhibitor remover column (Sigma, St. Louis, MO, U.S.A.). Ethylenediamine-N,N,N′,N′-tetraacetic acid disodium salt dehydrate (EDTA 2Na), formic acid, formaldehyde, tris(2-aminoethyl)amine (TREN), sodium hydroxide, acetone, methanol, dichloromethane, toluene (dehydrate), CuCl, CuCl2, 2-propanol (HPLC grade), α-chloro-pxylene, catecholamine derivatives, steroids, uracil, and phosphate buffer powder (66.7 mM, pH 7.0) were purchased from Wako Pure Chemicals (Osaka, Japan). Tris(2-N,N-dimethylaminoethyl)amine (Me6TREN) was synthesized according to the methods described in a previous report.41 ((Chloromethyl)phenylethyl)trimethoxysilane (mixed m,p isomers) was obtained from Gelest (Morrisville, PA, U.S.A.). A cellulose dialysis membrane (molecular weight cut off, 3847


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MWCO, 1 kDa) for removing ATRP catalysts was purchased from Spectrum Laboratories (Rancho Dominguez, CA, U.S.A.). Silica beads (diameter 5 μm, pore size 300 Å, and specific surface area 100 m2/g) were obtained from Chemco Scientific (Osaka, Japan). Proteins were obtained from Sigma. A stainless steel column (50 × 4.6 mm i.d.) was purchased from GL Science (Tokyo, Japan). Initiator Immobilization on Silica Beads Surface. ATRP initiator was immobilized onto silica beads surface through a silanization reaction, as shown in Figure 1. In this study, silica beads were used as base materials. In the past, we have investigated various stationary phases as base materials for thermoresponsive chromatography columns, such as polystyrene beads,42 poly(hydroxy methacrylate) beads,43 and monolithic silica rod columns.44 Silica beads exhibited a relatively high separation efficiency compared with those of polymer-based beads. Also, grafting of copolymer onto silica bead surfaces is relatively easy compared with grafting onto monolithic silica rod surfaces. Thus, in this study, we used silica beads as base materials and, in particular, beads with pore sizes 30 nm in diameter, because this relatively large pore diameter is suitable for the separation of large molecules, such as proteins. Additionally, grafting with polymer using surface-initiated ATRP tends to block pores,45 supporting the need to use silica beads with large pores. Silica beads (31.4 g) were washed with hydrochloric acid at 90 °C for 3 h. The beads were rinsed with large amounts of pure water until the pH of the supernatant became neutral. Beads were filtered and dried overnight in a vacuum oven at 150 °C. The washed beads (30.6 g) were then put into a roundbottomed 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 mM), and the solution (621 mL) was poured onto the beads in the flask. The suspension of beads was stirred for 16 h at room temperature. After the reaction, beads were filtered, rinsed sequentially with toluene, methanol, dichloromethane, and acetone, and dried for 3 h in a vacuum oven at 110 °C. Copolymer Brush Grafting Through ATRP. A thermoresponsive anionic copolymer brush possessing a strongly acidic moiety was grafted onto silica bead surfaces through surface-initiated ATRP. For ATRP, a 2-propanol/water 90:10 (v/v) mixture was used as the reaction solvent to resolve the AMPS. Additionally, the pH of the reaction solution was adjusted to 7.0 to avoid deactivation of the ATRP catalyst.46 The AMPS feed composition was varied from 0 to 10 mol % and tBAAm composition was set at 20 mol %. The total monomer concentrations were set at 0.3 and 0.6 M to prepare short and long copolymer brushes, respectively. The typical procedure for an AMPS feed composition of 5 mol %, was as follows. AMPS (1.34 g, 6.45 mmol) was dissolved in 21.4 mL of water, and the pH was adjusted to 7.0 by adding concentrated sodium hydroxide solution (10 M). IPAAm (2.19 g, 19.4 mmol, 75 mol %) and tBAAm (0.656 g, 5.16 mmol, 20 mol %) were dissolved in 38.5 mL of 2-propanol in a 100 mL flask, and 4.28 mL of the prepared AMPS solution was added to the monomer solution. Dissolved oxygen was removed by argon gas bubbling with stirring for 1 h. CuCl (84.7 mg, 0.86 mmol), CuCl2 (11.5 mg, 0.086 mmol), and Me6TREN (0.22 g, 0.959 mmol) were added to the reaction solution under an argon atmosphere. The initiator-modified beads (1.0 g) were placed into a 50 mL glass vessel. These and the sealed flask containing ATRP reaction solution were placed into a glovebag. Alternating cycles of vacuum and a flow of argon gas in the glovebag were repeated until the oxygen concentration in glovebag was 0.1%. The ATRP solution was then poured into the initiator beads within the glass vessel, and the latter was sealed in the glovebag. The reaction solution was continuously shaken for 16 h at 25 °C. After the reaction, the beads were washed sequentially with acetone, methanol, 50 mM EDTA solution, and water, and dried for 3 h in a vacuum oven at 25 °C. To compare the prepared beads with those possessing a weak acidic group, P(IPAAmco-AAc-co-tBAAm) brush-modified beads were prepared using the same procedure, except that AAc was used as the acidic monomer instead of AMPS. Characterization of Beads with Copolymer Brush. To determine the amounts of initiator and grafted copolymer on silica

bead surfaces, elemental analysis was performed with a CHNS elemental analyzer (PE 2400 series; PerkinElmer, Waltham, MA, U.S.A.). The amount of initiator on silica bead surfaces (g/m2) was estimated using the following equation.

immobilized initiator =

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

(1)

where %CI is the carbon percentage obtained from elemental analysis, %CI(calcd) is the calculated carbon percentage in the initiator, and S is the bead surface area (100 m2/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) − %C I/%C I(calcd)) × S

(2) 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. To determine the molecular weight and polydispersity index (PDI) of the grafted copolymer, the copolymer was recovered from the silica beads surface. The copolymer-grafted beads were immersed in 10 mol/L sodium hydroxide solution, with stirring, overnight. The solution was neutralized with hydrochloric acid and then filtered. The filtrate was dialyzed using a dialysis membrane (MWCO: 1 kDa) for 7 days by changing the water daily. After purification, copolymer was obtained by lyophilization. Number-averaged molecular weight (Mn) and PDI of the copolymer were measured using a GPC system (GPC8020 model II: columns TSKgel SuperAW2500, TSKgel SuperAW3000, and TSKgel SuperAW4000, Tosoh, Tokyo, Japan). DMF containing 50 mM lithium chloride was used as a mobile phase. A calibration curve was obtained using poly(ethylene glycol) and poly(N-isopropylacrylamide) standards. Elution profiles of the copolymers were observed by refractometer. The grafted copolymer density was obtained by 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 Mn of the grafted copolymer. The surface elemental composition was measured by XPS (K-Alpha, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Peak deconvolution of carbon C 1s signals was performed to obtain information about the chemical bonds. Copolymer Synthesis by ATRP. To characterize the copolymer properties, solution-phase ATRP was performed using similar ATRP conditions, except that α-chloro-p-xylene (50 μL, 53.3 mg, 379 μmol) was used instead of the initiator-modified beads. After ATRP, the reaction solution was dialyzed through a dialysis membrane (MWCO: 1 kDa) against EDTA solution for 2 days by changing the EDTA solution daily and against pure water for 5 days to remove the ATRP catalyst. The purified solution was lyophilized and copolymer was determined. Copolymer Characterization. Molecular weight and PDI of the copolymer was measured by the GPC system. The composition of each monomer in the copolymer was obtained by 1H NMR (UNITYINOVA 400 MHz spectrometer, Varian, Palo Alto, CA, U.S.A.) using deuterium oxide as a solvent, according to previous reports.47,48 The phase transition profile of the copolymer was measured by observing the transmittance of the copolymer solution. Copolymer solution (10 mg/mL) was prepared by dissolving the copolymer into 66.7, 33.3, and 16.7 mM phosphate buffer solutions (pH 7.0). Transmittance of the copolymer at 600 nm was monitored using a UV/visible spectrometer (V-530; JASCO, Tokyo, Japan) while 3848


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Table 1. Characterization of Thermoresponsive Strong Acidic Copolymer P(IPAAm-co-AMPS-co-tBAAm) LCSTd

IPAAm/AMPS/tBAAm (molar ratio) code

a

0.3IPAStB-0 0.3IPAStB-1 0.3IPAStB-3 0.3IPAStB-5 0.3IPAStB-7 0.3IPAStB-10 0.6IPAStB-0 0.6IPAStB-1 0.6IPAStB-3 0.6IPAStB-5 0.6IPAStB-7 0.6IPAStB-10

b

in feed

in copolymer

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 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

82.1/0/17.9 82.5/0.33/17.2 77.9/1.83/20.3 76.3/2.14/21.5 76.5/5.59/17.9 74.2/7.91/17.9 80.7/0/19.3 n.d. n.d. n.d. n.d. 73.1/8.95/18.0

Mn

c

Mw/Mn

5200 5500 5400 5600 5500 4400 7500 8500 8500 9100 9300 9600

1.30 1.28 1.31 1.27 1.28 1.27 1.32 1.29 1.24 1.28 1.27 1.33

c

in 66.7 mM PB

in 33.3 mM PB

in 16.7 mM PB

15.7 16.0 20.0 22.0 23.6 28.0 17.2 18.8 21.6 23.8 n.d. n.d.

17.4 18.6 22.5 25.8 31.3 34.7 18.3 19.4 22.2 25.4 28.9 n.d.

18.9 19.5 23.1 27.3 30.2 35.0 17.9 20.1 22.3 25.7 29.8 35.5

All samples were named using the monomer abbreviation and feed molar composition of AMPS. “IP”, “AS”, and “tB” represent IPAAm, AMPS, and tBAAm, respectively. bDetermined by 1H NMR measurement using deuterium oxide as an NMR solvent. cMeasured by GPC using DMF containing 50 mM LiCl with poly(ethylene glycol) standards. dDefined as the temperature where the sample solution had a transmittance of 90%. a

Figure 2. Phase transition profile of poly(IPAAm-co-AMPS-co-tBAAm) at various phosphate buffer concentrations (pH 7.0). (A, B, and C) 0.3IPAstB copolymers (Table 1) in 66.7, 33.3, and 16.7 mM PB, respectively. (D, E, and F) 0.6IPAstB copolymers in 66.7, 33.3, and 16.7 mM PB, respectively. heating the solution at 0.20 °C/min. The LCST value was determined as the temperature where there was 90% transmittance. Temperature-Modulated Elution of Biomolecules. The copolymer modified beads were packed into a stainless steel column (50 × 4.6 mm i.d.) by flowing water/methanol mixture solvent at 350 kg/cm2 for 1 h, as previously reported.18 The column was connected to an HPLC system controlled by Borwin software (JASCO). Catecholamine derivatives (DOPA, adrenalin, and tyramine) were used as basic small molecular samples to investigate the electrostatic interactions with acidic copolymer brushes. DOPA (1.26 mg), adrenaline (14.4 mg), and tyramine (26.6 mg) were dissolved in 10

mL of phosphate buffer solution containing 2.7 mg/mL Na2SO3. Hydrophobic steroids (hydrocortisone and dexamethasone) were used as hydrophobic small molecule samples to investigate the hydrophobic interactions with the copolymer brush. Hydrocortisone (4.47 mg) and dexamethasone (5.58 mg) were dissolved in 10 mL of ethanol. The properties of these analytes are shown in Table S1. Phosphate buffer solutions (pH 7.0) at various concentrations (66.7, 33.3, 16.7 mM) were used as mobile phases at a flow rate of 1.0 mL/min. Column temperature was modulated by a thermostated bath with a water circulator (CTA400; Yamato, Tokyo, Japan). Elution behaviors of catecholamine derivatives and hydrophobic steroids were observed at 3849


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Table 2. Elemental Analyses of P(IPAAm-co-AMPS-co-tBAAm) Brush-Grafted Silica Beads by an XPS Take-Off Angle of 90° atomc (%) code

a

C

initiator modified 0.3IPAStB-1 0.3IPAStB-3 0.3IPAStB-5 0.3IPAStB-7 0.3IPAStB-10 0.3IPACtB-5 0.6IPAStB-1 0.6IPAStB-3 0.6IPAStB-5 0.6IPAStB-7 0.6IPAStB-10 0.6IPACtB-5 calcd of IPAAmb calcd of AMPSb calcd of tBAAmb calcd of AAcb

19.0 58.4 55.6 54.7 52.1 53.2 59.7 67.0 67.7 67.1 67.2 66.1 66.6 75.0 53.8 77.8 60

± ± ± ± ± ± ± ± ± ± ± ± ±

N 0.37 1.25 1.28 4.59 0.74 1.14 2.50 5.43 0.51 1.47 3.98 0.75 0.78

0.47 8.67 7.79 6.46 7.41 7.03 4.90 8.92 10.2 9.80 9.98 9.78 10.1 12.5 8.33 11.1 0

± ± ± ± ± ± ± ± ± ± ± ± ±

O 0.30 0.18 0.09 2.24 0.54 0.86 0.35 0.76 0.47 0.78 0.45 0.44 0.39

49.1 21.0 23.6 28.1 26.1 25.8 27.7 16.9 15.2 16.3 16.2 17.0 16.1 12.5 0.33 11.1 40

± ± ± ± ± ± ± ± ± ± ± ± ±

Si 0.72 0.87 0.84 8.28 0.64 1.30 0.23 5.74 0.87 0.77 0.87 0.28 0.13

29.7 10.8 11.4 10.6 13.2 11.4 12.4 4.89 5.03 5.06 4.43 4.26 5.92

± ± ± ± ± ± ± ± ± ± ± ± ±

Cl 0.49 0.65 1.02 1.96 0.27 0.61 0.19 0.18 0.21 0.51 2.87 0.33 0.18

1.77 0.77 1.30 n.d. 0.73 1.17 0.86 1.18 1.25 0.85 0.95 0.96 1.33

S

± 0.55 ± 0.37 ± 0.85 ± ± ± ± ± ± ± ± ±

0.44 0.11 0.37 0.59 0.09 0.47 0.22 0.13 0.25

n.d. 0.37 0.39 0.21 0.54 1.46 0.10 1.10 0.60 0.81 1.30 1.91 n.d.

± ± ± ± ± ± ± ± ± ± ±

N/C ratio 0.64 0.67 0.37 0.97 0.77 0.17 0.41 0.57 0.70 0.53 0.46

0.0556

0.0247 0.148 0.140 0.118 0.142 0.132 0.137 0.133 0.151 0.146 0.149 0.148 0.151 0.167 0.155 0.143

All samples were named using the monomer abbreviation and feed molar composition of AMPS or AAc. “IP”, “AS”, “AC”, and “tB” represent IPAAm, AMPS, AAc, and tBAAm, respectively. bEstimated atomic composition of each monomer. cData from three separate experiments are shown as mean ± SD. a

Table 3. Characterization of P(IPAAm-co-AMPS-co-tBAAm) Brush-Modified Beads elemental composition (%)

code

a

initiator modified 0.3IPAStB-1 0.3IPAStB-3 0.3IPAStB-5 0.3IPAStB-7 0.3IPAStB-10 0.3IPActB-5 0.6IPAStB-1 0.6IPAStB-3 0.6IPAStB-5 0.6IPAStB-7 0.6IPAStB-10 0.6IPActB-5

C

b

H

4.74 ± 0.02 17.1 16.4 16.4 16.1 16.0 16.2 18.5 18.2 18.0 17.9 18.2 18.2

± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.07 0.02 0.05 0.19 0.08 0.14 0.07 0.17 0.06 0.56 0.19

b

N

0.37 ± 0.10 2.31 2.11 2.01 2.08 2.17 2.28 2.61 2.64 2.56 2.64 2.71 2.59

± ± ± ± ± ± ± ± ± ± ± ±

0.12 0.05 0.01 0.17 0.08 0.02 0.02 0.03 0.03 0.02 0.08 0.03

b

S

0.11 ± 0.03

n.d.

± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.00 0.05 0.20 0.44 0.08 0.13 0.19 0.24 0.36 0.77 0.12

2.74 2.61 2.70 2.51 2.46 2.10 2.90 2.74 2.67 2.65 2.65 2.43

immobilized initiatorc (μmol/m2)

b

0.05 0.05 0.12 0.07 0.03 0.01 0.03 0.06 0.03 0.04 0.09 0.08

grafted copolymerc (mg/m2)

Mnd

Mw/Mnd

graft density (chains/nm2)

2.66 2.48 2.49 2.40 2.40 2.41 3.04 2.97 2.93 2.91 3.02 2.95

10900 11900 11600 12000 10800 9650 12500 12500 12000 14600 14500 10600

1.32 1.53 1.62 1.85 1.26 1.30 1.37 1.39 1.56 1.33 1.60 1.37

0.147 0.126 0.128 0.120 0.134 0.150 0.146 0.143 0.146 0.120 0.125 0.167

4.60 ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.05 0.04 0.03 0.01 0.02 0.03 0.03 0.02 0.06 0.03

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

254 nm. All basic or neutral proteins (bradykinin, α-chymotrypsin, αchymotrypsinogen A, cytochrome c, hemoglobin, lysozyme, myoglobin, papain, ribonuclease A, and trypsin; 3.0 mg) were dissolved in 6 mL of PB. Various concentrations of PB (66.7, 33.3, and 16.7 mM) were used as mobile phases at a flow rate of 1.0 mL/min. Elution behaviors of these proteins were observed at 280 nm.

the total monomer concentration and y is the feed composition of AMPS; “IP”, “AS”, and “tB” denote IPAAm, AMPS, and tBAAm, respectively. 1H NMR measurements showed that the compositions of AMPS and tBAAm were slightly lower compared with the feed composition. Our previous report regarding copolymerization through ATRP indicated that compositions of acrylamide derivatives were almost the same as their feed compositions.32 However, the composition of each monomer in P(IPAAm-co-AMPS-co-tBAAm) differs slightly. We attribute the difference in composition to increased conversion with water. The ATRP in the study required 10 vol % water in the reaction solvents to dissolve AMPS and adjust pH, leading to a higher conversion and variable composition of these monomers.45,49 The composition of copolymer prepared at 0.6 M monomer concentration was not

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RESULTS AND DISCUSSION Characterization of Thermoresponsive Acidic Copolymer. Prepared thermoresponsive copolymers, P(IPAAm-coAMPS-co-tBAAm), were characterized by 1H NMR, GPC, and observing phase transition profiles in solution. Table 1 summarizes these data. The copolymers were named using the monomer abbreviation, monomer concentration in feed, and monomer feed composition, such as xIPAStB-y, where x is 3850


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Figure 3. Chromatograms of catecholamine derivatives separated by HPLC, for which the packing materials were P(IPAAm-co-AMPS-co-tBAAm) brush-grafted silica beads at various temperatures ((A) 0.3IPAStB-5, (B) 0.3IPAStB-3, (D) 0.6IPAStB-5, and (E) 0.6IPAStB-3 in Table 2), and P(IPAAm-co-AAc-co-tBAAm) brush-grafted silica beads ((C) 0.3IPACtB-5, (F) 0.6IPACtB-3) for comparison. Mobile phase was 66.7 mM phosphate buffer (pH 7.0). Peak Nos. 1, 2, and 3 represent DOPA, adrenaline, and tyramine, respectively.

Molecular weights of copolymers prepared at a 0.3 M monomer concentration were smaller than those prepared at 0.6 M, indicating that the molecular weight of the copolymer was modulated by changing the monomer concentration during ATRP. Although the monomer concentrations were relatively small compared with our previous report using a 1 M monomer concentration and 2-propanol as a solvent, a relatively large molecular weight of copolymer was obtained. This is attributed to an accelerated ATRP process and increased conversion in the presence of water in the reaction solvents.45,49 Phase transition profiles of the copolymer are shown in Figure 2. Phase transition profiles indicate that the phase transition temperature increased with increasing AMPS monomer composition and decreasing PB concentration. Additionally, copolymer containing a larger amount of AMPS did not exhibit total phase transition, which is attributed to the sulfonic acid moiety in the copolymer. The strong acid moieties in the copolymer conferred hydrophilicity on the copolymer, leading to restriction of dehydration. Additionally, inter- and intracopolymer electrostatic repulsions also restricted phase transition and aggregation of polymers. Thus, an excessive amount of AMPS in the copolymer is not suitable for thermal modulation of copolymer properties.

able to be determined because of low resolution in the NMR spectrum. GPC measurements showed that the copolymer was successfully synthesized with relatively controlled polymerization. We investigated various ATRP conditions for copolymerization of AMPS. ATRP using 2-propanol as a reaction solvent, which our previous reports used as an appropriate solvent for preparation of PIPAAm and its copolymer brush modified beads, was not sufficient for the copolymerization of P(IPAAm-co-AMPS-co-tBAAm), because of the lower solubility of AMPS in 2-propanol. Thus, we added water to 2-propanol and the mixed solvents (2-propanol:water 9:1 (v/v)) successfully dissolved all monomers and polymerization occurred. However, the molecular weight of the obtained copolymer was relatively low and decreased with increasing AMPS composition, probably because of deactivation of the ATRP catalysis by AMPS. Thus, we used an AMPS aqueous solution at pH 7.0. A previous report recommended more alkaline conditions, such as pH 8.5.46 However, alkaline conditions would dissolve the silica bead surface when grafting the copolymer. Thus, we used a neutral AMPS solution for copolymerization. As a result, using these ATRP conditions, the copolymerization was successfully performed and the molecular weight was independent of AMPS feed composition. 3851


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Figure 4. Temperature-dependent retention time changes of catecholamine derivatives on (A) a short P(IPAAm-co-AMPS-co-tBAAm) brush-grafted, silica bead packed column (0.3IPAStB-5 in Table 2), (B) a short P(IPAAm-co-AMPS-co-tBAAm) brush-grafted, silica bead packed column (0.3IPAStB-3 in Table 2), and (C) a short P(IPAAm-co-AAc-co-tBAAm) brush-grafted, silica bead packed column (0.3IPACtB-5 in Table 2) for comparison. The open circles represent DOPA; the closed triangles, adrenaline; the closed diamonds, tyramine. Numbers −1, −2, and −3 represent data using 66.7, 33.3, and 16.7 mM phosphate buffers (pH 7.0), respectively, as mobile phases.

Characterization of Thermoresponsive Beads. Prepared beads were characterized by XPS measurement. The elemental compositions of the beads are summarized in Table 2. The prepared beads were named the same way as copolymers in Table 1. Peak deconvolutions of C 1s peaks are shown in Figure S1. An additional peak in the spectrum of copolymer-modified beads, attributed to the CO bond, was observed in the higher binding energy region, while there was no peak observed for initiator-modified beads. This suggests that silica bead surfaces were modified with copolymer containing CO bonds. Regarding elemental composition, a larger carbon composition was observed in copolymer-modified beads compared with initiator-modified beads, indicating that copolymer was grafted onto the silica bead surface through surface-initiated ATRP, because the carbon element was attributed to the copolymer. Furthermore, silicon composition

decreased after ATRP, because grated copolymer on the silica bead surfaces blocked the detection of silica base materials. Sulfate composition, attributed to AMPS, was not correlated to AMPS feed composition, probably because of the small amount of sulfur in the copolymer compared with other elements. AMPS includes only one sulfur group in the monomer and the copolymer includes AMPS at less than 10 mol %. Thus, quantitative detection of sulfur in the copolymer by XPS analysis was difficult. CHNS elemental analysis of the beads was performed and is summarized in Table 3. Larger carbon contents were observed in all copolymer-modified beads, compared with initiatormodified beads, indicating that the copolymer was grafted onto the beads. Additionally, a larger carbon content was observed in copolymer prepared with a 0.6 M monomer solution compared with that prepared with a 0.3 M monomer solution. This result 3852


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small shoulder peaks, which could possibly be attributed to grafted copolymer inside the silica bead pores (Figure S2) Grafted copolymer density of the beads exceeded 0.10 chains/nm2, indicating that surface-initiated ATRP prepared a dense copolymer brush, possessing a strong acid moiety, on the silica bead surface.51 Elution Behavior of Basic Biomolecules. P(IPAAm-coAMPS-co-tBAAm) modified beads were evaluated as thermoresponsive chromatographic materials. The beads were packed into a stainless steel column and the elution behaviors of basic or hydrophobic biomolecules were observed by changing column temperature. Elution behavior of catecholamine was observed using 0.3IPAStB-5, 0.3IPAStB-3, 0.6IPAStB-5, and 0.6IPAStB-3 columns, because a higher AMPS composition (above 5 mol %) would not be sensitive to external temperature changes, as shown by the phase transition profiles of the copolymers. However, a lower AMPS composition, below 3 mol %, has only weakly acidic properties. Thus, 5 mol % or 3 mol % AMPS composition would be suitable for thermal modulation of beads surface properties. In addition, P(IPAAmco-AAc-co-tBAAm) brushes possessing weak acid moieties grafted onto beads and packed into columns (0.3IPACtB-5 and 0.6IPACtB-5) were used for comparison. Figure 3 shows temperature-dependent elution behavior of catecholamine on all columns using 66.7 mM PB as the mobile phase. Figures S3 and S4 show elution behaviors of catecholamine using 33.3 and 16.7 mM PB, respectively. In addition, temperature-dependent retention time alterations of catechol amines on short copolymer brush-modified bead columns 0.3IPAStB-5, 0.3IPAStB-3, and 0.3IPACtB-5 are shown in Figure 4. Those on the long copolymer brush-modified bead column are shown in Figure S5. All catecholamine derivatives were retained on the columns and retention of the times of these samples increased with the order of basicity. Additionally, larger AMPS composition columns, 0.3IPAStB-5 and 0.6IPAStB-5, exhibited longer retention times for catecholamine derivatives compared with 0.3IPAStB-3 and 0.6IPAStB-3 columns. These results indicate that the catecholamine derivatives were mainly retarded through electrostatic interaction between the sulfonic acid groups in AMPS and the amino group of catecholamine. Additionally, the retention time of tyramine increased with increasing temperature because of hydrophobic interactions between the copolymer and analytes. Grafted copolymer on silica bead surfaces was dehydrated and became hydrophobic with increasing temperature, leading to prolonged retention times of the relatively hydrophobic tyramine. Comparing the retention profiles of 0.3IPAStB-5 and 0.3IPACtB-5, longer retention times and higher resolution separations were obtained on 0.3IPAStB-5 than on 0.3IPACtB-5. This is attributed to the strongly acidic properties of the sulfonic acid group in AMPS compared with the carboxyl group in acrylic acid. The strongly acidic properties of the sulfonic acid group leads to strong electrostatic interactions between the grafted copolymer and analytes, which enables high resolution separation of basic analytes. Additionally, comparing elution behavior between short and long copolymer grafted beads, the latter exhibited longer retention times for catecholamine and wider peaks in the chromatograms. Smaller molecule analytes tend to diffuse further into the long copolymer brush than into the short copolymer brush, and analytes interact inside the brush, leading to longer retention times and broadened elution.18 Retention time increased with decreasing phosphate buffer concentration,

Figure 5. Chromatograms of hydrophobic steroids separated by HPLC, for which the packing materials were P(IPAAm-co-AMPS-cotBAAm) brush-grafted silica beads at various temperatures ((A) 0.3IPAStB-5, (B) 0.3IPAStB-3, (D) 0.6IPAStB-5, and (E) 0.6IPAStB-3 in Table 2) and P(IPAAm-co-AAc-co-tBAAm) brush-grafted silica beads ((C) 0.3IPACtB-5, (F) 0.6IPACtB-3) for comparison. Mobile phase was 66.7 mM phosphate buffer (pH 7.0). Peak No. 1 represents hydrocortisone; No. 2 represents dexamethasone.

indicates that a longer copolymer was grafted on 0.6IPAStB beads than on 0.3IPAStB beads. In addition, the sulfur content in the beads increased with increasing AMPS feed composition, indicating that AMPS composition in the grafted copolymer increased with increasing feed composition. The amount of grafted copolymer was calculated by carbon content. A larger amount of grafted copolymer was observed on the silica bead surface compared with those previously reported as prepared by conventional radical polymerization.50 To measure the molecular weight of grafted copolymer, copolymer was recovered from the silica beads. The molecular weight of the grafted copolymer on 0.6IPAStB beads was larger than that on 0.3IPAStB beads. The polydispersity index of retrieved copolymer was higher than that of copolymer prepared in solution ATRP (as shown in Table 1). This is probably because of the porous structure of the silica beads. Monomer and catalyst diffusion inside the pore is restricted compared with those outside the pores. Additionally, the polymerization proceeded at a relatively high rate, because water in the reaction solvent enhances catalytic activity. The enhanced polymerization rate would lead to a difference in molecular weight of grafted copolymer between the interior and exterior of the pores, leading to a relatively large PDI. Note that the GPC charts of recovered copolymer appear to show additional 3853


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Figure 6. Temperature-dependent retention time changes of hydrophobic steroids on (A) a short P(IPAAm-co-AMPS-co-tBAAm) brush-grafted, silica bead packed column (0.3IPAStB-5 in Table 2), (B) a short P(IPAAm-co-AMPS-co-tBAAm) brush-grafted, silica bead packed column (0.3IPAStB-3 in Table 2), and (C) a short P(IPAAm-co-AAc-co-tBAAm) brush-grafted, silica bead packed column (0.3IPACtB-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 mM phosphate buffer (pH 7.0) as mobile phases.

0.3IPAStB-5 column compared with those on the 0.3IPAStB3 and 0.3IPACtB-5 columns. This is because the larger amount of sulfonic acid in the 0.3IPAStB-5 copolymer imparted hydrophilic properties to the copolymer. Incorporation of ionic monomers into the copolymer makes it more hydrophilic, leading to a reduction in hydrophobic interactions with (hydrophobic) steroids. On longer copolymer brush-grafted beads, a relatively short retention time was observed compared with that on short copolymer-brush grafted beads, indicating that the former have relatively higher hydrophilic properties. Our previous reports indicated that longer PIPAAm or its copolymer brush tends to hydrate and become hydrophilic.52 In the same manner, a longer P(IPAAm-co-AMPS-co-tBAAm) brush tended to hydrate and become hydrophilic. Longer copolymer-brush bead columns exhibited broader peaks in the chromatograms. Steroids also tend to diffuse further inside long

because electrostatic interactions between the copolymer and analytes were enhanced at lower ion concentrations. To investigate the hydrophobic properties of the copolymer brushes, elution behaviors of hydrophobic steroids were observed. Figure 5 shows the temperature-dependent elution behaviors of hydrophobic steroids using 66.7 mM PB as the mobile phase. Figures S6 and S7 show the elution profiles using 33.3 and 16.7 mM PB, respectively. Figure 6 shows temperature-dependent retention time changes of hydrophobic steroids on short copolymer brushes. Figure S8 shows retention time changes on long copolymer brushes. On all columns, the retention time of steroids increased with increasing temperature. These results indicate that the grafted copolymer was dehydrated and become hydrophobic at elevated temperatures, leading to hydrophobic interactions between steroids and the copolymer. On short copolymer brush-grafted beads, slightly shorter steroid retention times were observed on the 3854


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Figure 7. Temperature-dependent peak area changes of proteins eluted from 0.3IPAStB-5: (A) bradykinin, (B) α-chymotrypsin, (C) αchymotrypsinogen A, (D) cytochrome c, (E) hemoglobin, (F) lysozyme, (G) myoglobin, (H) papain, (I) ribonuclease A, and (J) trypsin. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mM phosphate buffers (pH 7.0), respectively, as mobile phases.

Figure 8. Temperature-dependent peak area changes of proteins eluted from 0.3IPAStB-3: (A) bradykinin, (B) α-chymotrypsin, (C) αchymotrypsinogen A, (D) cytochrome c, (E) hemoglobin, (F) lysozyme, (G) myoglobin, (H) papain, (I) ribonuclease A, and (J) trypsin. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mM phosphate buffers (pH 7.0), respectively, as mobile phases.

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Figure 9. Temperature-dependent peak area changes of proteins eluted from 0.3IPACtB-5: (A) bradykinin, (B) α-chymotrypsin, (C) αchymotrypsinogen A, (D) cytochrome c, (E) hemoglobin, (F) lysozyme, (G) myoglobin, (H) papain, (I) ribonuclease A, and (J) trypsin. The open circles, the closed triangles, and the closed diamonds represent data using 66.7, 33.3, and 16.7 mM phosphate buffers (pH 7.0), respectively, as mobile phases.

Figure 10. Temperature-dependent elution of proteins from 0.3IPAStB-5; (A) α-chymotrypsinogen A, (B) lysozyme, (C) papain, and (D) myoglobin, obtained using 66.7 mM PB as the mobile phase.

0.6IPACtB-5, respectively. The peak areas of several proteins, α-chymotrypsinogen A, lysozyme, and papain, decreased with increasing temperature, indicating that these proteins adsorbed more onto copolymer brushes with increasing temperature. These results indicate that hydrophobic interactions attributed to the dehydration of copolymer at elevated temperatures contributed to adsorption of proteins. Further, protein adsorption was enhanced with decreasing PB concentration, indicating that electrostatic interactions between proteins and

copolymer brushes than into short copolymer brushes, leading to broadened elution peaks. To investigate protein adsorption properties relevant to protein purification applications, elution behaviors of neutral and basic proteins were observed. Figures 7, 8, and 9 show temperature-dependent peak area changes of proteins using 0.3IPAStB-5, 0.3IPAStB-3, and 0.3IPACtB-5, respectively. Figures S9, S10, and S11 show temperature-dependent peak area changes of proteins using 0.6IPAStB-5, 0.6IPAStB-3, and 3856


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suitable for thermal modulation of copolymer properties. CHNS elemental analysis of copolymer-modified beads and GPC measurement of grafted copolymer indicated that the copolymer was grafted onto the silica bead surface with a densely packed structure, exceeding 0.1 chains/nm2. Elution profiles of catecholamine derivatives indicate that the grafted copolymer brush interacts with basic analytes through strong electrostatic interactions, compared with the copolymer brushes possessing weak acidic groups. Hydrophobic interactions between the copolymer brush and analytes were modulated by changing temperature, leading to control of retention time and separation efficiency. Adsorption of basic proteins onto the copolymer brush was promoted by temperature increase, while adsorbed proteins were successfully recovered by reducing the temperature. These results indicate that P(IPAAm-co-AMPS-cotBAAm) brush-modified beads will be useful as thermoresponsive chromatography materials for separation of basic biomolecules.

Figure 11. Protein recoveries from the 0.3IPAStB-5 column by reducing column temperature. (A) lysozyme, (B) α-chymotrypsinogen A.

copolymer also contributed to adsorption. Thus, protein adsorption onto a P(IPAAm-co-AMPS-co-tBAAm) brush occurred through both electrostatic and hydrophobic interactions between proteins and the copolymer brush. In addition, changes in the peak area on longer copolymer-brush beads were smaller than those on shorter copolymer-brush columns. This was probably caused by the relatively hydrophilic properties of the long copolymer brush compared with the short copolymer brush, while the hydrophobicity of the copolymer brush was insufficient to cause protein adsorption. Figure 10 shows a temperature-dependent chromatogram for the proteins. The elution behavior of the neutral protein myoglobin is shown for comparison. Elution of α-chymotrypsinogen A and lysozyme was not observed at high temperature. However, a papain elution peak was observed at elevated temperature, although the peak width at high temperature was decreased, compared with that seen at low temperature. A previous report regarding chromatographic separation of papain indicated that papain eluted in two peaks; one being the native protein and the other denatured.53 In this case, one component adsorbed onto the copolymer brush and the other did not. Figure 11 shows the elution of adsorbed proteins with reducing column temperature. The results indicate that the P(IPAAm-coAMPS-co-tBAAm) brush modulated protein adsorption and desorption through changes in external temperature, which is expected to be useful in the purification of basic proteins. These results demonstrate that P(IPAAm-co-AMPS-cotBAAm) exhibited stronger electrostatic interactions with basic biomolecules, attributed to the presence of the sulfonic acid group. Hydrophobic interactions between the copolymer brush and analytes were modulated by changing temperature. In addition, adsorption/desorption of basic proteins onto the copolymer brush can be modulated simply by changing temperature. Thus, copolymer brush-modified beads are expected to be useful for thermally modulated cation exchange chromatography matrices for the separation of basic biomolecules.

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ASSOCIATED CONTENT

S Supporting Information *

Properties of the analytes, gel permeation chromatograms of the copolymers, XPS peak deconvolutions of the C 1s peaks, and chromatograms of catecholamine derivatives and hydrophobic steroids using low concentration PB as a mobile phase. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81-3-5367-9945, Ext. 6224, 6201. Fax: +81-3-33596046. E-mail: nagase.kenichi@ twmu.ac.jp. *E-mail: tokano@ twmu.ac.jp. Notes

The authors declare no competing financial interest.

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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; and subsidies from the Kumagai Foundation for Science and Technology.

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REFERENCES

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CONCLUSIONS Thermoresponsive copolymer brushes possessing sulfonic acid groups, P(IPAAm-co-AMPS-co-tBAAm), were successfully grafted onto a silica bead surface through surface-initiated ATRP to prepare strong anionic thermoresponsive chromatography matrices. Characterization of the copolymer showed that an excessive amount of AMPS restricted phase transition behavior of the copolymer because of its strongly hydrophilic properties. An approximately 5 mol % AMPS composition was 3857


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Thermoresponsive Anionic Copolymer Brushes Containing Strong

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