Preparation of Thermoresponsive Cationic Copolymer Brush Surfaces

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Preparation of Thermoresponsive Cationic Copolymer Brush Surfaces and Application of the Surface to Separation of Biomolecules 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, 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan, Department of Materials Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, and Department of Physical Pharmaceutical Chemistry, Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan Received December 28, 2007; Revised Manuscript Received February 8, 2008

We have prepared poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) brush-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) using the CuCl/CuCl2/Me6TREN catalytic system in 2-propanol at 25 °C for 16 h. The prepared temperature-responsive surfaces were characterized by chromatographic analysis using the modified silica beads as stationary phases. Chromatographic retention times for adenosine nucleotides in aqueous mobile phases were significantly increased compared to that previously reported for other cationic hydrogel surfaces, indicating that strong electrostatic cationic copolymer brush interactions occur between the surfaces and nucleotide analytes. Retention times for adenosine nucleotides significantly decreased with increasing column temperature, explained by the decreasing basicity in the copolymer with increasing temperature. Step-temperature gradients from 10 to 50 °C shorten ATP retention times. These results indicate that cationic copolymer brush surfaces prepared by ATRP can rapidly alter their electrostatic properties by changing aqueous temperature.

Introduction One of the most attractive stimuli-sensitive (“intelligent”) polymers is thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) and its derivatives. PIPAAm exhibits reversible temperature-dependent phase transition in aqueous solutions at its lower critical solution temperature (LCST) of 32 °C,1 and this intrinsic thermoresponsive property is widely used in biomedical applications, such as controlled drug and gene delivery systems,2–5 enzyme bioconjugates,6,7 microfluidics,8 cell culture substrates,9,10 and tissue engineering for regenerative medicine.11–15 Furthermore, a new form of hydrophobic chromatography utilizing PIPAAm on the stationary phase has been developed for thermally induced separation of bioactive compounds in aqueous milieu.16,17 This system is highly useful to control both stationary phase function and properties for highperformance liquid chromatography (HPLC) by changing only column temperature, with advantages in maintaining biological activity of peptides and proteins, and reduced pollution from organic mobile phases commonly used in reversed-phase chromatography. To improve separation efficiency for bioactive compounds, we have systematically investigated the preparation and characterization of a series of thermoresponsive polymer grafted surfaces as chromatographic stationary phases.16,17 PIPAAm-grafted silica bead surfaces have been prepared mainly by three kinds of grafting methods. First, by use of “grafting to” methods, PIPAAm-grafted silica beads were prepared by * Corresponding authors: Teruo Okano, [email protected]; Akihiko Kikuchi, [email protected]. † Tokyo Women’s Medical University. ‡ Tokyo University of Science. § Kyoritsu University of Pharmacy.

standard ester-amine coupling.18 This grafting method has the advantage of control over the molecular weights of grafted PIPAAm chains by adjusting relative ratios of monomers to chain transfer agent in polymerization. However, as in all “grafting to” methods, polymer graft density is limited because of sterically restricted reactivity limits with surface functional groups. A second “grafting from” method for PIPAAm uses surface-immobilized azo-initiator and crosslinker to prepare polymer layers with conventional radical polymerization.19 This method incorporates relatively large amounts of polymer onto the surfaces compared to “grafting to” methods. However, regulation of grafted polymer chain lengths (i.e., variable hydrogel layer thickness) is often difficult under these reaction conditions.19 Therefore, we have prepared well-defined PIPAAm-grafted surface on silica beads by surface-initiated atom transfer radical polymerization (ATRP).20,21 ATRP is an attractive polymer grafting method because it allows for preparation of surfaces with well-defined dense polymer brushe using surface-immobilized ATRP initiators.22–24 This method incorporates relatively large amounts of polymer onto surfaces compared to conventional methods, producing relatively strong interactions and stationary phase partitioning with analytes.21 Thus, densely PIPAAm-grafted chains allow improved control of strong hydrophobic interactions in aqueous milieu with changing temperature. Additionally, we have successfully separated bioactive ionic compounds, including catecholamines,25 angiotensin subtypes,26 and oligonucleotides27 with copolymer hydrogel modified surfaces and have introduced anionic charged monomer, acrylic acid (AAc)25,26 or cationic (N,N-dimethylamino)propylacrylamide (DMAPAAm) comonomers into PIPAAm.16,27 Previous

10.1021/bm701427m CCC: $40.75  2008 American Chemical Society Published on Web 03/21/2008

Thermoresponsive Cationic Copolymer Brush Surfaces

Biomacromolecules, Vol. 9, No. 4, 2008

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Figure 1. Scheme for preparation of P(IPAAm-co-DMAEMA) grafted silica bead surfaces using surface-initiated atom transfer radical polymerization (ATRP).

reports suggested a weakening of tertiary amine basicity in IPAAm-diethylamino(ethyl methacrylate) (DEAEMA) copolymers at elevated temperature, due to the decreased dielectric constant around the amino groups at higher temperature where the polymer loses hydration and becomes hydrophobic.28 Similarly, we have indicated that the charged density distribution of polymer-modified surfaces is reduced as polymer enters a hydrophobic, aggregated state at elevated temperature.25–27 We applied this behavior to modulate the electrostatic interactions from temperature-responsive ionic polymer-modified surfaces with charged bioactive compounds by temperature alteration alone. However, these surfaces were prepared by conventional radical polymerization, and the polymer graft amount of the surface was relatively low compared to surfaces prepared by ATRP. Thus, preparation of ionic copolymer-modified surfaces by ATRP would permit grafting of significantly larger amounts of ionic polymer onto surfaces compared to conventional radical polymerization. This would lead to improved control of strong electrostatic interactions with changing temperature and, hopefully, improved separations performance. In the present paper, we describe preparation of high-density thermoresponsive cationic copolymer brush comprising, P(IPAAm-co-2-(dimethylamino)ethylmethacrylate (DMAEMA)) on silica bead surfaces using a surface-initiated ATRP schematically shown in Figure 1. DMAEMA was used as a cationic monomer since the pKa of polyDMAEMA is 7.5,29 and the basicity of the copolymer would tend to decrease by temperature alteration at neutral pH. Characterization of the dense cationic copolymer brush surfaces on silica beads was investigated by chromatographic analysis using adenosine nucleotides and oligonucleotides as model analytes.

Experimental Section Materials. IPAAm was kindly provided by Kohjin Co. Ltd. (Tokyo, Japan) and recrystallized from n-hexane. DMAEMA, purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan), was purified by distillation at 46 °C (3 mmHg). CuCl and CuCl2 were purchased from Wako Pure Chemicals. Tris(2-aminoethyl)amine (TREN) was purchased from Acros Organics (Pittsburgh, PA). Formaldehyde, formic acid, and sodium hydroxide were purchased from Wako Pure Chemicals. Tris(2(N,N-dimethylamino)ethyl)amine (Me6TREN) was synthesized from TREN, according to previous reports.20,30 Silica beads (average diameter, 5 µm; pore size, 300 Å; specific surface area, 100 m2/g) were purchased from Chemco Scientific Co. Ltd. (Osaka, Japan). Hydrochloric acid, hydrofluoric acid, and ethylenediamine-N,N,N′,N′-tet-

raacetic acid disodium salt dehydrate (EDTA · 2Na) were purchased from Wako Pure Chemicals. 2-(m/p-Chloromethylphenyl)ethyltrichlorosilane was obtained from ShinEtsu Chemical Industry (Tokyo, Japan). 2-Propanol (HPLC grade) and toluene (dehydrate) were purchased from Wako Pure Chemicals. Adenosine nucleotides were purchased from Wako Pure Chemicals, and thymidine and adenine oligonucleotides were purchased from Operon (Tokyo, Japan). Preparation of ATRP Initiator-Immobilized Silica Beads. Adlayers of 2-(m/p-chloromethylphenyl)ethyltrichlorosilane as an ATRPinitiator were prepared as shown in the first step in Figure 1, according to previous reports.22,31 First, silica beads were washed in concentrated hydrochloric acid for 3 h at 90 °C and then rinsed with a large amount of distilled water repeatedly until washing water pH became neutral, followed by thorough drying under vacuum at 110 °C for 18 h. Formation of silane layers comprising the ATRP initiator on silica surfaces was performed as follows: Silica beads (12.4 g) were placed into a round-bottom flask and humidified at 60% relative humidity for 4.0 h, followed by the addition of 2.83 mL of 2-(m/p-chloromethylphenyl)ethyltrichlorosilane in 242 mL of dried toluene. Nitrogen gas was flowed over the reaction mixture for the first 5 min as HCl gas evolved and then the flask was sealed.22 The reaction proceeded at room temperature overnight under continuous stirring. ATRP initiatorimmobilized silica beads were collected by filtration and extensively rinsed with toluene, methanol, dichloromethane, and acetone and dried in a vacuum oven at 110 °C. Surface Modification of Silica Beads with Cationic Copolymer by ATRP. Cationic copolymer brushes composed of IPAAm and DMAEMA were prepared on ATRP-initiator immobilized silica beads by ATRP. A typical preparation procedure was as follows: The total monomer concentration was set at 1 mol/L with the following monomer composition in feed: IPAAm (4.62 g, 40.8mmol) and DMAEMA (338 mg, 2.15mmol) (monomer composition: DMAEMA 5 mol %) were dissolved in 42.9 mL of 2-propanol and deoxygenated by nitrogen gas bubbling for 30 min. Monomer composition in feed was changed as DMAEMA 5 mol %, 10 mol %, and 15 mol %, respectively. CuCl (84.7 mg, 0.86 mmol), CuCl2 (11.5 mg, 0.086 mmol), and Me6TREN (0.22 g, 0.959 mmol) were added under nitrogen atmosphere, and the solution was stirred for 20 min to form the CuCl/CuCl2/Me6TREN catalyst system. ATRP initiator-immobilized silica beads (1.0 g) were placed into a clean dry 50 mL glass vessel. Both monomer solution and the silica beads were placed into a glovebag purged with dry nitrogen gas by repeated vacuum and nitrogen flush (three times). The monomer solution was then poured into the glass vessel containing silica beads and sealed under nitrogen. The ATRP reaction proceeded for 16 h at 25 °C under continuous shaking on a shaker (SN-M40S, NISSIN, Tokyo, Japan). Copolymer-grafted silica beads were washed by ultrasonication in acetone for 30 min followed by centrifugation to

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remove unreacted monomers and ungrafted copolymers. This washing process by ultrasonication was repeated twice. Copolymer-grafted silica beads were further washed by sequential centrifugation and resuspension in methanol, 50 mM EDTA solution, and finally with Milli-Q water (prepared under ultrapure water purification systems, synthesis A10, Millipore (Billerica, MA)). Modified silica beads were filtered and rinsed with Milli-Q water and acetone and dried in a high vacuum oven at 50 °C for 5 h. Characterization of Initiator Immobilized Silica and Grafted PIPAAm. In order to determine the amount of ATRP-initiator and grafted copolymer on silica beads, the silica beads were subjected to elemental analysis using a CHN elemental analyzer varioEL (Elementar, Hanau, Germany). ATRP-initiator and copolymer (milligrams per square meter) on silica beads were calculated using the equations

ATRP-initiator )

%CI %CI(calcd)(1 - %CI ⁄ %CI(calcd))S

(1)

grafted copolymer ) %CC (2) %CC(calcd)(1 - %CC ⁄ %CC(calcd) - %CI ⁄ %CI(calcd))S where %C is the percent carbon increase as determined by elemental analysis, %C(calcd) is the calculated weight percent of carbon in initiator or copolymers, S is the specific surface area of the silica support in meters squared per gram (per manufacture’s data, i.e., 100 m2/g) and subscripts I and C denote initiator and copolymer, respectively. Grafted copolymer on silica bead surfaces was also retrieved and analyzed by gel permeation chromatography (GPC) to determine both the molecular weight and polydispersity index (PDI). Copolymer grafted silica bead surfaces were treated with concentrated hydrofluoric acid for 3 h and neutralized by the addition of sodium carbonate. The solution was filtered and dialyzed against Milli-Q water using a dialysis membrane (Spectra/Por standard regenerated cellulose dialysis membrane, molecular weight cut off (MWCO) 1000, Spectrum Laboratories Inc., Rancho Dominguez, CA) for 3 days with water changed daily, and the copolymer was recovered by freeze-drying. Number-average molecular weights and PDI values of the polymer were determined using the GPC system (Tosoh, Tokyo, Japan; columns TSKgel G3000H and TSKgel G4000H) controlled with an SC-8020 controller. A calibration curve was obtained using poly(ethylene glycol) standards. The flow rate was 1.0 mL/min using N,N-dimethylformamide (DMF) containing 100 mM LiCl as an eluent, column temperature was controlled at 45 °C using a column oven (CO-8020, Tosoh), and elution profiles were monitored with a refractometer (RI-8022, Tosoh). Graft density of PIPAAm on the silica beads surfaces was estimated using the equation

graft density )

mCNA Mn

(3)

where mc is the amount of grafted copolymer on the silica bead surfaces per unit area (g/m2), NA is Avogadro’s number, and Mn is the number average molecular weight of the grafted copolymer. Synthesis of Cationic Copolymer by ATRP. To characterized the cationic copolymer, P(IPAAm-co-DMAEMA) products with varying feed ratios (monomer composition in feed ranged from DMAEMA 5 mol %, 10 mol %, 15 mol %) were synthesized in solution-phase ATRP under similar conditions as copolymer grafting onto silica bead surfaces. Copolymerization was performed by the same protocol as grafting copolymer onto silica except that R-chloro-p-xylene (53.4 mg, 3.80 µmol) was added as an initiator in the reaction solution in place of initiator-immobilized silica beads. After the copolymerization, the solution was dialyzed against EDTA solution using a dialysis membrane (Spectra/Por standard regenerated cellulose dialysis membrane, MWCO 1000) for 3 days changing EDTA solution every day, followed by dialysis against Milli-Q water for 2 days, and the copolymer was obtained by lyophilization. Homopolymerization of PIPAAm and PDMAEMA was also performed by the similar protocols as in the previous report of ATRP of PIPAAm32 except that R-chloro-p-xylene

was used as the initiator. Typical preparation procedures were as follows: IPAAm (9.0 g, 79.6 mmol) or DMAEMA (12.5 g, 79.6 mmol) was dissolved in 11.5 mL of 2-propanol, and the solution was deoxygenated by nitrogen gas bubbling for 30 min. CuCl (39.4 mg, 0.40 mmol) and Me6TREN (91.5 mg, 0.40 mmol) were added under nitrogen atmosphere, and the solution was stirred for 20 min to form the CuCl/Me6TREN catalyst system. The reaction solution was divided into three glass vessel, then ATRP initiator, R-chloro-p-xylene (18.7 mg, 0.133 mmol), was added into the each reaction solutions. The ATRP reaction proceeded for a predetermined time period at 25 °C under continuous shaking. PIPAAm or PDMAEMA was obtained after dialysis and lyophilization. Characterization of Cationic Copolymer. Phase transitions of the cationic copolymer solutions in pure water were observed by optical transmittance changes. Solutions of P(IPAAm-co-DMAEMA) with varying DMAEMA amounts were prepared using 66.7 mM phosphate buffer at pH 7.0 (10 mg/mL). Optical transmittance changes of the polymer solutions were monitored at 600 nm with a UV-vis spectrometer (V-530, JASCO. Co. Ltd., Tokyo, Japan). The sample cell was thermostated with a Peltier-effect cell holder (EHC-477, JASCO) with a heating rate of 0.15 °C/min. The LCST was defined as the temperature at 90% transmittance of solution. DMAEMA content in the copolymers was determined by 1H NMR (UNITYINOVA 400 MHz spectrometer; Varian, CA) using D2O, calculated from the peak area of the singlet at 4.4 ppm from methylene protons adjacent to oxygen in DMAEMA and the overlapped peak at 1.2 ppm attributed to the resonance of the dimethyl protons of isopropyl groups in IPAAm units and methyl protons adjacent to main chain in DMAEMA. Temperature Modulated Elution of Anionic Bioactive Compounds. P(IPAAm-co-DMAEMA) grafted silica beads were packed into a stainless steel column (50 mm × 4.6 mm i.d.). A slurry of copolymer-grafted silica beads in water/methanol mixed solvents (1: 1) was poured into a slurry reservoir (TOSOH Co., Tokyo, Japan) connected to a stainless steel column. Water/methanol mixed solvent (1:1) was flowed through the slurry reservoir using an HPLC pump (PU-980 JASCO) at 350 kg/cm2 for 1 h, followed by equilibration with Milli-Q water for at least 12 h. Copolymer-grafted bead-packed columns were connected to an HPLC system (PU-980 and UV-970, JASCO) controlled by a personal computer with Borwin analysis software version 1.21 (JASCO). Adenosine nucleotide, adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) were all dissolved in 66.7 mM phosphate buffer solution (PBS, pH 7.0). Sample concentrations were 0.064 mg/mL for AMP, 0.112 mg/mL for ADP, and 0.24 mg/mL for ATP, respectively. All sample solutions were mixed together to produce chromatograms. PBS (66.7 mM, pH 7.0) was used as a mobile phase. Thermoresponsive elution behavior for adenosine nucleotides was monitored at 254 nm with a flow rate of 1.0 mL/min. Column temperature was controlled with a deviation of (0.1 °C using a thermostated water bath (RE206, Lauda, Germany). Adenosine oligonucleotides, p(dA)2, p(dA)3, p(dA)4, p(dA)5 were dissolved with PBS at concentrations of 0.0592, 0.0922, 0.125, and 0.158 mg/mL, respectively. All sample solutions were mixed together to produce chromatograms. Thymidine oligonucleotides p(dT)2, p(dT)3, p(dT)4, and p(dT)5 were dissolved in PBS at concentrations of 0.0573, 0.0894, 0.121, and 0.153 mg/mL, respectively. All sample solutions were also mixed together. Thermoresponsive elution behavior for oligonucleotides was monitored at 260 nm.

Results and Discussion Characterization of Thermoresponsive Cationic Copolymer. Characteristics of cationic copolymers of P(IPAAm-coDMAEMA) are summarized in Table 1. Prepared copolymers are abbreviated as ID-x where x represents the mole fraction of DMAEMA in the copolymer. Mole fraction of DMAEMA in the copolymer was larger than the feed composition, which was also observed in the copolymer prepared by conventional radical



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Table 1. Characterization of P(IPAAm-co-DMAEMA) Cationic Copolymer IPAAm/DMAEMA (molar ratio) code

in feed

in copolymera

Mnb

Mw/Mnb

LCSTc

ID-17 ID-20 ID-37 IP

95.0/5.0 90.0/10.0 85.0/15.0 100/0

83.0/17.0 79.7/20.3 63.1/36.9

6000 5600 3800 7200

1.36 1.37 1.55 1.19

40.7 56.1 64.6 31.5

a Determined by 1H NMR measurement. b Measured by GPC using DMF containing 100 mM LiCl with PEG standards. c Defined as the temperature at 90% transmittance in 66.7 mM phosphate buffer solution (pH 7.0).

Figure 2. Reaction-time-dependent changes in the molecular weight of PIPAAm and PDMAEMA: open circle, PIPAAm homopolymer; closed triangle, PDMAEMA homopolymer. Table 2. Characterization of PIPAAm and PDMAEMA Homopolymers

PIPAAm PDMAEMA

a

reaction time (h)

Mn

Mw/Mna

1 3 5 1 3 5

5200 11600 13600 9800 11700 12200

1.13 1.07 1.09 2.46 2.60 2.74

Measured by GPC using DMF containing 100 mM LiCl.

copolymerization.5 This is due to the higher reactivity ratio of DMAEMA compared to IPAAm.5 Furthermore, polydispersity of the copolymers increased and number averaged molecular weight decreased with increasing feed ratio of DMAEMA. Here, we used the well-known CuCl/CuCl2/Me6TREN catalyst system with 2-propanol as the solvent because this system was reported to produce controlled ATRP of IPAAm.32 In the previously reported ATRP polymerization procedure of DMAEMA, different amine ligands were used to control polymerization, such as N,N,N′,N′,N″-pentamethyldiethylentriamine (PMDETA),33 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA),34 and 2,2′-bipyridine (bpy).35 Thus, Me6TREN has not been used as an appropriate amine ligand in ATRP for DMAEMA. To investigate the control of polymerization of DMAEMA using this catalysis system, polymerizations of PIPAAm and PDMAEMA were performed using the CuCl/Me6TREN catalyst with the same polymerization conditions as for PIPAAm.32 Time-dependent changes in number averaged molecular weight of PIPAAm and PDMAEMA are shown in Figure 2. The number-averaged molecular weight and the polydispersity index of the obtained polymers are summarized in Table 2. At the onset of the ATRP reaction, the number averaged molecular weight of PDMAEMA was larger than that of PIPAAm, which indicates that the monomer reactivity of DMAEMA is higher than that of IPAAm. However, the molecular weight of

Figure 3. Phase transition profiles for P(IPAAm-co-DMAEMA) in 66.7 mM phosphate buffer solution (pH 7.0): closed circle, PIPAAm homopolymer; open triangle, ID-17; closed diamond, ID-20; open square, ID-37.

PDMAEMA leveled off after 3 h of polymerization while that of PIPAAm increased monotonously throughout the polymerization duration. In addition, polydispersity for PDMAEMA was significantly higher than that for PIPAAm. These results suggest that the ATRP catalyst (CuCl/Me6TREN in 2-propanol) was inadequate for controlled polymerization of DMAEMA. Thus, the polydispersity of P(IPAAm-co-DMAEMA) increases while molecular weight decreases with increasing feed ratio of DMAEMA as shown in Table 1. LCST of the copolymers increased with DMAEMA content in the copolymer as seen in Figure 3. This is because DMAEMA in the copolymer prevents dehydration of the copolymer, similarly to previously reported anion pH/temperature-sensitive copolymers containing acrylic acid (P(IPAAm-co-AAc)).25 The phase transition temperature of the copolymer was relatively high compared to the (P(IPAAm-co-AAc)) copolymers.25 Higher phase transition temperature is not suitable for temperature-responsive chromatography since the column temperature must be elevated to modulate the electrostatic property. In our previous reports, the hydrophobic monomers, n-butylmethacrylate (BMA)16,27 or tert-bultylacrylamide (tBAAm),26 were incorporated into the copolymers to modulate bulk polymer hydrophobicity while reducing the polymer phase transition temperature. However, we believe that the phase transition temperature of the grafted cationic copolymers would be reduced by dense grafting to surfaces. We have already shown that dense polymer grafts on silica bead surfaces contribute increased hydrophobicity compared to polymers in dilute solutions.21,31 Thus, the dense grafting of cationic copolymers on the silica bead surfaces should make the surfaces hydrophobic, and introduction of additional hydrophobic comonomers would not be required. Characterization of Initiator Surfaces and CopolymerGrafted Silica Beads. Initiator-immobilized silica beads and copolymer-grafted silica beads were characterized by elemental analyses. Detected elements (C, H, N) and amounts of immobilized initiator are summarized in Table 3. We prepared thermoresponsive copolymer grafted silica bead surfaces via surface-initiated ATRP. These surfaces are abbreviated as IDB-x where x represents feed composition of DMAEMA. B in IDB denotes the “brush”. Amounts of grafted copolymer were almost 10 times greater than that of polymer hydrogel-modified silica beads prepared by conventional radical polymerization we reported previously.26 This is due to the graft configuration of polymer brush prepared by surface-initiated ATRP. Polymer brushes prepared by surface-initiated ATRP form densely packed configurations compared to that by other radical polymerizations since the initiation efficiency of ATRP is quite high. Thus, P(IPAAm-co-DMAEMA) is densely grafted on

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Table 3. Characterization of P(IPAAm-co-DMAEMA) Grafted Silica Beads elemental composition (%)a code

C

H

N

immobilized initiator (µmol/m2)

initiator immobilized silica IDB-5 IDB-10 IDB-15 IPB-0

4.0

0.5

0.3

3.97

18.3 17.8 18.3 19.7

3.0 2.9 3.1 3.3

2.8 2.6 2.7 3.3

a

Determined by elemental analyses.

b

grafted polymer (mg/m2)

3.20 3.06 3.23 3.59

Mnb

Mw/Mnb

graft density (chains/nm2)

13700

3.67

0.14

19500

3.74

0.11

Determined by GPC using DMF containing 100 mM LiCl.

Figure 4. Chromatograms of adenosine nucleotides separated on HPLC columns at various temperatures: (a) IDB-5, (b) IDB-10, and (c) IDB-15 columns at various temperatures. Mobile phase is 66.7 mM phosphate buffer solution (pH 7.0). Peaks are as follows: 1, AMP; 2, ADP; 3, ATP.

silica bead surfaces, leading to significantly large amounts of grafted copolymer on these surfaces. In order to characterize the chain lengths and graft densities of copolymers on the silica surfaces, we confirmed the molecular weight of grafted copolymer by GPC after cleaving chains from silica beads with hydrofluoric acid. These data are also summarized in Table 3 (GPC charts of cleaved polymer are shown in Supporting Information). The polydispersity index of the cleaved copolymer was larger than that of copolymers prepared in solution. We have suggested that the larger polydispersity was attributed to the porous geometry of the silica beads.21,31 Polymerization reaction inside the pores is limited by monomer transport compared to outer exposed surfaces. In addition, propagation of the polymer chains from the initiator inside the pores is also restricted to the pore diameter. These factors lead to large polydispersity of grafted copolymers on porous silica bead surfaces. ElutionBehaviorofAdenosineNucleotidesfromP(IPAAmco-DMAEMA) Brush Surfaces. To investigate the interfacial electrostatic properties of the P(IPAAm-co-DMAEMA) brush surfaces, we observed elution behavior of the adenosine nucleotides from the copolymer grafted silica beads used as chromatographic stationary phases. Parts a-c of Figure 4 show chromatograms for adenosine nucleotides at various temperatures for IDB-5, IDB-10, and IDB-15 bead-packed columns, respectively, using PBS (66.7 mM, pH 7.0) as a mobile phase. Figure 5 show changes in retention times with temperatures on these columns. These analytes (AMP, ADP, and ATP) have the

same adenosine nucleotide chemistry but different numbers of anionic phosphate units. Retention times increased with increasing number of phosphate units in the analytes indicating that adenosine nucleotides interact with P(IPAAm-co-DMAEMA) brush surfaces primarily through electrostatic interactions. Retention times for adenosine nucleotides were significantly longer compared to that previously reported for a different cationic hydrogel surface,16 indicating that strong electrostatic interactions occur between these surfaces and analytes. This may be due to the larger amount of ion-exchange groups on the silica bead surfaces. As mentioned above, amounts of grafted copolymer were almost 10 times greater than surfaces prepared by conventional radical polymerization.26 Thus, larger amounts of amino groups on the silica bead surfaces interact with adenosine nucleotide analytes, leading to significantly longer retention times. Retention times for adenosine nucleotides significantly decreased with increasing column temperature. The behavior is explained by the decrease in the copolymer basicity with increasing temperature. Previous reports suggested a weakening of amine basicity in IPAAm copolymers with increasing temperature due to decreased dielectric constant around the dehydrated polymer amino groups at higher temperature where the polymer becomes hydrophobic.28 To investigate temperaturedependent surface hydrophilic/hydrophobic alterations, we observed elution behavior of hydrophobic steroids, hydrocortisone and prednisolone, from the surfaces at various temperatures. Figure 6 shows the van’t Hoff plots for these steroids, which exhibit a relationship between analyte retention and column temperature. The retention factor k′ value was defined as k′ ) (Rt - R0)/R0, where Rt is the retention time for known sample at predetermined temperature and R0 is the retention time of uracil as the initial standard. The ln k′ values remained almost constant on IDB columns with increasing temperature while the ln k′ values decreased on unmodified silica beads columns. This behavior was explained by the gradual increase in hydrophobicity on the IDB surface with increasing temperature and hydrophobic interaction between surface and analyte. In the case of unmodified silica beads, ln k′ decreased with decreasing 1/T (increasing temperature), since the adsorption of molecules on the stationary phase decreases, while the solubility of analyte in the mobile phase increases with increasing temperature.18 On the contrary, our previous reports suggested that retention times for steroids using PIPAAmmodified columns increased with increasing temperature, due to dehydration of grafted PIPAAm and increase in its hydrophobic interactions with steroids.16 Similarly, the cationic copolymer brushes on these bead surfaces were dehydrated with increasing temperature, and hydrophobic interactions from the surfaces retarded the steroids, although the hydrophobicity of cationic copolymer brush was relatively weak compared to the PIPAAm brush. Thus, the cationic copolymer brush surfaces gradually become hydrophobic with increasing temperature, and



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Figure 5. Temperature-dependent retention time changes of adenosine nucleotides on (a) IDB-5, (b) IDB-10, and (c) IDB-15 columns: closed circle, AMP; open triangle, ADP; closed diamond, ATP.

Figure 7. Chromatograms of (a) thymidine olgionucleotides and (b) adenosine oligonucleotides separated on ID-10 columns at various temperatures. Mobile phase is 66.7 mM phosphate buffer solution (pH 7.0). Peaks: (a) 1, p(dT)2; 2, p(dT)3; 3, p(dT)4; 4, p(dT)5; (b) 1, p(dA)2; 2, p(dA)3; 3, p(dA)4; 4, p(dA)5.

Figure 6. van’t Hoff plots of steroids on (a) IDB-5, (b) IDB-10, and (c) IDB-15 columns: open circle, hydrocortisone; closed triangle, prednisolone.

the temperature responsive hydrophobicity alteration on the surfaces leads to changes in the dissociation of amino groups in the copolymer brush. Elution Behavior of Oligonucleotides from P(IPAAm-coDMAEMA) Brush Surfaces. The cationic copolymer brush columns separated anionic adenosine nucleotides through relatively strong electrostatic interactions. This implies that the cationic copolymer brush columns would also separate large anionic molecules such as oligonucleotides with strong electrostatic interactions. Figure 7 shows chromatograms for thymidine oligonucleotides and adenine oligonucleotides at various temperatures for IDB-10 columns, using PBS (66.7 mM, pH 7.0) as the mobile phase. Retention times for both oligonucleotides were related to the number of repeating units, namely, the larger the molecular weight of oligonucleotides the longer the retention times. Thus, separation of oigonucleotides was achieved through electrostatic interactions rather than a size exclusion mode. Retention times for oligonucleotides decreased with increasing temperature though decrease in retention time

was small compared to that for adenosine nucleotides. Temperature-responsive oligonucletoide elution behavior is different from that of the smaller adenosine nucleotides. This is probably due to enhanced hydrophobic interactions with oligonucleotides on the cationic surfaces. Our previous report dealing with terminally modified copolymer surfaces (i.e., poly(IPAAm-co-DMAPAAm-co-BMA) sparsely grafted surface) suggested that oligonucleotides were separated with hydrophobic interactions, since retention times increased with increasing column temperature (i.e., dehydration of copolymer).27 In the present study, oligonucleotides interacted with IDB surfaces through both electrostatic and hydrophobic interactions. Electrostatic interaction decreases while hydrophobic interaction increases with increasing temperature, and overall retention time of oligonucleotides decreased slightly with temperature. Active Modulation of Analyte Elution through a StepTemperature Gradient. Cationic copolymer brush surfaces separate anionic bioactive compounds with strong electrostatic interactions, since a large amount of ion-exchange groups are contained in the copolymer brush surfaces. However, this also increases total elution times. As the surface electrostatic property alteration with temperature is reversible phenomena, steptemperature gradient effects were exploited to shorten total analysis time. Figure 8 shows effects of step temperature gradient application on elution of adenosine nucleotides on IDB10 columns. After identical elution of AMP and ADP through relatively stronger electrostatic interaction at 10 °C, column temperature was elevated to 50 °C by changing the thermostated water bath set at 50 °C. Retention time for ATP shortened with narrower peak widths. This result indicated the effectiveness of the thermoresponsive electrostatic property of P(IPAAm-co-

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can reversibly modulate interfacial electrostatic properties for these cationic copolymer brush surfaces and produce the most effective smart surface for aqueous thermally modulated separations to date. Acknowledgment. Part of the present research was financially supported by the Development of New Environmental Technology Using Nanotechnology Project of the National Institute of Environmental Science (NIES), commissioned from the Ministry of Environment, Japan. We appreciate Professor D. W. Grainger, University of Utah, for his technical comments and English editing. K.N. acknowledges research fellowship from the NIES. Figure 8. Effect of step-temperature gradient on adenosine nucleotide elution from the IDB-10 column. Mobile phase is 66.7 mM phosphate buffer solution (pH 7.0). Peaks: 1, AMP; 2, ADP; 3, ATP.

DMAEMA) brush surfaces. In conventional ion-exchange chromatography, elution of ionic compounds is modulated by increasing salt concentration in the mobile phase.36 However, this leads to several unwanted issues such as inactivation of analytes, a desalting process, and tedious operations. By contrast, using this thermoresponsive cationic column, elution of analytes can be modulated only by changing column temperatures without changing eluent composition. Baselines for the chromatograms immediately become steady after elevation of column temperature. This may be due to the graft configuration of the cationic copolymer. Our previous report indicated that PIPAAm-hydrogel surfaces required long equilibration times after changing temperatures, due to the restriction of graft chain mobility and hysteresis issues.17 On the contrary, in the case of cationic copolymer brush surfaces, polymer chains were not restricted and tend to collapse on the surface, leading to rapid alteration of surface properties, especially as cationic charges are restored and become repulsive. Thus, P(IPAAm-coDMAEMA) brush surfaces are seem as a most effective separation surface, rapidly altering their electrostatic properties solely with temperature.

Conclusions Silica beads grafted with cationic copolymer brushes comprising P(IPAAm-co-DMAEMA) were prepared by surface-initiated ATRP, and characterized for their thermoresponsive electrostatic interaction with analyte by aqueous chromatographic analysis using them as a stationary phase. Amounts of grafted cationic copolymer were significantly increased and retention times for adenosine nucleotides were significantly longer than those of our previously reported hydrogel-modified silica beads, since ATRP provides a densely graft configuration of copolymer and large amounts of amino group included in the grafted copolymer. Retention times for adenosine nucleotides significantly decreased with increasing temperature due to the decrease in the copolymer basicity with increasing copolymer hydrophobicity. Decreases in retention times for oligonucleotides by increasing temperature were relatively small compared to that for adenosine nucleotides. This is because the oligonucleotides were retarded by both the polymer hydrophobic and electrostatic interactions, and these effects counteract each other. Sudden increases in column temperature significantly shorten analyte retention times for ATP, producing a longer total analysis time. These results suggest that rapid temperature changes

Supporting Information Available. GPC molecular weight chart for PIPAAm cleaved from silica bead surfaces. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Preparation of Thermoresponsive Cationic Copolymer Brush Surfaces

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