Dehydration of

Sep 10, 2008 - UniVersity, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan. ReceiVed June 20, 2008. ReVised Manuscript ReceiVed July 31, 2008...
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Langmuir 2008, 24, 10981-10987

10981

Influence of Graft Interface Polarity on Hydration/Dehydration of Grafted Thermoresponsive Polymer Brushes and Steroid Separation Using All-Aqueous Chromatography Kenichi Nagase,† Jun Kobayashi,† Akihiko Kikuchi,*,‡ Yoshikatsu Akiyama,† Masahiko Annaka,§ 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, Department of Chemistry, Kyushu UniVersity, 6-10-1 Hakozaki, Higashi, Fukuoka 812-8581, Japan, and Faculty of Pharmacy, Keio UniVersity, 1-5-30 Shibakoen, Minato, Tokyo 105-8512, Japan ReceiVed June 20, 2008. ReVised Manuscript ReceiVed July 31, 2008 We have prepared poly(N-isopropylacrylamide) (PIPAAm) brush-grafted surfaces with varied temperature-responsive hydrophobic properties through surface-initiated atom transfer radical polymerization (ATRP). These temperatureresponsive surfaces were characterized by chromatographic analysis using modified silica beads as a chromatographic stationary phase in aqueous mobile phase. Mixed silane self-assembled monolayers (SAMs) comprising ATRP initiator and silanes with various terminal functional groups were formed on the silica bead surfaces. IPAAm was then polymerized by ATRP using the CuCl/CuCl2/Me6TREN catalyst system in 2-propanol at 25 °C for 16 h. The chromatographic retention behavior of steroids on the resulting PIPAAm brushes made on more polar silane components was distinct from that on more apolar silane interfaces. Retention times for steroids on PIPAAm mixed apolar silane graft interfaces were significantly longer than those on analogous polar silane interfaces due to enhanced dehydration of PIPAAm brushes on apolar silane-grafted surfaces. Changes in retention factor, k′, on polar silane PIPAAm-grafted interfaces were relatively large compared to that on apolar PIPAAm grafted interfaces due to larger hydration/dehydration alterations of grafted PIPAAm brushes on the former surfaces. Applied step-temperature gradients from 50 to 10 °C show that PIPAAm brushes on polar silane interfaces tend to hydrate more, leading to shorter retention times. In conclusion, the polarity of the grafted interface significantly influences the grafted PIPAAm brush hydration/dehydration characteristics and subsequently also the temperature-modulated separation of bioactive compounds in all-aqueous chromatography.

Introduction Temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), modified surfaces have been widely applied in several biomedical research areas.1-4 PIPAAm is well-known to exhibit reversible temperature-dependent phase transitions in aqueous solutions at its lower critical solution temperature (LCST) of 32 °C.1 PIPAAm’s intrinsic thermoresponsive property is widely exploited in biomedical applications, such as controlled drug and gene delivery systems,5,6 enzyme bioconjugates,7,8 microfluidics,9,10 cell culture substrates,11,12 and tissue engineer* Corresponding author. Phone: +81-3-5367-9945 Ext. 6201. Fax: +813-3359-6046. E-mail: [email protected] or [email protected]. † Tokyo Women’s Medical University. ‡ Tokyo University of Science. § Kyushu University. | Keio University.

(1) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Part A 1968, 2, 1441–1455. (2) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 207, 1165–1193. (3) Shimizu, T.; Yamato, M.; Kikuchi, A.; Okano, T. Biomaterials 2003, 32, 2309–2316. (4) Yamato, M.; Okano, T. Mater. Today 2004, 7, 42–47. (5) Cammas, S.; Suzuki, K.; Sone, C.; Sakurai, Y.; Kataoka, K.; Okano, T. J. Controlled Release 1997, 48, 157–164. (6) Kurisawa, M.; Yokoyama, M.; Okano, T. J. Controlled Release 2000, 69, 127–137. (7) Chilkoti, A.; Chen, G.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 1994, 5, 504–507. (8) Matsukata, M.; Takei, Y.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Bioconjugate Chem. 1994, 116, 682–686. (9) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 1958–1961. (10) Idota, N.; Kikuchi, A.; Kobayashi, J.; Sakai, K.; Okano, T. AdV. Mater. 2005, 17, 2723–2727.

ing for regenerative medicine.13-15 Additionally, we have previously introduced PIPAAm and its derivatized chains onto chromatographic stationary phases to separate different bioactive analyte classes in aqueous milieu.2,16-19 To improve the separation efficiency of bioactive compounds, we have systematically investigated the preparation and characterization of a series of thermoresponsive polymer-grafted surfaces as chromatographic stationary phases.2,16 We have reported that polymer grafting appears to be an important determinant affecting these separations because PIPAAm graft configurations produced from different grafting methods greatly influence temperature-dependent aqueous wettability changes and solute elution behaviors.2,16-18 Terminally PIPAAm grafted surfaces showed larger contact angle changes than multipoint attached surfaces over a narrow (11) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571–576. (12) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506–5511. (13) Shimizu, T.; Yamato, M.; Isoi, Y.; Akutsu, T.; Setomaru, T.; Abe, K.; Kikuchi, A.; Umezu, M.; Okano, T. Circ. Res. 2002, 90, e40–e48. (14) Nishida, K.; Yamato, M.; Hayashida, Y.; Watanabe, K.; Yamamoto, K.; Adachi, E.; Nagai, S.; Kikuchi, A.; Maeda, N.; Watanabe, H.; Okano, T.; Tano, Y. N. Engl. J. Med. 2004, 351, 1187–1194. (15) Ohashi, K.; Yokoyama, T.; Yamato, M.; Kuge, H.; Kanehiro, H.; Tsutsumi, M.; Amanuma, T.; Iwata, H.; Yang, J.; Okano, T.; Nakajima, Y. Nat. Med. 2007, 13, 880–885. (16) Kikuchi, A.; Okano, T. Macromol. Symp. 2004, 207, 217–227. (17) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100–105. (18) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Anal. Chem. 1999, 71, 1125–1130. (19) Kanazawa, H.; Nishikawa, M.; Mizutani, A.; Sakamoto, C.; Morita-Murase, Y.; Nagata, Y.; Kikuchi, A.; Okano, T. J. Chromatogr., A 2008, 1191, 157–161.

10.1021/la801949w CCC: $40.75  2008 American Chemical Society Published on Web 09/10/2008

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Figure 1. Scheme for preparation of mixed silane SAMs comprising ATRP initiator and coadsorbed alkylsilanes with different terminal groups, and grafting of PIPAAm from the surfaces through ATRP.

temperature range, due to their mobility and dynamics motion of the grafted polymer chains.20,21 Additionally, we have previously demonstrated an accelerated shrinking behavior of thermoresponsive hydrogels by introducing PIPAAm graft chains with freely mobile ends into the cross-linked PIPAAm gels.22,23 Introduction of freely mobile PIPAAm graft chains to PIPAAm multipoint attached surfaces also results in increased temperature sensitivity, leading to larger wettability changes in response to temperature changes.21 Recently, we have prepared thermoresponsive surfaces by grafting PIPAAm using controlled free radical polymerization techniques,24,25 such as atom transfer radical polymerization (ATRP).26-29 ATRP is an attractive polymer grafting method allowing preparation of surfaces with well-defined polymer brushes using surface immobilized ATRP initiators.26-29 The methodology allows for control of graft chain length by varying the duration of the polymerization,24 and regulation of graft density by varying the concentration of ATRP initiator on surfaces.25 The PIPAAm brush grafted chromatographic stationary phase, prepared through surface initiated ATRP, exhibited strong temperature modulated hydrophobic interaction with analyte, due to the densely grafted configuration of PIPAAm.24 Additionally, we have also reported that grafted PIPAAm hydration/dehydration is significantly influenced by the interfacial polarity of the substrate, based on cell adhesion/detachment behavior on PIPAAm hydrogel modified polystyrene surfaces.12 This indicates that temperature-dependent aqueous wettability changes of PIPAAm grafted surfaces are influenced not only by graft configuration but also by the surface properties of the grafted substrate. Thus, in the cases of PIPAAm brushes used as a chromatographic stationary phase, the substrate interfacial polarity should also influence the phase transition behavior of PIPAAm brushes, observed as alterations in the separation of biomolecules. In this regard, we have investigated the effects of graft interfacial chemistry on PIPAAm hydration/dehydration properties observed through the separation of bioactive compounds at various temperatures in all-aqueous chromatography. Selfassembled silane monolayers, comprising mixed ATRP initiators and other silane coadsorbates with various terminal groups, were formed on silica bead surfaces. PIPAAm was grafted onto these surfaces through surface initiated ATRP. Characterization of the resulting PIPAAm brushes grafted onto mixed SAM interfaces

was investigated by using them as thermoresponsive aqueous chromatographic stationary phases for steroid separations.

Experimental Section Materials. IPAAm was kindly provided by Kohjin Co. Ltd. (Tokyo, Japan) and recrystallized from n-hexane. 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 a previous report.24,30 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′-tetraacetic acid disodium salt dehydrate (EDTA · 2Na) were purchased from Wako Pure Chemicals. (Chloromethylphenyl)ethyltrimethoxysilane (CMPETMS; ATRP - initiator), 3-aminopropyltrimethoxysilane (APTMS), N-methylaminopropyltrimethoxysilane (MAPTMS), (3glycidoxypropyl)trimethoxysilane (GPTMS), and n-propyltrimethoxysilane (PTMS) were obtained from Gelest Inc. (Morrisville, PA). 2-Propanol (HPLC grade) and toluene (dehydrate) were purchased from Wako Pure Chemicals. Steroids and uracil were also purchased from Wako Pure Chemicals. Formation of Mixed SAM Adlayers on Silica Bead Surfaces. Mixed SAMs comprising 2-(m,p-chloromethylphenyl)ethyltrimethoxysilane as a silane ATRP-initiator, and other alkylsilanes were prepared to modulate the interfacial polarity of PIPAAm-grafted interfaces (Figure 1). (3-Glycidoxypropyl)trimethoxysilane will (20) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Macromolecules 1994, 27, 6163–6166. (21) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Langmuir 1998, 14, 4657–4662. (22) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240–242. (23) Kaneko, Y.; Sakai, K.; Kikuchi, A.; Yoshida, R.; Sakurai, Y.; Okano, T. Macromolecules 1995, 28, 7717–7723. (24) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir 2007, 23, 9409–9415. (25) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir 2008, 24, 511–517. (26) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. ReV. 2004, 33, 14–22. (27) Xiao, D.; Wirth, M. J. Macromolecules 2002, 35, 2919–2925. (28) Tu, H.; Heitzman, C. E.; Braun, P. V. Langmuir 2004, 20, 8313–8320. (29) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J., II.; Lopez, G. P. Langmuir 2003, 19, 2545–2549. (30) Ciampolini, M.; Nardii, N. Inorg. Chem. 1966, 5, 41–44.

PIPAAm Brushes on Polar Silane Interfaces present hydrophilic (secondary hydroxyl) groups.31,32 3-Aminopropyltrimethoxysilane and N-methylaminopropyltrimethoxysilane represent relatively hydrophilic silanes,32,33 and n-propyltrimethoxysilane was used as a hydrophobic silane.34,35 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 the wash water pH became neutral, followed by thorough drying under vacuum at 110 °C for 18 h. Formation of mixed silane SAMs comprising ATRP initiator and 3-glycydoxypropyltrimethoxyslilane on the silica surfaces was carried out as follows: 3.2 g of the silica beads was placed into a round-bottomed flask and humidified at 60% relative humidity for 4.0 h; then the 0.647 mL (2.57 mmol) of ATRP initiator and 0.151 mL (0.856 mmol) of 3-glycydoxypropyltrimethoxysilane were mixed in 64 mL of dried toluene (the feed ratios of initiator to other silanes was 3:1) and poured into the flask and closed. Silanization reactions proceeded at room temperature overnight under continuous stirring. Mixed SAM immobilized silica beads were collected by vacuum filtration and extensively rinsed with toluene, methanol, dichloromethane, and finally acetone, followed by drying in a vacuum oven at 110 °C. Other mixed SAMs comprising ATRP initiator and all other silanes (e.g., 3-aminopropyltrimethoxysilane, N-methylaminopropyltrimethoxysilane, and n-propyltrimethoxysilane) were formed on silica bead surfaces in a similar manner. To compare the immobilized initiator and PIPAAm densities, control SAMs comprising silane ATRP initiator alone were also formed on silica bead surfaces. Surface Modification of Silica Beads with PIPAAm by ATRP. PIPAAm grafting from mixed SAM surfaces was performed in a similar manner to previous reports.25 IPAAm (4.86 g, 42.9 mmol) was dissolved in 42.8 mL of 2-propanol and deoxygenated with nitrogen gas bubbling for 30 min. 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 a nitrogen atmosphere, and the solution was stirred for 20 min to form the CuCl/CuCl2/Me6TREN catalytic system. Mixed SAM silane-immobilized silica beads (1.0 g) were placed into a 50 mL glass vessel. Both the monomer solution and the silica beads were placed into a glovebag and purged with dry nitrogen gas by vacuum and subsequent flushing with nitrogen three times. The monomer solution was then poured into the glass vessel containing the silica beads, and the flask was finally sealed under nitrogen. The ATRP reaction proceeded for 16 h at 25 °C under vigorous shaking. PIPAAm-grafted silica beads were then washed by ultrasonication in acetone for 30 min followed by centrifugation to remove unreacted monomer and ungrafted polymer. Washing was repeated two additional times. PIPAAm-grafted silica beads were further washed by sequential centrifugation and resuspension in methanol, 50 mM EDTA solution, and finally Milli-Q water (prepared with an ultrapure water purification systems, synthesis A10, Millipore (Billerica, MA)). Modified silica beads were filtered and finally rinsed with Milli-Q water and acetone, and dried under vacuum at 50 °C for 5 h. Characterization of Mixed SAM Modified Silica Beads and Grafted PIPAAm. Elemental analyses of ATRP initiator in mixed SAMs on silica beads were performed using organic halogens and sulfur analysis (Yanako, Kyoto, Japan) and with an ion chromatography system ICA-2000 (TOA DKK, Tokyo, Japan). The amount of immobilized ATRP initiator on mixed silica beads (µmol/m2) was calculated using the following equation:

immobilized ATRP initiator ) %Cl(106) (1) MI × %Cl(calcd) × (1 - %Cl/%Cl(calcd)) × S where %Cl is the percent chloride as determined by elemental analysis, %Cl(calcd) is the calculated weight percent of chloride in (31) Tsukruk, V. V.; Luzinov, I.; Julthongpiput, D. Langmuir 1999, 15, 3029– 3032. (32) Lee, M. H.; Brass, D. A.; Morris, R.; Composto, R. J.; Ducheyne, P. Biomaterials 2005, 26, 1721–1730. (33) Siqueira Petri, D. F.; Wenz, G.; Schunk, P.; Schimmel, T. Langmuir 1999, 15, 4520–4523. (34) Karch, K.; Sebastian, I.; Halasz, I. J. Chromatogr. 1976, 122, 3–16. (35) Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. 1992, 64, 2783–2786.

Langmuir, Vol. 24, No. 19, 2008 10983 initiator subtracted by the weight of methoxy groups, MI is the molecular weight of initiator, and S is the specific surface area of the silica support in m2/g (per manufacture’s data). In order to determine the amount of grafted PIPAAm, silica beads were analyzed using the CHNS elemental analyzer 2400II (PerkinElmer Inc., MA). The concentration of SAMs and PIPAAm (mg/m2) on silica beads was calculated using the following equations:

%Cs(103) %Cs(calcd) × (1 - %Cs ⁄ %Cs(calcd)) × S (2)

modified SAMs )

grafted PIPAAm ) %Cp(103) %CP(calcd) × (1 - %Cp/%Cp(calcd) - %Cs/%Cs(calcd)) × S (3) where %C is the percent carbon increase as determined by elemental analysis, %C(calcd) is the calculated weight percent of carbon in SAMs subtracted by the weight of methoxy groups or monomer, and the subscripts s and p denote SAMs and PIPAAm, respectively. To determine PIPAAm graft density on silica bead surfaces, grafted PIPAAm on the bead surfaces was chemically cleaved, retrieved, and analyzed by gel permeation chromatography (GPC) to determine both molecular weight and polydispersity index (PDI). PIPAAmgrafted 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, while the water was changed every day, and the copolymer was recovered by freeze-drying. Number-average molecular weights and PDI values of recovered graft polymers were determined using a 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) molecular weight standards. The GPC flow rate was 1.0 mL/min using N,Ndimethylformamide (DMF) containing 100 mM LiCl as a mobile phase; the 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). The graft density of PIPAAm on the silica bead surfaces was estimated using the follow equation24,25

graft density )

mCNA Mn

(4)

where mc is the amount of grafted PIPAAm 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 PIPAAm. Temperature Modulated Elution of Steroid Analytes. PIPAAm grafted silica beads were packed into a stainless steel column (50 mm × 4.6 mm i.d.). Slurries of PIPAAm grafted silica beads in water/methanol mixed solvents (1:1) were poured into a slurry reservoir (TOSOH Co., Tokyo, Japan) connected to a stainless steel column. Water/methanol mixed solvents (1:1) were 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. PIPAAm 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). Urasil was dissolved with Milli-Q water at a concentration of 0.083 mg/mL, and used as a marker analyte. All steroids (5 mg) were completely dissolved with 3 mL of ethanol, and then 20 mL of Milli-Q water was added to yield a concentration of 0.217 mg/ mL. Thermoresponsive elution behavior for steroids was monitored at 254 nm with a flow rate of 1.0 mL/min. The column temperature

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Table 1. Characterization of Mixed SAM Adlayers Comprising an ATRP Initiator with Various Silanes, and the Resulting Grafted PIPAAm on Silica Beads elemental compositiona (%) code

silane component

C

H

N

Cl

IG75 IMA75 IA75 IP75 I100 IG75-IP IMA75-IP IA75-IP IP75-IP I100-IP

initiator/GPTMS initiator/MAPTMS initiator/APTMS initiator/PTMS initiator initiator/GPTMS initiator/MAPTMS initiator/APTMS initiator/PTMS initiator

1.18 ( 0.02 3.29 ( 0.09 3.09 ( 0.09 3.42 ( 0.22 3.59 ( 0.22 10.47 ( 0.18 17.23 ( 0.35 18.48 ( 0.10 19.92 ( 0.24 20.90 ( 0.26