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Effects of Graft Densities and Chain Lengths on Separation of Bioactive Compounds by Nanolayered Thermoresponsive Polymer Brush Surfaces 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 June 21, 2007. In Final Form: August 18, 2007 We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surfaceinitiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)ethyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 °C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by temperature-dependent elution behavior of hydrophobic steroids from the surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of solutes with the PIPAAm-grafted surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAAm at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAAm brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAAm graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAAm in dense-pack brush surfaces.
Introduction Modification with thin polymer layers is widely used in biomedical applications to provide functionality to surfaces. Using this method, various bioinert surfaces have been prepared with grafting of highly hydrophilic polymers, such as poly(ethylene glycol) (PEG),1,2 polyacrylamide (PAAm),3,4 and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).5,6 These surfaces are known to be resistant to nonspecific protein adsorption and cell adhesion, and are able to maintain the functionality of the * Corresponding author. Phone: +81-3-3353-8111, ext. 30233. Fax: +813-3359-6046. E-mail:
[email protected] (T.O.); kikuchia@ rs.noda.tus.ac.jp (A.K.). † Tokyo Women’s Medical University. ‡ Tokyo University of Science. § Kyoritsu University of Pharmacy. (1) Razatos, A.; Ong, Y.-L.; Boulay, F.; Elbert, D. L.; Hubbell, J. A.; Sharma, M. M.; Georgiou, G. Langmuir 2000, 16, 9155-9158. (2) Winblade, N. D.; Nikolic, I. D.; Hoffman, A. S.; Hubbell, J. A. Biomacromolecules 2000, 1, 523-533. (3) Ikada, Y.; Iwata, H.; Horii, F.; Matsunaga, T.; Taniguchi, M.; Suzuki, M.; Taki, W.; Yamagata, S.; Yonekawa, Y.; Handa, H. J. Biomed. Mater. Res. 1981, 15, 697-718. (4) Bamford, C. H., Al-Lamee, K. G. Polymer 1996, 37, 4885-4889. (5) Ishihara, K.; Ziats, N. P.; Tierney, B. P.; Nakabayashi, N.; Anderson, J. M. J. Biomed. Mater. Res. 1991, 25, 1397-1407. (6) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Langmuir 2005, 21, 59805987.
modified biomaterials for applications as membranes, bioimplants, and sensors.7,8 On the contrary, several attractive smart surfaces have been prepared by modifications using the thermoresponsive polymer poly(N-isopropylacrylamide) (PIPAAm) and its derivatives.9,10 PIPAAm exhibits a reversible temperaturedependent phase transition in aqueous solutions at its lower critical solution temperature (LCST) of 32 °C.11 PIPAAm’s unique thermoresponsive property is widely used in biomedical applications, such as controlled drug delivery systems12,13 and enzyme bioconjugates.14,15 Additionally, PIPAAm-modified surfaces have also been applied in biomedical research areas, (7) Kikuchi, A.; Okano, T. J. Controlled Release 2005, 101, 69-84. (8) Kato, K.; Uchida, E.; Kang, E. T.; Uyama, Y.; Ikada, Y. Prog. Polym. Sci. 2003, 28, 209-259. (9) Kikuchi, A.; Okano, T. Prog. Polym. Sci. 2002, 27, 1165-1193. (10) Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173-1222. (11) Heskins, M.; Guillet, J. E. J. Macromol. Sci. Chem. A 1968, 2, 14411455. (12) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Phys. 1990, 28, 923-936. (13) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y.; Bae, Y. H.; Kim, S. W. J. Biomater. Sci., Polym. Ed. 1991, 3, 155-162. (14) Chilkoti, A.; Chen, G.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 1994, 5, 504-507. (15) Matsukata, M.; Takei, Y.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Bioconjugate Chem. 1994, 116, 682-686.
10.1021/la701839s CCC: $40.75 © 2008 American Chemical Society Published on Web 12/18/2007
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such as microfluidics,16,17 cell culture substrates,18-20 and tissue engineering for regenerative medicine.21,22 Furthermore, we have previously introduced PIPAAm chains onto chromatographic stationary phases for the separation of different bioactive analyte classes in aqueous milieu.9,23-29 These systems are highly useful for controlling both stationary phase functions and properties for high-performance liquid chromatography (HPLC) by changing only column temperature, with the advantages of maintaining the biological activity of peptides and proteins and reducing pollution by organic mobile phases commonly used in reversephase chromatography. By systematic investigation into the preparation and characterization of a series of thermoresponsive polymer-modified surfaces as chromatographic stationary phases,9,23 we have recognized that polymer grafting appears to be an important determinant in these separations, since graft configurations of PIPAAm produced from different methods greatly influences temperature-dependent aqueous wettability changes and solute elution behaviors.9,23-26,29,30 Until now, we have prepared PIPAAm-grafted silica bead surfaces mainly with two kind of grafting methods. First, using “grafting to” methods, PIPAAm-grafted silica beads were activated by standard esteramine coupling. Terminally carboxylated PIPAAm was synthesized by semitelechelic polymerization and, as activated ester groups, reacted with amino groups on the silanized silica surfaces.24 This grafting method has the advantage of allowing for control of the molecular weights of grafted PIPAAm chains by adjusting the relative ratios of monomers to chain transfer agents in the bulk 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 a surfaceimmobilized azo-initiator and cross-linker to prepare polymer layers with conventional radical polymerization.25 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.25 Therefore, these methods offer little control over graft densities and chain lengths, which are believed to be two key parameters in determining the separation efficiency of biological compounds. Recently, we have prepared thermoresponsive surfaces by grafting PIPAAm to surfaces using controlled free radical polymerization techniques,26,29 such as atom transfer radical (16) Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 1958-1961. (17) Idota, N.; Kikuchi, A.; Kobayashi, J.; Sakai, K.; Okano, T. AdV. Mater. 2005, 17, 2723-2727. (18) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571-576. (19) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243-1251. (20) Akiyama, Y.; Kikuchi, A.; Yamato, M.; Okano, T. Langmuir 2004, 20, 5506-5511 (21) Yamato, M.; Okano, T. Mater. Today 2004, 7, 42-47. (22) Yang, J.; Yamato, M.; Okano, T. MRS Bull. 2005, 30, 189-193. (23) Kikuchi, A.; Okano, T. Macromol. Symp. 2004, 207, 217-227. (24) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100-105. (25) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Anal. Chem. 1999, 71, 1125-1130. (26) Idota, N.; Kikuchi, A.; Kobayashi, J.; Akiyama, Y.; Sakai, K.; Okano, T. Langmuir 2006, 22, 425-430. (27) Kobayashi, J.; Kikuchi, A.; Sakai, K.; Okano, T. Anal. Chem. 2001, 73, 2027-2033. (28) Yamanaka, H.; Yoshizako, K.; Akiyama, Y.; Sota, H.; Hasegawa, Y.; Shinohara, Y.; Kikuchi, A.; Okano, T. Anal. Chem. 2003, 75, 1658-1663. (29) Nagase, K.; Kobayashi, J.; Kikuchi, A.; Akiyama, Y.; Kanazawa, H.; Okano, T. Langmuir 2007, 23, 9409-9415. (30) Yakushiji, T.; Sakai, K.; Kikuchi, A.; Aoyagi, T.; Sakurai, Y.; Okano, T. Langmuir 1998, 14, 4657-4662.
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polymerization (ATRP).31-34 ATRP is an attractive polymer grafting method because it allows for preparation of surfaces with well-defined dense polymer brushes by using surfaceimmobilized ATRP initiators35-41 compared to the polymer brush surfaces prepared by conventional radical polymerizations.42 Furthermore, chain lengths of the grafted polymer can be regulated by varying the duration of the ATRP reactions.37,41 We have already prepared densely PIPAAm-grafted surfaces on silica beads as chromatographic matrices by surface-initiated ATRP,29 and this method incorporates relatively large amounts of polymer onto surfaces compared to conventional methods, producing relatively strong interactions and partitioning with analytes.29 On the other hand, other reports have suggested that polymer graft density can also be regulated by varying the concentration of ATRP initiator on surfaces, performed either by changing the composition of ATRP initiator in a self-assembled monolayer (SAM)43,44 or by deactivation of ATRP initiator with UV irradiation.45 These methodologies cannot be performed by conventional radical polymerization since initiator efficiency is relatively low compared to that of ATRP initiator. Thus, polymer grafting by ATRP can provide significant insight into the effects of the graft density and chain length of PIPAAm on the separation of bioactive compounds in thermoresponsive chromatography. In the present report, we investigated the effects of PIPAAm graft densities and chain lengths on the separation of bioactive compounds. PIPAAm graft density on silica bead surfaces was modulated by changing the composition of ATRP initiator in SAMs, and the chain lengths of grafted PIPAAm was controlled by changing the reaction time of ATRP. Characterization of the resulting PIPAAm brush surfaces on silica beads was investigated as a thermoresponsive aqueous chromatographic stationary phase for the separation of steroids. 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 Industries, Ltd. (Osaka, Japan). 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,Ndimethylamino)ethyl)amine (Me6TREN) was synthesized from TREN, according to previous reports.26,46 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 (31) Edmondson, S.; Osborne, V. L.; Huck, W. T. S. Chem. Soc. ReV. 2004, 33, 14-22. (32) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921-2990. (33) Masci, G.; Giacomelli, L.; Crescenzi V. Macromol. Rapid Commun. 2004, 25, 559-564. (34) Xia, Y.; Yin, X.; Burke, N. A. D.; Sto¨ver, H. D. H. Macromolecules 2005, 38, 5937-5943. (35) Huang, X.; Wirth, M. J. Anal. Chem. 1997, 69, 4577-4580. (36) Huang, X.; Doneski, L. J.; Wirth, M. J. Anal. Chem. 1998, 70, 40234029. (37) Xiao, D.; Wirth, M. J. Macromolecules 2002, 35, 2919-2925. (38) Tu, H.; Heitzman, C. E.; Braun, P. V. Langmuir 2004, 20, 8313-8320. (39) Bontempo, D.; Tirelli, N. AdV. Mater. 2002, 14, 1239-1241. (40) Balamurugan, S.; Mendez, S.; Balamurugan, S. S.; O’Brien, M. J., II; Lopez, G. P. Langmuir 2003, 19, 2545-2549. (41) Iwata, R.; Suk-In, P.; Hoven, V. P.; Takahara, A.; Akiyoshi, K.; Iwasaki, Y. Biomacromolecules 2004, 5, 2308-2314. (42) Zhao, B.; Brittain, W. J. Prog. Polym. Sci. 2000, 25, 677-710. (43) Jones, D. M.; Brown, A. A.; Huck, W. T. S. Langmuir 2002, 18, 12651269. (44) Feng, W.; Zhu, S.; Ishihara, K.; Brash, J. L. Biomaterials 2006, 27, 847855. (45) Yamamoto, S.; Ejaz, M.; Tsujii, Y.; Fukuda, T. Macromolecules 2000, 33, 5608-5612. (46) Ciampolini, M.; Nardii, N. Inorg. Chem. 1966, 5, 41-44.
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Figure 1. Schematic illustration of the preparation of mixed SAMs comprising ATRP initiator and dilute PIPAAm-grafted silica beads surfaces. from Wako Pure Chemicals. 2-(m/p-Chloromethylphenyl)ethyltrichlorosilane was obtained from ShinEtsu Chemical Industry (Tokyo, Japan). Phenethyltrichrolosilane was purchased from Fluorochem, Ltd. (Azusa, CA). 2-Propanol (HPLC grade) and toluene (dehydrate) were purchased from Wako Pure Chemicals. Steroids were also purchased from Wako Pure Chemicals. Preparation of ATRP Initiator-Immobilized Silica Beads. Mixed SAMs comprising 2-(m,p-chloromethylphenyl)ethyltrichlorosilane, as an ATRP-initiator, and phenethyltrichrolosilane were prepared to control the graft density of PIPAAm (Figure 1), according to previous reports.35,37 First, silica beads were washed in concentrated hydrochloric acid for 3 h at 90 °C, then rinsed repeatedly with distilled water, followed by thorough drying under vacuum at 110 °C for 18 h. For the formation of SAMs comprising 100% initiator on the silica surfaces, 11.0 g of the silica beads was placed into a round-bottomed flask and incubated at 60% relative humidity for 4.0 h, then 2.57 mL of 2-(m,p-chloromethylphenyl)ethyltrichlorosilane in 220 mL of dried toluene was poured into the flask. N2 gas was flowed over the reaction mixture for the first 5 min to flush out evolved HCl gas, and the flask was then sealed.37 The reaction proceeded at room temperature overnight under continuous stirring. ATRP-initiator 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. The formation of SAMs comprising 50% initiator on the silica surfaces was carried out as follows: 11.0 g of the silica beads was placed into a round-bottomed flask and humidified at 60% relative humidity for 4.0 h, then 1.28 mL of 2-(m,p-chloromethylphenyl)ethyltrichlorosilane and 1.14 mL of phenethyltrichrolosilane were added to 220 mL of dried toluene. The reaction proceeded in the same manner as described above. For the preparation of the 25% SAM, the mixed SAM was formed on silica beads surfaces in a similar manner except that the feed ratio of initiator to phenethyltrichrolosilane was 1:3. Surface Modification of Silica Beads with PIPAAm by ATRP. 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 nitrogen atmosphere, and the solution was stirred for 20 min to form the CuCl/CuCl2/Me6TREN catalytic system. ATRP initiator-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 glove bag 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 a predetermined period at 25 °C under vigorous shaking. PIPAAmgrafted silica beads were 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 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 under vacuum at 50 °C for 5 h. Characterization of Mixed SAMs and Grafted PIPAAm. Elemental analyses of ATRP initiator in mixed SAMs on silica beads were performed using organic halogens and a sulfur analyzer (Yanako, Kyoto, Japan) and with the ion chromatography system ICA-2000 (TOA DKK, Tokyo, Japan). Immobilized ATRP initiator on the silica beads (g/m2) was calculated from the chloride composition of initiator-immobilized silica beads using the following equation: Immobilized ATRP initiator ) %Cl (1) %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 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
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grafted PIPAAm, silica beads were analyzed using the CHN elemental analyzer varioEL (Elementar, Hanau, Germany). The amount of modified SAMs on silica beads (g/m2) was calculated using the following equation: Modified SAMs )
%Cs %Cs(calcd) × (1 - %Cs/%Cs(calcd)) × S (2)
Table 1. Characterization of Mixed SAMs on Silica Beads Comprising ATRP Initiator and Phenethyltrichlorosilane elemental compositiona code
C [%]
H [%]
N [%]
Cl [%]
immobilized initiator (µmol/m2)
INI100 INI50 INI25
3.1 3.5 3.5
0.4 0.4 0.4
