Langmuir 1998, 14, 4657-4662
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Graft Architectural Effects on Thermoresponsive Wettability Changes of Poly(N-isopropylacrylamide)-Modified Surfaces Taiji Yakushiji† and Kiyotaka Sakai† Department of Applied Chemistry, Faculty of Science and Technology, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8555, Japan
Akihiko Kikuchi,‡ Takao Aoyagi,‡ Yasuhisa Sakurai,‡ and Teruo Okano*,‡ Institute of Biomedical Engineering, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku, Tokyo 162-8666, Japan Received January 23, 1998. In Final Form: May 18, 1998 Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its critical temperature in aqueous media. When PIPAAm molecules are covalently attached to a solid surface, the graft configuration greatly affects the thermoresponsive wettability changes of PIPAAm-modified surfaces. Three types of temperature-responsive surfaces were prepared using PIPAAm grafts of different molecular architectures: PIPAAm terminally grafted surfaces, PIPAAm looped chain grafted surfaces using a copolymer of IPAAm and N-acryloxysuccinimide, and PIPAAm terminally grafted onto immobilized PIPAAm loops. These surfaces were prepared by changing the graft architecture as well as the density of PIPAAm chains to investigate temperature-responsive wettability changes. All surfaces showed temperature-responsive hydrophilic/hydrophobic surface property alterations demonstrated by observed large and discontinuous wettability changes. On both surfaces bearing terminally grafted PIPAAm, surface wettability changed dramatically over the range 32-35 °C, a temperature corresponding to the phase-transition temperature for PIPAAm in aqueous media. This implies that terminally grafted PIPAAm chains retain a highly mobile nature and respond rapidly to temperature changes. The loop-grafted surface showed relatively large wettability changes but had a slightly lower transition temperature (∼27 °C). This reduced transition temperature is likely due to restricted conformational transitions for this multipoint grafted PIPAAm. Combination of both loops and terminally grafted chains showed the largest surface free energy changes among three surfaces. We conclude that PIPAAm graft architecture strongly influences surface wettability responses to temperature changes due to differences in the dynamic motion of the grafted polymer chains.
Introduction Surface-grafted polymers have been shown to change their structure under the influence of external stimuli, specifically temperature,1-10 electric field,11 and pH.12,13 Such surfaces exhibit surface properties modulated by external stimuli with specific magnitudes and sensitivity. * To whom correspondence should be addressed. E-mail: tokano@ lab.twmc.ac.jp. † Phone: +81-3-5286-3216. Fax: +81-3-3209-7957. ‡ Phone: +81-3-3353-8111. Fax: +81-3-3359-6046. (1) Okahata, Y.; Noguchi, H.; Seki, T. Macromolecules 1986, 19, 493. (2) Yamada, N.; Okano, T.; Sakai, H.; Karikusa, F.; Sawasaki, Y.; Sakurai, Y. Makromol. Chem., Rapid Commun. 1990, 11, 571. (3) Gewehr, M.; Nakamura, K.; Ise, N.; Kitano, H. Makromol. Chem. 1992, 193, 249. (4) Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. J. Biomed. Mater. Res. 1993, 27, 1243. (5) Yoshioka, H.; Mikami, M.; Nakai, T.; Mori, Y. Polym. Adv. Technol. 1994, 6, 418. (6) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. Macromolecules 1994, 27, 6163. (7) Hosoya, K.; Kimata, K.; Araki, T.; Tanaka, N.; Frechet, J. M. J. Anal. Chem. 1995, 67, 1907. (8) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297. (9) Kanazawa, H.; Yamamoto, K.; Matsushima, Y.; Takai, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 100. (10) Kanazawa, H.; Kashiwase, Y.; Yamamoto, K.; Matsushima, Y.; Kikuchi, A.; Sakurai, Y.; Okano, T. Anal. Chem. 1997, 69, 823. (11) Osada, Y.; Hondo, K.; Ohta, M. J. Membr. Sci. 1986, 27, 327. (12) Iwata, H.; Oodate, M.; Uyama, Y.; Amemiya, H.; Ikada, Y. J. Membr. Sci. 1991, 55, 119. (13) Nagasaki, Y.; Kataoka, K. Trends Polym. Sci. 1996, 4, 59.
Thus, they can be used to change the interaction with adsorbing substances.2,4,5,7-10 Poly(N-isopropylacrylamide) (PIPAAm) exhibits thermally reversible soluble/insoluble changes in response to temperature changes across a lower critical solution temperature (LCST) at 32 °C in aqueous solution.14 Polymer chains of IPAAm show a hydrated, expanded conformation in water below the LCST, changing to compact forms above the LCST by sudden dehydration and inter- and intramolecular hydrophobic interaction. Utilizing this thermoresponsive property for PIPAAm, we have reported PIPAAm hydrogels as drug delivery matrixes15-17 to regulate on-off drug release, and bioconjugates18,19 as thermally recyclable bioreactor components. Recently, we have also investigated thermal modulation of hydrophilic/hydrophobic properties for PIPAAm-grafted surfaces.2,4,6,8 PIPAAm-grafted surfaces are hydrophilic (14) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. 1968, A2, 1441. (15) Bae, Y. H.; Okano, T.; Kim, S. W. J. Polym. Sci. Polym. Phys. 1990, 28, 923. (16) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y.; Bae, Y. H.; Kim, S. W. J. Biomater. Sci. Polym. Ed. 1991, 3, 155. (17) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. J. Biomater. Sci. Polym. Ed. 1992, 3, 243. (18) Matsukata, M.; Takei, Y.; Aoki, T.; Sanui, K.; Ogata, N.; Sakurai, Y.; Okano, T. J. Biochem. 1994, 116, 682. (19) Matsukata, M.; Aoki, T.; Sanui, K.; Ogata, N.; Kikuchi, A.; Sakurai, Y.; Okano, T. Bioconjugate Chem. 1996, 7, 96.
S0743-7463(98)00090-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/11/1998
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at temperatures below its LCST, and as temperature increases, a sudden and dramatic discontinuous water contact angle increase is observed, indicative of the increased surface hydrophobicity. Temperature-responsive surface property changes for terminally grafted polymer surfaces were kinetically more rapid and significant than that seen for multipoint grafted PIPAAm surfaces. Cells generally adhere and grow on hydrophobic surfaces but not on highly hydrophilic surfaces. Cultured cells on hydrophobic PIPAAm-grafted surfaces at 37 °C easily detached and were recovered by lowering the temperature.2,4,8 PIPAAm-grafted surfaces showed thermal modulation to control cell attachment and detachment without any deterioration of cellular function. We have also used thermoresponsive polymer-grafted surfaces for novel hydrophobic liquid chromatography matrixes9,10 where temperature control modulates separation and solutesurface partitioning. From these results, we have emphasized the importance of surface chemical composition and molecular architecture to regulate the grafted polymer hydration/dehydration as well as surface interactions. In particular, surfaces with well-designed architectures are important to achieve effective surface property changes that control surface interactions with molecules and/or cells. In this study, we have prepared three types of PIPAAmmodified surfaces (shown in Figure 1) in order to investigate the effect of grafted polymer architecture on the mode and the magnitude of thermoresponsive wettability changes. The three polymer grafts studied are (1) PIPAAm terminally grafted surfaces, (2) a copolymer of IPAAm with N-acryloxysuccinimide, P(IPAAm-co-ASI), a multipoint attached surface, and (3) PIPAAm terminally grafted on the P(IPAAm-co-ASI) surface using residual active ester groups. The PIPAAm graft chain density as well as the graft chain configuration greatly affect temperatureinduced surface wettability changes. Polymers grafted by a single terminal have a strong influence on thermoresponsive hydrophilic/hydrophobic surface property alterations because of the highly mobile nature of these polymer chains. Experimental Section Materials. All procedures for reagent purification and polymer syntheses were performed in a ventilated draft chamber to avoid inhalation of toxic materials. N-Isopropylacrylamide (IPAAm) was kindly provided by KOHJIN (Tokyo, Japan) and was purified by recrystallization from n-hexane and dried at 25 °C in vacuo. N-Acryloxysuccinimide (ASI) and dibenzoyl peroxide (BPO) were obtained from Kanto Chemicals (Tokyo, Japan). N,N-Dimethylformamide (DMF), tetrahydrofuran (THF), and toluene were obtained from Kanto Chemicals and purified by conventional methods. 2,2′Azobisisobutyronitrile (AIBN; Wako Pure Chemicals, Osaka, Japan) was purified by recrystallization from ethanol and dried at 25 °C in vacuo. N,N′-Dicyclohexylcarbodiimide (DCC), diethyl ether, perchloric acid standard solution (0.1 M acetic acid solution), and acetic acid were purchased from Wako Pure Chemicals. 3-Mercaptopropionic acid (MPA; Tokyo Chemical Industry, Tokyo, Japan) was distilled under reduced pressure, and the fraction boiling at 95 °C (5 mmHg) was used. 2-Mercaptoethylamine hydrochloride (MEAHC; Tokyo Chemical Industry) was purified by recrystallization from methanol and dried at 25 °C in vacuo. Isopropylamine (IPA) and triethylamine (TEA) were obtained from Tokyo Chemical Industry and distilled under atmospheric pressure. N-Hydroxysuccinimide (HOSu) was purchased from Tokyo Chemical Industry. (3-Aminopropyl)triethoxysilane (APTES; ShinEtsu Chemical Industry, Tokyo, Japan) was distilled under reduced pressure and the fraction boiling at 85 °C (8 mmHg) was used. Glass cover slips (24 mm
Yakushiji et al.
Figure 1. Three types of PIPAAm-modified surfaces. × 50 mm, average thickness 0.21 mm) were obtained from Matsunami Glass Industry (Tokyo, Japan), and glass beads (average diameter 50 µm) were purchased from Towa Science (Tokyo, Japan). Sulfo-succinimidyl-4-o-(4,4′-dimethoxytrityl)butyrate (s-SDTB) was obtained from Pierce (Rockford, IL). Preparation of Carboxyl- or Amino-Terminated PIPAAm. Semitelechelic IPAAm polymers were prepared by radical telomerization using thiol compounds as telogens.3,20,21 Preparation of PIPAAm bearing single carboxyl end groups proceeded as follows: IPAAm (34 g, 0.30 mol) was dissolved in DMF (100 mL). To this solution was added MPA (0.32 g, 3.0 mmol) and AIBN (80 mg, 0.49 mmol) as a chain-transfer agent and initiator, respectively. The reaction mixture was degassed by subjecting it to freeze-thaw cycles, and the ampule containing the mixture was sealed under reduced pressure. Polymerization then proceeded at 70 °C for 2 h. After the reaction solution was concentrated by evaporation, the reactant was poured into diethyl ether to precipitate the polymer. The polymer was further purified by repeated precipitation from THF into diethyl ether twice. After it was dried at 25 °C in vacuo for 12 h, the polymer was dissolved in distilled water to a 5.0 wt % solution for dialysis, using a cellulose ester dialysis membrane tube (molecular weight cutoff 500, SPECTRUM, California), and dialyzed against distilled water at 5 °C for 3 days. The polymer was recovered by freeze-drying. The polymer molecular weight was determined by gel permeation chromatography (GPC) (TSKgel G3000HHR + TSKgel G4000HHR; TOSOH GPC system; TOSOH, Tokyo, Japan) at 40 °C using DMF containing 20 mM LiBr solution as an eluent. Polystyrene was used as a molecular weight standard. The presence of terminal carboxyl groups was confirmed by an aqueous acid-base titration with standardized 0.05 M NaOH using a pH meter with continuous N2 gas bubbling. PIPAAm having single amino end groups was prepared as follows: IPAAm (34 g, 0.30 mol) was dissolved in DMF (100 mL). To this solution was dissolved MEAHC (0.34 g, 3.0 mmol) as a chain-transfer agent and BPO (0.12 g, 0.50 mmol) as an initiator. The reaction mixture was treated and reacted in the same way as that for carboxyl-terminated PIPAAm. The polymer was purified by repeated precipitation from THF to diethyl ether and dialysis against water. As the polymer is obtained as the hydrochloric acid salt, it was treated with TEA in THF to convert it to the free amine. After removal of precipitated triethylamine hydrochloride by filtration, the solution was concentrated by evaporation. The polymer was recovered as a precipitate from dry diethyl ether. The molecular weight of the polymer was determined by GPC using polystyrene standards. The presence (20) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y. Bioconjugate Chem. 1993, 4, 42. (21) Kaneko, Y.; Sakai, K.; Kikuchi, A.; Yoshida, R.; Sakurai, Y.; Okano, T. Macromolecules 1995, 28, 7717.
Thermoresponsive Wettability Changes
Figure 2. Preparation scheme for the PIPAAm terminally grafted surface (model surface A). of terminal amino groups was confirmed by nonaqueous potentiometric titration with a standardized 0.05 M perchloric acid/ acetic acid solution using a potentiometer. Polymers are abbreviated as PIPAAm-X where X represents the terminal functional group (COOH or NH2). Preparation of PIPAAm with Active Ester Side Chains. IPAAm copolymer with active ester side chains was prepared by radical copolymerization of IPAAm with ASI in DMF. A typical procedure was as follows: IPAAm (20 g, 0.18 mol) and ASI (9.3 g, 55 mmol) were dissolved in DMF (300 mL). AIBN (0.12 g, 0.73 mmol) was used as an initiator. The reaction mixture was degassed, and the reaction proceeded at 70 °C for 2 h. The copolymer was recovered from diethyl ether. The copolymer molecular weight was determined by GPC. The amount of active ester groups in the copolymer was determined by a spectrometric assay measuring the ultraviolet absorption of liberated succinimidyl anion at 260 nm in methanolic NH4OH.22 The copolymer is abbreviated as P(IPAAm-co-ASI). Amination of Glass Surfaces. Glass cover slips were cleaned by treating with 3% hydrofluoric acid for a few seconds, followed by immersion in 10% hydrogen peroxide for 30 min and washing with distilled water. These cover slips were then thoroughly dried at 40 °C for 12 h in vacuo and further dried at 40 °C for 30 min under vacuum in a flask. The flask was purged with dry nitrogen gas so that the inner pressure was returned to atmospheric pressure. Toluene (250 mL) and APTES (5 mL) were poured into the flask under a nitrogen gas stream. The silanization reaction proceeded under reflux at 100 °C for 5 days. These cover slips were finally washed with toluene, methanol, and distilled water, consecutively, and dried at 40 °C for 12 h in vacuo. These were stored in a desiccator under nitrogen atmosphere. Modification of Aminated Glass Surfaces with PIPAAmCOOH (Model Surface A). Carboxyl terminal groups on PIPAAm-COOH were activated with HOSu using DCC as a condensing agent. PIPAAm-COOH, HOSu, and DCC were dissolved in DMF (polymer concentration 0.10 g/mL) at mole ratios of 1:2:2, respectively, and the reaction proceeded with stirring at 25 °C for 12 h. After the precipitated N,N′dicyclohexylurea was removed by filtration, the mixture was concentrated by evaporation. The end active-esterified PIPAAm was recovered by precipitation from dry diethyl ether. The presence of the succinimidyl group at the end of the polymer was confirmed by an ultraviolet absorption at 260 nm in methanolic NH4OH.22 Modification of aminated glass surfaces with active ester terminated PIPAAm was carried out through amide bond formation between polymer active ester and surface amine.6 PIPAAm (2.0 g) with terminal active ester was dissolved in 200 mL of distilled water in the separable flask, and the solution was stirred gently with nitrogen bubbling for 30 min at 25 °C. Aminated cover slips were then immersed in the solution, and the flask was sealed. The reaction proceeded at 25 °C for 12 h. This process was repeated three times. Modified cover slips were washed with cold distilled water and dried at 25 °C for 12 h in vacuo. Figure 2 shows the schematic representation of the preparation of these PIPAAm terminally grafted surfaces (model surface A). (22) Miron, T.; Wilchek, M. Anal. Biochem. 1982, 126, 433.
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Figure 3. Preparation scheme for the looped PIPAAm chain grafted surface (model surface B). Modification of Aminated Glass Surfaces with IPAAm Copolymer (Model Surface B). To investigate the effect of graft configuration on thermoresponsive wettability changes of P(IPAAm-co-ASI) laterally grafted surfaces, surface modification with P(IPAAm-co-ASI) was carried out in two different solvent systems. P(IPAAm-co-ASI) was reacted with aminated glass surfaces in either dioxane (good solvent) or dioxane/toluene mixed solvent (relatively poor solvent). P(IPAAm-co-ASI) (2.0 g) was dissolved in 200 mL of dioxane in a separable flask equipped with a reflux condenser, and aminated cover slips were immersed in this solution. The condensation reaction proceeded between succinimidyl ester groups in polymer side chains and amino groups on the surface at 80 °C for 12 h. PIPAAm-modified cover slips were washed with dioxane to remove unreacted polymer and dried at 25 °C for 12 h in vacuo. Residual active esters on PIPAAm-modified cover slips were substituted into isopropylamide by reacting with 20% isopropylamine (IPA) in THF at 25 °C for 12 h under nitrogen atmosphere. When dioxane/toluene mixed solvent was used, the condensation reaction proceeded as follows: P(IPAAm-co-ASI) (2.0 g) was dissolved in 160 mL of dioxane in a 4-mouth separable flask equipped with a reflux condenser. Aminated cover slips were then immersed in the solution. After the solution was heated to 80 °C, 40 mL of toluene was added to the flask (final mixing ratio of the solvent was 8 volumes of dioxane to 2 volumes of toluene). The reaction proceeded at 80 °C for 12 h. Modified cover slips were washed with dioxane to remove unbound polymer and dried at 25 °C for 12 h in vacuo. Half of these modified cover slips were treated with IPA to transform active esters to isopropylamide groups. The other half of the cover slips were used for the introduction of PIPAAm-NH2 following the procedure described in the next section. Figure 3 illustrates the modification of the glass surface with P(IPAAm-co-ASI) (model surface B). Introduction of PIPAAm-NH2 on a Copolymer-Modified Surface (Model Surface C). Introduction of PIPAAm-NH2 onto copolymer-modified surfaces was carried out by utilizing residual copolymer succinimidyl ester groups immobilized on the polymergrafted glass surface. In a solution of PIPAAm-NH2 (2.0 g) dissolved in 200 mL of dioxane in a 4-mouth separable flask were immersed copolymer-modified cover slips. The reaction proceeded at 80 °C for 12 h to form model surface C (Figure 4). These cover slips were washed with dioxane, dried at 25 °C for 12 h in vacuo, and treated with IPA. Confirmation of Polymer Introduction onto Glass Surfaces. s-SDTB reacts quantitatively with an amino group on the solid surface through amide bond formation.23 Addition of perchloric acid liberates the dimethoxytrityl cation from the support. Dimethoxytrityl cation has a maximum absorption at 498 nm. Thus, the introduction of polymer on an aminated surface is confirmed by measuring the consumption of surface amino groups with s-SDTB. For this purpose, glass beads were used because of the limited surface area of a glass plate for quantification. Aminated glass beads (average diameter 50 µm) were modified with polymers and copolymers according to the same procedure as described in the previous section. The amount of amino groups on glass beads was determined according to the (23) Gaur, R. K.; Gupta, K. C. Anal. Biochem. 1989, 180, 253.
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Yakushiji et al. Table 1. Poly(N-isopropylacrylamide) Grafting to Aminopropyl Silanized Glass Surfaces PIPAAm-COOH introductiona
model surface (µmol/m2) A B C
(%)
20 85 unmodified unmodified
P(IPAAm-co-ASI) surface amine PIPAAm-NH2 consumptiona introductionb (µmol/m2)
(%)
unmodified 23 98 23 98
N/Cc atomic (µmol/m2) (%) ratio unmodified unmodified 62 44
0.17 0.15 0.15
a Estimated from the consumption of primary amino groups on solid surfaces. b Estimated from the consumption of active ester groups on surface B. c Determined by ESCA measurement (cf. theoretical IPAAm N/C ) 0.17).
Figure 4. Preparation scheme for the looped PIPAAm chain surfaces grafted with freely mobile PIPAAm chains (model surface C). method described by Gaur and Gupta.23 A half gram of modified glass beads was allowed to react with an excess amount of s-SDTB. The amount of polymer on the surface was estimated from the consumption of surface amino groups by comparing polymermodified surfaces with unmodified surfaces. For P(IPAAm-co-ASI) copolymer-modified surfaces, the amount of active ester groups on the surface was determined by UV absorption of the succinimidyl group in alcoholic NH4OH.22 The quantity of end-grafted PIPAAm on the P(IPAAm-co-ASI) surface was calculated from the consumption of active ester groups on the surface by introduction of PIPAAm-NH2. Elemental analyses of these modified surfaces utilized electron spectroscopy for chemical analysis (ESCA; ESCA750, Shimadzu, Kyoto, Japan) with a takeoff angle of 90°. The proportion of nitrogen to carbon was calculated for the surface region. These modified surfaces were also observed using atomic force microscopy (AFM, NanoScope III, Digital Instruments, Inc., Santa Barbara, CA) with tapping mode AFM in a dry state. Dynamic Contact Angle Measurement. Dynamic contact angle (DCA) changes for these PIPAAm-modified surfaces were measured by the Wilhelmy plate technique at various temperatures. The DCA measuring apparatus (DCA-20, ORIENTEC, Tokyo, Japan) was placed in a chromato chamber, and the measuring cell unit was connected to the COOLNICS circulator (CTE42A, Yamato-KOMATSU, Tokyo, Japan) so that every measurement was carried out at constant temperature with a deviation of (0.5 °C.
Results and Discussion Syntheses and Characterization of Functionalized PIPAAm. For the modification of glass surfaces with thermoresponsive polymers, three types of functionalized PIPAAms were synthesized. PIPAAm with a carboxyl end and PIPAAm having an amino terminal were each synthesized by radical telomerization using 3-mercaptopropionic acid and 2-mercaptoethylamine, respectively, as telogens. Laterally functionalized PIPAAm was prepared by radical copolymerization of IPAAm with ASI. Polymer molecular weights were determined by either GPC or end functional group titration. The numberaveraged molecular weight of P(IPAAm-co-ASI) was 7900 (Mw/Mn ) 3.19), as determined by GPC measurement. The copolymer contained approximately 11.9 mol % succinimide groups according to UV measurement of liberated succinimidyl anion. The number-averaged molecular weight of PIPAAm-NH2 was 13 000 (Mw/Mn ) 3.26) from GPC measurement. From both potentiometric titration of amino groups and the molecular weight obtained by GPC measurement, one of four PIPAAm
molecules contains an amino terminal. The numberaveraged molecular weight of PIPAAm-COOH was 4100 (Mw/Mn ) 3.92) by GPC. Acid-base titration data showed that three of the four PIPAAm-COOH’s have a carboxyl group at the chain end. Modification of Aminated Surfaces by Thermoresponsive Polymers. P(IPAAm-co-ASI) was reacted with aminated glass surfaces in either dioxane or dioxane/ toluene mixed solvent to investigate the effect of molecular conformation on wettability changes. In both solvents, nearly the same amount of surface amino groups reacted with active ester groups on P(IPAAm-co-ASI) side chains. After the polymer introduction, residual succinimidyl groups on the surface were quantified by UV absorption of liberated succinimidyl anion in alkaline solution. Aminated glass beads were used for this assay. When dioxane was used as the reaction solvent, the quantity of active ester groups on the beads was 140 µmol/m2, while 141 µmol/m2 of active esters remained when dioxane/ toluene mixed solvent was used. Unreacted active ester groups on both surfaces were converted to isopropylamide by reacting with isopropylamine, resulting in only 5.7 µmol/m2 of active ester remaining on the surface. Thus, residual active ester groups on the surfaces were converted into isopropylamide with 96% efficiency. Table 1 summarizes the results of the surface analyses for three types of temperature-responsive surfaces. Polymer surface coverage was estimated from the consumption of amino groups on the surface after reaction with polymer active ester groups. Terminally grafted PIPAAm amounted to 20 µmol/m2 coverage on surface A. Almost equal quantities of amino groups on the surfaces were consumed by reaction with the polymers P(IPAAm-coASI) (model surface B) and PIPAAm-COOH (model surface A). Residual active ester groups (141 µmol/m2) on the P(IPAAm-co-ASI) surface (surface B) reacted with 44% efficiency with PIPAAm-NH2 to form model surface C. The yield of grafted polymer chains introduced onto model surface C was 62 µmol/m2. The amount of terminally grafted PIPAAm chains per unit area on model surface C was three times larger than that on model surface A. Although 44% of the residual active esters on model surface B were reacted with PIPAAm-NH2, the increased amount of terminally grafted chains on model surface C over model surface A is probably due to the distribution of active esters three-dimensionally on surface C. As P(IPAAm-co-ASI) is attached to the surface in a globular conformation, the total reactive groups per unit area on model surface B increased. At the same time, steric hindrance prevents PIPAAm-NH2 from efficiently reacting with active ester groups, resulting in less than half the active ester consumption. Introduction of PIPAAm to aminated glass surfaces was also confirmed by ESCA measurement (takeoff angle of 90°, sampling depth ) 90 Å). The N/C ratios calculated
Thermoresponsive Wettability Changes
Figure 5. Temperature-dependent advancing contact angle changes for P(IPAAm-co-ASI)-grafted surfaces modified in dioxane (open circles) and in dioxane/toluene (8/2) mixed solvent (closed circles).
from the data obtained by ESCA were almost identical for all three model surfaces and compare well to the theoretical N/C value calculated from the composition for IPAAm (see Table 1). Therefore, these data support almost complete surface coverage by PIPAAm for all three surfaces. These modified surfaces were observed using TappingMode AFM in a dry state for estimation of surface roughness. The base glass surface was very smooth, showing a root-mean-square (rms) roughness of 0.48 nm. The root-mean-square roughness is the standard deviation of the Z values within the observed area of 1.0 µm2. Model surface A was rougher with a rms roughness of 15.8 nm. Model surface B was slightly rougher with a rms roughness of 3.55 nm, probably due to globular PIPAAm chain incorporation. The degree of roughness on model surface B was extremely smaller than the range of molecular chain length of P(IPAAm-co-ASI) estimated from its molecular weight: polymer chains were attached to the surface with multipoint anchoring and shrank up in a dry state. Model surface C was more rough with a rms roughness of 8.44 nm due to grafting new PIPAAm chains. The extent of roughness was influenced by the configuration and density of modified PIPAAm chains on surfaces. All surfaces were concluded to be flat with a roughness below 0.1 µm. Therefore, the effect of roughness on contact angle is negligible.24 Effect of the Reaction Solvent on TemperatureResponsive Surface Wettability Changes of PIPAAm Multipoint Grafted Surfaces. As described in the previous section, P(IPAAm-co-ASI) was grafted to aminated glass surfaces in either dioxane or dioxane/toluene mixed solvent to investigate the effects of molecular conformation on surface wettability changes with temperature changes. Figure 5 shows temperature-dependent dynamic contact angle changes for PIPAAm multipoint grafted surfaces modified in either dioxane or dioxane/ toluene mixed solvent. Both surfaces became more hydrophobic as temperature was increased. A larger contact angle change with temperature was observed for (24) Andrade, J. D.; Smith, L. M.; Gregonis, D. E. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, p 249.
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the glass surface modified in mixed solvent over that for the surface modified in dioxane. Discontinuous aqueous wettability changes were observed for the glass surface modified in mixed solvent at 27 °C, while the other solvent system showed a more continuous change in contact angle. Since functional group analysis and ESCA revealed that both surfaces were almost completely covered with PIPAAm, the difference observed in thermoresponsive surface property changes is attributed to differences in PIPAAm molecular mobility as well as graft density. Since dioxane is a good solvent for P(IPAAm-co-ASI), polymer chains exist in a relatively expanded conformation in solution, and it seems to react with the amino surface, maintaining this expanded conformation. The loop lengths of the fixed polymer chains begin to approximate the distance between polymer binding points on the surface. The flexibility of the polymer chain becomes smaller, restricting the dynamic motion of the grafted polymer chains. Therefore, the temperature-dependent wettability change is smaller and the discontinuity near the polymer transition temperature is not apparent. In the dioxane/toluene mixed solvent, the polymer chain is expected to exist in a relatively globular conformation, since toluene is a poor solvent for P(IPAAm-co-ASI). This globular conformation in the mixed solvent allows the grafted polymer to exist longer than the average distance between binding points on the glass surface. Longer polymer loops retain a relatively larger mobility and larger dynamic motion with temperature changes caused by temperature-dependent polymer hydration/dehydration. This results in an observed discontinuous and larger surface wettability change with temperature. We have previously compared the temperature-dependent wettability changes between two PIPAAmmodified surfaces: terminally grafted surfaces and multipoint attached surfaces.6 Both were modified in cold water. Terminally grafted surfaces showed larger contact angle changes than multipoint attached surfaces over a narrow temperature range. The P(IPAAm-co-ASI) chain existed in a relatively expanded conformation in water at 4 °C and was fixed on the surface maintaining this conformation. Therefore, smaller contact angle changes were observed for PIPAAm multipoint anchored surfaces.6 These results clearly demonstrate that surface wettability is strongly influenced by the dynamic motion of modified PIPAAm chains on the PIPAAm multipoint anchored surfaces. Thermoresponsive hydrophilic/hydrophobic surface properties are regulated by choosing the surface modification route, controlling the grafted molecule’s mobility. The PIPAAm-modified surface with longer loop chain lengths should effectively amplify surface property alterations with temperature changes. In the following experiments, we used multipoint attached PIPAAm surfaces modified in dioxane/toluene mixed solvent. Introduction of Freely Mobile PIPAAm Chains and Its Influence on Temperature-Dependent Surface Wettability Changes. Terminally grafted PIPAAm chains on surfaces show a highly mobile nature compared with that of PIPAAm chains attached using a multipoint configuration. 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.21,25 Freely mobile, grafted PIPAAm chains respond to temperature changes rapidly, and these dehydrated chains then become (25) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature 1995, 374, 240.
4662 Langmuir, Vol. 14, No. 16, 1998
Figure 6. Temperature-dependent advancing contact angle changes for three types of PIPAAm-modified surfaces (surface A, open triangles; surface B, closed circles; surface C, open squares).
hydrophobic cores within the hydrogels. The increased hydrophobic aggregation forces between the cores accelerate the dehydration. Thus the shrinking rate of the entire gels accelerates at elevated temperatures. Therefore it is reasonable to conclude that the conformation and the attachment modes for PIPAAm chains on surfaces greatly affect thermoresponsive surface property alterations. We then compared multipoint polymer-modified surfaces with two other surfaces having terminally grafted PIPAAm chains in terms of thermoresponsive wettability changes. Three types of PIPAAm-modified surfaces were prepared: (1) PIPAAm terminally grafted surfaces where PIPAAm was introduced directly onto aminated glass surfaces via amide bond formation (surface A), (2) P(IPAAm-co-ASI) multipoint modified surfaces where polymer chains maintained a relatively flexible looped conformation on the surface (surface B), and (3) PIPAAm terminally grafted onto surface B using residual active ester groups (surface C). Temperature-dependent wettability changes for all three grafted surfaces were estimated by aqueous dynamic contact angle measurements, and results are shown in Figure 6. All three surfaces showed temperatureresponsive hydrophilic/hydrophobic surface property alterations: hydrophilic at lower temperature and hydrophobic at elevated temperature. Large and discontinuous wettability changes were observed for all surfaces. Surface A shows an advancing contact angle (θA) of 21° (cos θA ) 0.93) at 5 °C and θA ) 70° (cos θA ) 0.34) at 40 °C, respectively. Surface B shows θA ) 24° (cos θA ) 0.91) at 5 °C and θA ) 93° (cos θA ) -0.05) at 40 °C. For surface C, θA ) 26° (cos θA ) 0.90) at 5 °C and θA ) 93° (cos θA ) -0.05) at 40 °C. On terminally grafted surfaces A and C, surface wettabilities change dramatically near 32 °C. On surface B, the surface wettability changed discontinuously at 27 °C. PIPAAm on surface B has a restricted
Yakushiji et al.
conformation due to the multipoint graft configuration described in the previous section. This may be the reason for the observed lower transition temperature of 27 °C for hydrophilic to hydrophobic surface wettability changes. Surface C was prepared to both introduce freely mobile PIPAAm chain ends and increase the density of PIPAAm at the surface. In fact, similar to surface A, surface C shows drastic and discontinuous surface wettability changes over the temperature range 32-35 °C. This temperature range corresponds to the PIPAAm LCST in aqueous media. Therefore, terminally grafted PIPAAm chains on surfaces A and C respond rapidly to small temperature changes due to their highly mobile nature. A larger temperature-responsive surface free energy change for surface C over that for surface A is observed. This is attributed to the increased amount of freely mobile PIPAAm chains per unit area as well as the increased density of basal PIPAAm-looped chains, both of which are capable of inducing stronger hydrophobic aggregation at elevated temperature. As a consequence, PIPAAm graft architecture is noted to strongly influence temperature-responsive surface wettability changes. Introduction of freely mobile PIPAAm graft chains to the PIPAAm multipoint attached surfaces results in both increased apparent density and increased temperature-sensitivity, leading to larger surface wettability changes in response to temperature changes. Conclusions We prepared three types of PIPAAm-grafted surfaces to investigate the effects of the grafted PIPAAm configuration on thermoresponsive aqueous wettability changes. PIPAAm graft architecture and density show a strong influence on surface wettability changes in response to temperature changes. Hydrophilic/hydrophobic surface property alterations could be regulated by choosing surface modification conditions as well as the PIPAAm graft configuration. Introduction of PIPAAm chains with freely mobile ends effectively alters surface properties within a narrow temperature range. These results indicate the importance of surface design with the polymer as a switching element. Utilizing these thermoresponsive surface property alterations, we are able to modulate the interactions of PIPAAm-grafted surfaces with soluble surface-active substances such as proteins and cells in an aqueous milieu. This has led us to develop new hydrophobic chromatography systems9,10 for separation and purification of bioactive compounds and cells using grafted PIPAAm surfaces. Different grafting strategies will provide many modes for thermally modulated separations. Acknowledgment. Part of this work was financially supported by the Ministry of Education, Science, Sports, and Culture, Japan (Grant No. 07408030), Japan High Polymer Center (JHPC), and Proposal-Based Advanced Industrial Technology R&D Program, New Energy and Industrial Technology Development Organization (NEDO). The authors are grateful to Dr. David W. Grainger, Colorado State University, for his valuable discussions and comments as well as critical reading of this manuscript. LA980090+
