Correlation between the Structure of Water in the Vicinity of

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Correlation between the Structure of Water in the Vicinity of Carboxybetaine Polymers and Their Blood-Compatibility Hiromi Kitano,*,† Susumu Tada,† Takayuki Mori,† Kohei Takaha,† Makoto Gemmei-Ide,† Masaru Tanaka,‡ Mitsuhiro Fukuda,§ and Yoshiyuki Yokoyama| Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan, Creative Research Initiative “Sousei” (CRIS), Hokkaido University, Sapporo 060-0812, Japan, Textile Materials Science Laboratory, Hyogo University of Teacher Education, Yashiro-cho, Hyogo 673-1494, Japan, and Central Research Institute, Toyama Industrial Technology Center, Takaoka 933-0981, Japan Received June 13, 2005. In Final Form: September 30, 2005 The structure and hydrogen bonding of water in the vicinity of carboxybetaine homopolymer (poly[1carboxy-N,N-dimethyl-N-(2′-methacryloyloxyethyl)methanaminium inner salt] (PolyCMB), and a random copolymer of CMB and n-butyl methacrylate, Poly(CMB-r-BMA), with various molecular weights were analyzed in their aqueous solutions and thin film with contours of O-H stretching of Raman and attenuated total reflection infrared (ATR-IR) spectra, respectively. The relative intensity of the collective band (C value) corresponding to a long-range coupling of O-H stretchings of the Raman spectra for aqueous solution of Poly(CMB-r-BMA) was very close to that for pure water, which is in contrast with the smaller C value in aqueous solution of ordinary polyelectrolytes. The number of hydrogen bonds collapsed by the presence of one monomer residue (Ncorr value) of PolyCMB and Poly(CMB-r-BMA) (CMB, 45 mol %) (Mw, 1.14 × 104 and 1.78 × 104, respectively) could be calculated from the C value. The Ncorr values were much smaller than those for ordinary polyelectrolytes and close to those for nonionic water-soluble polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone). Furthermore, a water-insoluble Poly(CMB-r-BMA) with a large BMA content (Mw ) 347 kD, CMB 27 mol %) could be cast as a thin film (thickness, ca. 10 µm) on a ZnSe crystal for the ATR-IR analyses. At an early stage of sorption of water into the Poly(CMB-r-BMA) film, the O-H stretching band of IR spectra for the water incorporated in the film was similar to that for free water, which is in contrast with the drastic change in the O-H stretching band of water incorporated in polymer films such as poly(methyl methacrylate) (PMMA) and poly(n-butyl methacrylate) (PBMA). The theoretical vibrational frequency for water molecules hydrating a betaine molecule calculated by using a density functional method supported the experimental results. The adhesion of human platelets to Poly(CMB-r-BMA) films was much less than that to PMMA and PBMA. With an increase in the content of CMB residue, the number of platelets adhered to the Poly(CMB-r-BMA) film drastically decreased and then gradually increased, probably due to the increase in the roughness of the film surface. These results suggest that the carboxybetaine monomer residues with a zwitterionic structure do not significantly disturb the hydrogen bonding between water molecules in both aqueous solution and thin film systems, resulting in the excellent blood-compatibility of the carboxybetaine polymers.

Introduction Water is ubiquitous on Earth and plays an important, often crucial, role in our living bodies. Hydrogen bonding (abbreviated as H-bonding, hereafter) between water molecules is induced by the electrostatic interaction between partially the negative oxygen atom on water molecule and the partially positive hydrogen atom of a neighboring water molecule.1,2 The H-bonding and nonlinear bent structure of water molecules induce a fluctuated H-bonded network in the liquid state and give * To whom correspondence should be addressed. E-mail: [email protected]. † Toyama University. ‡ Hokkaido University. § Hyogo University of Teacher Education. | Toyama Industrial Technology Center. (1) Scheiner, S. in Hydrogen Bonding: A Theoretical Perspective; Oxford University Press: New York, 1997. (2) (a) Eisenberg, D.; Kauzmann, W. in The Structure and Properties of Water; Clarendon Press: Oxford, U.K., 1969. (b) Walrafen, G. E. in Water - A Comprehensive Treatise, Vol. 1; Franks, F., Ed.; Plenum Press: New York, 1972. (c) Walrafen, G. E. in Structure of Water and Aqueous Solutions; Luck, W. A. P., Ed.; Verlag Chemie: Weinheim, Germany, 1974.

anomalous physical properties of liquid water among ordinary liquids (heat capacity, heat of solidification, heat of vaporization, thermal conductivity, etc.). In aqueous polymer systems, interaction between water and a polymer chain is highly important to determine the physical properties of the systems in wide concentration regions. For example, the interaction between water and a polymer chain influences the conformation of the polymer chains in dilute polymer solutions. Whereas, water sorbed into polymer solids from the air or the aqueous medium often causes a significant change in mechanical properties of the polymers.3 Furthermore, the interaction causes many interesting phenomena. For example, water bound to polymer chains is not frozen even much below the usual freezing point of bulk water,4 and some kinds of polymers show a coil(3) (a) Kawagoe, M.; Tekeshima, M.; Nomiya, M.; Qiu, J.; Morita, M.; Mizuno, W.; Kitano, H. Polymer 1999, 40, 1371. (b) Kawagoe, M.; Hashimoto, S.; Nomiya, M.; Morota, M.; Qiu, J.; Mizuno, W.; Kitano, H. J. Raman Spectrosc. 1999, 30, 913. (4) (a) Murase, N.; Gonda, K.; Watanabe, T. J. Phys. Chem. 1986, 90, 5420. (b) Ohno, H.; Shibayama, M.; Tsuchida, E. Makromol. Chem. 1983, 184, 1017.

10.1021/la0515571 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/08/2005

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globule transition phenomenon upon raising the temperature of their aqueous solutions.5 The properties of water in polymer systems have extensively been investigated by various techniques such as DSC,4 NMR,6 sound velocity mesurement,7a pulsedfield gradient NMR,7b neutron scattering,7c dielectric measurement,7d etc. Time-averaged structures of water have been categorized in three types: (1) I-structure (incidental structure), (2) V-structure (vibration-averaged structure), and (3) D-structure (diffusion-averaged structure). Molecular vibrations used as a probe in vibrational spectroscopy (IR and Raman methods) have smaller relaxation times (τ ) 10-13-10-14 s) than those of rotational rearrangement of water molecules in liquid phase (τ ) 10-11-10-12 s), and therefore, these methods are quite useful to analyze the V-structure of water which reflects the orientation of water molecules.2,8 Numerous researchers have reported synthesis and solution behavior of zwitterionic polymers.9 It is known that the solution behavior of zwitterionic polymers is often opposite to that of typical polyelectrolytes, exhibiting “antipolyelectrolyte” behavior. However, the structure of water in the vicinity of zwitterionic polymers has not been reported so much. Previously, we reported that zwitterionic polymers such as poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC, a phospholipid analogue (phosphobetaine) polymer) and poly[3-sulfo-N,N-dimethyl-N-(3′-methacrylamidopropyl)propanaminium inner salt] (PSPB, a so-called “sulfobetaine” polymer) did not disturb the H-bonded structure of surrounding water in their aqueous solutions significantly using a polarized Raman spectroscopic analysis of the O-H stretching vibration band (2800-3800 cm-1) of water.10,11 As for the water sorbed into polymer thin films, we have been using an attenuated total reflection infrared (ATR-IR) technique to extract the information about the structure of water incorporated in various kinds of polymer films.12 It was revealed that, even at an early stage of contact with liquid water, the water in a thin film of poly(2-methoxyethyl acrylate) (PMEA) has a similar H-bonded network structure to that of free water.13

Quite recently, Raman and ATR-IR analyses of the O-H stretching vibration band of water have revealed that the structure of water in the vicinity of Poly(MPCr-BMA) both in aqueous solution and thin film systems, respectively, is similar to that of bulk water.14 Such analyses could be pursued because a random copolymer of MPC and n-butyl methacrylate (BMA) (Poly(MPC-rBMA)) with a small molecular weight is water-soluble, whereas that with a larger molecular weight is not.15 Mention should be made here that both Poly(MPC-r-BMA) and PMEA have been reported to be highly bloodcompatible.15,16 Grafted sulfobetaine polymer chains on the solid surface have been found to be blood-compatible, too.17 In this report, Raman and ATR-IR analyses of O-H stretching vibration band of water in the vicinity of other kinds of zwitterionic polymers, carboxybetaine homo- and copolymers, have been carried out. These polymers could be prepared much more easily and cheaply than phosphobetaine and sulfobetaine polymers. Similar to Poly(MPC-r-BMA), a random copolymer of 1-carboxy-N,Ndimethyl-N-(2′-methacryloyloxyethyl)methanaminium inner salt (CMB) and BMA (Poly(CMB-r-BMA)) with a small molecular weight is water-soluble, whereas that with a larger molecular weight is not. Therefore, the structure of water in the vicinity of Poly(CMB-r-BMA) has been analyzed in both aqueous solution and thin film systems using Raman and ATR-IR techniques, respectively. BMA has been adopted as a comonomer of CMB, because as Lowe et al. pointed out (1) the copolymer obtained has a film-forming characteristic and (2) the applied coatings are stable in an aqueous environment.18 The structure of water in the vicinity of a homopolymer of CMB (PolyCMB) has also been investigated for comparison. Furthermore, the blood-compatibility of the carboxybetaine copolymer films has been examined. By the compilation of these analytical data, we are further convinced of the usability of vibrational spectroscopy for a first screening step to check blood-compatibility of any polymeric materials without contact with human blood itself.

(5) Terada, T.; Inaba, T.; Kitano, H.; Maeda, Y.; Tsukida, N. Macromol. Chem. Phys. 1994, 195, 3261. (6) (a) Katayama, S.; Fujiwara, S. J. Am. Chem. Soc. 1979, 101, 4485. (b) Quinn, F. X.; Kampf, E.; Smyth, G. McBriety, V. J. Macromolecules 1988, 21, 3191. (7) (a) Gekko, K.; Noguchi, H. Biopolymers 1971, 10, 1513. (b) Rarnes, A. C.; Bieze, T. W. N.; Enderby, J. E.; Leyte, J. C. J. Phys. Chem. 1994, 98, 11527. (c) Bieze, T. W. N.; Barnes, A. C.; Huige, C. J. M.; Leyte, J. C. J. Phys. Chem. 1994, 98, 6568. (d) Shinyashiki, N.; Matsumura, Y.; Miura, N.; Yagihara, S.; Mashimo, S. J. Phys. Chem. 1994, 98, 13612. (8) (a) Maeda, Y.; Kitano, H. Spectrochim. Acta 1995, A51, 2433. (b) Maeda, Y.; Kitano, H. Trends Phys. Chem. 1997, 6, 269. (c) Maeda, Y.; Ide, M.; Kitano, H. J. Mol. Liquids 1999, 80, 149. (d) Terada, T.; Maeda, Y.; Kitano, H. J. Phys. Chem. 1993, 97, 3619. (e) Maeda, Y.; Tsukida, N.; Kitano, H.; Terada, T.; Yamanaka, J. J. Phys. Chem. 1993, 97, 13903. (f) Tsukida, N.; Maeda, Y.; Kitano, H. Macromol. Chem. Phys. 1996, 197, 1681. (9) (a) Lowe, A. B.; McCormick, C. L. Chem. Rev. 2002, 102, 4177. (b) Thomas, D. B.; Vasilieva, Y. A.; Armentrout, R. S.; McCormick, C. L. Macromolecules 2003, 36, 9710. (10) Kitano, H.; Sudo, K.; Ichikawa, K.; Ide, M.; Ishihara, K. J. Phys. Chem. B 2000, 104, 11425. (11) Kitano, H.; Imai, M.; Sudo, K.; Ide, M. J. Phys. Chem. B 2002, 106, 11391. (12) (a) Ide, M.; Yoshikawa, D.; Maeda, Y.; Kitano, H. Langmuir 1999, 15, 926. (b) Kitano, H.; Ichikawa, K.; Ide, M.; Fukuda, M.; Mizuno, W. Langmuir 2001, 17, 1889. (c) Ichikawa, K.; Mori, T.; Kitano, H.; Fukuda, M.; Mochizuki, A.; Tanaka, M. J. Polym. Sci., Part B, Polym. Phys. 2001, 39, 2175. (13) (a) Kitano, H.; Ichikawa, K.; Fukuda, M.; Mochizuki, A.; Tanaka, M. J. Colloid Interface Sci. 2001, 242, 133. (b) Ide, M.; Mori, T.; Ichikawa, K.; Kitano, H.; Tanaka, M.; Mochizuki, A.; Oshiyama, H.; Mizuno, W. Langmuir 2003, 19, 429.

A. Materials. A carboxybetaine monomer, 1-carboxy-N,Ndimethyl-N-(2′- methacryloyloxyethyl)methanaminium inner salt (CMB) was kindly donated by Osaka Organic Chemical Industries, Ltd., Osaka, Japan (commercial name of CMB, GLBT). Other reagents were commercially available. Milli Q grade water was used for preparation of sample solutions. B. Polymers. (i) PolyCMB (Scheme 1). CMB (5 g) was polymerized using 2,2′-azobisisobutyronitrile (AIBN) and 2-mercaptoethanol (ME) as radical initiator and chain transfer reagent, respectively, (CMB: AIBN: ME ) 100: 2:10) in ethanol (25 mL)

Experiments

(14) Kitano, H.; Imai, M.; Mori, T.; Gemmei-Ide, M.; Yokoyama, Y.; Ishihara, K. Langmuir 2003, 19, 10260. (15) (a) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355. (b) Ueda, T.; Oshida, H.; Kurita, K.; Ishihara, K.; Nakabayashi, N. Polym. J. 1992, 24, 1259. (c) Ishihara, K.; Ishikawa, E.; Iwasaki, Y.; Nakabayashi, N. J. Biomater. Sci., Polym. Ed. 1999, 10, 1047. (d) Ishihara, K. Sci. Technol. Adv. Mater. 2000, 1, 131. (16) (a) Tanaka, M.; Motomura, T.; Kawada, M.; Anzai, T.; Kasori, Y.; Shiroya, T.; Shimura, K.; Onishi, M.; Mochizuki, A. Biomaterials 2000, 21, 1471. (b) Tanaka, M.; Mochizuki, A.; Ishii, N.; Motomura, T.; Hatakeyama, T. Biomacromolecules 2002, 3, 36. (c) Tanaka, M.; Mochizuki, A. J. Biomed. Mater. Res. 2004, 68A, 684. (17) (a) Yuan, Y. L.; Ai, F.; Zhang, J.; Zang, X. B.; Shen, J.; Lin, S. C. J. Biomater. Sci., Polym. Ed. 2002, 13, 1081. (b) Jun, Z.; Youling, Y.; Kehua, W.; Jian, S.; Sicong, L. Colloids Surfaces B: Biointerfaces 2003, 28, 1. (c) Yuan, J.; Mao, C.; Zhou, J.; Shen, J.; Lin, S. C.; Zhu, W.; Fang, J. L. Polym. Int. 2003, 52, 1869. (d) Yuan, Y.; Zang, X.; Ai, F.; Zhou, J.; Shen, J.; Lin, S. Polym. Int. 2004, 53, 121. (18) Lowe, A. B.; Vamvakaki, M.; Wasall, W. A.; Wong, L.; Billingham, N. C.; Armes, S. P.; Lloyd, A. W. J. Biomed. Mater. Res. 2000, 52, 88.

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Scheme 1. Chemical Structure of Polymers Examined: (a) PolyCMB, (b) Poly(CMB-r-BMA), (c) PMMA, (d) PBMA

Scheme 2. Schematic of the Hydration of Betainea

a The number corresponds to that of the water molecule in Table 3.

Table 1. Characteristics of Carboxybetaine Polymers Examined

polymer PolyCMB Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA) Poly(CMB-r-BMA)

feeding composition ratio ratio yield Mw CMB:BMA CMB:BMA (g) (kD) Mw/Mn 100:0 50:50 5:95 10:90 15:85 20:80 25:75 30:70 35:65 40:60

100:0 45:55 4:96 7:93 14:86 17:83 23:77 27:73 31:69 34:66

3.64 3.50 0.84 0.36 2.46 1.82 2.47 2.41 3.01 2.66

11.4 17.8 330 429 280 249 213 347 169 163

1.43 1.47 4.31 3.06 3.86 4.19 4.40 2.31 3.78 4.52

at 70 °C for 24 h. After evaporation, the viscous oil was dialyzed against water for several days (membrane, Spectra/Por, Spectrum Laboratories, Inc.; exclusion limit, 1 kD (1000 daltons ) 1 kD)), and the polymer product was finally lyophilized (3.64 g, Mw ) 11.4 kD, Mw/Mn ) 1.43; Table 1). (ii) Poly(CMB-r-BMA) (Scheme 1). CMB (3.28 g) and BMA (2.23 mL, freshly distilled in vacuo) were polymerized using AIBN (23.1 mg) and ME (49.3 µL) (CMB: BMA: AIBN: ME ) 50:50: 0.5:2.5) in ethanol (25 mL) at 70 °C for 24 h. After evaporation, the viscous oil was precipitated in n-hexane. The obtained polymer product was dissolved in water and dialyzed against water for several days (membrane, exclusion limit, 3.5 kD), and the polymer product was finally lyophilized (3.5 g, CMB: BMA ) 45:55, Mw

Figure 1. Raman spectra of the O-H stretching region of pure water. (a) I| and I⊥ spectra of pure water at 25 °C. I⊥/FO-H spectra was also shown with a dotted line. (b) The collective band of water. ) 17.8 kD, Mw/Mn ) 1.47). In a similar manner, a water-insoluble copolymer of CMB and BMA was prepared at a molar ratio CMB: BMA:AIBN ) 30:70:0.05. After dialysis of the polymer product against methanol, the polymer was precipitated in n-hexane (2.41 g, CMB: BMA ) 27:73, Mw ) 347 kD, Mw/Mn ) 2.31; Table 1). The molecular weight of the water-soluble polymers was determined by gel permeation chromatography (GPC; a 0.1 M aqueous NaBr solution was used as mobile phase (standard, pullulan, Showa Denko, Tokyo, Japan)). In the case of waterinsoluble random copolymers, the molecular weight was determined by GPC (a 0.1 M LiCl dissolved in chloroform-methanol (6:4) was used as mobile phase; standard, poly(methyl methacrylate), Showa Denko), too. Other polymers, poly(methyl methacrylate) (PMMA, Mw ) 1.58 × 105, Mw/Mn ) 1.38) and poly(n-butyl methacrylate) (PBMA, Mw ) 2.06 × 105, Mw/Mn ) 1.24) (Scheme 1), were prepared as described elsewhere.13b C. Raman Spectroscopy. The Raman spectra of water in various aqueous polymer solutions were recorded by using the polarization method in the region between 2500 and 4000 cm-1 (Figure 1) (for details, see the Supporting Information). The component of O-H stretching band of water centered at 3250 cm-1 was highly polarized and diminished in the spectra at perpendicular position. The polarized O-H stretching band of water, which is called the collective band, is ascribed to H2O molecule executing ν1 vibrations all in phase with each other but vibrational amplitude varying from molecule to molecule in water clusters which are strongly hydrogenbonded.19-21 Theoretical calculations of random network model,19,22 which is characterized by the fluctuated defects in water-water hydrogen bonds in a distorted tetrahedral network, support the interpretation. The intensity of the collective band (Ic) observed around 3250 cm-1 was separated from the spectra using eq 1 (Figure 1). (19) (a) Sceats, M. G.; Rice, S. A. In Water - A Comprehensive Treatise, Vol. 7; Franks, F., Ed.; Plenum Press: New York, 1982. (b) Stillinger, F. H. Science 1980, 209, 451. (c) Tao, N. J. In Water and Biological Macromolecules; Westhof, E., Ed.; MacMillan Press: New York, 1993; p 266. (20) (a) Green, J. L.; Lacey, A. R.; Sceats, M. G. J. Phys. Chem. 1986, 90, 3958. (b) Green, J. L.; Lacey, A. R.; Sceats, M. G.; Henderson, S. J.; Speedy, R. J. J. Phys. Chem. 1987, 91, 1684. (c) Green, J. L.; Lacey, A. R.; Sceats, M. G. J. Chem. Phys. 1987, 86, 1841. (d) Green, J. L.; Lacey, A. R.; Sceats, M. G. Chem. Phys. Lett. 1987, 134, 385. (e) Green, J. L.; Lacey, A. R.; Sceats, M. G. Chem. Phys. Lett. 1987, 137, 527. (f) Green, J. L.; Sceats, A. R.; Lacey, A. R. J. Chem. Phys. 1987, 87, 3603. (g) Karger, N.; Lu¨demann, H.-D.; Sceats, M. G. Ber. Bunsen-Ges. Phys. Chem. 1995, 99, 1104. (h) Annaka, M.; Motokawa, K.; Sasaki, S.; Nakahira, T.; Kawasaki, H.; Maeda, H.; Amo, Y.; Tominaga, Y. J. Chem. Phys. 2000, 113, 5980. (21) (a) Hare, D. E.; Sorensen, C. M. J. Chem. Phys. 1990, 93, 25. (b) Hare, D. E.; Sorensen, C. M. J. Chem. Phys. 1990, 93, 6954. (22) Belch, A.; Rice, S. A. J. Chem. Phys. 1983, 78, 4817.

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Ic ) I| - I⊥/FO-H

(1)

where I⊥ and I| are the intensities of the spectra observed with the polarizer oriented perpendicular and parallel to the electric vector of the incident laser beam, respectively. A depolarization ratio, F O-H, is expressed as F O-H ) I⊥/I|. Since the intensities of Raman spectra are not absolute, the area of Ic was normalized as in eq 2:

C)

∫I (w) dw ∫I (w) dw c

(2)

|

where w is the Raman shift in cm-1. D. ATR-IR Measurements. ATR-IR measurements on the ZnSe element were carried out as described in the Supporting Information. The IR difference spectra of sorbed water to the polymer films with a thickness of 10 µm were collected at appropriate time intervals after onset of the flowing of humid air (50 ( 5% RH). For the investigation of water cluster within the film, the film was covered with 40 µL of pure water using a microsyringe. Sixty minutes after onset of the experiment in the humid air system, the ATR-IR spectra of water almost no longer changed. The difference ATR-IR spectra between the spectra for the films saturated with the water from humid air and those on which the liquid water was deposited for 20 s were also obtained. E. Calculation of Vibrational Frequencies Using A Density Functional Method. Vibrational frequencies for water molecules around betaine (1-carboxy-N,N,N-trimethylmethanaminium inner salt) were calculated with a density-functional method (see the Supporting Information). F. Viscometric Measurements. The intrinsic viscosity, [η]

[η] ) lim ) ηsp/φ, φf0

ηsp ) (η - ηo)/ηo dL-1,

for various where φ is the concentration of polymer in g kinds of aqueous polymer solutions at 25 °C, was determined by using an Ubbelohde dilution type viscometer (Type 0B; Kusano, Tokyo, Japan). G. Thermogravimetric Analysis. The amount of water sorbed into various polymer films was evaluated by a thermogravimetric analyzer (Pyris 1 TGA, Perkin-Elmer Instruments) as described before.13b The mass change of the hydrated polymer film (∆m) by heating the film from 25 to 180 °C (heating rate ) 10 °C.min-1) was recorded. H. Platelet Adhesion Test.16a A procedure to examine platelet adhesion to various polymer films was described in the Supporting Information. I. Contact Angle Measurement.14 Static contact angles, θ, of both air bubble and sessile drop of water on the surface of various polymer films were measured as described in the Supporting Information.

Results and Discussion A. Structure of Water in Aqueous Polymer Solutions. The O-H stretching Raman band of liquid water gave a broad band composed of several overlapping components corresponding to the unperturbed O-H stretching band by intra- and intermolecular vibrational coupling of O-H oscillators (Figure 1). The broad and asymmetric band shape of decoupled O-H stretching which can be typically observed in D2O-H2O mixture2,8d indicates that electromagnetic field around individual O-H oscillator, which is mainly determined by H-bond strength, has a broad distribution. The relative intensities of the collective bands (C) are reduced by the decoupling of O-H oscillators, (1) when a stretching frequency of an O-H oscillator is largely different from that of the O-H oscillator which is combined by H-bond with the former one (bond defect) and (2) when the H-bond between coupled O-H oscillators is broken by

translational or rotational rearrangement of water molecules (network defect).19 In a pure water, the values of C are reduced with an increase in temperature by intrinsic H-bond defects caused by thermal motion.20 As previously reported, the values of C for polymer solutions were almost constant in a relatively low Mw region and decreased with an increase in Mw at the region where the Mw values were larger than some critical values. In this region, the polymer chains are considered to contact with each other for a sufficiently long time to affect the long-range coupling of O-H oscillators19 (“pseudonetwork structure” of the polymer chains).23 In solutions of polymers with relatively lower Mw, on the contrary, the H-bond defects can be considered to locate principally in the hydration shell around the polymer chain and, in the case of polyelectrolytes, their counterions. In other words, only water molecules that are in contact with or near the polymer chain or counterions are removed from H-bond network of water. In this region, therefore, C values are independent of Mw of the polymers. In a dilute solution, polymer coils are separated from each other, whereas when the polymer concentration (φ) approaches a critical value (φ*), the polymer coils begin to overlap and make a pseudonetwork. In the concentration region φ > φ*, the polymer solutions are called “semidilute” solutions.23 Graessley suggested a concentration criterion between low and high-concentration regimes for random coils in good solvent as 1 < φ [η] < 10.24 Therefore, the value of φ* can be roughly related to the intrinsic viscosity [η] as eq 3

1/[η] = φ*

(3)

From the value of [η] for aqueous solutions of carboxybetaine homo- and copolymers at 25 °C, the φ* values of the homopolymer (11.4 kD) and copolymer (CMB, 45%; 17.8 kD) were determined to be 16.7 (pX ) 0.014) and 31.1 g/dL (pX ) 0.030), respectively. These values indicated that almost all the polymers examined here give dilute solutions at pX ) 0.01 (pX, mole fraction of monomer residue in the aqueous solution). B. Evaluation of Ncorr Value for Poly(CMB-r-BMA). In aqueous solutions, water molecules interact both with hydrophilic or hydrophobic solutes and change their positions and orientations in the hydration shell around the solutes. Consequently, intensities of the collective band of aqueous solutions at certain temperature T (CX(T)) are reduced or risen by breakage or enhancement of waterwater H-bonding in comparison with those of pure water (CW(T)) at the same temperature. The defect probability, Pd, that an O-H oscillator is excluded from H-bonded network of water molecules because of unfavorable position or orientation is defined as eq 4

Pd )

CW(T) - CX(T) CW(T)

(4)

The C values of aqueous solutions of simple electrolytes20d and polar substances were smaller than those of pure water at the same temperature,20d whereas those of hydrophobic substances such as tert-butyl alcohol20e and tetra-n-butylammonium hydroxide20f were higher than that of pure water, due to an enhancement of the H-bond between water molecules in hydrophobic hydration shells around the hydrocarbon moiety. (23) de Gennes, P. G. In Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, NY, 1979. (24) Graessley, W. W. Adv. Polym. Sci. 1974, 16, 1.

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To elucidate the effects of properties of the monomeric unit of various polymers, the number of H-bond defects existing in the H-bonded network structure of water per monomer unit of a polymer, N, were calculated from the defect probability (Pd), by using eq 5

N ) Pd/fX

(5)

where fX is the number of monomer units per one O-H oscillator. Similar to small molecular solutes, ionized and polar groups in water-soluble polymer chains and counterions may disturb the H-bond between water molecules and raise the N value, whereas hydrophobic moieties such as hydrocarbon chains may enhance the H-bond and reduce the N value. The exposure area of the chemical groups to water is also important to the structure of water around them. Mention should be made here that an intrinsic defect of H-bonding in pure water exists at a certain temperature in comparison with the perfectly H-bonded ice.20a At the evaluation of Pd values, this factor is not necessary to be considered, because the Pd values are calculated by the ratio of (CW - CX) and CW values. However, the number of O-H oscillators H-bonded in pure liquid water at a certain temperature should be smaller than the total number of O-H oscillators, and therefore, the N values were simply corrected by this factor as Ncorr ) N(CW/Cice). The Ncorr values obtained for dilute solutions of various polymers with relatively lower Mw, in which H-bond defects are expected to be localized in the hydration shell of polymer chains and their counterions, were determined and listed in Table 2. The Ncorr values of ordinary polyelectrolytes which have one kind of ionic group (sodium polyacrylate,8e sodium poly(ethylenesulfonate), and poly-L-lysine HBr salt)10 are larger than those of nonionic water-soluble polymers (PEG and PVPy), indicating that ionic groups and counterions of polyelectrolytes strongly disturb the structure of water in their hydration shells. Previously, Tao et al. evaluated the N value of aqueous DNA solution to be around 30 using eq 425 (the Ncorr value could be calculated to be around 20). On the contrary, the Ncorr values for carboxybetaine homopolymer (PolyCMB, Mw ) 11.4 kD) and copolymer (Poly(CMB-r-BMA), Mw ) 17.8 kD) were very small (-0.27 and +0.02, respectively). The pKa values for the carboxyl group in PolyCMB and Poly(CMB-r-BMA) are 2.9 and 3.0, respectively, and therefore, these polymers are almost in a charge-balanced state (in other words, zwitterionic like PMPC) at neutral pH (the pH values of the aqueous solutions of PolyCMB (15.4 mg/mL) and Poly(CMB-rBMA) solutions (25.1 mg/mL) were 5.4 and 5.6, respectively). Previously, the pKa value for another carboxybetaine polymer, poly[4-(N,N-diallyl-N-methylammonio)butanoate], was reported to be 3.6 and is similar to that of PolyCMB.25 The relatively nonpolar BMA residues in the copolymers examined in this work seem to hide behind the hydrophilic CMB residues, resulting in the absence of the effect of BMA residues on the structure of water. As indicated above, the Ncorr value for zwitterionic polymer, PMPC, was very small (-0.7 and -0.1 for PMPC 4.3 and 8.3 kD, respectively),10 and that for Poly(MPC-r-BMA) (Mw ) 1.3 × 104, MPC 40 mol %) was very small, too.14 Previously, we reported that the C value for aqueous amino acid solutions under neutral conditions (where both R-amino and R-carboxyl groups of amino acids are ionized (25) Tao, N. J.; Lindsay, S. M.; Rupprecht, A. Biopolymers 1989, 28, 1019.

(zwitterionic)) were slightly smaller than that for pure water and almost constant despite the difference in hydrophobicity of the side chain.26 In other words, the Ncorr values for amino acids at neutral pH are small positive values and do not depend on the hydrophobicity of the side chain significantly. The absolutely small Ncorr values for the zwitterionic PolyCMB, Poly(CMB-r-BMA), PMPC, and Poly(MPC-r-BMA) are not inconsistent with this. The partially negative oxygen atom of the water molecule is toward the positively charged ions in solution, whereas the negatively charged ions attract the partially positive H-atoms of water, creating their hydration shells.28 However, the anionic carboxyl group and cationic quaternary ammonium group, which are in close proximity each other in the CMB residue, might counteract such an electrostatic hydration. Høiland reported that hydration sheaths for ionizable groups in close proximity are overlapped (e.g., oxalic acid, malonic acid, and succinic acid), and an additivity rule for the hydration of each component is not applicable.29 Quite recently, we have reported that the Ncorr values for R,ω-amino acids increased with an increase in the distance between the amino and carboxyl goups (from glycine to 8-aminooctanoic acid).30 These results support the tendency observed in this work. Recently, a sum frequency generation (SFG) method has often been adopted to investigate the structure of water at air-water interface.31 This is due to an ability of SFG to selectively detect information of molecules without a center of symmetry at an interface between two isotropic media. Gragson et al. for example obtained a sum frequency spectrum at the air-water interface in the presence of ionic surfactants.32 The spectrum of water was strongly enhanced by the presence of charged surfactants such as sodium dodecyl sulfate (SDS) and dodecylammonium chloride (DAC), whereas, there is no water signal for the 1:1 mixture of SDS and DAC, showing that the water molecules in the vicinity of the mixed monolayer (electric charges in the monolayer were neutralized) do not have an inversion center, which is similar to the bulk water. Using the same technique, it was shown that zwitterionic 1,2-dilauroyl-sn-phosphatidylcholine headgroups, which are adsorbed and aligned parallel to the aqueous-CCl4 interface, can stabilize in-plane water molecules.33 These results are consistent with the tendency observed in this work, too. Meanwhile, the Ncorr values for nonionic water-soluble polymers such as PEG and PVPy were very small (Table 2). Using a time domain reflectometry, it was reported that oligo(ethylene glycol)s (degree of polymerization 4-7) dissolves in water without much perturbation to water structure.34 PEG and PVPy are used in pharmaceutical aids as ointment, suppository base, and dispersing and suspending reagent of medicines. In addition, PEG is nonimmunogenic, showing that PEG does not perturb selfprotection system in living bodies. Furthermore, liposomes (26) (a) Thomas, D. B.; Vasiliev, Y. A.; Armentrout, R. S.; McCormick, C. L. Macromolecules 2003, 36, 9710. (b) Kathman, E. E.; White, L. A.; McCormick, C. L. Polymer 1997, 38, 879. (27) Ide, M.; Maeda, Y.; Kitano, H. J. Phys. Chem. 1997, 101, 7022. (28) Garrett, R. H.; Grisham, C. M. Biochemistry; Saunders: Fort Worth, TX, 1995; p 36. (29) Høiland, H. J. Chem. Soc., Faraday Trans. I 1975, 71, 797. (30) Kitano, H.; Takaha, K.; Gemmei-Ide, M. J. Colloid Interface Sci. 2005, 283, 452. (31) Richmond, G. L. Chem. Rev. 2002, 102, 2693. (32) Gragson, D. E.; McCarty, B. M.; Richmond, G. L. J. Am. Chem. Soc. 1997, 119, 6144. (33) Walker, R. A.; Gragson, D. E.; Richmond, G. L. Colloids Surf. A 1999, 154, 175. (34) Sato, T.; Niwa, H.; Chiba, A. J. Chem. Phys. 1998, 108, 4138.

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Table 2. Ncorr Values for Various Polymers at 25 °Ca polymer polyacrylateb

sodium poly(ethylene glycol)b poly(N-vinylpyrrolidone)b sodium polyethylenesulfonatec poly-L-lysine HBrc PMPCc Poly(MPC-r-BMA) (40:60)d PolyCMBe Poly(CMB-r-BMA) (45:55)e

Mw (in kD)

Ncorr value

5.0 2.0 2.3 2.2 2.7 4.3 13 11.4 17.8

5.9 0.7 0.6 5.1 5.5 -0.7 -0.2 -0.27 0.02

a In H O. The N 2 corr values are for the polymers with the smallest Mw examined except for PolyCMB and Poly(CMB-r-BMA). The uncertainties of Ncorr values are within 1.0. b Reference 8e. c Reference 10. d Reference 14. e This work.

coated with PEG chains can avoid the attack by the reticulo-endothelial systems (RES).35 It has been pointed out that Poly(MPC-r-BMA) has an excellent blood-compatibility (no thrombus formation).14 An aqueous solution of carboxybetaine copolymer has very often been used as a hair liquid. Taking into account the practical usage of these polymers in biomedical fields, the present result seems to suggest that, from the viewpoint of physical chemistry, polymeric materials without perturbation of the structure of surrounding water have a blood-compatibility, though many factors such as morphology and steric stabilization effect affect the bloodcompatibility of polymeric materials in a complicated manner.36 It should be noted here that the Ncorr value for gelatin (Mw ) 104), a highly bio-compatible denatured protein with many cationic and anionic charges (at least 11.4% of the amino acid residues had an ionizable side chain), was 0 at 20 °C and 10 wt %,8f supporting the hypothesis described above. Recent computer simulations provide useful information on the structure and properties of water in hydration shells of individual groups on polymer chains.37 Using the same method, the structure of water around the polymers with zwitterionic pendent groups will be clarified. C. Structure of Water in Poly(CMB-r-BMA) Film. To examine the effect of a polymer matrix on the structure of water, the ATR-IR spectra of the O-H stretching band for the water sorbed into the polymer film with a thickness of 10 µm was observed in this section. By this procedure, the water inside of the polymer film could exclusively be observed, and the water at the outer surface of the polymer film was not detected, because the films were too thick for the evanescent field to reach the film-air interface (assuming that the refractive index of the copolymer is the same as that of PMMA, the penetration depth of the evanescent wave (dp) is calculated as 0.54 µm at 3000 cm-1).12,13 The ordinary transmission IR band of water sorbed into polymer films deposited on the transparent plate such CaF2, on the contrary, provides the information of water locating both inside and at the surface of the films. Figure 2 shows the ATR-IR bands for the sorbed water from (A) humid air (RH ≈ 50%) and (B) liquid water existing in the matrix of various polymer films 20 s after onset of the sorption measurement. In the liquid water (35) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235. (36) (a) Biomedical Applications of Polymeric Materials; Tsuruta, T., Hayashi, T., Kataoka, K., Kimura, Y., Ishihara, K., Eds.; CRC Press: Boca Raton, FL, 1993. (b) Water in Biomaterials Surface Science; Morra, M., Ed.; Wiley: Chichester, U.K., 2001. (37) (a) Tamai, Y.; Tanaka, H.; Nakanishi, K. Macromolecules 1996, 29, 6750. (b) Tamai, Y.; Tanaka, H.; Nakanishi, K. Macromolecules 1996, 29, 6761.

Figure 2. ATR-IR spectra of water sorbed into various films. (A) Obtained by the humid air method. (B) Obtained by the liquid water method (40 µL of water was added). (a) Poly(CMBr-BMA): Mw ) 347 kD. CMB, 27 mol %. (b) PMMA: Mw ) 158 kD. (c) PBMA: Mw ) 206 kD. In the liquid water experiment, 40 µL of water was dropped above the film. Twenty seconds after dropping liquid water, ATR-IR spectra was obtained.

system examined here, liquid water was attached dropwise to the film to avoid a gradual penetration of liquid water into the space between the polymer film and the ZnSe crystal at the end of the film upon swelling. Such a troublesome phenomenon was significant in the PMMA and PBMA systems. Many tiny peaks in the range of 37004000 cm-1 correspond to the O-H stretching of water molecule in the air. It should be mentioned here that, both in humid air and liquid water methods, the O-H stretching for water sorbed to Poly(CMB-r-BMA) was quite similar to that for free water (Figure 2). Previously, it was shown that the ATR spectra for the sorbed water from the humid air correspond to the water H-bonded to polar group such as hydroxyl, carboxyl and amide groups in the polymeric films (primarily hydrating water). The shape of spectra obtained by the humid air method indicated that PMMA and PBMA, which had ester groups in the side chains, had two peaks around 37003600 and 3600-3500 cm-1, and Poly(CMB-r-BMA), which had betaine moieties in the side chains, had large and broad peaks around 3700-3100 cm-1. Whereas, that for the liquid water reflects the absorbance of both primarily hydrating water and water surrounding the primary hydration layer.12b,c,13a Therefore, by the subtraction of the intensity of primary hydrating water (sorbed water from the humid air) from that of liquid water, we could roughly extract the absorption band for the water existing within the polymer matrix as the “surrounding” water.13a We assumed here that the amount of water primarily hydrating to the film was the same in both humid air and liquid water systems. In Figure 3, the O-H stretching bands for “surrounding” water in three kinds of polymer films Poly(CMB-r-BMA), PMMA, and PBMA are shown altogether with the O-H stretching band for free liquid water. The figure clearly

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Kitano et al. Table 4. Amount of Water Sorbed into Various Polymer Filmsa polymer

Poly(CMB-r-BMA)b PMMA

sorbed water (humid air) sorbed water (liquid) difference a

0.117 1.18 1.003

PBMA

0.0050 0.00050 0.0089 0.0017 0.0039 0.0012

In g/g polymer. b Mw ) 347 kD. Table 5. Contact Angles for Various Polymer Films contact angle, θ (degree)

Figure 3. ATR-IR difference spectra of water sorbed into various polymer films. (a) Poly(CMB-r-BMA): Mw ) 347 kD. CMB, 27 mol %. (b) PMMA: Mw ) 158 kD. (c) PBMA: Mw ) 206 kD. (d) free water. Table 3. Calculated Wavenumbers for the O-H Stretching of Water Molecules Hydrating Betaine as a Model Compound of Carboxybetaine Polymera density functional B3LYP/6-31++G(d,p)SCRF model hydration model

νas

νs

Iνas/Iνs

(H3C)3N+CH2COO- H2O 3520.35 3366.15 279.155/768.589 3488.29 3264.89 251.534/1132.07 - HOH-H2O - HOH-H2O 3509.78 3341.44 236.873/828.583 - HOH-HOH-H2O 3499.26 3427.71 698.998/310.999 - HOH-HOH-H2O 3490.01 3295.58 178.446/1342.72 - HOH-HOH-H2O 3514.64 3348.34 241.771/650.944

no.b 1 2 3 4 5 6

a Values were obtained by multiplying the shift parameter 0.9578. The wavenumber for the strong peak intensity is shown in bold letters. b The number corresponds to that of water molecule in Scheme 2.

shows that the structure of water kept in the matrix of Poly(CMB-r-BMA) film was similar to that of free water except the slightly larger absorbance at the lower frequency region in the former case. In contrast with the Poly(CMB-r-BMA) film, the structure of water in the matrix of PMMA and PBMA films was clearly different from that of free liquid water. The calculated frequencies for water molecules surrounding the betaine molecule with a density-functional method are shown in Table 3. The table clearly shows that the theoretical frequencies for water molecules around the betaine molecule are mainly around 3200-3500 cm-1 and are qualitatively in agreement with the experimental results. It should be emphasized here that the ATR-IR spectra of water at an early stage of sorption into polymer thin films were analyzed in this section. At that stage, the aqueous region (secondary hydration area + bulk water) outside of the primary hydration region may not be so large, and therefore, the effect of polymeric material on the structure of secondary hydrating area can be preferentially obtained. It is a matter of course that, a long time after the onset of the sorption measurement, the O-H stretching band of water sorbed into any kinds of polymer films, which are largely swollen, shows a similar shape to that of free water. Table 4 shows that the amount of water sorbed into Poly(CMB-r-BMA) from the humid air was the largest among polymer films examined in this work. Similarly, the amount of water kept in the matrix of Poly(CMB-rBMA) (surrounding water) was the largest among the polymer films examined. As discussed above, the hydrogenbonded network structure of water, which is widely accepted as the basic structure of ordinary liquid water,2,19

sample

sessile drop

air-in-water

sum

PBMA PMMA Poly(CMB-r-BMA)a

89.4 68.2 85.2

96.8 109.1 150.4

186.2 177.3 235.6

a

Mw ) 347 kD.

was more significantly disturbed in the matrix of PMMA and PBMA than in the matrix of Poly(CMB-r-BMA). It should be mentioned here that the amounts of water sorbed onto PMMA and PBMA films in both humid air and liquid water measurements were very small. Table 5 shows contact angle (θ) of water droplet or air bubble at the surface of various polymer films. As for homogeneous polymer films, the θ values indicated the degree of hydrophilicity of the polymer materials: The more hydrophilic, the smaller the θ value measured by the sessile drop method. The opposite tendency was observed by the air-in-water method, and the sum of two kinds of contact angles was approximately 180°. However, the θ value by the sessile drop method indicated that Poly(CMB-r-BMA) film was very hydrophobic, whereas the θ value by the air-in-water method indicated that the film was hydrophilic, and the sum of two kinds of contact angles was much larger than 180°. The same tendency was obtained for the copolymer films with various CMB contents (see the Supporting Information, Table S-1). These results showed that the composition of the copolymer film at its surface was changed with the contacting medium to minimize the surface free energy: In water, a CMB rich domain directs to the surface, whereas a BMA rich one directs to the surface in contact with the air. Such a medium-responsiveness was previously observed in the Poly(MPC-r-BMA) and Poly(SPBr-BMA) systems, too.14,38 D. Blood-Compatibility of Carboxybetaine Polymer Films. As described in the Introduction, it was reported that the number of blood cells adhered to the Poly(MPC-r-BMA) film was much smaller than that to the PBMA film,15 and the structure of water incorporated in the copolymer film was similar to that of free water.14 Quite recently, the same tendency has been observed for the sulfobetaine copolymer film, Poly(SPB-r-BMA).38 Previously, we reported that the structure of water in the matrix of a PMEA film, which shows an excellent bloodcompatibility, was similar to that of bulk water, too.13a,b However, the structure of water primarily sorbed to the polymer film does not reflect the blood-compatibility of the film (the structure of water sorbed into Poly(MPCr-BMA) and Poly(SPB-r-BMA) from the humid air was quite similar to that of free water, whereas that into PMEA was not).12b,c,13a,b,38 Tanaka et al. reported a cold crystallization (CC) phenomenon (freezing at ca -40 °C upon heating after a cooling below -100 °C) for the water incorporated in the PMEA film.16b They attributed the excellent blood-compatibility of PMEA to the presence of (38) Kitano, H.; Mori, T.; Takeuchi, Y.: Tada, S.; Gememei-Ide, M.; Yokoyama, Y.; Tanaka, M. Macromol. Biosci. 2005, 5, 314.

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Figure 5. Number of platelets adhered onto surface of various polymer films. (a) Hydrophilic glass, (b) PBMA, (c) PMMA, (d) Poly(CMB-r-BMA); CMB, 31 mol %. The number of platelets adhered to polymer films was normalized by the number of platelets adhered to PBMA film.

Figure 4. Effect of the content of CMB residue in the Poly(CMB-r-BMA) film on the number of adhered platelets. Polymer films were prepared by (A) the cast method and (B) the spin coat method (solvent: chloroform or chloroform/methanol mixture). The inset shows the data for spin coat films prepared with methanol. The number of platelets adhered to the polymer films was normalized by the number of platelets adhered to PBMA film.

CC phenomenon. A similar phenomenon was also observed in the aqueous concentrated solution of PMPC.39 The presence or absence of cold crystallization phenomenon in carboxybetaine polymer will be examined and reported in a forthcoming paper. As mentioned above, the structure of water incorporated within the polymer matrix (surrounding water) seemed to correlate with the difference in the blood-compatibility of the polymeric materials. In sections B and C, it has been indicated that the number of H-bonds between water molecules collapsed by the presence of one monomer residue (Ncorr value) of zwitterionic copolymer, Poly(CMBr-BMA), was nearly zero, and the O-H stretching band of water incorporated in the Poly(CMB-r-BMA) film is similar to that of free water, showing that the copolymer does not disturb the H-bonded network structure of neighboring water molecules. Therefore, we examined the blood-compatibility of the carboxybetaine copolymers. Figure 4 shows the number of platelets adhered to Poly(CMB-r-BMA) films with various contents of CMB. The copolymer film indicated a better blood-compatibility with an increase in the content of CMB residues. However, at CMB content of 14-17%, the number of adhered platelets reached a minimum, and after that, gradually increased. The optical micrograph showed that the polymer films prepared by the cast method had a large roughness. By the spin coating, the number of platelets adhered to the copolymer films at higher BMA content became smaller. However, the images by an atomic force microscope (AFM) indicated that the roughness of the films still exists (data (39) Tanaka, M. Private communication.

not shown), and the increase in the number of adhered platelets with an increase in CMB content above 14-17 % was observed, though its tendency was becoming relatively smaller. Moreover, we prepared other spincoated thin films using methanol in which Poly(CMB-rBMA) with a higher CMB content could be well dissolved. The spin coating gave a flatter surface, and the number of platelets adhered to the films became smaller (Figure 4 inset). Therefore, a strong correlation of the bloodcompatibility with the CMB content in the film definitely existed. Since the film showed changes in the contact angle with the medium and the θ values for all of the CMBBMA copolymers in the air-in-water system were quite similar (Supporting Information, Table S-1), the surface of the Poly(CMB-r-BMA) seemed to be fully covered with the CMB residues. Figure 5 showed the number of platelets adhered to various kinds of polymer films. The blood-compatibility of the copolymer films is better than other kinds of films. Taking account of the structure of water in the polymer films, a strong correlation between the absence of disturbing effect of any polymer on the H-bonded network structure of water and the blood-compatibility of the polymer seems to be highly probable. Needless to say, the effect of the polymer chain on the O-H stretching band of water would be decreased with an increase in the distance between the water molecule and the polymer chain. The distance needed to gradate from the primarily bound water to bulk water might depend on the chemical structure of polymers, and if the distance is very short, the polymeric materials might be blood-compatible. Recently, it was reported that zwitterionic self-assembled monolayers (SAMs) resist against the nonspecific adsorption of various proteins from a buffer solution.40 A 1:1 mixture of cationic and anionic SAM-forming compounds accumulated on a gold surface was also highly resistant to the nonspecific adsorption of proteins.40 We found that a SAM of CMB telomer is also highly resistant against the nonspecific adsorption of proteins.41 The absence of nonspecific adsorption of serum proteins has been considered as an essential factor for usability of polymeric materials in biomedical fields.15,16 Therefore, it is highly probable that the structure of water at the surface of the zwitterionic compounds plays an essential role in the resistance against nonspecific adsorption of proteins, (40) Erik Holmlin, R.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 2841. (41) Kitano, H.; Kawasaki, A.; Kawasaki, H.; Morokoshi, S. J. Colloid Interface Sci. 2005, 282, 340.

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resulting in both the absence of adhesion and activation of platelets and, consequently, the appearance of biocompatibility. Previously, it was found that linear and branched polyethyleneimines modified with acrylic acid showed no acute toxicity in mice up to dosage of 1 g kg-1.42 The zwitterionic form was attributed to the nontoxic nature of the amino acid type polymers, supporting the tendency observed in this work. Conclusion Structural characterization of aqueous solutions and thin films of homo- and copolymers of carboxybetaine could be carried out using Raman spectroscopy and ATR-IR spectroscopy, respectively. In contrast with ordinary polyelectrolytes which had one kind of ionized group, the carboxybetaine homo- and copolymers with zwitterionic moieties had a very small effect on the structure of hydrogen-bonded network of water molecules in both (42) Kobayashi, S.; Gross, L.; Muacevic, G.; Ringsdorf, H. Macromol. Chem. 1983, 184, 793.

Kitano et al.

aqueous solution and thin film systems. The zwitterionic polymeric materials seem to be blood-compatible in general. Acknowledgment. This research was supported by Grants-in-Aid (16205015 and 17750105) from the Japan Society for the Promotion of Science. The authors are grateful to Osaka Organic Chemicals for the gift of CMB monomer. The authors are indebted to Professor H. Shinohara of this department for allowing us to use the atomic force microscope. The authors thank Mr. Kazutaka Tachimoto of our laboratory for his technical assistance with the AFM measurements. Supporting Information Available: Detailed procedures of Raman and ATR-IR spectroscopic measurements, platelet adhesion test, contact angle measurements and calculation of vibrational frequency using a density-functional method. This material is available free of charge via the Internet at http://pubs.acs.org. LA0515571