Structure of Water in the Vicinity of Phospholipid Analogue

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Structure of Water in the Vicinity of Phospholipid Analogue Copolymers As Studied by Vibrational Spectroscopy† Hiromi Kitano,*,‡ Makoto Imai,‡ Takayuki Mori,‡ Makoto Gemmei-Ide,‡ Yoshiyuki Yokoyama,§ and Kazuhiko Ishihara| Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan, Central Research Institute, Toyama Industrial Technology Center, Takaoka 933-0981, Japan, and Department of Materials Engineering, The University of Tokyo, Tokyo 113-8656, Japan Received June 3, 2003. In Final Form: September 25, 2003 The structure and hydrogen bonding of water in the vicinity of phospholipid analogue random copolymers [poly(2-methacryloyloxyethyl phosphorylcholine-r-n-butyl methacrylate), Poly(MPC-r-BMA)] with various molecular weights were analyzed in their aqueous solutions and thin films 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 the aqueous solution of Poly(MPC-r-BMA) was very close to that for pure water, which is in contrast with the smaller C value in the aqueous solution of ordinary polyelectrolytes. The number of hydrogen bonds collapsed by the presence of one monomer residue (Ncorr value) of Poly(MPC-r-BMA) (Mw 1.3 × 104, 3.0 × 104, and 9.3 × 104) was much smaller than those for ordinary polyelectrolytes and close to those for neutral polymers such as poly(ethylene glycol) and poly(N-vinylpyrrolidone). Furthermore, water-insoluble Poly(MPC-r-BMA) with a large molecular weight (4.2 × 105) could be cast as a thin film (thickness, ca. 10 µm) on a ZnSe crystal for the ATR-IR spectroscopy. At an early stage of sorption of water into the Poly(MPC-r-BMA) film, the O-H stretching band of the 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(2-hydroxyethyl methacrylate), poly(methyl methacrylate), and poly(n-butyl methacrylate). These results suggest that the phospholipid analogue monomer residues with a zwitterionic structure do not significantly disturb the hydrogen bonding between water molecules in either the aqueous solution or the thin film systems.

Introduction Hydrogen bonding (abbreviated as H bonding, hereafter) between water molecules is induced by the electrostatic interaction between the partially positive hydrogen atom on the water molecule and the partially negative oxygen atom of the 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 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 has a significant importance to determine the physical properties of the systems in wide concentration regions. For example, water sorbed into polymer solids from the air or the aqueous solution often causes a significant change in the mechanical properties of the polymers.3 Whereas in dilute polymer solutions, * Author to whom correspondence should be addressed. † Partly presented at the 50th Polymer Symposium at Waseda University in September 2001. ‡ Toyama University. § Toyama Industrial Technology Center. | The University of Tokyo. (1) Scheiner, S. Hydrogen Bonding: A Theoretical Perspective; Oxford University Press: New York, 1997. (2) Eisenberg, D.; Kauzmann, W. The Structure and Properties of Water; Clarendon Press: Oxford, 1969. (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.

the interaction between water and the polymer chain influences the conformation of the polymer chains. Furthermore, the interaction causes many interesting phenomena. For example, water bound to polymer chains is not frozen even below the usual freezing point of bulk water,4 and some kinds of polymers show a coil-globule transition phenomenon upon dehydration in their aqueous solutions.5 The properties of water in polymer systems have been extensively investigated by various techniques such as differential scanning calorimetry,4 NMR,6 and so forth.7 Three kinds of time-averaged structures of water have been defined as (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 shorter relaxation times (τ ) 10-1310-14 s) than those of rotational rearrangement of water molecules in the liquid phase (τ ) 10-11-10-12 s), and, therefore, these methods are quite useful to analyze the (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. (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 1994, 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, 13169.

10.1021/la0349673 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/01/2003

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V structure of water, which reflects the orientation of the water molecules.2,8 Previously, we reported that a phospholipid analogue polymer [poly(2-methacryloyloxyethyl phosphorylcholine)], PMPC) in its aqueous solution did not disturb the H-bonded structure of surrounding water significantly using polarized Raman spectroscopy.9 A similar tendency has been observed in an aqueous solution of another zwitterionic polymer, poly[N,N-dimethyl-N-(3′-sulfopropyl)-3-methacrylamidopropanaminium inner salt], a socalled “sulfobetaine” polymer.10 As for the water incorporated in the polymer thin films, we have been using an attenuated total reflection infrared (ATR-IR) technique to extract the information about the structure of water within water pools existing in various kinds of polymer films.11 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 H-bonded network structure similar to that of free water.12 Mention should be made here that both PMPC and PMEA have been reported to be highly bloodcompatible.13,14 In this report, Raman and ATR-IR analyses of the O-H stretching vibration band (2800-3800 cm-1) of water were carried out to reveal the structure of water in various kinds of polymer systems, including phospholipid analogue copolymers. It has been recently reported that a random copolymer of 2-methacryloyloxyethyl phosphorylcholine (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.13 Therefore, on the basis of the knowledge accumulated about the analysis of water in the polymer system, the structure of water in the vicinity of Poly(MPC-r-BMA) could be analyzed both in the aqueous solution and in the thin film systems using Raman and ATR-IR techniques, respectively. By the compilation of these analytical data, we will be able to establish a technique to predict the blood compatibility of any polymeric materials without contact with human blood itself in the near future. Experiments A. Materials. Three kinds of poly(2-methacryloyloxyethyl phosphorylcholine-r-n-butyl methacrylate) [Poly(MPC-r-BMA) 30, 93, and 420 kDa (1000 Da ) kDa); percent of MPC residues (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. Liq. 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) Kitano, H.; Sudo, K.; Ichikawa, K.; Ide, M.; Ishihara, K. J. Phys. Chem. B 2000, 104, 11425. (10) Kitano, H.; Imai, M.; Sudo, K.; Ide, M. J. Phys. Chem. B 2002, 106, 11391. (11) (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. (12) (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. (13) (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. (14) (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.

Chart 1. Chemical Structures of the Polymers Examined

in the polymers, 30% (30 and 93 kDa) and 31% (420 kDa)] were prepared by the radical copolymerization of MPC and BMA (purified by distillation under reduced pressure) in ethanol with tert-butyl peroxyneodecanoate as the initiator at 60 °C for 6 h (30 and 93 kDa) or 45 °C for 24 h (420 kDa) and purified by precipitation from n-hexane and ether.13 Poly(MPC-r-BMA) with a smaller molecular weight (13 kDa; percent of MPC residues in the polymer, 40%) was prepared at 70 °C for 24 h using 2,2′azobis-isobutyronitrile (AIBN) and 2-mercaptoethanol as the initiator and chain transfer reagent, respectively. The polymer was dissolved in ethanol, precipitated in n-hexane, and classified between 103 and 104 by dialysis and ultrafiltration using a cellulose membrane (Spectrum, molecular weight cutoff 1000) and Amicon membrane (YM10), respectively. The molecular weight of the random copolymers was determined by gel permeation chromatography [0.5% LiBr dissolved in chloroform-methanol (6:4) and an aqueous 0.1 M NaBr solution were used as mobile phase for 30 and 93 kDa (standard, poly(methyl methacrylate), PMMA) and for 13 kDa (standard, pullulan), respectively]. Milli-Q grade water was used for the preparation of the sample solutions. Other polymers, PMMA (Mw ) 1.58 × 105, Mw/Mn ) 1.38), poly(2-hydroxyethyl methacrylate) (PHEMA, Mv ) 6.4 × 104) and PBMA (Mw ) 2.06 × 105, Mw/Mn ) 1.24), were prepared by the conventional radical polymerization using AIBN as the initiator and purified by precipitation from acetone in methanol (PMMA and PBMA) or from methanol in ether (PHEMA). The chemical structures of the polymers examined are shown in Chart 1. B. Raman Spectroscopy. The Raman spectra of various aqueous polymer solutions were recorded on a NR-1100 spectrophotometer (Japan Spectroscopic Co., Tokyo, Japan; light source, Argon laser 488.0 nm) with a band resolution of 5 cm-1. For the polarization geometries X(ZZ)Y (parallel position, I|) and X(ZX)Y (perpendicular position, I⊥), a polarizer plate was rotated by exactly 90° in front of the slit, where X and Y are the directions of laser beam and observation, respectively. The electric vector of the laser beam was maintained in the vertical Z direction for both geometries. A polarization scrambler was used between the slit and the polarizer. The O-H stretching Raman band of water in various polymer solutions was recorded in the region between 2500 and 4000 cm-1 by using the polarization method, as exemplified in Figure 1. The component of the O-H stretching band of water centered at 3250 cm-1 was highly polarized and diminished in the spectra at the perpendicular position. The polarized O-H stretching band of water, which is called the collective band, is ascribed to the H2O molecule executing ν1 vibrations all in phase with each other but vibrational amplitudes varying from molecule to molecule in

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Figure 1. Raman spectra of the O-H stretching region. (a) I| and I⊥ spectra of pure water at 25 °C. The I⊥/FO-H spectrum was also shown with a dotted line. (b) The collective band of water. water clusters that are strongly hydrogen-bonded.15 Theoretical calculations of the random network model,15 which is characterized by the fluctuating defects in water-water hydrogen bonds in a distorted tetrahedral network, support the interpretation. To clarify the effect of polymers on the structure of water, the intensity of the collective band (Ic) observed around 3250 cm-1 was separated from the spectra using eq 1 (Figure 1):

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 incident laser beam, respectively. A depolarization ratio, FO-H, is an indicator of symmetry of the vibration mode, and is expressed as FO-H ) I⊥/I|. The FO-H value is determined to minimize the high-frequency contribution to Ic. Because the intensities of the Raman spectra are not absolute, the area of Ic was normalized according to eq 2:

C)

∫I (ν) dν/∫I (ν) dν c

|

(2)

where ν is the Raman shift in cm-1. C. ATR-IR Measurements. A ZnSe element (80 × 10 × 5 mm, Trough Plate ZnSe 45°; Pike Technologies, Madison, WI) was used for the ATR-IR measurements. An ATR cell was tightly sealed, except for the portals for the inlet and outlet of the dry N2 and humid air of a constant relative humidity (RH). Polymer films with a thickness of 10 µm were prepared on the ZnSe element from the chloroform solution (50 mg‚L-1) under dry N2 and dried at 25 °C under a vacuum for 24 h. The thickness of the film was determined from the diffraction of visible light measured by a fiber-optic multichannel photodetection system (MCPD1100, Otsuka Electronics, Hirakata, Japan) coupled with an optical microscope (Optiphot, Nikon, Tokyo, Japan). The humid air of 50 ( 5% RH was prepared by passing the air at 1.8 mL‚s-1 through a saturated aqueous NaBr solution.11,12 The RH value was monitored at 25 °C with a model SU-610 Humidity Meter, Testoterm, Inc., Yokohama, Japan. The backgrounds were repeatedly measured while the dry N2 flowed through the portals into the holder. When the background was equilibrated, the IR difference spectra of sorbed water to the polymer films were collected at appropriate time intervals after onset of the flowing of humid air. The IR spectra of the films were recorded on a System 2000 FT-IR (Perkin-Elmer) with a TGS detector. All spectra between 2500 and 4000 cm-1 were collected with a resolution of 4 cm-1 and 128 scans at 25 °C. (15) (a) Sceats, M. G.; Rice, S. A. In Water - A Comprehensive Treatise; Frank, F., Ed.; Plenum Press: New York, 1971; Vol. 7. (b) Stillinger, F. H. Science 1980, 209, 451. (c) Walrafen, G. E. In Structure of Water and Aqueous Solutions; Luck, W. A. P., Ed.; Verlag Chemie: Weinheim, 1974.

For the investigation of the water cluster within the film, the film was covered with 1 mL of pure water using a microsyringe. At an appropriate time, the ATR-IR absorption spectra between 2500 and 4000 cm-1 were measured. Thirty minutes after onset of the experiment, 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 vapor and those on which the liquid water was deposited were also obtained. D. Viscometric Measurements. The intrinsic viscosity, [η], ([η] ) limφf0 ) ηsp/φ, ηsp ) (η - η0)/η0, and φ is the concentration of polymer in g‚dL-1) for various kinds of aqueous polymer solutions at 25 °C was determined by using an Ubbelohde dilution type viscometer (Type 0B; Kusano, Tokyo, Japan). E. 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.12b The mass change of the hydrated polymer film (∆m) by heating the film from 25 to 180 °C (heating rate ) 10 °C/min) was recorded. F. Contact Angle Measurement. Static contact angles, θ, of air bubbles on the surface of various polymer films were measured at room temperature by the air-in-water method (CAD, Kyowa Interface Science, Tokyo, Japan).16 The polymer film (thickness, ca. 3 µm) was prepared by the conventional method: a polymer solution (5 mg/mL chloroform or ethanol) was cast on a slide glass in a desiccator and dried at room temperature overnight. Sixty seconds after immersing the polymer film into water, an air bubble (10 µL) was attached to the film surface, and the θ value was quickly measured. Using the same apparatus, static contact angles, θ, of water droplets (10 µL) on the surface of various polymer cast films (30 s after the contact with the water droplet) were measured, too. The θ values were determined four times to give a reliable average value.

Results and Discussion A. Structure of Water in Aqueous Polymer Solutions. The O-H stretching Raman band of liquid water gave a broad band (Figure 1) composed of several overlapping components, which were attributed to the unperturbed O-H stretching band by the intra- and intermolecular vibrational coupling of O-H oscillators. The broad and asymmetric band shape of decoupled O-H stretching, which can be typically observed in a D2OH2O mixture,2,8d indicates that the electromagnetic field around an 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 the H bond between coupled O-H oscillators is broken by the translational or rotational rearrangement of water molecules and (2) when a stretching frequency of an O-H oscillator is largely different from that of the O-H oscillator, which is combined by H bonding with the former one.17 In pure water, values of C at a certain temperature T [CW(T)] are reduced with an increase in the temperature by intrinsic H-bond defects caused by thermal motion.18 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 (16) (a) Tezuka, T.; Araki, A. Langmuir 1994, 10, 1865. (b) Kitano, H.; Fukui, N.; Ohhori, K.; Maehara, Y.; Kokado, N.; Yoshizumi, A. J. Colloid Interface Sci. 1999, 212, 1999. (17) (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.

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where the Mw values were larger than some critical value. 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 the O-H oscillators17 (“pseudonetwork structure” of the polymer chains).19 In solutions of polymers with relatively lower Mw, on the contrary, 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 a polymer chain or counterions are removed from H-bond network of water. Therefore, in this region, the C values are independent of Mw of the polymers. In a dilute solution, polymer coils are separated from each other and intermolecular interaction between the coils is sufficiently weak that the solution can be described as a nonideal gas. When the polymer concentration (φ) approaches a critical value (φ*), the polymer coils begin to overlap and make a pseudo-network. In the concentration region φ > φ*, the polymer solutions are called “semidilute” solutions.19 Graessley suggested a concentration criterion between low- and high-concentration regimes for random coils in a good solvent as 1 < φ[η] < 10.20 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 phospholipid analogue copolymers at 25 °C, it was concluded that almost all the polymers examined here give dilute solutions at pX ) 0.01 (pX is the mole fraction of the monomer residue in the aqueous solution): The φ* values of the copolymers 13, 30, and 93 kDa were 58.9 g/dL (pX ) 0.051), 26.9 g/dL (pX ) 0.025), and 18.1 g/dL (pX ) 0.017), respectively, at least. B. Estimation of the Ncorr Value for Poly(MPC-rBMA). 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 a certain temperature T [CX(T)] are reduced or raised by the breakage or enhancement of water-water H bonding in comparison with those of pure water [CW(T)] at the same temperature. The probability, Pd, that an O-H oscillator is excluded from the H-bonded network of water molecules because of an 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 electrolytes17d and polar substances were smaller than those of pure water at the same temperature,17d whereas those of hydrophobic substances such as tert-butyl alcohol17e and tetra-n-butylammonium hydroxide17f were higher than that of pure water as a result of an enhancement of the H bond between water molecules in hydrophobic hydration shells around the hydrocarbon moiety. To elucidate the effects of the properties of a monomeric unit of various polymers, the number of H-bond defects (18) (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. (19) de Gennes, P. G. Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, 1979. (20) Graessley, W. W. Adv. Polym. Sci. 1974, 16, 1.

Table 1. N and Ncorr Values for Various Polymers at 25 °Ca polymer

Mw (in kDa)

N value

Ncorr value

sodium poly(ethylenesulfonate) poly-L-lysine HBr sodium polyacrylate PEG PVPy PMPC Poly(MPC-r-BMA) Poly(MPC-r-BMA) Poly(MPC-r-BMA)

2.2 2.7 5.0 2.0 2.3 4.3 13 30 93

7.5b 8.1b 8.7c 1.0b 0.9b -1.1b -0.3d -0.6d 1.7d

5.1 5.5 5.9 0.7 0.6 -0.7 -0.2 -0.4 1.1

a In H O. The N and N 2 corr values are for the polymers with the smallest Mw examined, except for Poly(MPC-r-BMA). The uncertainties of the N and Ncorr values are within 1.0 and 0.7, respectively. b Reference 9. c Reference 8e. d This work.

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. In a similar way to small molecular solutes, ionized and polar groups in water-soluble polymer chains and counterions may disturb the H bond between water molecules and increase 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. The N 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 are listed in Table 1. 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 [for example, C ) 0.365 for pure water at 25 °C (CW); C ) 0.54 for the perfectly H-bonded ice (Cice)].17a For the evaluation of the Pd values, this factor was not considered, because the Pd values are calculated by the ratio of CW - CX to 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) and are also compiled in Table 1. The Ncorr values of ordinary polyelectrolytes that had one kind of ionic group (sodium polyacrylate,8e sodium poly(ethylenesulfonate), and poly-L-lysine HBr salt)9 are larger than those of water-soluble neutral polymers (poly(ethylene glycol), PEG, and poly(N-vinylpyrrolidone), 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 the aqueous DNA solution to be around 30 using eq 521 (the Ncorr value could be calculated to be around 20). On the contrary, the Ncorr value for the zwitterionictype polyelectrolyte, PMPC, was very small (-0.7 and -0.1 for PMPC 4.3 and 8.3 kDa, respectively).9 Similarly, those for three kinds of Poly(MPC-r-BMA)s (Mw ) 1.3 × 104, 3.0 × 104, and 9.3 × 104) were very small, too. These slightly negative Ncorr values are probably due to the small (21) Tao, N. J.; Lindsay, S. M.; Rupprecht, A. Biopolymers 1989, 28, 1019.

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error associated with the separation of the collective band from the OH stretching band, and, therefore, they can be regarded as 0 approximately. The relatively nonpolar BMA residues in the copolymers seem to hide behind the hydrophilic MPC residues, resulting in the absence of the effect of BMA residues on the structure of water. 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 (zwitterionic)] were slightly smaller than that for pure water and almost constant despite the difference in the hydrophobicity of the side chain.22 In other words, the Ncorr values for amino acids at a neutral pH are small positive values and do not depend on the hydrophobicity of the side chain significantly (Asn, 3.7; Ser, 2.9; Thr, 4.2; Gly, 2.6; Ala, 2.6; and Val, 2.3).10 The absolutely small Ncorr values for zwitterionic Poly(MPC-r-BMA)s are not inconsistent with this. The anionic phosphate group and cationic quatenary ammonium group, which are in close proximity to each other in the MPC residue, might counteract the 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.23 Recently, a sum frequency generation (SFG) method has been frequently adopted to investigate the structure of water at the air-water interface.24 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.25 The spectrum of water was strongly enhanced by the presence of charged surfactant 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.26 These results are consistent with the tendency observed in this work. Meanwhile, the Ncorr values for electrically neutral polymers were very small. However, it does not mean that these polymers do not interact with water at all. In the aqueous solution of PEG, for example, the number of hydrating water molecules was reported to be 2-6 per monomer unit of PEG.27-29 Using a time domain reflectometry, on the other hand, it was reported that oligo(ethylene glycol)s (degree of polymerization 4-7) dissolve in water without much perturbation to the water structure.30 (22) Ide, M.; Maeda, Y.; Kitano, H. J. Phys. Chem. 1997, 101, 7022. (23) Høiland, H. J. Chem. Soc., Faraday Trans. 1 1975, 71, 797. (24) Richmond, G. L. Chem. Rev. 2002, 102, 2693. (25) Gragson, D. E.; McCarty, B. M.; Richmond, G. L. J. Am. Chem. Soc. 1997, 119, 6144. (26) Walker, R. A.; Gragson, D. E.; Richmond, G. L. Colloids Surf., A 1999, 154, 175. (27) Hager, S. L.; MacRury, T. B. J. Appl. Polym. Sci. 1980, 25, 1559. (28) de Vringer, T.; Joosten, J. G. H.; Junginger, H. E. Colloid Polym. Sci. 1986, 264, 623. (29) Tilcock, C. P. S.; Fisher, D. Biochim. Biophys. Acta 1982, 688, 645. (30) Sato, T.; Niwa, H.; Chiba, A. J. Chem. Phys. 1998, 108, 4138.

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It has been pointed out that Poly(MPC-r-BMA) has an excellent blood compatibility (no thrombus formation).13 PEG and PVPy are used in pharmaceutic aids as ointment, a suppository base, and the dispersing and suspending reagent of medicines. In addition, PEG is nonimmunogenic, showing that PEG does not perturb the selfprotection system in living bodies. Furthermore, liposomes coated with PEG chains can avoid the attack by the reticulo-endothelial systems.31 Taking account of 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 the morphology and steric stabilization effect affect the blood compatibility of polymeric materials in a complicated manner.32 It should be noted here that the Ncorr value for gelatin (Mw ) 104), a highly biocompatible 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 previously. Recent computer simulations provide useful information on the structure and properties of water in hydration shells of individual groups on polymer chains. For example, simulations using molecular dynamics showed that the average H-bond numbers for water in the vicinity of hydrophilic groups of polymers are smaller by 1-1.9 than those in pure water.33 Using the same method, the structure of water around the polymers with zwitterionic pendent groups will be clarified. C. Structure of Water in the Poly(MPC-r-BMA) Film. To examine an effect of the 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 was exclusively 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 0.59 µm at 3333 cm-1].11,12 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 located both inside and at the surface of the films. Figure 2 shows the ATR-IR bands for the sorbed water vapor and liquid water existing in the matrix of Poly(MPC-r-BMA) film 30 min after onset of the sorption measurement. Many tiny peaks in the range of 37004000 cm-1 correspond to the O-H stretching of the water molecule in air. In addition, the negative-going intensity in the 3000-2800 cm-1 region might be caused by the hydration of the hydrocarbons (CH and CH2 groups) in the polymer chain.34 (31) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. FEBS Lett. 1990, 268, 235. (32) (a) Biomedical Applications of Polymeric Materials; Tsuruta, T., Hayashi, T., Kataoka, K., Kimura, Y., Ishihara, K., Eds.; CRC Press: Boca Raton, FL, 1993. (b) Jo¨nsson, B.; Lindman, B.; Hokmberg, K.; Kronberg, B. Surfactants and Polymers in Aqueous Solution; John Wiley & Sons Press: New York, 1999. (33) (a) Tamai, Y.; Tanaka, H.; Nakanishi, K. Macromolecules 1996, 29, 6750. (b) Tamai, Y.; Tanaka, H.; Nakanishi, K. Macromolecules 1996, 29, 6761. (34) (a) Mizuno, K.; Ochi, T.; Shindo, Y. J. Chem. Phys. 1998, 109, 9502. (b) Maeda, Y.; Higuchi, T.; Ikeda, I. Langmuir 2000, 16, 7503. (c) Scheiner, S. Hydrogen Bonding: A Practical Approach; Oxford University Press: London, 1997; p 99.

Structure of Water

Figure 2. ATR-IR spectra of water sorbed into the Poly(MPCr-BMA) film (a) obtained by the water vapor method and (b) by the liquid water method (1 mL of water was added). (c) Difference spectrum of spectra b and a. (d) Bulk water.

Figure 3. ATR-IR difference spectra of water sorbed into various polymer films: (a) free water; (b) Poly(MPC-r-BMA), Mw ) 420 kDa, MPC, 31 mol %; (c) PHEMA, Mv ) 64 kDa; (d) PMMA, Mw ) 158 kDa; and (e) PBMA, Mw ) 206 kDa. In the liquid water experiment, 40 µL of water was dropped above the film. Twenty seconds after dropping liquid water, the ATR-IR spectrum was obtained.

Previously, it was shown that the ATR spectra for the sorbed water vapor correspond to the water H-bonded to a polar group such as hydroxyl, carboxyl, and amide groups in the polymeric films (primarily hydrating water), whereas that for the liquid water reflects the absorbance of both primarily hydrating water and water surrounding the primary hydration layer.11b,c,12a Therefore, by the subtraction of the intensity of primary hydrating water (sorbed water vapor) from that of liquid water, we could roughly extract the absorption band for the water existing within the polymer matrix as the “surrounding” water.12a We assumed here that the amount of water primarily hydrating to the film was the same in both water vapor and liquid water systems. The dotted line in Figure 2 shows the subtracted IR bands for the water within the polymer matrix. In Figure 3, the O-H stretching bands for “surrounding” water in four kinds of polymer films Poly(MPC-r-BMA), PBMA, PMMA, and PHEMA are shown together with the O-H stretching band for free liquid water. In the liquid water system examined here, 40 µL of liquid water was added dropwise onto 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 is very significant in the cases of PBMA and PMMA. Figures 2 and 3 clearly show that the structure of water kept in the matrix of the Poly(MPC-r-BMA) film was similar to that of free water, except for the slightly larger absorbance at the lower frequency region in the former case. In contrast with this, the structure of water in the matrix of the PHEMA film was clearly different from that of free liquid water (three broad peaks in the PHEMA film system

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correspond to the O-H stretching of water molecules binding to the ester and hydroxyl groups in PHEMA),11c,12a and, furthermore, those in the PBMA and PMMA films were extremely different. 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 the 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 2 shows that the amount of water vapor sorbed into Poly(MPC-r-BMA) was the largest and that to PHEMA was the second among polymer films examined in this work. Similarly, the amount of water kept in the matrix of Poly(MPC-r-BMA) was the largest among the polymer films examined. As discussed previously, the hydrogen-bonded network structure of water, which is widely accepted as the basic structure of ordinary liquid water,2,16 was more significantly disturbed in the matrix of PHEMA, PMMA, and PBMA than in the matrix of Poly(MPC-r-BMA). In the cases of PMMA and PBMA, though the diffusion coefficient of water vapor in the film was larger than that of PHEMA,11c the amounts of sorbed water in both the water vapor and the liquid water measurements were very small, and the structure of water kept in the PMMA and PBMA matrixes was disturbed to a large extent. 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 the polymers, and if the distance is very short, the polymeric materials might be blood-compatible. It has been reported that the number of blood cells adhered to the Poly(MPC-r-BMA) film was much smaller than those to PBMA and PHEMA films.13 Quite recently, we have reported that the structure of water in the matrix of the PMEA film, which shows an excellent biocompatibility, was quite similar to that of bulk water.12a,b The amount of water sorbed into the PMEA film is also shown in Table 2. Tanaka et al. reported a cold crystallization phenomenon (freezing at ca. -40 °C upon heating after a cooling below -100 °C) for the water incorporated in the PMEA film.14 A similar phenomenon was also observed in the aqueous concentrated solution of PMPC.35 Therefore, it is highly probable that the “cold-crystallization phenomenon” will be observed in the aqueous Poly(MPCr-BMA) system, too. As mentioned previously, the structure of water incorporated within the polymer matrix seemed to correlate with the blood compatibility of the polymeric materials. As discussed in section B, the number of H bonds between water molecules collapsed by the presence of one monomer residue (Ncorr value) of the zwitterionic copolymer, Poly(MPC-r-BMA), was nearly zero, showing that the copolymer does not disturb the H-bonded network structure of water molecules. Because this polymer has been frequently reported to be highly biocompatible,13 a strong correlation between the absence of the disturbing effect of any polymer (35) Tanaka, M. Private communication.

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Table 2. Amount of Water Sorbed into Various Polymer Films polymer sorbed water (vapor) (g/g polymer) sorbed water (liquid) (g/g polymer) a

Poly(MPC-r-BMA)a

PHEMA

PMMA

PBMA

PMEA

0.118 0.860

0.054b

0.0050 0.0089

0.00050 0.0017

0.0065b 0.030b

Mw ) 420 kDa. b Calculated from the data in ref 12b. c This value was comparable with the literature data in refs 14b and 36.

Table 3. Contact Angles of the Water Droplets and Air Bubbles above Various Polymer Films sample Poly(MPC-r-BMA)a PHEMA PMMA PBMA PMEA a

0.443b,c

sessile drop (degree) air-in-water (degree) 96.8 (( 1.8) 26.2 (( 2.7) 68.2 (( 1.4) 89.4 (( 1.1) 52.1 (( 1.9)

152.2 (( 1.9) 150.4 (( 2.1) 109.1 (( 1.5) 96.8 (( 1.7) 135.7 (( 2.0)

Mw ) 420 kDa.

on the hydrogen-bonded network structure of water and the biocompatibility of the polymer seems to be highly probable. Table 3 shows contact angles (θ) of the water droplets and air bubbles at the surfaces of various polymer films. The θ values by the sessile drop method were in the order Poly(MPC-r-BMA) > PBMA . PMMA > PMEA > PHEMA and mostly consistent with the polarity of the polymers. As for Poly(MPC-r-BMA) in the dry state, the BMA residues might jut out into the exterior to give a rough surface,37 resulting in the slightly larger θ value than that for PBMA. In the air-in-water system, the hydrophilic surface in general shows a larger θ value than the hydrophobic ones, in a manner opposite that of the ordinary sessile drop method.16 Therefore, the order of θ values by the air-in-water method (PHEMA > PMEA . PMMA > PBMA) was quite understandable. The large θ value for Poly(MPC-r-BMA) by the air-in-water method suggests that the copolymer, which is hydrophobic in the dry state, becomes highly hydrophilic in water. In addition, the θ value for the copolymer did not change with time for 30 min after the immersion into water noticeably, indicating that the copolymer quickly and drastically changed its conformation upon contact with water. The MPC residues, which did not perturb the hydrogen-bonded structure of water as discussed in section B, might (36) Kermis, H. R.; Rao, G.; Barbari, T. A. J. Membr. Sci. 2003, 212, 75. (37) (a) Wenzel, R. N. J. Phys. Colloid Chem. 1949, 53, 1466. (b) Wenzel, R. N. Ind. Eng. Chem. 1936, 28, 988.

promptly appear at the polymer-water interface.16a This environmental response seemed to enable the copolymer to show the excellent blood compatibility.13 At the end of this section, it should be emphasized that the amount of sorbed water in the polymer matrix does not strongly correlate with the structure of water (the amount of water sorbed was in the order Poly(MPC-rBMA) > PHEMA > PMEA, while the resemblance of the structure of sorbed water to that of free water was in the order Poly(MPC-r-BMA) ≈ PMEA > PHEMA).11b,c,12a,b In addition, the structure of water vapor primarily sorbed into the polymer film does not reflect the biocompatibility of the film (the structure of water vapor sorbed into Poly(MPC-r-BMA) was quite similar to that of free water, whereas those into PMEA and PHEMA were not).11b,c,12a,b Conclusion The structural characterization of aqueous solutions and thin films of phospholipid analogue copolymers could be carried out using Raman spectroscopy and ATR-IR spectroscopy, respectively. Important information about the interaction between water and polymer chains could be obtained from the structure of water. In contrast with ordinary polyelectrolytes that had one kind of ionized group, the phospholipid analogue copolymers with zwitterionic residues had a very small effect on the structure of the hydrogen-bonding network of water molecules in both the aqueous solution and the thin film systems. Acknowledgment. This research was supported by a Grant-in-Aid (12450381, 13022225, and 13555260) from the Ministry of Education, Science, Sports and Culture, Japan. The authors wish to thank Mr. Kurao Sudo, Toyama University, for his technical assistance. The authors are grateful to Dr. Masaru Tanaka, Research Institute for Electronic Science, Hokkaido University, for his kind suggestion concerning the cold crystallization phenomenon of PMPC. LA0349673