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Studies on bound water restrained by poly(2-methacryloyloxyethyl phosphorylcholine): Comparison with polysaccharide–water systems
Acta Biomaterialia 6 (2010) 2077–2082
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Studies on bound water restrained by poly(2-methacryloyloxyethyl phosphorylcholine): Comparison with polysaccharide–water systems T. Hatakeyama a,*, M. Tanaka b, H. Hatakeyama c a
Lignocel Research, 73-8 Yatsumata, Fukui 910-3558, Japan Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan c Graduate School of Engineering, Fukui University of Technology, 3-6-1 Gakuen, Fukui 910-8505, Japan b
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 2 November 2009 Accepted 7 December 2009 Available online 11 December 2009 Keywords: Poly(2-methacryloyloxyethyl phosphorylcholine) Polysaccharides Non-freezing water Freezing bound water Differential scanning calorimetry
a b s t r a c t The structural change of water restrained by poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was investigated by differential scanning calorimetry (DSC), since the biocompatibility of PMPC and related biopolymers is affected by the structure of water on the polymer surface. The phase transition behaviour of PMPC–water systems with a water content (Wc = mass of water/mass of dry sample, g g1) in the range 0–2.0 was measured in the temperature range 150 to 50 °C. Glass transition, cold crystallization and melting were observed. Cold crystallization, which has been suggested as an index of biocompatibility, was detected for PMPC with a Wc in the range 0.5–0.9. The amounts of two types of bound water, non-freezing water and freezing bound water, were calculated from the melting enthalpy. The amount of non-freezing water of PMPC was 0.48. It was found that the phase transition behaviour and amount of bound water of PMPC were quite similar to those of water-soluble polysaccharide electrolytes. The results indicate that the bound water, not the free water, is restrained by PMPC. Ó 2010 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.
1. Introduction In order to develop new biocompatible polymers various factors affecting the biocompatibility of foreign materials, such as chemical groups on the surface, ionic charge, other physical factors such as morphological features and surface evenness, have been examined [1–3]. It is appropriate to consider the structural changes of water on the surface of the polymer as a major factor, since it is reasonable to expect that water strongly restrained by biomaterials shows unique behaviour, different from bulk water [2,4–12]. The molecular mobility of water molecules surrounding biomaterials has been investigated by nuclear magnetic relaxation studies [13,14]. It is thought that three different types of water [15] can be categorized from the relaxation time of water, i.e. the correlation time (sc) calculated from the spin–lattice (T1) and spin–spin relaxation times (T2) of water directly restrained by biomaterials is in the range 105 to 106 s, with a second layer of water with a correlation time of 108 s. These values are greater than that of the outer layer of water molecules, whose sc is 1012 s. Water species can be identified from nuclear magnetic relaxation measurements, however, the amount of each type of water is difficult to estimate using only nuclear magnetic relaxation measurements. The number of water molecules restrained by the matrix surface * Corresponding author. Tel./fax: +81 776 89 2885. E-mail address: [email protected] (T. Hatakeyama).
as a first layer has been calculated from a water sorption isotherm obtained in gravimetric experiments in the 1930s [16]. The Brunauer–Emett–Teller equation [16] can be applied to quantify the number of bound water molecules, even though more elaborate methods, such as the quartz crystal microbalance method, have been introduced and applied to polymer–protein biopolymer– water interactions [17,18]. From mass measurements precise results can be obtained for the initial stage of water sorption onto the surface of the polymer. At the same time, it is notable that the method has certain limitations in determining the exact amount of the second and other outer layer of waters during the stage of cluster formation. Among various techniques to investigate the structural changes of water restrained by polymers [8], we have paid particular attention to thermal analysis, since the phase transition behaviour of water can be easily detected by this method [9]. Although thermogravimetry can be used to quantify the amount of water by a vaporization technique [19], differential scanning calorimetry (DSC) has been paid special attention. When hydrated hydrophilic polymers, such as polysaccharides, are measured by DSC, three kinds of water are identified from the temperature and enthalpy of transition peaks. Hatakeyama and coworkers have classified hydrated water (water in the polymer) as follows: freezing (crystallizable) water which can be observed as the first order phase transition and grouped into (1) free water which crystallizes at 0 °C and is slightly affected by the matrix polymer and (2)
1742-7061/$ - see front matter Ó 2010 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2009.12.018
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freezing bound water which crystallizes during the heating process at a temperature lower than 0 °C and is strongly affected by the matrix polymer. In addition to freezing water there exists (3) non-freezing water, i.e. water which is non-crystallizable due to strong molecular interactions with the matrix polymer. Both freezing bound water and non-freezing water are categorized as bound water [2,9,20]. A large number of polymers having a biomembrane-like surface have been investigated by many researchers in order to develop new biomedical materials [21,22]. In our previous studies it was found that poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility, i.e. the amount of protein adsorbed onto the surface of PMEA was much lower than that of other conventional polymers [3,17,18,23–25]. It is noteworthy that the degree of denaturation of proteins remained at a low level on the surface of PMEA. Because of this, PMEA has already been applied as a coating material for a blood oxygenator which showed reliable performance under the designated conditions both in vitro and in vivo [26,27]. Furthermore, it was observed that the activity of blood components was reduced when a cardio-pulmonary bypass was coated with PMEA [27–29]. In our previous studies the melting behaviour of a hydrated PMEA analogue poly(methacrylate) having different side chain structures was systematically investigated and the blood compatibility of each polymer was correlated with of the amount of bound water [2]. It was concluded that the freezing bound water, detected as cold crystallization in DSC heating curve, plays a crucial role in blood compatibility. The above facts suggest that the presence of freezing bound water can be utilized as an index in choosing biocompatible polymers. In this study we pay particular attention to poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a large number of studies have been concerned with PMPC co-polymers, which are reported to reduce protein adsorption [5,30,31]. PMPC is a watersoluble polymer, so hydrophobic polymers are introduced as a co-polymerized component or chemically cross-linked [33] in order to obtain water insoluble polymers. Various types of PMPC co-polymers have been synthesized, taking into consideration the surface structure of biomembranes [34]. It has been reported that PMPC co-polymers show reasonable blood compatibility, such as suppression of protein adsorption and platelet adhesion, and they have been applied as surface modifiers of all sorts of medical devices in order to improve blood compatibility [32]. In order to confirm the role of water restrained by PMPC, in this study the phase transition behaviour of hydrated PMPC is extensively investigated. It is also intended to make a comparison with the thermal behaviour of water-soluble polysaccharides, which are accepted as representative water-soluble biopolymers. 2. Materials and methods 2.1. Samples The chemical structure of PMPC is shown in Fig. 1. PMPC in powder form was obtained from NOF Co., Tokyo. The molecular mass of PMPC was 1.1 105 g mol1 and the number average molecular mass was 0.7 105. Details of the preparation method can be found elsewhere [5].
H
CH3
C
C
H
C
n
O
O-
OCH2CH2OPOCH2CH2N+(CH3)3 O Fig. 1. Chemical structure of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).
samples were held at 150 °C for 10 min and then heated to 60 °C at 10 °C min1 (first heating run). The samples were cooled to 150 °C at a predetermined rate of between 2 and 50 °C min1 (second cooling run) and then heated at 10 or 2 °C min1 (second heating run). In order to obtain PMPC with various water contents, the following procedure was carried out. Samples (3–5 mg) were placed in pans and a small amount of deionized water was added using a micro-syringe. The water was evaporated until a predetermined mass of water was attained. The sample pans were then hermetically sealed using an auto-sealer. The samples with added water were weighed. These samples were kept at room temperature overnight and reweighed in order to confirm that no mass loss had occurred. After DSC measurements the pan was pierced, annealed at 110 °C for 1 h in an electric oven and then weighed. The amount of water was evaluated using Eq. (1). Although the water content (Wc) of hydrated polymers has been defined by various equations, in this study Wc is defined as follows:
W c ¼ mw =ms
ð1Þ
where ms is the mass of the dry sample and mw is the mass of water in the system. The glass transition temperature (Tg) was defined as the temperature at which the extrapolated baseline before the transition intersects the tangent drawn at the point of greatest slope of the heat capacity change step due to glass transition. Temperature and enthalpy of transition was calibrated using pure water as a standard [34]. The heat capacity difference at Tg (DCp) was calculated using the total mass of the sample (m0 + mw) [35,36]. The peak temperature of melting was designated the Tm. When two melting peaks of water were observed the high temperature side peak was designated Tmh and the low temperature side peak Tml. In this study cold crystallization was defined as an exothermic transition that occurred in the temperature range between Tg and Tm. The peak temperature of the exothermic peak was defined as Tcc. The melting enthalpy (DHm, J g1), cold crystallization enthalpy (DHcc, J g1) and melting enthalpy (DHm, J g1) were calculated using the mass of water (mw) in the PMPC–water systems. The enthalpy of the melting of water (334 J g1) was used for calculation. The amount of freezing water was calculated from DHm, calculated from the second heating curve. As described in the above, enthalpies of transitions were calculated from the peak area of each transition. When two melting peaks were observed the enthalpy of each peak was calculated. In this study bound water was calculated as follows:
W c ¼ W f þ W fb þ W nf
ð2Þ
2.2. Differential scanning calorimetry
W b ¼ W fb þ W nf
ð3Þ
A Seiko Instruments differential scanning calorimeter DSC 200C equipped with a cooling apparatus was used. The nitrogen gas flow rate was 30 ml min1. The samples were heated to 60 °C and then cooled to 150 °C at a rate of 10 °C min1 (first cooling run). The
where Wf is the amount of free water calculated from DHm of the high temperature side melting peak, Wfb is the freezing bound water calculated from DHm of the low temperature side melting peak and Wb is the total amount of bound water. The amount of
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non-freezing water (Wnf) can be calculated using Eq. (2), as Wc was obtained from Eq. (1).
3. Results and discussion 3.1. Phase diagram of PMPC–water systems PMPC–water systems with a water content (as defined in Eq. (1)) ranging from 0 to 2.0 were measured by DSC, in the temperature from 150 to 80 °C. Fig. 2 shows representative stacked DSC heating curves of PMPC–water systems with various water contents. As shown in the figure, a heat capacity change due to glass transition was observed for all samples. Cold crystallization was clearly seen as a broad exothermic peak in the sample with Wc = 0.55. Cold crystallization was scarcely observed when Wc exceeded 1.0. Besides the main peak of melting, a melting peak with a shoulder on the low temperature side was observed. From DSC curves the temperature of each transition, i.e. Tg, Tcc, Tmh, and Tml, could be obtained. Phase diagrams of PMPC–water systems were obtained based on the DSC heating curves shown in Fig. 2. Tg decreased markedly in the initial stage, reached a minimum and then increased to reach equilibrium. Glass transition of PMPC–water systems was observed over wide water content and temperature ranges, as shown in Fig. 3. It is known that glass transition is not observed for dry PMPC [38]. Glass transition of dry polyelectrolytes is ordinarily not observed due to strong intermolecular interactions. When a small amount of water is added to the polyelectrolytes molecular motion of the main chain can be detected. When PMPC–water systems with Wc values ranging from 0 to 0.7 were studied by DSC in the temperature range 150 to 60 °C the glass transition temperature varied in a complex manner depending on the water content, as shown in Fig. 3. It is reasonable to suppose that main chain motion of dry PMPC is strongly restricted due to intermolecular bonding. However, when a small amount of water is added to PMPC, water molecules diffuse between the PMPC molecules, break the inter-
Fig. 2. Stacked DSC heating curves of PMPC–water systems (2nd heating run). Numerals in the figure show Wc (g g1). Heating rate 10 °C min1.
Fig. 3. Phase diagram of the PMPC–water system. Tg, glass transition temperature; Tcc, cold crystallization temperature; Tm, melting temperature.
molecular bonding and main chain molecular motion is enhanced. The baseline change due to glass transition is clearly observed for PMPC with Wc = 0.44. Generally the water diffusion constant in a hydrophilic polymer from the surface to the inner part is between 0.5 and 1.5 105 cm2 s1 [1]. Here it is appropriate to consider that water molecules are homogeneously restrained by PMPC molecules. The heat capacity difference at Tg of the sample with Wc = 0.45 was 0.5 J g1 K1, which is far larger than the ordinal polymer, suggesting that both water molecules and PMPC initiated movement at the same time [10,35]. The glass transition temperature (Tg) decreased, reaching its lowest temperature (Tg min) of approximately 100 °C, then increased slightly and reached a constant value (Tg plateau) of approximately 40 °C with increasing water content. Molecular motion was disturbed in the presence of free water due to presence of ice. Glass transition of water-soluble polyelectrolytes is thought to reflect the cooperative molecular motion of polyelectrolyte molecules and amorphous ice frozen without crystallization [10,35,38,39]. This was confirmed by the fact that the Tg of the system (Tg system) was coincident with the calculated values, assuming an additive relationship of the Tg of amorphous ice in the system and the Tg of the dry sample [40], although in many polyelectrolyte systems the Tg of the completely dry system is an assumed value, obtained by extrapolation from the Tg values of samples with varying amounts of water. The above consideration leads to the conclusion that the glass transition behaviour of PMPC is similar to that of water-soluble polyelectrolytes, such as sodium polystyrene sulfonate [41], among others [9]. Nuclear magnetic resonance (NMR) spectrometry 1H relaxation times (sc) for the bound water in polyelectrolyte–water systems were between 108 and 106 s at approximately 50 °C. The above values were calculated assuming that the inverse longitudinal relaxation 1 (T 1 1 ) and transverse relaxation times (T 2 ) of bound water were 1 different but in the case of free water T 1 = T 1 2 , since extreme narrowing conditions were fulfilled [7,41]. The above data suggest that the water molecules are categorized as non-rigid solid, since the typical NMR pattern due to splitting of 1H spins could be observed at low temperature [41,42]. The NMR results indicate that the water molecules are frozen in a glassy state together with matrix polymers. Two melting peak temperatures, Tml and Tmh, were observed at Wc values higher than 0.5. Tml was observed at a constant temperature of approximately 30 °C, regardless of Wc. Tmh was observed at a temperature below 0 °C in the initial stage and then maintained a constant value. Tcc was observed in the water content range 0.5–0.8 at around 60 °C. It was noticed that the temperature of the transition peak depended on the sample mass and thermal history. Accordingly, the exact values can only be defined when the experimental conditions are strictly defined. Since
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deconvolution of DSC curves is not theoretically available in this study, the peak was employed as an index of the melting temperature. Because of this, when values of Tmh and Tml of different samples were compared the values always necessarily involved a certain degree of error. In DSC the peak temperature and the width of the melting peak depend on the matrix biopolymer and also the amount of co-existing water. The fact that the temperature difference between Tml and Tmh was 30 °C in the PMPC–water system indicates that the characteristics of freezing water bound to PMPC is markedly different from that of free water. In the case of watersoluble hydrophilic polymers, such as polysaccharide electrolytes, similar phase diagrams can be observed [9]. The phase diagrams of PMPC–water systems are characterized by a large temperature difference between Tml and Tmh compared with that of polysaccharide electrolyte–water systems.
Table 1 Non-freezing water in various kinds of hydrophilic polymer and polyelectrolyte.
a b c
Sample
Wnf
References
PMPC Gum Arabic (Acacia senegal) Hyaluronan Sodium cellulose sulfate Sodium carboxymethyl cellulose Sodium alginate Xanthan gum Guar gum Tara guma Locust bean gum Celluloseb,c Methylcellulose
0.48 0.55 0.50 0.43 0.85 0.43 0.55 0.75 0.5 0.70 0.2–0.4 0.88
This work [44,45] [36] [46,47] [48] [49] [50] [37] [37] [37] [9,19,51] [52]
Wnf does not level off until Wc = 3.8. Water insoluble. Wnf depends on crystallinity.
3.2. Non-freezing water of PMPC–water systems The enthalpy of transition can be calculated from the area under each peak of DSC heating curves shown in Fig. 3, assuming the melting enthalpy of ice is 334 J g1. As described in Section 2, each transition enthalpy was determined using the second heating curve and the amount of bound water was calculated using Eqs. (2) and (3). Fig. 4 shows Wb, Wfb, and Wnf for the PMPC–water systems as a function of Wc. It can clearly be seen that the Wnf of PMPC increases with increasing Wc and levels off at around 0.5, while Wfb is observed at Wc = 0.5 and decreases with increasing Wc. A slight increase in Wfb at Wc = 0.5 can be explained by the fact that the number of water molecules available to form clusters which freeze to form hexagonal ice [43] was insufficient for the system with Wc = 0.5, since the major part of bound water presented as Wnf [43]. When a sufficient amount of free water was supplied to the system a part of Wfb was included as free water and the enthalpy of the low temperature side melting peak decreased slightly. Table 1 shows the value of Wnf calculated from the melting enthalpy of water restrained by various kinds of biopolymers. The calculation was carried out using Eqs. (2) and (3), as described in Section 2. The value of Wnf obtained for the equilibrium state of PMPC and various other kinds of biopolymers were obtained under similar experimental conditions by DSC. As can be clearly seen in Table 1, the value of Wnf for PMPC was similar to the values for polysaccharides electrolytes and other natural polysaccharides. It should be noted that water molecules are strongly restrained by ionic groups on the 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. The results in Table 1 indicate that the surface of PMPC is not covered with free water, as Ishihara and coworkers sug-
gested [5]. The results shown in Table 1 strongly suggest that the Wnf value of PMPC and its co-polymer is similar to that of other polysaccharides. The equilibrium water content (EWC) is an important index in the biomedical application of various kinds of polymers. EWC is generally measured by weighing a water absorbing sample film after immersion in water or saline solution. Although the value is affected by the experimental conditions, such as immersion temperature, time, and thickness of film, reliable EWC values can be obtained with a certain precision. The method is only applicable when the sample is water insoluble but swells by a certain amount in water. Ishihara reported that the EWC of a PMPC co-polymer with a hydrophobic component (butylmethacrylate) was 0.36 when the water content is defined as mw/(mw + ms). In this study the above value corresponds to 0.56 when the water content is defined as shown in Eq. (1). The above EWC value accords well with the maximum Wnf value of PMPC, as shown in Fig. 4 and Table 1. This indicates that the non-freezing water of PMPC co-polymers is mainly attached to hydrophilic groups of the MPC component. The polysaccharides shown in Table 1 are water soluble except for cellulose. Natural cellulose obtained from various plants is water insoluble and mainly obtained as a fibre or in powder form. Because of this it is inappropriate to obtain EWC in a similar manner to film samples. Rather than EWC, the water content of cellulose samples saturated in a 100% relative humidity atmosphere at a determined temperature is used as the criterion of hydrophilicity. The water content obtained by the above method is defined as the water regain, used in the field of applications. We have reported that the water regain of cellulose from various plants having different crystallinities accords well with 1/2 of the Wnf value determined by DSC [53]. The above results strongly suggest that the value of Wnf is an index of the hydrophilicity of hydrophilic polymers such as PMPC and polysaccharides.
3.3. Cold crystallization and freezing bound water
Fig. 4. Relationship between Wb, Wfb, Wnf, and Wc of the PMPC–water system. Wb, bound water content; Wfb, freezing bound water content; Wnf, non-freezing water content.
Crystallization of amorphous materials at a temperature between the glass transition and melting is termed cold crystallization. Cold crystallization can be frequently observed in glassy polymers and detected as an exothermic event in DSC heating curves. In a previous study on the phase transition behaviour of polyethylene glycol–water systems [10] distinct cold crystallization was found over a wide range of water contents. From the enthalpy balance of cold crystallization, crystallization and melting it can be concluded that the glassy state formed amorphous ice and polymer cooperatively, involving cold crystallization of the system [10,54].
T. Hatakeyama et al. / Acta Biomaterialia 6 (2010) 2077–2082 Table 2 Temperature and water content range of cold crystallization observed in polymer– water systems. Sample
PMPC Poly(ethylene glycol) Hyaluronan Gum Arabic Cellulose sulfate Carboxymethylcellulose Methylcellulose Sodium alginate Pectin Xanthan gum Guar gum Tara gum Locust bean gum
Peak temperature (Tcc) (°C) 65 48 50 55 55 40 50 30 50 60 35 25 25
to to to to to to to to to to to to to
60 44 20 20 23 38 20 20 41 20 25 20 20
Water content range (g g1)
References
0.5–0.9 0.5–1.2 0.7–1.4 0.5–0.8 0.5–1.1 0.8–1.7 0.8–1.5 0.5–1.2 0.4–0.6 0.5–1.1 0.4–2.5 0.6–1.0 0.6–0.7
This work [10] [36] [44,45] [46] [48] [52] [49] [55] [50] [37] [37] [37]
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certain amount of freezing bound water is masked by the DSC melting peak. This indicates that the Wfb value at around a Wc equal to the EWC is equivalent to or more than the Wfb of PEMA [4]. 4. Conclusions Structural changes of water on the surface of biomembranes are important factors when designing new biopolymers. The levels of the two types of bound water, non-freezing water and freezing bound water, were calculated from the melting enthalpy and the bound water content of PMPC by DSC. It was found that the amount of non-freezing water in PMPC was approximately 0.5, which is similar to the values for various kinds of polysaccharides. The amount of freezing bound water in PMPC was in the range 0.05–0.1, depending on the water content. The results indicate that the bound water on the surface of biopolymers plays a crucial role in the biocompatibility of biopolymers. Acknowledgements
Table 2 shows the temperature and water content ranges over which cold crystallization was observed in various kinds of biopolymer–water systems. In this table the water content at which the cold crystallization peak was observed is also indicated. The peak temperature of cold crystallization (Tcc) depends on water content, and generally Tcc increases slightly with increasing water content. In the case of PMPC, Tcc was observed at approximately 30 °C. The enthalpy of cold crystallization showed a maximum value at the Wc at which Wnf plateaued for almost all polysaccharides–water systems. Ishihara and coworkers have reported that PMPC co-polymers show excellent biocompatibility [5,32]. In order to explain the characteristic features of these polymers they focused on the role of water molecules in PMPC co-polymers [30,33]. According to Ishihara and coworkers the mobility of water on the surface of membranes plays a crucial role. The above description does not accord with the results obtained in this study, since they described how a large amount of free water in the polymers was responsible for the excellent cytocompatibility of MPCs [5]. They defined free water as ‘‘water” showing a first order phase transition, even though the melting peak starts to deviate in the endothermic direction at temperatures below 0 °C. Furthermore, it is notable that the amount of free water in PMPC depends only on concentration, i.e. the EWC of PMPC is difficult to define. In the case of MPC–hydrophobic co-polymers the value of Wnf is defined as the amount bound to the MPC fraction, while the remaining water at EWC can be categorized as freezable water [33]. It is thought that freezable water as defined by Ishihara and coworkers includes freezing bound water when the definition of our study is employed, as shown in Eq. (2). Tanaka and coworkers showed that the biocompatible nature of PMEA is attributable to the amount of freezing bound water [2,4,25]. They paid attention to the cold crystallization which is observed in the DSC heating curves of PMEA–water systems. Tanaka and coworkers stated that the cold crystallization observed in the DSC heating curves of PMEA–water systems shows a significant relationship with biocompatibility [2]. Various homologues of PMEA, whose cold crystallization has been observed by DSC, showed improved results when examined in biological tests. Ice formed by cold crystallization melts at a temperature lower than the melting temperature of free water. In this study the above water corresponded to the lower temperature side peak, characterized as Tml and DHml. Wfb was calculated from DHml. As shown in Fig. 4, the Wfb of PMPC showed a maximum value around Wc, where Wnf plateaued. It is considered that the hydrophilicity of the MPC fraction of the various co-polymers synthesized by Ishihara and coworkers have a role as water restraining sites and that a
The authors are grateful to Professor Emeritus Teiji Tsuruta, University of Tokyo, and Professor Clive S. Langham, Nihon University, for their comments and Ms Shizuka Horita for her helpful assistance. References [1] Ichikawa K, Mori T, Kintano H, Fukuda M, Mochizuki A, Tanaka M. Fourier transform infrared study on the sorption of water to various kinds of polymer thin films. J Polym Sci Polym Phys 2001;39:2176–82. [2] Tanaka M, Mochizuki A. Effect of water structure on blood compatibility – thermal analysis of water in poly(methacrylate). J Biomed Mater Res A 2004;68:684–95. [3] Morimoto N, Watanabe A, Iwasaki Y, Akiyoshi K, Ishihara K. Nano-scale surface modification of a segmented polyurethane with a phospholipid polymer. Biomaterials 2004;25:5353–61. [4] Tanaka M, Motomura T, Ishii N, Shimura K, Onishi M, Mochizuki A, et al. Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate). Polym Int 2000;49:1709–13. [5] Ishihara K, Nomura H, Mihara T, Kurita K, Iwasaki Y, Nakabayashi N. Why do phospholipid polymers reduce protein adsorption? J Biomed Mater Res 1998;39:323–30. [6] Morra M, editor. Water in biomaterial surface science. New York: Wiley; 1999. p. 2001. [7] Hatakeyama H, Hatakeyama T. Nuclear magnetic relaxation studies of water– cellulose and water–sodium cellulose sulfate system. In: Kennedy JF, Phyllips GO, Williams PA, editors. Cellulose: structural and functional aspects. Chichester, UK: Ellis Horwood; 1990. p. 131–6. [8] Hatakeyama T, Hatakeyama H. Molecular relaxation of cellulosic polyelectrolytes with water. In: Glasser WG, Hatakeyama H, editors. Viscoelasticity of biomaterials. ACS symposium series, vol. 489. Washington, DC: ACS; 1992. p. 329–40. [9] Hatakeyama H, Hatakeyama T. Interaction between water and hydrophilic polymers. Thermochim Acta 1998;308:3–22. [10] Hatakeyama T, Kasuga H, Tanaka M, Hatakeyema H. Cold crystallization of poly(ethylene glycol)–water systems. Thermochim Acta 2007;465:59–66. [11] Hoffman K, Hatakeyama H. 1H n.m.r. relaxation studies and line shape analysis of aqueous sodium carboxymethylcellulose. Polymer 1994;35:2749–58. [12] Kitano H, Ichikawa K, Fukuda M, Mochizuki A, Tanaka M. The structure of water sorbed to polymethylmethacrylate film as examined by FT-IR spectroscopy. J Colloid Interface Sci 2001;242:133–40. [13] Sterling C, Masuzawa M. Gel/water relationships in hydrophylic polymers: nuclear magnetic resonance. Macromol Chem 1968;116:140–5. [14] Quinn FX, Kampff E, Smyth G, McBrierty VJ. Water in hydrogels. 1. A study of water in poly(N-vinyl-2-pyrrolidone/methyl methacrylate) copolymer. Macromolecules 1988;21:3192–8. [15] Shporer M, Frommer MA. Magnetic resonance studies of the properties of water in cellulose acetate membranes. J Macromol Sci Phys 1974;B10:529–43. [16] Brunauer S, Emett PH, Teller E. Adsorption of gases in multimolecular layers. J Am Chem Soc 1938;60:309–19. [17] Tanaka M, Mochizuki A, Shiroya T, Motomura T, Shimura K, Onishi A, et al. Study on kinetics of protein adsorption and desorption on poly(2methoxyethyl acrylate) (PMEA) surface. Colloids Surface A 2002;203:195–204. [18] Tanaka M, Mochizuki A, Motomura T, Shimura K, Onishi A, Okahata Y. In situ studies on protein adsorption onto a poly(2-methoxyethyl acrylate) surface by a quartz crystal microbalance. Colloids Surface A 2001;193:145–52. [19] Hatakeyama T, Nakamura K, Hatakeyama H. Vaporization of bound water associated with cellulose fibres. Thermochim Acta 2000;352–353:233–9.
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[20] Hatakeyama T, Hatakeyama H. Thermal properties of green polymers and biocomposites. Dordrecht, The Netherlands: Kluwer Academic; 2004. [21] Tsuruta T. Contemporary topics in polymeric materials for biomedical applications. Adv Polym Sci 1996;126:1–51. [22] Tsuruta T. Reactive and functional polymers. Changing features but conception unchanged. React Funct Polym 2007;67:1105–14. [23] Tanaka M, Motomura T, Kawada M, Anzai T, Kasori Y, Shiroya T, et al. Blood compatible aspects of poly(2-methoxyethylacrylate) (PMEA). Relationship between protein adsorption and platelet adhesion on PMEA surface. Biomaterials 2000;21:1471–81. [24] Tanaka M, Motomura T, Kawada M, Anzai T, Kasori Y, Shimura K, et al. A new blood compatible surface prepared by poly(2-methoxyethylacrylate) (PMEA) coating protein adsorption on PMEA surface. Jpn J Artif Organs 2000;29:209–16. [25] Tanaka M, Mochizuki A, Ishii N, Motomura T, Hatakeyama T. Study on blood compatibility of poly(2-methoxyethyl acrylate). Relationship between water structure and platelet compatibility in poly(2-methoxyethyl acrylate–co-2hydroxyethylmethacrylate). Biomacromolecules 2002;3:36–41. [26] Kido T, Nojiri C. Evaluation of the biocompatibility of an in vitro test using a poly(2-methoxyathy acrylate) coated oxygenators. Jpn J Artif Organs 2000;9:73–7. [27] Baykut D, Bernet F, Wehrle J, Weichelt K, Schwartz P, Zerkowski HR. New surface biopolymers for oxygenators: an in vitro hemocompatibility test of poly(2-methoxyethylacrylate). Eur J Med Res 2001;30:297–305. [28] Saito N, Motoyam S, Sawamoto J. Effects of new polymer coated extracorporeal circuits on biocompatibility during cardiopulmonary bypass. Artif Organs 2000;24:547–54. [29] Suhara H, Sawa Y, Nishimura M, Oshiyama H, Yokoyama K, Saito N, et al. Efficacy of a new coating material, PMEA, for cardiopulmonary bypass circuits in a porcine model. Ann Thorac Surg 2001;71:1603–8. [30] Ishihara K, Oshida H, Endo Y, Ueda T, Watanabe A, Nakabayashi N. Hemocompatibility of human whole blood on polymers with a phospholipid polar group and its mechanism. J Biomed Mater Res 1992;26:1543–52. [31] Iwasaki Y, Ishihara K, Nakabayashi N, Khang G, Jeon JH, Lee JW, et al. Platelet adhesion on the gradient surfaces grafted with phospholipid polymer. J Biomater Sci Polym 1998;9:801–16. [32] Iwasaki Y, Nakabayashi N, Ishihara K. In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers. J Artif Organs 2003;6:260–6. [33] Morisaku T, Watanabe J, Konno T, Takai M, Ishihara K. Hydration of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis. Polymer 2008;49:4652–7. [34] Xu Y, Takai M, Ishihara K. Protein adsorption and cell adhesion on cationic, neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces. Biomaterials 2009;30:4930–8. [35] Hatakeyama T, Quinn FX. Thermal analysis fundamentals and applications to polymer science. 2nd ed. Chichester, UK: John Wiley; 1999. [36] Yoshida H, Hatakeyama T, Hatakeyama H. Effect of water on the main chain motion of polysaccharide hydrogels. In: Glasser WG, Hatakeyama H, editors. Viscoelasticity of biomaterials. ACS symposium series, vol. 489. Washington, DC: ACS; 1992. p. 218–30.
[37] Naoi S, Hatakeyama T, Hatakeyama H. Phase transition of locust bean gum–, tara gum– and guar gum–water systems. J Therm Anal Calorim 2002;70:841–52. [38] Inoue Y, InoueY J, Takai M, Yusa SI, Ishihara K. Synthesis of sequencecontrolled copolymers from extremely polar and apolar monomers by living radical polymerization and their phase-separated structures. J Polym Sci Polym Chem 2005;43:6073–83. [39] Ghormley JA. Thermal behavior of amorphous ice. J Chem Phys 1950;25:599. [40] Sugisaki M, Suga H, Seki S. Calorimetric study of the galssy state. IV. Heat capacities of glassy water and cubic ice. Bull Chem Soc Jpn 1968;41:2500–91. [41] Hatakeyama T, Hatakeyama H. Thermal and nuclear magnetic relaxation studies of water–sodium polystyrene sulfonate systems. Polym Adv Technol 1990;1:305–10. [42] Hatakeyama H, Hatakeyama T. Nuclear magnetic relaxation studies of water– cellulose and water–sodium cellulose sulfate systems. In: Kennedy JF, Phyllips GO, Williams PA, editors. Cellulose: structural and functional aspects. Chichester, UK: Ellis Horwood; 1989. p. 131–6. [43] Thorne JM, Slaughter H. Liquid water cluster sizes. Thermochim Acta 1972;3:181–8. [44] Takigami S, Takigami M, Phyillips GO. Effect of preparation method on the hydration characteristics of hylan and comparison with another highly crosslinked polysaccharide, gum Arabic. Carbohydr Polym 1995;26:11–8. [45] Phillips GO, Takigami S, Takigami M. Hydration characteristics of the gum exudate from Acacia segenal. Food Hydrocolloids 1996;10:11–9. [46] Hatakeyama T, Yoshida H, Hatakeyama H. A differential scanning calorimetry study of the phase transition of the water–sodium cellulose sulfate system. Polymer 1987;28:1282–6. [47] Onishi T, Hatakeyama H, Hatakeyama T. DSC and AFM studies of chemically cross-linked sodium cellulose sulfate hydrogels. In: Hu TQ, editor. Characterization of lignocellulosic materials. Cambridge, MA: Blackwell; 2008. p. 329–39. [48] Nakamura K, Hatakeyama T, Hatakeyama H. DSC studies on monovalent and divalent cation salts of carboxymethylcellulose in highly concentrated aqueous solutions. In: Kennedy JF, Phyllips GO, editors. Wood and cellulose. Chichester, UK: Ellis Horwood; 1987. p. 97–103. [49] Nakamura K, Hatakeyama T, Hatakeyama H. Formation of the glassy state and mesophase in the water–sodium alginate system. Polym J 1991;23:253–8. [50] Yoshida H, Hatakeyama T, Hatakeyama H. Phase transitions of the water– xanthan system. Polymer 1990;31:693–8. [51] Nakamura K, Hatakeyama T, Hatakeyama H. Relationship between official regain and bound water in cellulose. Sen-i Gakkaishi (J Fiber Sci Technol Jpn) 1981;37:T-533–5. [52] Hatakeyama H, Onishi T, Endo T, Hatakeyama T. Gelation of chemically crosslinked methylcellulose studied by DSC and AFM. Carbohydr Polym 2007;69:792–8. [53] Nakamura K, Hatakeyama T, Hatakeyama H. Studies on bound water of cellulose by differential scanning calorimetry. Text Res J 1981;51:607–13. [54] Hatakeyama T, Naoi S, Hatakeyama H. Liquid crystallization of glassy guar gum with water. Thermochim Acta 2004;416:121–7. [55] Iijima M, Nakamura K, Hatakeyama T, Hatakeyama H. Phase transition of pectin with sorbed water. Carbohydr Polym 2000;41:101–6.
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Studies on bound water restrained by poly(2-methacryloyloxyethyl phosphorylcholine): Comparison with polysaccharide–water systems T. Hatakeyama a,*, M. Tanaka b, H. Hatakeyama c a
Lignocel Research, 73-8 Yatsumata, Fukui 910-3558, Japan Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan c Graduate School of Engineering, Fukui University of Technology, 3-6-1 Gakuen, Fukui 910-8505, Japan b
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 2 November 2009 Accepted 7 December 2009 Available online 11 December 2009 Keywords: Poly(2-methacryloyloxyethyl phosphorylcholine) Polysaccharides Non-freezing water Freezing bound water Differential scanning calorimetry
a b s t r a c t The structural change of water restrained by poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was investigated by differential scanning calorimetry (DSC), since the biocompatibility of PMPC and related biopolymers is affected by the structure of water on the polymer surface. The phase transition behaviour of PMPC–water systems with a water content (Wc = mass of water/mass of dry sample, g g1) in the range 0–2.0 was measured in the temperature range 150 to 50 °C. Glass transition, cold crystallization and melting were observed. Cold crystallization, which has been suggested as an index of biocompatibility, was detected for PMPC with a Wc in the range 0.5–0.9. The amounts of two types of bound water, non-freezing water and freezing bound water, were calculated from the melting enthalpy. The amount of non-freezing water of PMPC was 0.48. It was found that the phase transition behaviour and amount of bound water of PMPC were quite similar to those of water-soluble polysaccharide electrolytes. The results indicate that the bound water, not the free water, is restrained by PMPC. Ó 2010 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.
1. Introduction In order to develop new biocompatible polymers various factors affecting the biocompatibility of foreign materials, such as chemical groups on the surface, ionic charge, other physical factors such as morphological features and surface evenness, have been examined [1–3]. It is appropriate to consider the structural changes of water on the surface of the polymer as a major factor, since it is reasonable to expect that water strongly restrained by biomaterials shows unique behaviour, different from bulk water [2,4–12]. The molecular mobility of water molecules surrounding biomaterials has been investigated by nuclear magnetic relaxation studies [13,14]. It is thought that three different types of water [15] can be categorized from the relaxation time of water, i.e. the correlation time (sc) calculated from the spin–lattice (T1) and spin–spin relaxation times (T2) of water directly restrained by biomaterials is in the range 105 to 106 s, with a second layer of water with a correlation time of 108 s. These values are greater than that of the outer layer of water molecules, whose sc is 1012 s. Water species can be identified from nuclear magnetic relaxation measurements, however, the amount of each type of water is difficult to estimate using only nuclear magnetic relaxation measurements. The number of water molecules restrained by the matrix surface * Corresponding author. Tel./fax: +81 776 89 2885. E-mail address: [email protected] (T. Hatakeyama).
as a first layer has been calculated from a water sorption isotherm obtained in gravimetric experiments in the 1930s [16]. The Brunauer–Emett–Teller equation [16] can be applied to quantify the number of bound water molecules, even though more elaborate methods, such as the quartz crystal microbalance method, have been introduced and applied to polymer–protein biopolymer– water interactions [17,18]. From mass measurements precise results can be obtained for the initial stage of water sorption onto the surface of the polymer. At the same time, it is notable that the method has certain limitations in determining the exact amount of the second and other outer layer of waters during the stage of cluster formation. Among various techniques to investigate the structural changes of water restrained by polymers [8], we have paid particular attention to thermal analysis, since the phase transition behaviour of water can be easily detected by this method [9]. Although thermogravimetry can be used to quantify the amount of water by a vaporization technique [19], differential scanning calorimetry (DSC) has been paid special attention. When hydrated hydrophilic polymers, such as polysaccharides, are measured by DSC, three kinds of water are identified from the temperature and enthalpy of transition peaks. Hatakeyama and coworkers have classified hydrated water (water in the polymer) as follows: freezing (crystallizable) water which can be observed as the first order phase transition and grouped into (1) free water which crystallizes at 0 °C and is slightly affected by the matrix polymer and (2)
1742-7061/$ - see front matter Ó 2010 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. doi:10.1016/j.actbio.2009.12.018
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freezing bound water which crystallizes during the heating process at a temperature lower than 0 °C and is strongly affected by the matrix polymer. In addition to freezing water there exists (3) non-freezing water, i.e. water which is non-crystallizable due to strong molecular interactions with the matrix polymer. Both freezing bound water and non-freezing water are categorized as bound water [2,9,20]. A large number of polymers having a biomembrane-like surface have been investigated by many researchers in order to develop new biomedical materials [21,22]. In our previous studies it was found that poly(2-methoxyethyl acrylate) (PMEA) shows excellent blood compatibility, i.e. the amount of protein adsorbed onto the surface of PMEA was much lower than that of other conventional polymers [3,17,18,23–25]. It is noteworthy that the degree of denaturation of proteins remained at a low level on the surface of PMEA. Because of this, PMEA has already been applied as a coating material for a blood oxygenator which showed reliable performance under the designated conditions both in vitro and in vivo [26,27]. Furthermore, it was observed that the activity of blood components was reduced when a cardio-pulmonary bypass was coated with PMEA [27–29]. In our previous studies the melting behaviour of a hydrated PMEA analogue poly(methacrylate) having different side chain structures was systematically investigated and the blood compatibility of each polymer was correlated with of the amount of bound water [2]. It was concluded that the freezing bound water, detected as cold crystallization in DSC heating curve, plays a crucial role in blood compatibility. The above facts suggest that the presence of freezing bound water can be utilized as an index in choosing biocompatible polymers. In this study we pay particular attention to poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a large number of studies have been concerned with PMPC co-polymers, which are reported to reduce protein adsorption [5,30,31]. PMPC is a watersoluble polymer, so hydrophobic polymers are introduced as a co-polymerized component or chemically cross-linked [33] in order to obtain water insoluble polymers. Various types of PMPC co-polymers have been synthesized, taking into consideration the surface structure of biomembranes [34]. It has been reported that PMPC co-polymers show reasonable blood compatibility, such as suppression of protein adsorption and platelet adhesion, and they have been applied as surface modifiers of all sorts of medical devices in order to improve blood compatibility [32]. In order to confirm the role of water restrained by PMPC, in this study the phase transition behaviour of hydrated PMPC is extensively investigated. It is also intended to make a comparison with the thermal behaviour of water-soluble polysaccharides, which are accepted as representative water-soluble biopolymers. 2. Materials and methods 2.1. Samples The chemical structure of PMPC is shown in Fig. 1. PMPC in powder form was obtained from NOF Co., Tokyo. The molecular mass of PMPC was 1.1 105 g mol1 and the number average molecular mass was 0.7 105. Details of the preparation method can be found elsewhere [5].
H
CH3
C
C
H
C
n
O
O-
OCH2CH2OPOCH2CH2N+(CH3)3 O Fig. 1. Chemical structure of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).
samples were held at 150 °C for 10 min and then heated to 60 °C at 10 °C min1 (first heating run). The samples were cooled to 150 °C at a predetermined rate of between 2 and 50 °C min1 (second cooling run) and then heated at 10 or 2 °C min1 (second heating run). In order to obtain PMPC with various water contents, the following procedure was carried out. Samples (3–5 mg) were placed in pans and a small amount of deionized water was added using a micro-syringe. The water was evaporated until a predetermined mass of water was attained. The sample pans were then hermetically sealed using an auto-sealer. The samples with added water were weighed. These samples were kept at room temperature overnight and reweighed in order to confirm that no mass loss had occurred. After DSC measurements the pan was pierced, annealed at 110 °C for 1 h in an electric oven and then weighed. The amount of water was evaluated using Eq. (1). Although the water content (Wc) of hydrated polymers has been defined by various equations, in this study Wc is defined as follows:
W c ¼ mw =ms
ð1Þ
where ms is the mass of the dry sample and mw is the mass of water in the system. The glass transition temperature (Tg) was defined as the temperature at which the extrapolated baseline before the transition intersects the tangent drawn at the point of greatest slope of the heat capacity change step due to glass transition. Temperature and enthalpy of transition was calibrated using pure water as a standard [34]. The heat capacity difference at Tg (DCp) was calculated using the total mass of the sample (m0 + mw) [35,36]. The peak temperature of melting was designated the Tm. When two melting peaks of water were observed the high temperature side peak was designated Tmh and the low temperature side peak Tml. In this study cold crystallization was defined as an exothermic transition that occurred in the temperature range between Tg and Tm. The peak temperature of the exothermic peak was defined as Tcc. The melting enthalpy (DHm, J g1), cold crystallization enthalpy (DHcc, J g1) and melting enthalpy (DHm, J g1) were calculated using the mass of water (mw) in the PMPC–water systems. The enthalpy of the melting of water (334 J g1) was used for calculation. The amount of freezing water was calculated from DHm, calculated from the second heating curve. As described in the above, enthalpies of transitions were calculated from the peak area of each transition. When two melting peaks were observed the enthalpy of each peak was calculated. In this study bound water was calculated as follows:
W c ¼ W f þ W fb þ W nf
ð2Þ
2.2. Differential scanning calorimetry
W b ¼ W fb þ W nf
ð3Þ
A Seiko Instruments differential scanning calorimeter DSC 200C equipped with a cooling apparatus was used. The nitrogen gas flow rate was 30 ml min1. The samples were heated to 60 °C and then cooled to 150 °C at a rate of 10 °C min1 (first cooling run). The
where Wf is the amount of free water calculated from DHm of the high temperature side melting peak, Wfb is the freezing bound water calculated from DHm of the low temperature side melting peak and Wb is the total amount of bound water. The amount of
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non-freezing water (Wnf) can be calculated using Eq. (2), as Wc was obtained from Eq. (1).
3. Results and discussion 3.1. Phase diagram of PMPC–water systems PMPC–water systems with a water content (as defined in Eq. (1)) ranging from 0 to 2.0 were measured by DSC, in the temperature from 150 to 80 °C. Fig. 2 shows representative stacked DSC heating curves of PMPC–water systems with various water contents. As shown in the figure, a heat capacity change due to glass transition was observed for all samples. Cold crystallization was clearly seen as a broad exothermic peak in the sample with Wc = 0.55. Cold crystallization was scarcely observed when Wc exceeded 1.0. Besides the main peak of melting, a melting peak with a shoulder on the low temperature side was observed. From DSC curves the temperature of each transition, i.e. Tg, Tcc, Tmh, and Tml, could be obtained. Phase diagrams of PMPC–water systems were obtained based on the DSC heating curves shown in Fig. 2. Tg decreased markedly in the initial stage, reached a minimum and then increased to reach equilibrium. Glass transition of PMPC–water systems was observed over wide water content and temperature ranges, as shown in Fig. 3. It is known that glass transition is not observed for dry PMPC [38]. Glass transition of dry polyelectrolytes is ordinarily not observed due to strong intermolecular interactions. When a small amount of water is added to the polyelectrolytes molecular motion of the main chain can be detected. When PMPC–water systems with Wc values ranging from 0 to 0.7 were studied by DSC in the temperature range 150 to 60 °C the glass transition temperature varied in a complex manner depending on the water content, as shown in Fig. 3. It is reasonable to suppose that main chain motion of dry PMPC is strongly restricted due to intermolecular bonding. However, when a small amount of water is added to PMPC, water molecules diffuse between the PMPC molecules, break the inter-
Fig. 2. Stacked DSC heating curves of PMPC–water systems (2nd heating run). Numerals in the figure show Wc (g g1). Heating rate 10 °C min1.
Fig. 3. Phase diagram of the PMPC–water system. Tg, glass transition temperature; Tcc, cold crystallization temperature; Tm, melting temperature.
molecular bonding and main chain molecular motion is enhanced. The baseline change due to glass transition is clearly observed for PMPC with Wc = 0.44. Generally the water diffusion constant in a hydrophilic polymer from the surface to the inner part is between 0.5 and 1.5 105 cm2 s1 [1]. Here it is appropriate to consider that water molecules are homogeneously restrained by PMPC molecules. The heat capacity difference at Tg of the sample with Wc = 0.45 was 0.5 J g1 K1, which is far larger than the ordinal polymer, suggesting that both water molecules and PMPC initiated movement at the same time [10,35]. The glass transition temperature (Tg) decreased, reaching its lowest temperature (Tg min) of approximately 100 °C, then increased slightly and reached a constant value (Tg plateau) of approximately 40 °C with increasing water content. Molecular motion was disturbed in the presence of free water due to presence of ice. Glass transition of water-soluble polyelectrolytes is thought to reflect the cooperative molecular motion of polyelectrolyte molecules and amorphous ice frozen without crystallization [10,35,38,39]. This was confirmed by the fact that the Tg of the system (Tg system) was coincident with the calculated values, assuming an additive relationship of the Tg of amorphous ice in the system and the Tg of the dry sample [40], although in many polyelectrolyte systems the Tg of the completely dry system is an assumed value, obtained by extrapolation from the Tg values of samples with varying amounts of water. The above consideration leads to the conclusion that the glass transition behaviour of PMPC is similar to that of water-soluble polyelectrolytes, such as sodium polystyrene sulfonate [41], among others [9]. Nuclear magnetic resonance (NMR) spectrometry 1H relaxation times (sc) for the bound water in polyelectrolyte–water systems were between 108 and 106 s at approximately 50 °C. The above values were calculated assuming that the inverse longitudinal relaxation 1 (T 1 1 ) and transverse relaxation times (T 2 ) of bound water were 1 different but in the case of free water T 1 = T 1 2 , since extreme narrowing conditions were fulfilled [7,41]. The above data suggest that the water molecules are categorized as non-rigid solid, since the typical NMR pattern due to splitting of 1H spins could be observed at low temperature [41,42]. The NMR results indicate that the water molecules are frozen in a glassy state together with matrix polymers. Two melting peak temperatures, Tml and Tmh, were observed at Wc values higher than 0.5. Tml was observed at a constant temperature of approximately 30 °C, regardless of Wc. Tmh was observed at a temperature below 0 °C in the initial stage and then maintained a constant value. Tcc was observed in the water content range 0.5–0.8 at around 60 °C. It was noticed that the temperature of the transition peak depended on the sample mass and thermal history. Accordingly, the exact values can only be defined when the experimental conditions are strictly defined. Since
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deconvolution of DSC curves is not theoretically available in this study, the peak was employed as an index of the melting temperature. Because of this, when values of Tmh and Tml of different samples were compared the values always necessarily involved a certain degree of error. In DSC the peak temperature and the width of the melting peak depend on the matrix biopolymer and also the amount of co-existing water. The fact that the temperature difference between Tml and Tmh was 30 °C in the PMPC–water system indicates that the characteristics of freezing water bound to PMPC is markedly different from that of free water. In the case of watersoluble hydrophilic polymers, such as polysaccharide electrolytes, similar phase diagrams can be observed [9]. The phase diagrams of PMPC–water systems are characterized by a large temperature difference between Tml and Tmh compared with that of polysaccharide electrolyte–water systems.
Table 1 Non-freezing water in various kinds of hydrophilic polymer and polyelectrolyte.
a b c
Sample
Wnf
References
PMPC Gum Arabic (Acacia senegal) Hyaluronan Sodium cellulose sulfate Sodium carboxymethyl cellulose Sodium alginate Xanthan gum Guar gum Tara guma Locust bean gum Celluloseb,c Methylcellulose
0.48 0.55 0.50 0.43 0.85 0.43 0.55 0.75 0.5 0.70 0.2–0.4 0.88
This work [44,45] [36] [46,47] [48] [49] [50] [37] [37] [37] [9,19,51] [52]
Wnf does not level off until Wc = 3.8. Water insoluble. Wnf depends on crystallinity.
3.2. Non-freezing water of PMPC–water systems The enthalpy of transition can be calculated from the area under each peak of DSC heating curves shown in Fig. 3, assuming the melting enthalpy of ice is 334 J g1. As described in Section 2, each transition enthalpy was determined using the second heating curve and the amount of bound water was calculated using Eqs. (2) and (3). Fig. 4 shows Wb, Wfb, and Wnf for the PMPC–water systems as a function of Wc. It can clearly be seen that the Wnf of PMPC increases with increasing Wc and levels off at around 0.5, while Wfb is observed at Wc = 0.5 and decreases with increasing Wc. A slight increase in Wfb at Wc = 0.5 can be explained by the fact that the number of water molecules available to form clusters which freeze to form hexagonal ice [43] was insufficient for the system with Wc = 0.5, since the major part of bound water presented as Wnf [43]. When a sufficient amount of free water was supplied to the system a part of Wfb was included as free water and the enthalpy of the low temperature side melting peak decreased slightly. Table 1 shows the value of Wnf calculated from the melting enthalpy of water restrained by various kinds of biopolymers. The calculation was carried out using Eqs. (2) and (3), as described in Section 2. The value of Wnf obtained for the equilibrium state of PMPC and various other kinds of biopolymers were obtained under similar experimental conditions by DSC. As can be clearly seen in Table 1, the value of Wnf for PMPC was similar to the values for polysaccharides electrolytes and other natural polysaccharides. It should be noted that water molecules are strongly restrained by ionic groups on the 2-methacryloyloxyethyl phosphorylcholine (MPC) unit. The results in Table 1 indicate that the surface of PMPC is not covered with free water, as Ishihara and coworkers sug-
gested [5]. The results shown in Table 1 strongly suggest that the Wnf value of PMPC and its co-polymer is similar to that of other polysaccharides. The equilibrium water content (EWC) is an important index in the biomedical application of various kinds of polymers. EWC is generally measured by weighing a water absorbing sample film after immersion in water or saline solution. Although the value is affected by the experimental conditions, such as immersion temperature, time, and thickness of film, reliable EWC values can be obtained with a certain precision. The method is only applicable when the sample is water insoluble but swells by a certain amount in water. Ishihara reported that the EWC of a PMPC co-polymer with a hydrophobic component (butylmethacrylate) was 0.36 when the water content is defined as mw/(mw + ms). In this study the above value corresponds to 0.56 when the water content is defined as shown in Eq. (1). The above EWC value accords well with the maximum Wnf value of PMPC, as shown in Fig. 4 and Table 1. This indicates that the non-freezing water of PMPC co-polymers is mainly attached to hydrophilic groups of the MPC component. The polysaccharides shown in Table 1 are water soluble except for cellulose. Natural cellulose obtained from various plants is water insoluble and mainly obtained as a fibre or in powder form. Because of this it is inappropriate to obtain EWC in a similar manner to film samples. Rather than EWC, the water content of cellulose samples saturated in a 100% relative humidity atmosphere at a determined temperature is used as the criterion of hydrophilicity. The water content obtained by the above method is defined as the water regain, used in the field of applications. We have reported that the water regain of cellulose from various plants having different crystallinities accords well with 1/2 of the Wnf value determined by DSC [53]. The above results strongly suggest that the value of Wnf is an index of the hydrophilicity of hydrophilic polymers such as PMPC and polysaccharides.
3.3. Cold crystallization and freezing bound water
Fig. 4. Relationship between Wb, Wfb, Wnf, and Wc of the PMPC–water system. Wb, bound water content; Wfb, freezing bound water content; Wnf, non-freezing water content.
Crystallization of amorphous materials at a temperature between the glass transition and melting is termed cold crystallization. Cold crystallization can be frequently observed in glassy polymers and detected as an exothermic event in DSC heating curves. In a previous study on the phase transition behaviour of polyethylene glycol–water systems [10] distinct cold crystallization was found over a wide range of water contents. From the enthalpy balance of cold crystallization, crystallization and melting it can be concluded that the glassy state formed amorphous ice and polymer cooperatively, involving cold crystallization of the system [10,54].
T. Hatakeyama et al. / Acta Biomaterialia 6 (2010) 2077–2082 Table 2 Temperature and water content range of cold crystallization observed in polymer– water systems. Sample
PMPC Poly(ethylene glycol) Hyaluronan Gum Arabic Cellulose sulfate Carboxymethylcellulose Methylcellulose Sodium alginate Pectin Xanthan gum Guar gum Tara gum Locust bean gum
Peak temperature (Tcc) (°C) 65 48 50 55 55 40 50 30 50 60 35 25 25
to to to to to to to to to to to to to
60 44 20 20 23 38 20 20 41 20 25 20 20
Water content range (g g1)
References
0.5–0.9 0.5–1.2 0.7–1.4 0.5–0.8 0.5–1.1 0.8–1.7 0.8–1.5 0.5–1.2 0.4–0.6 0.5–1.1 0.4–2.5 0.6–1.0 0.6–0.7
This work [10] [36] [44,45] [46] [48] [52] [49] [55] [50] [37] [37] [37]
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certain amount of freezing bound water is masked by the DSC melting peak. This indicates that the Wfb value at around a Wc equal to the EWC is equivalent to or more than the Wfb of PEMA [4]. 4. Conclusions Structural changes of water on the surface of biomembranes are important factors when designing new biopolymers. The levels of the two types of bound water, non-freezing water and freezing bound water, were calculated from the melting enthalpy and the bound water content of PMPC by DSC. It was found that the amount of non-freezing water in PMPC was approximately 0.5, which is similar to the values for various kinds of polysaccharides. The amount of freezing bound water in PMPC was in the range 0.05–0.1, depending on the water content. The results indicate that the bound water on the surface of biopolymers plays a crucial role in the biocompatibility of biopolymers. Acknowledgements
Table 2 shows the temperature and water content ranges over which cold crystallization was observed in various kinds of biopolymer–water systems. In this table the water content at which the cold crystallization peak was observed is also indicated. The peak temperature of cold crystallization (Tcc) depends on water content, and generally Tcc increases slightly with increasing water content. In the case of PMPC, Tcc was observed at approximately 30 °C. The enthalpy of cold crystallization showed a maximum value at the Wc at which Wnf plateaued for almost all polysaccharides–water systems. Ishihara and coworkers have reported that PMPC co-polymers show excellent biocompatibility [5,32]. In order to explain the characteristic features of these polymers they focused on the role of water molecules in PMPC co-polymers [30,33]. According to Ishihara and coworkers the mobility of water on the surface of membranes plays a crucial role. The above description does not accord with the results obtained in this study, since they described how a large amount of free water in the polymers was responsible for the excellent cytocompatibility of MPCs [5]. They defined free water as ‘‘water” showing a first order phase transition, even though the melting peak starts to deviate in the endothermic direction at temperatures below 0 °C. Furthermore, it is notable that the amount of free water in PMPC depends only on concentration, i.e. the EWC of PMPC is difficult to define. In the case of MPC–hydrophobic co-polymers the value of Wnf is defined as the amount bound to the MPC fraction, while the remaining water at EWC can be categorized as freezable water [33]. It is thought that freezable water as defined by Ishihara and coworkers includes freezing bound water when the definition of our study is employed, as shown in Eq. (2). Tanaka and coworkers showed that the biocompatible nature of PMEA is attributable to the amount of freezing bound water [2,4,25]. They paid attention to the cold crystallization which is observed in the DSC heating curves of PMEA–water systems. Tanaka and coworkers stated that the cold crystallization observed in the DSC heating curves of PMEA–water systems shows a significant relationship with biocompatibility [2]. Various homologues of PMEA, whose cold crystallization has been observed by DSC, showed improved results when examined in biological tests. Ice formed by cold crystallization melts at a temperature lower than the melting temperature of free water. In this study the above water corresponded to the lower temperature side peak, characterized as Tml and DHml. Wfb was calculated from DHml. As shown in Fig. 4, the Wfb of PMPC showed a maximum value around Wc, where Wnf plateaued. It is considered that the hydrophilicity of the MPC fraction of the various co-polymers synthesized by Ishihara and coworkers have a role as water restraining sites and that a
The authors are grateful to Professor Emeritus Teiji Tsuruta, University of Tokyo, and Professor Clive S. Langham, Nihon University, for their comments and Ms Shizuka Horita for her helpful assistance. References [1] Ichikawa K, Mori T, Kintano H, Fukuda M, Mochizuki A, Tanaka M. Fourier transform infrared study on the sorption of water to various kinds of polymer thin films. J Polym Sci Polym Phys 2001;39:2176–82. [2] Tanaka M, Mochizuki A. Effect of water structure on blood compatibility – thermal analysis of water in poly(methacrylate). J Biomed Mater Res A 2004;68:684–95. [3] Morimoto N, Watanabe A, Iwasaki Y, Akiyoshi K, Ishihara K. Nano-scale surface modification of a segmented polyurethane with a phospholipid polymer. Biomaterials 2004;25:5353–61. [4] Tanaka M, Motomura T, Ishii N, Shimura K, Onishi M, Mochizuki A, et al. Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate). Polym Int 2000;49:1709–13. [5] Ishihara K, Nomura H, Mihara T, Kurita K, Iwasaki Y, Nakabayashi N. Why do phospholipid polymers reduce protein adsorption? J Biomed Mater Res 1998;39:323–30. [6] Morra M, editor. Water in biomaterial surface science. New York: Wiley; 1999. p. 2001. [7] Hatakeyama H, Hatakeyama T. Nuclear magnetic relaxation studies of water– cellulose and water–sodium cellulose sulfate system. In: Kennedy JF, Phyllips GO, Williams PA, editors. Cellulose: structural and functional aspects. Chichester, UK: Ellis Horwood; 1990. p. 131–6. [8] Hatakeyama T, Hatakeyama H. Molecular relaxation of cellulosic polyelectrolytes with water. In: Glasser WG, Hatakeyama H, editors. Viscoelasticity of biomaterials. ACS symposium series, vol. 489. Washington, DC: ACS; 1992. p. 329–40. [9] Hatakeyama H, Hatakeyama T. Interaction between water and hydrophilic polymers. Thermochim Acta 1998;308:3–22. [10] Hatakeyama T, Kasuga H, Tanaka M, Hatakeyema H. Cold crystallization of poly(ethylene glycol)–water systems. Thermochim Acta 2007;465:59–66. [11] Hoffman K, Hatakeyama H. 1H n.m.r. relaxation studies and line shape analysis of aqueous sodium carboxymethylcellulose. Polymer 1994;35:2749–58. [12] Kitano H, Ichikawa K, Fukuda M, Mochizuki A, Tanaka M. The structure of water sorbed to polymethylmethacrylate film as examined by FT-IR spectroscopy. J Colloid Interface Sci 2001;242:133–40. [13] Sterling C, Masuzawa M. Gel/water relationships in hydrophylic polymers: nuclear magnetic resonance. Macromol Chem 1968;116:140–5. [14] Quinn FX, Kampff E, Smyth G, McBrierty VJ. Water in hydrogels. 1. A study of water in poly(N-vinyl-2-pyrrolidone/methyl methacrylate) copolymer. Macromolecules 1988;21:3192–8. [15] Shporer M, Frommer MA. Magnetic resonance studies of the properties of water in cellulose acetate membranes. J Macromol Sci Phys 1974;B10:529–43. [16] Brunauer S, Emett PH, Teller E. Adsorption of gases in multimolecular layers. J Am Chem Soc 1938;60:309–19. [17] Tanaka M, Mochizuki A, Shiroya T, Motomura T, Shimura K, Onishi A, et al. Study on kinetics of protein adsorption and desorption on poly(2methoxyethyl acrylate) (PMEA) surface. Colloids Surface A 2002;203:195–204. [18] Tanaka M, Mochizuki A, Motomura T, Shimura K, Onishi A, Okahata Y. In situ studies on protein adsorption onto a poly(2-methoxyethyl acrylate) surface by a quartz crystal microbalance. Colloids Surface A 2001;193:145–52. [19] Hatakeyama T, Nakamura K, Hatakeyama H. Vaporization of bound water associated with cellulose fibres. Thermochim Acta 2000;352–353:233–9.
2082
T. Hatakeyama et al. / Acta Biomaterialia 6 (2010) 2077–2082
[20] Hatakeyama T, Hatakeyama H. Thermal properties of green polymers and biocomposites. Dordrecht, The Netherlands: Kluwer Academic; 2004. [21] Tsuruta T. Contemporary topics in polymeric materials for biomedical applications. Adv Polym Sci 1996;126:1–51. [22] Tsuruta T. Reactive and functional polymers. Changing features but conception unchanged. React Funct Polym 2007;67:1105–14. [23] Tanaka M, Motomura T, Kawada M, Anzai T, Kasori Y, Shiroya T, et al. Blood compatible aspects of poly(2-methoxyethylacrylate) (PMEA). Relationship between protein adsorption and platelet adhesion on PMEA surface. Biomaterials 2000;21:1471–81. [24] Tanaka M, Motomura T, Kawada M, Anzai T, Kasori Y, Shimura K, et al. A new blood compatible surface prepared by poly(2-methoxyethylacrylate) (PMEA) coating protein adsorption on PMEA surface. Jpn J Artif Organs 2000;29:209–16. [25] Tanaka M, Mochizuki A, Ishii N, Motomura T, Hatakeyama T. Study on blood compatibility of poly(2-methoxyethyl acrylate). Relationship between water structure and platelet compatibility in poly(2-methoxyethyl acrylate–co-2hydroxyethylmethacrylate). Biomacromolecules 2002;3:36–41. [26] Kido T, Nojiri C. Evaluation of the biocompatibility of an in vitro test using a poly(2-methoxyathy acrylate) coated oxygenators. Jpn J Artif Organs 2000;9:73–7. [27] Baykut D, Bernet F, Wehrle J, Weichelt K, Schwartz P, Zerkowski HR. New surface biopolymers for oxygenators: an in vitro hemocompatibility test of poly(2-methoxyethylacrylate). Eur J Med Res 2001;30:297–305. [28] Saito N, Motoyam S, Sawamoto J. Effects of new polymer coated extracorporeal circuits on biocompatibility during cardiopulmonary bypass. Artif Organs 2000;24:547–54. [29] Suhara H, Sawa Y, Nishimura M, Oshiyama H, Yokoyama K, Saito N, et al. Efficacy of a new coating material, PMEA, for cardiopulmonary bypass circuits in a porcine model. Ann Thorac Surg 2001;71:1603–8. [30] Ishihara K, Oshida H, Endo Y, Ueda T, Watanabe A, Nakabayashi N. Hemocompatibility of human whole blood on polymers with a phospholipid polar group and its mechanism. J Biomed Mater Res 1992;26:1543–52. [31] Iwasaki Y, Ishihara K, Nakabayashi N, Khang G, Jeon JH, Lee JW, et al. Platelet adhesion on the gradient surfaces grafted with phospholipid polymer. J Biomater Sci Polym 1998;9:801–16. [32] Iwasaki Y, Nakabayashi N, Ishihara K. In vitro and ex vivo blood compatibility study of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer-coated hemodialysis hollow fibers. J Artif Organs 2003;6:260–6. [33] Morisaku T, Watanabe J, Konno T, Takai M, Ishihara K. Hydration of phosphorylcholine groups attached to highly swollen polymer hydrogels studied by thermal analysis. Polymer 2008;49:4652–7. [34] Xu Y, Takai M, Ishihara K. Protein adsorption and cell adhesion on cationic, neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces. Biomaterials 2009;30:4930–8. [35] Hatakeyama T, Quinn FX. Thermal analysis fundamentals and applications to polymer science. 2nd ed. Chichester, UK: John Wiley; 1999. [36] Yoshida H, Hatakeyama T, Hatakeyama H. Effect of water on the main chain motion of polysaccharide hydrogels. In: Glasser WG, Hatakeyama H, editors. Viscoelasticity of biomaterials. ACS symposium series, vol. 489. Washington, DC: ACS; 1992. p. 218–30.
[37] Naoi S, Hatakeyama T, Hatakeyama H. Phase transition of locust bean gum–, tara gum– and guar gum–water systems. J Therm Anal Calorim 2002;70:841–52. [38] Inoue Y, InoueY J, Takai M, Yusa SI, Ishihara K. Synthesis of sequencecontrolled copolymers from extremely polar and apolar monomers by living radical polymerization and their phase-separated structures. J Polym Sci Polym Chem 2005;43:6073–83. [39] Ghormley JA. Thermal behavior of amorphous ice. J Chem Phys 1950;25:599. [40] Sugisaki M, Suga H, Seki S. Calorimetric study of the galssy state. IV. Heat capacities of glassy water and cubic ice. Bull Chem Soc Jpn 1968;41:2500–91. [41] Hatakeyama T, Hatakeyama H. Thermal and nuclear magnetic relaxation studies of water–sodium polystyrene sulfonate systems. Polym Adv Technol 1990;1:305–10. [42] Hatakeyama H, Hatakeyama T. Nuclear magnetic relaxation studies of water– cellulose and water–sodium cellulose sulfate systems. In: Kennedy JF, Phyllips GO, Williams PA, editors. Cellulose: structural and functional aspects. Chichester, UK: Ellis Horwood; 1989. p. 131–6. [43] Thorne JM, Slaughter H. Liquid water cluster sizes. Thermochim Acta 1972;3:181–8. [44] Takigami S, Takigami M, Phyillips GO. Effect of preparation method on the hydration characteristics of hylan and comparison with another highly crosslinked polysaccharide, gum Arabic. Carbohydr Polym 1995;26:11–8. [45] Phillips GO, Takigami S, Takigami M. Hydration characteristics of the gum exudate from Acacia segenal. Food Hydrocolloids 1996;10:11–9. [46] Hatakeyama T, Yoshida H, Hatakeyama H. A differential scanning calorimetry study of the phase transition of the water–sodium cellulose sulfate system. Polymer 1987;28:1282–6. [47] Onishi T, Hatakeyama H, Hatakeyama T. DSC and AFM studies of chemically cross-linked sodium cellulose sulfate hydrogels. In: Hu TQ, editor. Characterization of lignocellulosic materials. Cambridge, MA: Blackwell; 2008. p. 329–39. [48] Nakamura K, Hatakeyama T, Hatakeyama H. DSC studies on monovalent and divalent cation salts of carboxymethylcellulose in highly concentrated aqueous solutions. In: Kennedy JF, Phyllips GO, editors. Wood and cellulose. Chichester, UK: Ellis Horwood; 1987. p. 97–103. [49] Nakamura K, Hatakeyama T, Hatakeyama H. Formation of the glassy state and mesophase in the water–sodium alginate system. Polym J 1991;23:253–8. [50] Yoshida H, Hatakeyama T, Hatakeyama H. Phase transitions of the water– xanthan system. Polymer 1990;31:693–8. [51] Nakamura K, Hatakeyama T, Hatakeyama H. Relationship between official regain and bound water in cellulose. Sen-i Gakkaishi (J Fiber Sci Technol Jpn) 1981;37:T-533–5. [52] Hatakeyama H, Onishi T, Endo T, Hatakeyama T. Gelation of chemically crosslinked methylcellulose studied by DSC and AFM. Carbohydr Polym 2007;69:792–8. [53] Nakamura K, Hatakeyama T, Hatakeyama H. Studies on bound water of cellulose by differential scanning calorimetry. Text Res J 1981;51:607–13. [54] Hatakeyama T, Naoi S, Hatakeyama H. Liquid crystallization of glassy guar gum with water. Thermochim Acta 2004;416:121–7. [55] Iijima M, Nakamura K, Hatakeyama T, Hatakeyama H. Phase transition of pectin with sorbed water. Carbohydr Polym 2000;41:101–6.