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Summary: Hemocompatibility is an essential aspect of blood contacting polymers. Knowledge of the relationship between polymer structure and hemocompatibility is important in designing such polymers. In this work, the effect of swelling behavior and states of water on the hemocompatibility of poly(acrylonitrile-co-N-vinyl-2-pyrrolidone) (PANCNVP) films was studied. Platelet adhesion and plasma recalcification time tests were used to evaluate the hemocompatibility of the films. Considering the importance of surface properties on the hemocompatibility of polymers, static water contact angles were measured by both sessile drop and captive bubble methods. It was found that, on the film surface of PANCNVP with a higher NVP content, adhered platelets were remarkably suppressed and the recalcification time was longer. The total water content adsorbed on the PANCNVP film was determined through swelling experiments performed at temperatures of interest. Differential scanning calorimetry and thermogravimetric analysis were

used to probe the states of water in the films. Based on the results from these experiments, it was hypothesized that the better hemocompatibility of PANCNVP films with higher NVP contents was due to their higher free water content, because water molecule exchange at the polymer/liquid interface, facilitated by a high free water content, is unfavorable for the formation of surface bound water, which causes poor hemocompatibility.

Hemocompatibility of Poly(acrylonitrile-co-N-vinyl2-pyrrolidone)s: Swelling Behavior and Water States Ling-Shu Wan, Zhi-Kang Xu,* Xiao-Jun Huang, Zhen-Gang Wang, Peng Ye Institute of Polymer Science, Zhejiang University, Hangzhou 310027, P. R. China Fax: þ 86 571 8795 1773; E-mail: [email protected]

Received: October 7, 2004; Revised: December 18, 2004; Accepted: January 3, 2005; DOI: 10.1002/mabi.200400157 Keywords: biocompatibility; differential scanning thermogravimetric analysis (TGA); water structure

calorimetry

Introduction Blood contacting medical devices, such as the artificial kidney, heart valves, dialyzers and plasma separators, must meet the requirements of completely preventing the activation of the coagulation system and other biochemical systems. Although polymeric biomaterials have been used widely, improving the hemocompatibility of these devices continues to be a major challenge.[1] In general, it is accepted that the factors which influence the hemocompatibility of a polymer include the surface chemical structure, the hydrophilicity/hydrophobicity, the morphology and the topography.[2] Since there are so many influencing factors and because polymer/blood interactions are very complicated, the relationship between hemocompatibility and polymer structure/property is not yet well understood and many hypotheses have been presented. For example, it has been suggested that moderate interfacial free energy, hydrophilic/hydrophobic domains, volume restriction and Macromol. Biosci. 2005, 5, 229–236

(DSC);

poly(acrylonitrile-co-N-vinyl-2-pyrrolidone);

osmotic repulsion effects would be favorable for improving the hemocompatibility of the polymer surface.[3] Among the various polymers available, water-soluble or hydrophilic polymers, especially poly(N-vinyl-2-pyrrolidone) (PVP),[4] poly(ethylene glycol) (PEG)[5] and phospholipid analogous polymers,[6] possess excellent hemocompatibility. Therefore, to impart hemocompatibility to common polymers, much attention has been paid to incorporating these hemocompatible components into other polymers by copolymerization,[4a] surface coating,[7] grafting[4d] and blending.[4e] Due to the water-soluble or hydrophilic characteristics of these hemocompatible components, understanding the influence of water content and its states in polymers on the hemocompatibility has been attractive and challenging work from both a theoretical and practical point of view.[8–10] Water in polymers can be classified into non-freezable and freezable forms,[11] or more specifically as non-freezable bound water, freezable bound water and free water.[10] However, the number of

DOI: 10.1002/mabi.200400157

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different water types detected in a polymer depends on the technique used. Various methods, including nuclear magnetic resonance,[12,13] differential scanning calorimetry (DSC),[14–18] thermo-gravimetric analysis (TGA)[17] and infrared spectroscopy,[9,16] have been used to probe the water in swollen polymers. Thermal analysis, especially DSC, has been used extensively to study phase transition behavior, heat capacity and pore size distribution by characterizing water in water-containing systems.[10–22] Polyacrylonitrile (PAN) exhibits good mechanical properties and has been widely used as a separation membrane material.[23] In recent years, the usage of polyacrylonitrilebased membranes has been extended from traditional ultrafiltration to blood contacting processes by the incorporation of various comonomers.[24] In our previous work,[25] PEG was immobilized on poly(acrylonitrile-comaleic acid) membranes to improve their hemocompatibility. N-Vinyl-2-pyrrolidone (NVP), phospholipid analogues or sugar moieties were also incorporated into polyacrylonitrile by a copolymerization process for the same purpose. Especially for copolymers of acrylonitrile with NVP, applications in an artificial liver support system have been recently described.[26] However, it is unknown why NVP-containing polymers show good hemocompatibility normally. Herein, copolymers of acrylonitrile/Nvinyl-2-pyrrolidone (termed poly(acrylonitrile-co-N-vinyl2-pyrrolidone), PANCNVP) with various NVP contents were synthesized. The swelling behavior and water states of PANCNVP films are discussed and some efforts are made to understand the relationship between the hemocompatibility and the polymer structure/properties.

under a vacuum and their thickness was approximately 45
5 mm. Platelet Adhesion Examination Experiments were carried out with fresh platelet-enriched plasma (PRP) bought from the Blood Center of Hangzhou, China. First, the PANCNVP film was placed onto a piece of flat glass. Then, a sample of 20 mL of PRP was carefully dropped on the film center. After incubation for 30 min at room temperature (about 37 8C), the film was carefully rinsed several times in phosphate buffer solution (PBS, 19.1008 g Na2HPO4 and 1.8145 g KH2PO4 for 1 000 mL of buffer solution, pH ¼ 7.4). Adhered platelets on the film were preserved with 2.5% glutaraldehyde/PBS solution for 30 min, followed by a dehydration procedure using a series of ethanol-water mixtures (0, 30, 50, 70, 90, 100 vol.-% of ethanol) for 30 min. Samples were then air dried and investigated with a scanning electron microscope (Sirion, FEI) after gold sputtering. More than 4 micrographs for each sample were used to count the adhered platelets. Plasma Recalcification Time (PRT) Test The fresh human plasma from which Ca2þ was removed (Blood Center of Hangzhou, China) and the CaCl2 solution (0.025 mol  L1) were warmed up to 37 8C. Then 0.2 mL of each solution was taken onto the film. While stirring the recalcified plasma with a small stainless-steel hook, the process until the silky fibrin appeared was timed. The time was recorded as PRT. Five samples made from each copolymer were used to obtain a reliable value. Water Contact Angle Measurements

Experimental Part Sample Preparation PAN and PANCNVPs with various N-vinyl-2-pyrrolidone (NVP) contents were synthesized by water phase precipitation copolymerization in our laboratory.[25] Briefly, de-ionized water (50 mL), acrylonitrile (AN, 10.06 mL), NVP (1.82 mL) and the initiator system (20 mg of NaClO3, 45.6 mg of Na2S2O5) were added into a round flask with mechanical agitation at 60 8C under a nitrogen atmosphere. The copolymerization was continued for 3 h and then the precipitated copolymer was filtered and washed three times with de-ionized water and ethanol respectively. The resultant copolymer was dried under a vacuum for at least 6 h at 60 8C. The NVP contents in the copolymers calculated from 1H NMR spectra were 7%, 15%, 22% and 31% (w/w) and these copolymers were denoted PANCNVP07, PANCNVP15, PANCNVP22 and PANCNVP31 respectively. Film samples were prepared by casting DMSO solutions of the copolymers (8 wt.-%) onto clean glass plates. The films were fabricated by evaporation of the solvent in the atmosphere at room temperature for 8 h after being cast using a casting knife with a 150 mm gate opening. They were then were immersed in deionized water for 24 h. The resultant films were finally dried for another 24 h at 60 8C Macromol. Biosci. 2005, 5, 229–236

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Static water contact angles of the films were measured by both sessile drop and captive bubble methods at room temperature with a contact angle goniometer (Dataphysics, OCA20, Germany) equipped with a video camera. Using a typical sessile drop method, a water drop (5 mL) was added onto a dry film sample in air, an image was recorded after 10 s and a static water contact angle was then determined from the image with the imaging software. For the captive bubble method, an air/ water/film interface was formed by immersing a small film panel in a glass observation cell containing de-ionized water and releasing an air bubble beneath the film surface with a curved syringe. An image of the bubble was then recorded and used to calculate the contact angle. The samples for the captive bubble method were pre-immersed into deionized water for 12 h. At least ten measurements of different water drops were averaged to get a reliable value. The dynamic contact angle (the dependence of contact angle on time) was also recorded every 30 s in 30 min. Swelling Behavior Determination The dry film was cut into fragments of about 4 cm2 and weighed accurately. These films were then immersed in de-ionized water at designated temperatures (25, 37 or 50 8C) and left until ß 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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equilibrium was attained. The equilibrium water content, WE, was calculated by gravimetry according to the following equation:

WE ¼ ðW  W0 Þ=W

ð1Þ

where W is the weight of the fully swollen film and W0 is the weight of the dry film. The swelling behavior was followed by measuring the weight gain with the immersion time after wiping the surface with filter papers. The water uptake, Wc(t), at time t was also obtained using the following equation:

Wc ðtÞ ¼ ðWt  W0 Þ=W0

ð2Þ

where Wt is the weight of the swollen film at time t. DSC and TGA Analysis Differential scanning calorimetry (DSC) conducted on a STA409PC thermal analysis system was used to examine the states of water in the swollen films with different water contents. The samples were sealed in aluminum pans and cooled to 60 8C and then heated to 35 8C at a heating rate of 5 8C  min1 under a nitrogen gas flow. The bound water was determined by thermogravimetric analysis (TGA) through the heating of the samples from room temperature to 300 8C, at a heating rate of 5 8C  min1 under nitrogen. Samples of PANCNVP31 with different water contents were prepared by the room temperature evaporation of water from films previously swollen up to equilibrium. Each sample was frozen in liquid nitrogen to avoid vaporization before measurement.

Results and Discussion To determine the hemocompatibility of the copolymer films, platelet adhesion and plasma recalcification time were examined. When a foreign material comes into contact with blood, the initial blood response is the adsorption of blood proteins, followed by platelet adhesion and the activation of coagulation pathways, leading to thrombus formation.[27] As is well known, platelet adhesion has generally been applied to evaluate the hemocompatibility of materials. Figure 1 shows the number of adhered platelets on the copolymer film surfaces. Typical scanning electron micrographs of PAN and PANCNVP31 are shown in Figure 2. Micrographs for the other samples are available in the Supporting Information. It is obvious that the number of adhered platelets decreases sharply with increasing NVP content in the copolymer. Furthermore, not only the number but also the morphology of the adhered platelets must be taken into account.[28] Ko et al.[29] classified the shape change of activated platelets into five stages in the following sequence: discoid, dendritic (early pseudopodial), spreaddendritic (intermediate pseudopodial), spreading (late pseudopodial and hyaloplasm spreading) and fully spreading (hyaloplasm well spreading and no distinct pseudopodia). Since one of the platelet’s main roles in the vasculature is essentially to ‘‘plug holes’’ in blood vessels by adhering to Macromol. Biosci. 2005, 5, 229–236

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Figure 1. Number of adhered platelets on film samples with different NVP contents.

and covering sites of injury,[30] platelets fully spread to achieve the largest area coverage on the incompatible material surface. As can be seen from Figure 2 and the micrographs in the Supporting Information, on the film surface of PAN or PANCNVP samples with lower NVP contents (PANCNVP07 and PANCNVP15), the platelets are larger (fully spreading) and no distinct pseudopodia can be observed for the hyaloplasm spreading. At the same time, platelets have the tendency to aggregate and a lot of platelet fragments were found on these samples. However, on PANCNVP samples with a higher NVP content (PANCNVP22 and PANCNVP31), some of the platelets have extended pseudopodia, but many retain a discoid shape which is similar to the original shape of the platelet and indicates an unactivated state.

Figure 2. Typical scanning electron micrographs of adhered platelets on the film surface (a) PAN; (b) PANCNVP31 (10 000 for those marked by prime, 2 000 for the others). ß 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. Plasma recalcification time for film samples with different NVP contents.

Coagulation is a result of the cascading chemical reactions of plasma proteins (clotting factors).[30] When anticoagulated human plasma is added to Ca2þ (Factor IV), the endogenetic blood coagulation system will start to activate prothrombin (Factor II), converting it into thrombin. Thrombin will then initiate the formation of insoluble fibrin from fibrinogen. The duration of this procedure can be measured as plasma recalcification time (PRT). It can be seen from Figure 3 that, although there are fewer significant differences among samples compared with those found in the platelet adhesion test, the recalcification time increased gradually with increasing NVP content in the copolymer. The water contact angle was used to characterize the surface properties of the PANCNVP films. It is well known that hydrophilic groups at the polymer-air interface try to bury themselves into the bulk of films to minimize the interface tension, and these groups may rearrange in a water environment. Therefore, the time dependence of the water contact angle within 30 min was measured by the sessile drop method. It can be seen from Figure 4 that the contact angle for each sample decreased remarkably with the elongation of time. Both the rearrangement of polar groups and the penetration of water into the film matrix may be responsible for the decrease in the water contact angle. However, as shown in Figure 5, the values of static contact angle show no distinct differences among the studied samples with various NVP contents, within the experimental error. Contact angles from the air bubble method also remained nearly constant for the samples pre-immersed into water for 12 h before measurements. Furthermore, both the cyanic groups of PAN and the carbonyl groups of PVP are strong polar groups. Therefore, water absorption might play a key role in the decrease in the water contact angle in our case, which can be confirmed to some extent by the fact that the change in the contact angle (D(CA)) within 30 min became larger and larger with increasing NVP content in Macromol. Biosci. 2005, 5, 229–236

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Figure 4. Typical curves for the time dependence of water contact angle on the films measured by a sessile drop method: (}) PAN; (3) PANCNVP07; (~) PANCNVP15; (") PANCNVP22; (!) PANCNVP31. The inset shows the contact angle change (D(CA)) on the copolymer film within 30 min.

the copolymer film, as shown in the inset of Figure 4. Therefore, one can envisage that some relationship might exist between the hemocompatibility of PANCNVP films and the large water adsorption ability induced by a high NVP content in the copolymer. Swelling experiments were performed to determine the effect of total water content on the hemocompatibility of PANCNVP films. Figure 6 shows the kinetic behavior of the PANCNVP films at 37 8C. As shown in the Supporting Information, the swelling behavior at other temperatures (25 and 50 8C) was similar to that at 37 8C. It can be seen that the water uptake, Wc, increases with increasing NVP content of the copolymers. As compiled in Table 1, the equilibrium water content, WE, also increases with increasing NVP content in the copolymer, due to the hydrophilicity of

Figure 5. Static water contact angles on the films measured by sessile drop (&) and captive bubble (&) methods. ß 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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It is recognized that this melting peak originates from freezable water, so the content of non-freezable water, Wnf, can be calculated from Equation (3) by assuming the enthalpy change of the melting of the freezable water equals that of pure bulk water at any temperature.[17,31] However, just as pointed out by Higuchi and Lijima,[32] the heat of fusion of water in the films must be substantially lower than that of pure bulk water. They also suggested that, when the water content in films was very high, no evidently separated melting peaks were observed and the heats of fusion of water in different states may be identical to each other. This was confirmed in our experiments for fully swollen samples. The following equation can be approximately used to obtain Wnf: Wnf ¼ WE  ðWf þ Wfb Þ ¼ WE  Qendo =Qf Figure 6. Sorption isotherms for copolymer films with different compositions at 37 8C: (}) PAN; (3) PANCNVP07; (~) PANCNVP15; (")PANCNVP22; (!) PANCNVP31.

NVP.[14,20] Moreover, equilibrium for samples with low NVP contents (e.g., PANCNVP07) is achieved before that for ones with a higher NVP content (e.g., PANCNVP31). It can also be seen from Table 1 that the effect of the NVP content on water uptake is far more efficient than that of temperature. In other words, the swelling behavior of these films is not sensitive to temperature in these regimes. This is in agreement with other results on hydrogels reported by Roman et al.[20] As mentioned above, the incorporation of NVP into PAN is favorable to water uptake. However, for the hemocompatibility of hydrophilic materials, not only the total water content must be considered, but also the states of water adsorbed in the polymers. DSC and TGAwere used to determine the water content and its states in PANCNVP films. DSC is an indirect probe of water behavior in the sense that it monitors the heat capacity associated with the phase transition induced by temperature changes. With a few exceptions, DSC is insensitive to the thermal events of water below 70 8C because the change in the heat capacity is too small to be detected.[13] The PANCNVP samples were cooled down to about 70 8C and the heating to room temperature was recorded, as shown in Figure 7. One broad melting peak can be seen in the DSC curve for each sample.

ð3Þ

where WE is the equilibrium water content and Wf, Wfb and Wnf are the contents of free water, freezable bound water and non-freezable bound water in the swollen films respectively. Qendo (J  g1) is the heat which is obtained from the area of the DSC curve divided by the amount of water absorbed, while Qf (334 J  g1) is the phase transition heat of pure bulk ice reported in the literature. It can be seen that Wnf increases with increasing NVP content. Some researchers have reported that two distinct melting peaks induced by free water and freezable bound water can be seen in DSC curves at around 0 8C and 10 8C.[14,17] Galin et al.[18a] mentioned that the melting points induced by freezable bound water in some polymers, such as PVP or poly(p-hydroxystyrene), were distributed over a narrow temperature range close to 0 8C. A melting peak with a shoulder was also reported by Tanaka et al.[9,10]. In this case, the melting peak in Figure 7 also exhibits a visible shoulder, which may be assigned to the freezable bound water. The distinction between these two melting peaks may be influenced by several factors. Although the formation of bound water seems to be complicated and the data between water molecular mobility from NMR and heat capacity from DSC are sometimes not consistent with each other,[33] Katayama et al.[34] proposed that bound water is restricted in motion compared with free water and the restriction depends on the size of the cavities in the sample. Ping et al.[16] pointed out that the melting behavior of bound water in

Table 1. Equilibrium water content (WE) at different temperatures estimated with gravimetry and contents of non-freezable bound water (Wnf) and freezable water (Wf). Samples for DSC and TGA were prepared at 37 8C. Sample

PAN PANCNVP07 PANCNVP15 PANCNVP22 PANCNVP31

WE

Wnf

Wf

25 8C

37 8C

50 8C

TGA

DSC

TGA

DSC

TGA

0.287 0.302 0.432 0.539 0.567

0.297 0.306 0.433 0.555 0.583

0.268 0.285 0.421 0.554 0.602

0.284 0.348 0.412 0.556 0.612

0.046 0.052 0.096 0.160 0.193

0.034 0.058 0.102 0.210 0.248

0.251 0.254 0.337 0.395 0.390

0.250 0.290 0.310 0.346 0.364

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Figure 7. DSC thermograms of fully swollen film samples with different NVP contents as indicated in the diagram.

Figure 8. DSC thermograms for PANCNVP31 films with different water contents as indicated in the diagram.

polymers can be attributed to the effect of capillary condensation or/and the strong interactions of the water molecules with the polar groups of hydrophilic polymers. Cohen-Addad et al.[21] showed that melting point depression of liquid in polymers relates to two aspects. One is the volume contribution while the other is the water-polymer surface interaction. On the other hand, McBrierty et al.[22] found that reducing the scanning rate from 20 to 5 K  min1 produced no significant change in the recorded DSC thermograms and Huglin et al.[14] reported that, as the heating rate was increased from 2.5 K  min1, the wellresolved thermograms merged into one broad peak and shifted to higher temperature. Higuchi and Lijima[32] studied water in poly(vinyl alcohol) and its copolymer membranes systematically, and they proposed that the depression of phase transition temperature is usually ascribed to the interactions of the water with the polymer chains and/or capillary condensation in the membranes. They also found that both the heating rate during DSC analysis and the water content in membranes might influence this depression and therefore the shape of the melting peak. Therefore, the non-freezable water content can be estimated from the method mentioned above. However, since the fusion heat of freezable water is not a constant, the following method was adopted to ascertain the non-freezable bound water content. The DSC curves of PANCNVP31 samples with different water contents were recorded, as shown in Figure 8. No melting peak was found for the sample with WE ¼ 0.153, while distinct melting peaks were seen for those with WE of more than 0.343. The Wnf value for the PANCNVP31 sample calculated from the DSC curve was 0.193, which is located between 0.153 and 0.343.

TGA was also used to confirm the results from DSC measurements. Typical results are shown in Figure 9 and Table 1 and other TGA thermograms are available in the Supporting Information. It can be concluded that the data from TGA agree well with those from DSC and gravimetry. Several works concerning the effect of water in polymers on the hemocompatibility have been reported. Bruck et al.[7] found that a critical level of water exists in poly(2hydroxyethyl methacrylate) and poly(acrylamide) hydrogels to achieve hemocompatibility. Tanaka et al.[10] concluded that the main factor causing the excellent

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Figure 9. Typical TGA curve of a swollen PANCNVP31 film sample with WE ¼ 0.583 (WE, Wf, Wnf denote equilibrium water content, free water content and non-freezable bound water content, respectively). ß 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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compatibility of poly(2-methoxyethyl acrylate) was the freezable bound water which will prevent the blood components from contacting the polymer surface of nonfreezing bound water on the polymer surface. Ishihara et al.[8] suggested that fewer proteins are adsorbed and their original conformation is not changed on phospholipid polymer surfaces that possess a high free water fraction. These results are not consistent with each other and it seems that no simple relationship between the water content/ structure and the hemocompatibility of polymers has been found. Such discrimination may be contributed to by the differences between polymer chemical/physical structures and the interactions of polymer with blood components. However, water content/structure on a polymer surface is one of the key factors, which is well accepted. With regard to surface character, Tsuruta et al.[35] pointed out that the magnitude of the ‘‘defensive response in blood’’ depends on the interfacial properties between the polymer surface and water. Especially, strongly networked water on the polymer surface, such as non-freezing water, would cause poor hemocompatibility of the polymer. In view of the fact that swelling is a dynamic process, there should be a dynamic exchange of water molecules between different water states in the bulk of polymers, especially exchange between water molecules in polymers and in contacted liquids (e.g. blood) at the polymer/liquid interface.[36] The diffusion coefficient of water into films may give direct and quantitative characterization of these exchanges, which can be calculated from Fick’s law.[20] When a plot of water uptake versus t1/2 was created, however, Fickian behavior was not obeyed for systems in our regimes. Nevertheless, the larger diffusion ability of water molecules (or diffusion coefficient) can be qualitatively deduced for systems with a higher free water content, which was induced by higher NVP contents.[20] Therefore, based on the results obtained from the above experiments, we hypothesize that the better hemocompatibility of the PANCNVP film with higher a NVP content was mainly due to its higher free water content. Although the content of non-freezable bound water for PANCNVP increased with NVP content, it might mostly exist in the bulk of the film. On the other hand, as summarized in Table 1, the amount of Wf is much larger than that of Wnf. Water molecule exchange at the polymer/ liquid interface facilitated by high free water content might be unfavorable for the formation of surface bound water and then be beneficial to the hemocompatibility of a polymer. However, as summarized in the Introduction, other factors besides water content and its states may influence the hemocompatibility of polymers. Data for the contact angle shown in Figure 4 and in our previous works concerning PAN-based copolymers have not been well understood. Therefore, more detailed work on the surface physicochemical structures of a series of PAN-based copolymers has been carried out in our laboratory. Macromol. Biosci. 2005, 5, 229–236

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Conclusion PANCNVP films with different N-vinyl-2-pyrrolidone contents were prepared. Platelet adhesion and plasma recalcification time tests indicated a better hemocompatibility for films with a higher NVP content. The swelling behavior of the PANCNVP films at temperatures of interest showed that the equilibrium water content increased with increasing NVP content in the film. The content of non-freezable bound water determined by DSC and TGA together with the increase in the content of free water with NVP content. Based on these results, we hypothesize that water molecule exchange at the polymer/liquid interface facilitated by high free water content is unfavorable for the formation of surface bound water and therefore is beneficial to the hemocompatibility of PANCNVP. Therefore, the better hemocompatibility of the film with a higher NVP content might be due to its higher free water content. Acknowledgements: Financial support from the National Natural Science Foundation of China (Grant no. 50273032) and the National Basic Research Program of China (Grant no. 2003CB15705) is gratefully acknowledged.

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