Grafting of Zwitterion from Cellulose Membranes via ATRP for

Biomacromolecules 2009, 10, 2809–2816

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Grafting of Zwitterion from Cellulose Membranes via ATRP for Improving Blood Compatibility Ping-Sheng Liu,† Qiang Chen,*,†,‡ Xiang Liu,† Bo Yuan,† Shi-Shan Wu,† Jian Shen,*,†,§ and Si-Cong Lin† School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China, Jiangsu Engineering Research Center for Biomedical Function Materials, Nanjing Normal University, Nanjing 210097, People’s Republic of China, and High Technology Research Institute of Nanjing University, Changzhou 213164, People’s Republic of China Received June 9, 2009; Revised Manuscript Received August 25, 2009

A p-vinylbenzyl sulfobetaine was grafted from cellulose membrane (CM) using surface-initiated atom transfer radical polymerization for blood compatibility improvement. Surface structure, wettability, morphology, and thermal stability of the CM substrates before and after modification were characterized by attenuated total reflectance Fourier transform infrared spectra, X-ray photoelectron spectroscopy measurement, water contact angle measurement, atomic force microscopy, and thermogravimetric analysis, respectively. The results showed that zwitterionic brushes were successfully fabricated on the CM surfaces, and the content of the grafted layer increased gradually with the polymerization time. The blood compatibility of the CM substrates was evaluated by protein adsorption tests and platelet adhesion tests in vitro. It was found that all the CMs functionalized with zwitterionic brush showed improved resistance to nonspecific protein adsorption and platelet adhesion, even though the grafting polymerization was conducted for several minutes.

Introduction Cellulose and its derivatives have been widely used in biomedical fields, including controlled release, blood purification therapies, and tissue engineering due to their biocompatibility and good properties.1-10 However, the biocompatibility (especially the blood compatibility) of the raw cellulose is inadequate and needs to be improved before use. Over the past two decades, numerous attempts have been made to enhance the biocompatibility of cellulose and its derivatives by surface modification with biocompatible materials such as heparin,11,12 PEG,13,14 and phospholipid polymers,15 as well as formation of cellulose composites with synthetic polymers and biopolymers.16-18 Among these materials, both PEG and phospholipid polymers, which originated from the simulation of biomembranes, have been identified as two major synthetic nonfouling and nonthrombogenic materials.17,18 Based on molecular engineering, our group postulated the “normal conformation” hypothesis in 1984,19 and further suggested that polymers with zwitterionic structures could maintain normal conformation of biomacromolecules and improve biocompatibility.20 With the exception of the most frequently studied zwitterion, phosphobetaine, over the past few years our group reported the utilization of two other kinds of zwitterions, sulfobetaine and carboxybetaine.21-25 In recent years, Jiang’s group reported superlow fouling surfaces polymerized from these two zwitterions via surface-initiated atom transfer radical polymerization (ATRP).26-28 The samples obtained from this method were more biocompatible compared with those obtained from random radical polymerization.29 This * To whom correspondence should be addressed. Tel.: +86 25 83594933. Fax: +86 25 83594404. E-mail: [email protected] (J.S.); chem100@ nju.edu.cn (Q.C.). † Nanjing University. ‡ Nanjing Normal University. § High Technology Research Institute of Nanjing University.

may be due to the fact that ATRP could lead to well-defined polymers with narrow molecular weight distribution.30 Accordingly, the surface-initiated ATRP can bring about uniform functional brushes with fewer defects than those resulting from random radical polymerization affecting the topography, which is one of the most important parameters that influence protein adsorption and the host response.31 In addition, higher packing density of functional groups with regular structures could be obtained on the surface through this method.32 Recently, zwitterions have been directly grafted via ATRP from metallic and inorganic substrates (including gold chips,33 glass slides,34 and silicon wafers35), synthesized as zwitterionic polymer composites,16-18 and polymerized as block coating materials for biomedical applications.36,37 However, very few studies have been reported based on fabrication of zwitterions on polymeric membranes by this approach since Malmstrom’s first report.38 Protein adsorption on biomaterials’ surfaces is thought to be the first step of many undesired bioreactions and bioresponses,39 followed by platelet adhesion and activation of coagulation pathways, leading to thrombus formation.40 Although there are many studies that demonstrated surfaces with resistance to human fibrinogen or other single protein solutions such as bovine serum albumin and lysozyme, there have been few studies on the total protein adsorption from complex matrices,34 which are more close to the natural contact between surfaces and blood. In this study, we investigated the direct grafting of poly N,Ndimethyl-N-(p-vinylbenzyl)-N-(3-sulfopropyl) ammonium (PDMVSA) from CMs via surface-initiated ATRP. The surface structure, wettability, morphology and thermal stability of CMs were characterized by attenuated total reflectance Fourier transform infrared spectra (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), atomic force microscopy (AFM), and thermogravimetric analysis (TGA),

10.1021/bm9006503 CCC: $40.75  2009 American Chemical Society Published on Web 09/10/2009

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Figure 1. 1H NMR spectrum of DMVSA in deuterium oxide.

respectively. The blood compatibility of the CMs was evaluated by protein adsorption tests and platelet adhesion tests in vitro. The effect of polymerization time on the properties of CM substrates, as well as the relationship between protein adsorption and platelet adhesion, was studied.

Experimental Methods Materials. Cellulose membrane (CM) was purchased from SigmaAldrich and cut into circular pieces. 2-Bromoisobutyryl bromide (BIBB, 97%), 2-dimethylaminopryridine (DMAP, 97%), and 1,3-propanesulfone (1,3-PS, 99%) were purchased from Alfa Aesar. N-(4Vinylbenzyl)-N,N-dimethyl amine (90%) was purchased from Acros Organics. Copper(I) bromide (CuBr, 98.5%), 2,2′-bipyridine (BPY, 99.5%), triethylamine (TEA, 99%), chloroform (CHCl3, AR), and tetrahydrofuran (THF, AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. and purified before use. Whole blood was provided by Blood Center of Jiangsu Red Cross. Phosphate-buffered saline (PBS, 0.02 M phosphate, 0.15 M sodium chloride, pH 7.4) was purchased from Boster Biotechnology Co., Ltd. An enhanced BCA (Bicinchoninic acid) protein assay reagent kit and sodium dodecyl sulfate (SDS, 10 wt % in PBS) were purchased from Beyotime Institute of Biotechnology, China. Preparation of N,N-Dimethyl-N-(p-vinylbenzyl)-N-(3-sulfopropyl) Ammonium (DMVSA). DMVSA monomer was prepared as reported in our previous paper.21 Typically, 14.54 g (80 m mol) of N-(4vinylbenzyl)-N,N-dimethyl amine (dissolved in 100 mL chloroform) was added into a 250 mL flask equipped with magnetic stirring, before 12.14 g (96 m mol) of 1,3-propanesulfone (dissolved in 100 mL of chloroform) was added dropwise into the flask. The reaction was continued for more than 10 h at 30 °C. A white powdered crude product was obtained after filtration of the suspension. The typical yield of the product was about 60% after recrystallized in ethanol twice. 1H NMR was recorded on a Bruker spectrometer (300 MHz) using deuterium oxide as the solvent (Figure 1): δ 7.50 (q, 4H, aromatic), 6.75 (q, 1H, )CH), 5.85 (d, 1H, cis, )CH2), 5.30 (d, 1H, trans, )CH2), 4.42 (s, 2H, -CH2), 3.36 (m, 2H, CH2-N), 2.97 (s, 6H, -CH3), 2.88 (t, 2H, -CH2), 2.23 (m, 2H, -CH2-). Surface-Initiated ATRP from CM. A two-step process of the surface-initiated ATRP from CMs was performed. For the esterification of hydroxyl groups with BIBB, CM substrates were immersed in 50 mL of THF solution containing TEA (4.44 g, 44 m mol) and a catalytic

amount of DMAP. The reaction mixture was stirred under an ice bath and BIBB (9.2 g, 40 m mol) was then added into the mixture dropwise. The reaction proceeded at 20 °C for 24 h. After the reaction, the initiator functionalized CMs (hereinafter refer to CM-Br) were removed from the solution and thoroughly washed with dichloromethane and ethanol ultrasonically. For the surface-initiated ATRP, four to six CM-Br substrates and CuBr (286 mg, 1.0 m mol) were introduced into a dry flask. The flask was then evacuated and backfilled with nitrogen (five cycles). Degassed solution (methanol and distilled water in 1:1 volume ratio) containing DMVSA (2.28 g, 4.0 m mol) and BPY (312 mg, 1.0 m mol) was then added into the flask under nitrogen protection. The ATRP reaction proceeded at 25 °C for predetermined time period. After the reaction, the PDMVSA functionalized CMs (hereinafter refered to as CMG-n, where n means the grafting polymerization time, minute) were removed from the solution, thoroughly washed with PBS and distilled water ultrasonically, and dried under vacuum. Preparation of PDMVSA via Random Radical Polymerization. A random PDMVSA was prepared according to a previous report.41 Briefly, under a nitrogen protection, 710 mg DMVSA (2.5 m mol) was polymerized at 50 °C for 3 h and then at 80 °C for an additional 2 h using ammonium persulfate and sodium sulfite as a compounded redox initiator. The resulting reaction solution was precipitated into ethanol and redissolved into water repeatedly. After the separation and dried under vacuum at room temperature, a white powdered PDMVSA was obtained. Characterization of CMs. ATR-FTIR measurements of the CM substrates were performed on a Nicolet 170 sx FTIR equipped with an Omni sampler over 32 scans. The spectra were recorded with a resolution of 4 cm-1. XPS measurements were obtained on an ESCA Lab MK II (V.G. Scientific Co. Ltd., U.K.) equipped with a Mg KR radiation source (12 kV and 20 mA at the anode). The takeoff angle of the photoelectron was kept at 45°. The binding energy was referenced by setting the C1s hydrocarbon peak to be 285.0 eV. TGA results were obtained in nitrogen atmosphere with a Perkin-Elemer thermogravimetric analyzer (Pyris 1, U.S.A.). Samples were heated at a constant rate of 20 °C/min from ambient temperature to 750 °C. Water contact angles were measured and calculated in static mode on a DataPhysics Instrument (OCA-30, Germany) at ambient temperature. One drop of water (3 µL) was put on the surface of the film with an automatic piston syringe and photographed. Three spots were performed for each sample. The surface morphology of the substrates was observed by an atomic

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Scheme 1. Surface-Initiated ATRP from CM

force microscopy (SPI3800, Seiko Instruments, Japan). A piece of properly sized dry substrate was placed on the surface of a clean silicon wafer (fixed by silica gel film) and observed in tapping mode. Protein Adsorption. After being equilibrated with PBS overnight, the CM substrates were immersed in 2 mL of platelet-poor plasma (PPP)42 at 37 °C for 90 min and then rinsed with PBS three times. The adsorbed proteins were detached in 1% SDS for 60 min and the concentration of the adsorbed proteins was determined by BCA method39 at 562 nm. Independent measurements were performed in triplicate samples and the total amounts of the adsorbed proteins were calculated from the concentration of the standard protein solution. Platelet Adhesion.43 The CM substrates were placed in individual wells of 24-well tissue culture plate and equilibrated with PBS overnight. A total of 500 µL of platelet-rich plasma (PRP)42 was added into each well and incubated at 37 °C for 120 min under static conditions. After being rinsed with PBS, the substrates were immersed in 2.5% glutaraldehyde in PBS for 30 min, subjected to a series of graded alcohol-water solutions (25, 50, 75, 95, and 100%) for 20 min in each step, and dried under vacuum. Finally, the substrates were examined using a scanning electron microscopy (SEM, Shimadzu, SSX550) after coating with gold. Three different spots were observed on each sample.

Results and Discussion Immobilization of ATRP Initiators on the CMs. The initiator of ATRP was immobilized on the CM surface through esterification of hydroxyl groups with BIBB, as shown in Scheme 1. Attempts to analyze the immobilization of initiator by ATR-FTIR failed. The initiator (ester group) was undetectable because the detection limit of this method is micrometer scale,38 and the layer of ester groups is very thin in comparison. The surface composition variations of the pristine CM and CM-Br were characterized by XPS using the atomic detection of C, O, and Br. Table 1 lists the detailed data from XPS scans on different surfaces. After the reaction was conducted for 24 h, the content of carbon (C1s) increased, while the content of oxygen (O1s) decreased, and the total atomic ratio of O/C decreased from 0.63 to 0.57. In addition, a small amount of bromine (1.31%, Br3d) appeared (Figure 2c). As seen from the narrow scan spectra of C1s (Figure 2b,d), the C1s peaks in CM and CM-Br are mainly attributed to the overlap of binding energy peaks of C-H (containing C-C at ∼285.0 eV), C-O (∼286.6 eV), and -CdO (∼288.0 eV). Because CM has fewer C-H species than C-O species in its molecular chain, the peak at 285.0 eV is smaller than that at 286.5 eV. However, the peak of C-H species on the CM-Br surface is obviously larger than that on the pristine CM,

Table 1. Elemental Surface Composition of CM Substrates Determined from XPSa,b element (atom %) sample

C

O

CM CM-Br CMG-5 CMG-15 CMG-30 CMG-60 CMG-240 CMG-480 PDMVSA

61.22 62.68 65.52 68.48 70.35 69.54 70.31 72.28 (73.69)

38.78 36.01 27.10 23.51 21.53 22.43 21.02 18.85 (15.79)

S

atomic ratio N

Br 1.31

3.55 3.82 4.04 3.96 4.32 4.34 (5.26)

3.83 4.19 4.08 4.07 4.35 4.53 (5.26)

O/C 0.63 0.57 0.41 0.34 0.31 0.32 0.30 0.26 (0.21)

a The values in the parentheses are the theoretical atomic percentages of PDMVSA. b Data precision is ∼5%.

indicating that the initiator was indeed immobilized on the surface. This is because the initiator, BIBB, has no C-O species but several C-H species in its structure, and the immobilization of the initiator increased the content of C-H species and decreased the content of C-O species. Because the pristine CM is hydrophilic while the initiator is hydrophobic, the static water contact angles of CM and CMBr were measured using a water droplet method. As a result, the contact angle of initiator-immobilized CM substrate (CMBr) was sharply increased from 47 to 73°, showing a transformation from hydrophilic to hydrophobic, clearly visible from the images in Figure 3. Surface-Initiated ATRP from the CM-Br substrate. In the present study, the surface grafting reactions of DMVSA from the CM-Br were carried out by varying polymerization time. The grafted CMs with different polymerization time, as well as the pristine CM, were first characterized by ATR-FTIR, as shown in Figure 4. The ATR-FTIR spectrum of pristine CM (Figure 4a) shows the characteristic peaks of the saccharide structure (asymmetric stretching of C-O-C at 1159 cm-1, skeletal vibration involving the C-O stretching at 1058 cm-1 and 1024 cm-1).24 The characteristic peaks in the spectrum of CM-Br (Figure 4b) and PDMVSA grafted CMs with the polymerization time less than 240 min (Figure 4c-f) are almost unchanged as compared with those in the spectrum of pristine CM. This is because there was just a monolayer of the initiator or a very thin zwitterionic polymer immobilized on the surfaces. The thicknesses or the contents of these layers may be below the detection limit of ATR-FTIR. After the polymerization was proceeded for 240 min (Figure 4g), the characteristic absorption bands of -SO3-, originated

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Figure 2. Survey scan and C1s scan spectra of CM (a,b), CM-Br (c,d), and CMG-5 (e,f) substrates.

Figure 3. Water contact images of CM and CM-Br.

Figure 4. ATR-FTIR spectra of pristine CM (a), CM-Br (b), CMG-5 (c), CMG-15 (d), CMG-30 (e), CMG-60 (f), CMG-240 (g), and CMG480 (h).

from PDMVSA,21,24 appeared at 1197 cm-1 and 1031 cm-1; the latter was overlapped with the skeletal vibration of C-O at 1024 cm-1. The intensity of the 1197 cm-1 band increased when the polymerization time was prolonged to 8 h (Figure 2h), indicating that the thickness of the grafted zwitterionic layer increased with the polymerization time. According to the XPS data (Table 1), after the reaction had occurred for 5 min, the content of the carbon increased while

that of oxygen decreased, and the total atomic ratio of O/C decreased by 17 percentage points. Furthermore, the S and N components, with an approximate ratio of 1:1, were detected at 164.0 and 402.5 eV, respectively, while no corresponding peak was detected in the CM-Br (Figure 2e). As compared with the data in our previous study,24 the grafted layer resulted from ATRP had higher surface packing densities of functional zwitterionic groups (more than 4%) than those of the monolayers (less than 2%). As seen from the narrow scan spectra of C1s (Figure 2), there was a remarkable content variation of the different carbon species, indicating that the reactions indeed occurred. For CMBr, the peak at ∼286.6 eV is much larger than that at ∼285.0 eV, indicating that there were more C-O species than C-H species on the initiator immobilized surface. However, the peak at ∼285.0 eV is much larger than that at ∼286.6 eV in CMG5, showing that the C-O species was no longer the dominant species on the grafted surface. The explanation for this phenomenon may be similar to that of immobilization of the initiator: no C-O species but C-H, C-C, CdC species (whose binding energies are all around 285.0 eV) exists in the sulfobetaine structure. In addition, after the grafting polymerization had occurred for 5 min, the signal of the CdO species almost disappeared (Figure 2f), suggesting that the CM substrate may be basically covered by the grafted zwitterionic layer without the CdO species. Though there was no bromine signal (Br3d) on all the grafted surfaces, the atomic ratio of O/C further decreased with increasing polymerization time, and the rate of this decrease was obviously slowed down after the reaction was continued for more than 30 min. A similar tendency was observed in water contact angle measurements. As shown in Figure 5, at 30 min into the reaction, the water contact angle is sharply decreased from 73 to 36°, and it is slightly further deceased with the reaction time. According to Malmstrom’s study,44 the disappearance of the bromine content could be attributed to the entanglement of the chains on the surfaces; therefore, the living


Figure 5. Water contact angles of pristine CM, CM-Br, and CM substrates with different polymerization time (CM-Br is selected as the origin of horizontal coordinate, while CM is added as a control to show the obvious tendency). Data from three separated experiments are shown as mean ( SD.

chain-ends were not located directly in the outermost layer under vacuum during the XPS analysis. Although the signal of Br3d was almost no longer detectable on grafted surfaces, the chain propagation could also take place with the reaction time. As shown in the reaction rate law derived for ATRP in Matyjaszewski’s work,30 there is a directly proportional relationship between the reaction rate and the monomer concentration. Therefore, the reaction rate should slow down with the decrease of the monomer concentration during its conversion. The present study shows that the growth rate of the zwitterionic groups, as well as the variation rate of water contact angle, slowed down gradually in polymerization. In this study, although it is hard to accurately measure the thickness and the chain length of the grafted polymer because the surface of pristine CM is rough, the surface topologies of the pristine CM, CMG-60, and CMG-480 substrates were observed by AFM under dry conditions, using a tapping mode at a scan rate of 0.5 Hz over an area of 5 × 5 µm. Figure 6 shows the 2D (a-c) and the corresponding 3D (d-f) images of the substrates in an area of 25 µm2. Different topological changes are clearly observed between the pristine and grafted CM substrates. Figure 6a, b, and c are the height images of CM, CMG-60, and CMG-480, while d, e, and f are their corresponding 3D images, respectively. Comparing the height images a and b, together with their 3D images d and e, we can clearly see that the CMG-60 surface is rougher than that of the CM, and some cuspidal outshoots (belonging to the aggregation of grafted polymer chains) appear throughout the surface, indicating that the surface-initiated zwitterionic brushes were fabricated on the CM surface. In addition, the surface of CMG480 is still rougher than that of CM, with more uniform and less cuspidal outshoots than that of CMG-60, suggesting that a higher surface package density of zwitterionic groups resulted from the prolongation of the polymerization. As with the FTIR and XPS results discussed above, the content of the zwitterionic groups increased gradually in the course of polymerization, which is in good agreement with the AFM observation result. However, the maximum depth of the pristine CM is higher than that of CMG-60. This might be due to the fact that the impurity or defect, clearly visible from the SEM images in Figure 6, has a greater depth on the CM surface (in Figure 6a and d). To illustrate that the maximum depth of grafted surfaces


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are higher than that of pristine CM surface, a smaller area (2 × 2 µm) was selected for observation. The related images of the three surfaces (see Supporting Information) showed that the maximum depth of grafted surfaces (30 nm for CMG-60 and 60 nm for CMG-480) were indeed higher than that of pristine CM (20 nm). TGA of the pristine CM, the grafted CM, and the pure PDMVSA have also been investigated as shown in Figure 7. To reduce the experimental error, all samples had been dried under vacuum for 24 h and then kept in vacuum before use. As can be seen, all the samples underwent weight losses in three stages. The first stage is the loss of the moisture, while the others are attributed to the pyrolysis. For pristine CM, the temperature of the first stage ranges from room temperature (RT) to ∼160 °C. In contrast, for CMG-480, the temperature of this stage ranges from RT to ∼200 °C, and pure PDMVS has the widest temperature range in this stage, from RT to ∼250 °C. In addition, from the data listed in Table 2, CMG-480 has more weight loss than pristine CM, while pure PDMVS has the largest weight loss during this stage. This is attributed to the successful grafting of the zwitterionic polymer that introduces zwitterionic structures onto the surfaces and can form a hydration layer and bind more water molecules via electrostatic interactions.28 Table 2 lists the detailed data of the initial pyrolysis temperature (Ti) and percent char of the substrates at 750 °C. The pyrolysis of pristine CM begins at 161.8 °C, while pyrolysis of CMG-480 begins at 290.5 °C, and the percent char of CMG480 is about 1.5 percentage points higher than that of pristine CM at 750 °C. In reference to the related data of pure PDMVSA, we can conclude that this phenomenon may be due to a thin benzyl zwitterionic polymer layer grafted on the pristine CM surface, resulting in delay of pyrolysis and an increase of char because the C-C and CdC bonds from PDMVSA are more stable than those of C-O-C bonds from pristine CM. Plasma Protein Adsorption. Protein adsorption on the biomaterials’ surface is thought to be the first step of many undesired bioreactions and bioresponses.39,40 Therefore, the primary target for preparing biomaterials is to construct superlow fouling or even nonfouling surfaces. Many studies demonstrated surfaces with resistance to human plasma fibrinogen and other single proteins such as bovine plasma fibrinogen, bovine serum albumin, and lysozyme. In this paper, the fresh human PPP was selected as the protein source for total protein adsorption investigation. Figure 8 shows the results of the protein adsorption tests. It can be clearly seen from the figure that the amount of adsorbed proteins drastically increased after the surface immobilization of initiator. Jiang et al.29 obtained similar results in the study on the fabricating of sulfobetaine and carboxybetaine onto glass slides through ATRP. This increase may be attributed to the hydrophobic initiator being immobilized on the surface, resulting in the transformation from hydrophilic to hydrophobic. As expected, hydrophobic surfaces have greater interaction from the outer layer and possibly from the inner core of the protein45 and on which protein adsorption is even more energetically favorable.45,46 Moreover, the bromine group on the initiator may have a strong interaction with proteins, so there are more proteins adsorbed on the CM-Br compared with those on the pristine CM. When the zwitterionic brushes were fabricated on the surface, the amount of adsorbed proteins significantly decreased even less than that on pristine CM. Many researchers attributed this decrease to the zwitterionic structure bonding more water and having less interaction with proteins. It is believed that zwit-

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Figure 6. AFM images of pristine CM (a,d), CMG-60 (b,e), and CMG-480 (c,f).

Figure 7. TGA curves of pristine CM, CMG-480, and pure PDMVSA. Table 2. Thermal Analytical Data of Pristine CM, CMG-480 and Pure PDMVSA samples

weight lossa (%)

Tib (°C)

char (%)

CM CMG-480 PDMVSA

7.7 8.6 9.8

161.8 259.2 290.5

16.8 18.3 33.6

a Value for the first weight loss stage. b Temperature of the initial decomposition (the second weight loss stage).

terions form a hydration layer via electrostatic interactions in addition to hydrogen bonding and can bind a significant amount of water molecules (as shown in Table 2),28 which generates a strong repulsive force to protein at specific separation distances

Figure 8. Amount of protein adsorbed on the CM substrates. Data from three separate experiments are shown as mean ( SD.

or makes the protein contact with the material surface in a reverse manner without a significant conformation change.47 In other words, the zwitterionic surfaces can maintain the normal conformation of the biomacromolecules when they are close to or in contact with each other.19,20 Platelet Adhesion. Platelet spreading and aggregation are marks of platelet activation and are considered to be a major mechanism of thrombosis.48 Meanwhile, platelet adhesion is one of the intuitive methods to measure the blood compatibility of biomaterials.



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Figure 9. SEM photographs of CM (a), CM-Br (b), CMG-5 (c), CMG-15 (d), CMG-30 (e), CMG-60 (f), CMG-240 (g), and CMG-480 (h) after a 120 min incubation in the human PRP (magnifications are 2000, bars are 5 µm).

Figure 9 clearly shows the SEM images of platelet adhesion on different CM substrates. As expected, there were a few platelets adhering on the pristine CM (Figure 9a), while there were a lot of platelets adhering and aggregating with extended pseudopodia after the initiator was immobilized on the CM surface (Figure 9b). This may be because the higher protein adsorption on the hydrophobic surface (CM-Br) resulted in the higher activation and adhesion of platelets compared with that of pristine CM, because platelet adhesion is thought to be the next step to protein adsorption with a link by link relationship in thrombosis process.39,40 However, when the zwitterionic layer was fabricated on the surface, there was almost no platelet adhered on the zwitterion functionalized surface (Figure 9c) and no obvious difference was observed in platelet adhesion among zwitterionic surfaces with different polymerization times (Figure 9c-h), showing that all the zwitterionic surfaces have excellent resistance to platelet adhesion even though the grafting polymerization was conducted for several minutes. This is incomplete in agreement with the tendency of the protein adsorption above. There are two probable explanations for this phenomenon. First, fibrinogen has been identified as the key player of conveying the surface chemical feature to platelets in material-platelet interactions.49,50 However, the amount of the adsorbed protein in this study is the amount of the adsorbed total proteins after multiple competitive and cooperative reactions from human plasma, which contains more than 100 proteins, and fibrinogen is just one of the them. Second, and more importantly, it is believed that the aggregation and adhesion of platelets to materials may depend on the interaction between the exposed “receptor induced binding site” on conformationally changed fibrinogen and the “ligand induced binding site” on the platelet membrane,49 therefore, the conformation of the adsorbed fibrinogen, more than its amount, takes the primary responsibility for platelet adhesion and activation.43,49,50

Conclusions Zwitterionic sulfobetaine was grafted from CMs via surfaceinitiated ATRP with different polymerization times. FTIR result, XPS element analysis, and water contact angle measurement made it sure that the sulfobetaine was successfully grafted from CMs and its content increased gradually with polymerization

time. AFM observations indicated that the fabricated zwitterionic layers had a brush appearance, and the maximum depth also increased with the polymerization time. TGA results revealed that the grafted CM could maintain more water and be more thermally stable than the pristine CM under the same condition. Furthermore, all the CMs functionalized with zwitterionic brush showed improved resistance to nonspecific protein adsorption and platelet adhesion, even though the grafting polymerization was conducted for several minutes. This work provides a simple approach to fabricate superlow fouling and blood compatible brush layers on polymers, which possess wide biomedical application prospects. Acknowledgment. This research was supported by the National High Technology Research and Development Program of China (2006AA03Z445) and the Changzhou Science and Technology Research Project. Supporting Information Available. Water contact angle images of CM, CM-Br, and CMG-240, low magnification images (magnification are 600, bars are 20 µm) of platelet adhesion on the surfaces, as well as smaller range AFM images (2 × 2 µm) of CM, CMG-60, and CMG-480. This material is available free of charge via the Internet at http://pubs.acs.org.

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