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Crystalline TiO2 grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom-transfer radical polymerization
Applied Surface Science 257 (2010) 1596–1601
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Crystalline TiO2 grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom-transfer radical polymerization Yuancong Zhao, Qiufen Tu, Jin Wang, Qiongjian Huang, Nan Huang ∗ Key Lab. of Advanced Technology for Materials, Education Ministry, School of Material Science and Technology of Southwest Jiaotong University, Chengdu, Sichuan, China
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 25 August 2010 Accepted 25 August 2010 Available online 24 September 2010 Keywords: Poly(2-methacryloyloxyethyl phosphorylcholine) TiO2 film Chemical modification Platelet adhesion
a b s t r a c t Crystalline TiO2 films were prepared by unbalanced magnetron sputtering and the structure was confirmed by XRD. An organic layer of 11-hydroxyundecylphosphonic acid (HUPA) was prepared on the TiO2 films by self-assembling, and the HUPA on TiO2 films was confirmed by FTIR analysis. Simultaneously, hydroxyl groups were introduced in the phosphonic acid molecules to provide a functionality for further chemical modification. 2-Methacryloyloxyethyl phosphorylcholine (MPC), a biomimetic monomer, was chemically grafted on the HUPA surfaces at room temperature by surface-initiated atom-transfer radical polymerization. The surface characters of TiO2 films modified by poly-MPC were confirmed by FTIR, XPS and SEM analysis. Platelet adhesion experiment revealed that poly-MPC modified surface was effective to inhibit platelet adhesion in vitro. © 2010 Elsevier B.V. All rights reserved.
1. Introduction 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers, which contain a zwitterionic phospholipid group that is also present in cell membranes and possesses nonthrombogenic properties and high biocompatibility, have been widely used to construct non-biofouling surfaces in various biomedical applications as they have been shown to resist both protein adsorption and cell adhesion [1–3]. Phosphorylcholine (PC)-containing biomaterials are effective in reducing the adsorption of various proteins, cells, blood platelets, and bacteria [4–14]. The biomimetic strategy that uses PC-containing polymers to improve biocompatibility of biomaterials has received much attention over the past two decades [15–24]. Antithrombogenic biomaterials are one of the ultimate objectives for developing blood-contacting biomedical devices. Anticoagulant activity is very important to an artificial surface contacting with blood since it enhances the life time of medical devices and the patients health. Developments focus on minimizing the tendency of a material surface to activate blood platelets and to induce blood clotting, and reduce the risk of thrombosis. Many studies have aimed to improve hemocompatibility of biomaterials either by deposition of thin hemocompatible films [25,26] or by
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (N. Huang). 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.08.100
grafting biologically active molecules [27,28]. Recently, titanium oxide thin films were paid more attention because of their biocompatibility and bioinertness [29]. All kinds of crystalline TiO2 films, which are prepared by physical vapour deposition (PVD), plasma immersion ion implantation (PIII), vacuum arc deposition technology, plasma oxidation, and so on, possess favorable biocompatibility [30–35]. The composition, crystalline form and thickness of TiO2 films are factors to influence the biocompatibility [30–32]. The favorable TiO2 films can inhibit platelet adhesion and fibrinogen adsorption and activation effectively [33–35]. The evaluation in vivo shows that the TiO2 films posses higher ability to restraint thrombus formation [35]. A self-assembly of alkyl phosphonic acid on TiO2 has been reported to have physiological stability and suitable surface density [36–40]. To further enhance the anticoagulation ability of titanium oxide film, in this paper, 11-hydroxyundecylphosphonic acid (HUPA) was used to anchor on TiO2 film surface and to act as a linking layer. The hydroxyl group of HUPA was converted with 2-bromo-2-methylpropionyl bromide for chemical grafting of polymerized 2-methacryloyloxyethyl phosphorylcholine (MPC) on the surface by surface-initiated atom-transfer radical polymerization (ATRP) using oligomeric methoxy polyethylene glycol 2-bromoisobutyrate (OEGBr) as initiator and Cu(I)Br/2,2bipyridine (bpy) as catalyst at room temperature. Characterization of the grafting of poly-MPC by chemical methods on the metallic biomaterials for improvement of blood compatibility is reported. Platelet incubation studies indicate that the surface of TiO2 film modified by poly-MPC can effectively inhibit platelet adhesion in vitro.
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2. Experimental 2.1. Materials 2-Methacryloyloxyethyl phosphorylcholine (MPC, 96%) was supplied by the Joy-Nature Technology Institute (China) and used without further purification. Cu(I)Br (99.999%), 2,2-bipyridine (bpy, 99%), methoxy poly(ethylene glycol) (Mn ca. 200 g/mol), 11-bromoundecanol, triethyl phosphate (98%), 2-bromo-2methylpropionyl bromide (98%), were supplied by Alfa Aesar and used without further purification. Tetrahydrofuran (THF) was dried over metal Na, and distilled just prior usage. Acetonitrile and triethylamine (TEA) were distilled over calcium hydride just prior usage. 2.2. Film preparation TiO2 films were prepared on silicon (1 0 0) wafers using an unbalanced magnetron sputtering system. A Ti target (purity 99.9%) was mounted on the target source. The target-to-substrate distance was 10 cm. The ratio of O2 /Ar was sustained at 13/60 by a flow monitor system. The discharge current was kept constant at 3 A. The deposition time was 15 min. The film thickness is 300–450 nm measured by surface profilometer (AMBIOS Technology XP-2 Stylus Profiler). 2.3. Synthesis and characterization of HUPA 11-Hydroxyundecylphosphonic acid was prepared by the following procedure reported by Putvinski et al. [41]. Acetyl chloride (1.9 g, 0.024 mol) in 10.0 mL of dried H2 Cl2 was added dropwise to a mixture of 11-bromoundecanol (5.0 g, 0.02 mol) and triethylamine (2.2 g, 0.022 mol) in 100.0 mL of dried CH2 Cl2 at 0 ◦ C. The reaction mixture was stirred an additional 4 h in an ice bath. The solution was washed with saturated brine and H2 O until the aqueous layer was at pH 6. The organic layer was isolated, dried over anhydrous sodium sulfate, and to give white 11-bromoundecyl acetate, which was used in the following reaction directly without further purification. The crude product of the bromo acetate and triethyl phosphate (6.4 g, 0.025 mol) were heated at 150 ◦ C for 24 h. After the excess triethyl phosphite was removed under reduced pressure, concentrated HCl (8.0 mL) was added to a solution of diethyl (11-acetoxyundecyl)phosphonate in dioxane (20.0 mL), and the mixture was heated at 100 ◦ C for 3 days under argon. After cooling, the solvent was evaporated under reduced pressure. Ice water was added and the solution brought to pH 2 with aqueous HCl. The resulting precipitate was collected by filtration and recrystallized from ethyl acetate and petroleum ether (60–90) to give white (11-hydroxyundecyl)phosphonic acid (3.2 g, yield: 63.5% with respect to 11-bromoundecanol). 1 H NMR (CDC13 ): ı = 1.15–1.52 (m, 18H, CH2 ), 1.78 (m, 2H, CH2 P), 3.51 (m, 2H, CH2 O), 4.67 (s, 3H, OH); 31 P NMR: ı = 24.15. HR-MS (Calcd. for C11 H25 O4 P: 252.1490): 251.1414 (C− ). 2.4. Synthesis and characterization of OEGBr initiator Oligomeric methoxy polyethylene glycol 2-bromoisobutyrate (OEGBr) was prepared by the following procedure reported by Jin et al. [42]. To a solution of methoxyoligo(ethylene glycol) (10.0 g, 28.6 mmol) and triethylamine (6.0 mL, 42.8 mmol) in 50.0 mL of dried CH2 Cl2 cooled in an ice-water bath was added dropwise 2bromoisobutyryl bromide (10.1 g, 42.8 mmol) diluted in 50.0 mL of dried CH2 Cl2 . A white precipitate, triethylamine hydrogenbromide salt, was formed immediately. The mixture was magnetically stirred for 2 h at 0 ◦ C and then carried over night at room
Fig. 1. Schematic figure of the grafting process.
temperature for a complete conversion. The triethylamine hydrogenbromide salt was filtered off and washed with 10.0 mL of CH2 Cl2 three times. The yellow solution of the crude product was washed with 100.0 mL of 1.0 M NaHCO3 solution three times, followed with saturated brine and deionized water three times, respectively. The solution was dried over MgSO4 . The CH2 Cl2 was evaporated under reduced pressure, and the OEGBr was obtained. Yield: 72.0% (20.5 g) with respect to methoxyoligo(ethylene glycol). 1 H NMR (CDCl3 , 400 MHz): ı = 4.27 (t, 2H), 3.75 (t, 2H), 3.62 (m, 24H), 3.56 (t, 2H), 3.37 (s, 3H), 1.90 (s, 6H). 2.5. Grafting of poly-MPC on TiO2 film surface Poly-MPC was grafted on TiO2 film surface according to the procedures described below. The principle is presented in Fig. 1. TiO2 film was cut into 1 cm × 1 cm samples and ultrasonically cleaned with acetone, ethanol and hot distilled water. The samples were dried under vacuum. TiO2 films were dipped in a dried THF solution of HUPA (10.0 mM) for 6 h at ambient temperature and pressure; films were then removed from solution and warmed in an oven under vacuum at 120 ◦ C for 20 h. Samples were then cooled, dipped in a dried THF solution of HUPA again, operated repeatedly for five times. After the reaction, the films were rinsed with THF, acetone, ethanol under ultrasound and dried under vacuum. TiOHUPA was obtained. The TiO-HUPA films were placed in three-necked flat bottom flask cooled in an ice-water bath, and 40.0 mL of anhydrous THF and triethylamine (4.8 mL, 20.0 mmol) was added, then 2bromoisobutyryl bromide (4.7 g, 20.0 mmol) was added after 1 h. The reaction was carried over night. After the reaction, triethylamine hydrogenbromide salt and solvent were removed. TiO-BUP was obtained and rinsed in acetone, ethanol, deionized water under ultrasound and dried under vacuum. Poly-MPC was grafted on TiO-BUP by surface-initiated ATRP [43,44]. In a typical ATRP grafting polymerization reaction, Cu(I)Br (11.5 mg, 0.08 mmol), bpy (25.0 mg, 0.16 mmol), and MPC (2.4 g, 8 mmol) were added to a 25.0 mL two-neck pear-shape flask containing a magnetic stir bar. The flask was evacuated and purged with argon (four cycles). Methanol (15.0 mL bubbled for 1 h with argon before use) was then added to the flask via syringe. The resulting dark brown liquid was stirred under argon for 10 min and then cooled to −20 ◦ C. OEGBr initiator (20.05 mg, 0.04 mmol) in deaerated methanol was added to the mixture via syringe. The reaction mixture was homogenized by agitation for 2 min and then transferred to the three-necked flat bottom flask containing initiator-treated TiO-BUP films. Polymerization was carried at
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Fig. 4. DR-FTIR spectra of samples. Fig. 2. XRD patterns of the deposited TiO2 film (A, anatase; R, rutile).
room temperature for 48 h to allow complete monomer conversion. The polymerization was terminated by exposing to air, causing the dark brown reaction solution to turn blue. The TiO-MPC films were removed from the solution, cleaned ultrasonically with methanol and deionized water to remove any unattached poly-MPC, rinsed again with methanol under ultrasound and dried under vacuum. 2.6. Characterization Samples for XPS, FTIR analyses were prepared in size of 1 cm × 1 cm and 0.8 cm × 0.8 cm for contact angle analysis. The crystalline phases of the film were identified by X-ray diffraction ˚ at 45 kV/35 mA instrument using Cu K␣ radiation ( = 1.5406 A) on Philips X’Pert diffractometer in 2 range of 20–70◦ , in steps of 0.03◦ and with a counting time of 0.5 s per step. XPS analysis was performed on a KRATOS XSAM800 X-ray photoelectron spectrometer using a Mg K␣ (h = 1253.6 eV) line excitation source. The pressure in the chamber was below 2 × 10−9 Torr before the data were taken. The binding energy scale was referenced by setting Cls peak at 284.6 eV. The FTIR spectra were obtained using NICOLET 5700 infrared spectroscopy instrument with the diffuse reflectance mode for modified films. The contact angle was measured using a DSA100 Drop Shape Analyzer. At least 6 contact angles on different areas were measured and averaged. The 1 H NMR spectra were recorded in deuteriochloroform (CDCl3 ) with tetramethylsilane (TMS) as the internal standard at
ambient temperature on an INOVA-400 (Varian America) spectrometer. ESI-MS was recorded on Bruker Daltonics Bio TOF mass spectrometer. 2.7. Platelet adhesion Fresh human whole blood was centrifuged at 1500 rpm for 15 min to separate the blood corpuscles, and the resulting platelet rich plasma (PRP) was used for the platelet adhesion experiment. After incubation in PRP for 1 h, samples were rinsed with phosphate buffered solution (PBS) and reserved for analysis via SEM. These samples were treated by 2% glutaraldehyde for 2 h, and were dehydrated and dealcoholed followed with drying by critical point dryer (BAL-TEC CPD 030). The platelets attached surface was gold deposited and examined by SEM (Philips Quanta 200). 3. Results and discussion 3.1. Poly-MPC chemically grated on TiO2 films surface It has been reported that the crystalline TiO2 films possess better biocompatibility than amorphous TiO2 films, and the anatase and rutile are the main types of TiO2 for biomedical applications [36,45]. Fig. 2 shows the XRD patterns of the prepared TiO2 film. The diffraction peaks corresponded to anatase as referred to JCPDS file 21-1272 and 21-1276.
Fig. 3. SEM images of the films: (a) unmodified TiO2 film, (b) TiO-MPC.
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Fig. 5. XPS survey of the surface of samples.
The TiO2 film was grafted with 11-hydroxyundecylphosphonic acid assembling in which the phosphonic acid groups anchored on the TiO2 surface [46,47]. The presence of hydroxyl groups on the surface plays an important role in covalently grafting poly-MPC onto the TiO2 -HUPA film surface by surface-initiated atom-transfer radical polymerization through chemical methods. Fig. 3 shows SEM of unmodified TiO2 film and MPC grafted TiO2 film. The surface of TiO2 film was smooth before modification (Fig. 3a). The surface of TiO-MPC film had white substances due to grafting poly-MPC attached (Fig. 3b). But poly-MPC did not cover the surface uniformly and completely, indicating that the reaction of HUPA with TiO2 film was inhomogenous. FTIR, XPS and contact angle measurements were carried out to check if poly-MPC had been grafted on the TiO2 film surface. Fig. 4 shows FTIR spectra of films before and after HUPA assembling. It can be clearly seen that in the TiO2 films spectrum after HUPA assembling, there were new absorptions between 3000 cm−1 and 2500 cm−1 compared with those of TiO2 . In the spectrum of TiO2 -MPC, it can be seen that the peak at 2921.9 cm−1 and 2851.3 cm−1 were characteristic adsorption peaks of –CH2 –, the peak at 1735.5 cm−1 was ascribed to C O, the peaks at 1458.4 cm−1 was ascribed to P O, the peak at 1289.8 cm−1 was ascribed to P–O–CH2 , the peaks at 1054.2 cm−1 was ascribed to P–O, the peak at 989.2 cm−1 was ascribed to C–N. This fact indicates that ploy-MPC was grafted successfully on the surfaces of TiO2 films by chemical methods. Fig. 5a shows the XPS survey result for TiO-HUPA and TiO-MPC. It shows that phosphorus was introduced in HUPA self-assembling and MPC grafting processes, simultaneously nitrogen was introduced in MPC grafting process as new elements on the surface of TiO2 film. In the XPS full spectrum of TiO-MPC surface, the peak of bromine element was too weak to determine (it is not shown in the figure).
Fig. 5b shows the element high-resolution results of XPS survey of the TiO-MPC surface. The peak of 133.7 eV represents the binding energy of P2p . The peaks of 399.2 and 402.3 eV represent the binding energy of N1s , the peak of 402.3 eV was introduced by the –N+ (CH3 )3 group of poly-MPC, and the peak of 399.2 eV was introduced by N2 adsorbed on the TiO-MPC surface from the air. The peaks of 529.9, 531.7 and 532.9 eV represent the binding energy of O1s , which were introduced by TiO2 film and chemical substances grafted on the surface. XPS analysis indicates that the poly-MPC was grafted on the TiO2 film surface. The water contact angle of TiO2 film was 55.4◦ , while the contact angle of TiO2 -HUPA film increased to 75.5◦ , which was due to contributed mainly by the hydrophobicity of the long aliphatic carbochain of HUPA, though HUPA contains the hydrophilic hydroxyl end group. And the contact angle decreased dramatically to 61.3◦ after poly-MPC was grafted on the surface. It was because that polyMPC contains a large number of hydrophilic phosphorylcholine groups, and increased the hydrophilicity of the surface.
3.2. Platelet adhesion SEM of adhered platelets on the sample surface after 1 h incubation is shown in Fig. 6. Although the platelet rich plasma was equal for all samples, the distribution of the platelets on the different samples showed obvious differences. On the poly-MPC grafted TiO2 film surface, adhered platelets were significantly decreased and there is no obvious aggregation and pseudopodium compared with that of pure TiO2 film, or different, frequently used materials for bloodcontacting devices, such as stainless steel and pyrolytic carbon surface, where platelets showed significant adhesion and pseudopod formation. The adhered platelets showed that poly-MPC grafted TiO2 film surface was effective to inhibit platelet adhesion.
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Fig. 6. SEM of adhered platelets on the sample surface after 1 h: (a) TiO-MPC, (b) unmodified TiO2 film, (c) stainless steel, (d) pyrolytic carbon.
4. Conclusion Crystalline TiO2 film was fabricated by unbalanced magnetron sputtering, further engineered with HUPA self-assembling. 2Bromo-2-methylpropionyl group was introduced onto the TiO2 film surface by esterification and was used as linking point for polyMPC. Poly-MPC was chemically grafted on the TiO2 film surface by surface-initiated atom-transfer radical polymerization. XPS and FTIR confirmed the occurrence of the modification. The wettability of the surface changed with the surface chemistry. Although ploy-MPC did not cover completely on the surface of TiO2 film, in vitro evaluation proved that the poly-MPC modified TiO2 film decreased dramatically platelet adhesion and showed better blood compatibility compared to the TiO2 film, stainless steel or pyrolytic carbon. Acknowledgements This work was financially supported by Key Basic Research Project No. 2005CB623904 and National High Tech Program in China No. 2006AA02A139, Natural Science Foundation of China Nos. 50971107 and 30831160509, the Fundamental Research Funds for the Central Universities No. SWJTU09CX050. References [1] K. Ishihara, T. Ueda, N. Nakabayashi, Polym. J. 22 (1990) 355. [2] K. Ishihara, H. Nomura, T. Mihara, K. Kurita, Y. Iwasaki, N. Nakabayashi, J. Biomed. Mater. Res. 39 (1998) 323. [3] Y. Iwasaki, K. Ishihara, Anal. Bioanal. Chem. 381 (2005) 534. [4] K. Ishihara, H. Oshida, Y. Endo, T. Ueda, A. Watanabe, N. Nakabayashi, J. Biomed. Mater. Res. 26 (1992) 1543. [5] K. Ishihara, H. Oshida, Y. Endo, A. Watanabe, T. Ueda, N. Nakabayashi, J. Biomed. Mater. Res. 27 (1993) 1309. [6] T. Ueda, A. Watanabe, K. Ishihara, N. Nakabayashi, J. Biomater. Sci. Polym. Ed. 3 (1991) 185.
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Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Crystalline TiO2 grafted with poly(2-methacryloyloxyethyl phosphorylcholine) via surface-initiated atom-transfer radical polymerization Yuancong Zhao, Qiufen Tu, Jin Wang, Qiongjian Huang, Nan Huang ∗ Key Lab. of Advanced Technology for Materials, Education Ministry, School of Material Science and Technology of Southwest Jiaotong University, Chengdu, Sichuan, China
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 25 August 2010 Accepted 25 August 2010 Available online 24 September 2010 Keywords: Poly(2-methacryloyloxyethyl phosphorylcholine) TiO2 film Chemical modification Platelet adhesion
a b s t r a c t Crystalline TiO2 films were prepared by unbalanced magnetron sputtering and the structure was confirmed by XRD. An organic layer of 11-hydroxyundecylphosphonic acid (HUPA) was prepared on the TiO2 films by self-assembling, and the HUPA on TiO2 films was confirmed by FTIR analysis. Simultaneously, hydroxyl groups were introduced in the phosphonic acid molecules to provide a functionality for further chemical modification. 2-Methacryloyloxyethyl phosphorylcholine (MPC), a biomimetic monomer, was chemically grafted on the HUPA surfaces at room temperature by surface-initiated atom-transfer radical polymerization. The surface characters of TiO2 films modified by poly-MPC were confirmed by FTIR, XPS and SEM analysis. Platelet adhesion experiment revealed that poly-MPC modified surface was effective to inhibit platelet adhesion in vitro. © 2010 Elsevier B.V. All rights reserved.
1. Introduction 2-Methacryloyloxyethyl phosphorylcholine (MPC) polymers, which contain a zwitterionic phospholipid group that is also present in cell membranes and possesses nonthrombogenic properties and high biocompatibility, have been widely used to construct non-biofouling surfaces in various biomedical applications as they have been shown to resist both protein adsorption and cell adhesion [1–3]. Phosphorylcholine (PC)-containing biomaterials are effective in reducing the adsorption of various proteins, cells, blood platelets, and bacteria [4–14]. The biomimetic strategy that uses PC-containing polymers to improve biocompatibility of biomaterials has received much attention over the past two decades [15–24]. Antithrombogenic biomaterials are one of the ultimate objectives for developing blood-contacting biomedical devices. Anticoagulant activity is very important to an artificial surface contacting with blood since it enhances the life time of medical devices and the patients health. Developments focus on minimizing the tendency of a material surface to activate blood platelets and to induce blood clotting, and reduce the risk of thrombosis. Many studies have aimed to improve hemocompatibility of biomaterials either by deposition of thin hemocompatible films [25,26] or by
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (N. Huang). 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.08.100
grafting biologically active molecules [27,28]. Recently, titanium oxide thin films were paid more attention because of their biocompatibility and bioinertness [29]. All kinds of crystalline TiO2 films, which are prepared by physical vapour deposition (PVD), plasma immersion ion implantation (PIII), vacuum arc deposition technology, plasma oxidation, and so on, possess favorable biocompatibility [30–35]. The composition, crystalline form and thickness of TiO2 films are factors to influence the biocompatibility [30–32]. The favorable TiO2 films can inhibit platelet adhesion and fibrinogen adsorption and activation effectively [33–35]. The evaluation in vivo shows that the TiO2 films posses higher ability to restraint thrombus formation [35]. A self-assembly of alkyl phosphonic acid on TiO2 has been reported to have physiological stability and suitable surface density [36–40]. To further enhance the anticoagulation ability of titanium oxide film, in this paper, 11-hydroxyundecylphosphonic acid (HUPA) was used to anchor on TiO2 film surface and to act as a linking layer. The hydroxyl group of HUPA was converted with 2-bromo-2-methylpropionyl bromide for chemical grafting of polymerized 2-methacryloyloxyethyl phosphorylcholine (MPC) on the surface by surface-initiated atom-transfer radical polymerization (ATRP) using oligomeric methoxy polyethylene glycol 2-bromoisobutyrate (OEGBr) as initiator and Cu(I)Br/2,2bipyridine (bpy) as catalyst at room temperature. Characterization of the grafting of poly-MPC by chemical methods on the metallic biomaterials for improvement of blood compatibility is reported. Platelet incubation studies indicate that the surface of TiO2 film modified by poly-MPC can effectively inhibit platelet adhesion in vitro.
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2. Experimental 2.1. Materials 2-Methacryloyloxyethyl phosphorylcholine (MPC, 96%) was supplied by the Joy-Nature Technology Institute (China) and used without further purification. Cu(I)Br (99.999%), 2,2-bipyridine (bpy, 99%), methoxy poly(ethylene glycol) (Mn ca. 200 g/mol), 11-bromoundecanol, triethyl phosphate (98%), 2-bromo-2methylpropionyl bromide (98%), were supplied by Alfa Aesar and used without further purification. Tetrahydrofuran (THF) was dried over metal Na, and distilled just prior usage. Acetonitrile and triethylamine (TEA) were distilled over calcium hydride just prior usage. 2.2. Film preparation TiO2 films were prepared on silicon (1 0 0) wafers using an unbalanced magnetron sputtering system. A Ti target (purity 99.9%) was mounted on the target source. The target-to-substrate distance was 10 cm. The ratio of O2 /Ar was sustained at 13/60 by a flow monitor system. The discharge current was kept constant at 3 A. The deposition time was 15 min. The film thickness is 300–450 nm measured by surface profilometer (AMBIOS Technology XP-2 Stylus Profiler). 2.3. Synthesis and characterization of HUPA 11-Hydroxyundecylphosphonic acid was prepared by the following procedure reported by Putvinski et al. [41]. Acetyl chloride (1.9 g, 0.024 mol) in 10.0 mL of dried H2 Cl2 was added dropwise to a mixture of 11-bromoundecanol (5.0 g, 0.02 mol) and triethylamine (2.2 g, 0.022 mol) in 100.0 mL of dried CH2 Cl2 at 0 ◦ C. The reaction mixture was stirred an additional 4 h in an ice bath. The solution was washed with saturated brine and H2 O until the aqueous layer was at pH 6. The organic layer was isolated, dried over anhydrous sodium sulfate, and to give white 11-bromoundecyl acetate, which was used in the following reaction directly without further purification. The crude product of the bromo acetate and triethyl phosphate (6.4 g, 0.025 mol) were heated at 150 ◦ C for 24 h. After the excess triethyl phosphite was removed under reduced pressure, concentrated HCl (8.0 mL) was added to a solution of diethyl (11-acetoxyundecyl)phosphonate in dioxane (20.0 mL), and the mixture was heated at 100 ◦ C for 3 days under argon. After cooling, the solvent was evaporated under reduced pressure. Ice water was added and the solution brought to pH 2 with aqueous HCl. The resulting precipitate was collected by filtration and recrystallized from ethyl acetate and petroleum ether (60–90) to give white (11-hydroxyundecyl)phosphonic acid (3.2 g, yield: 63.5% with respect to 11-bromoundecanol). 1 H NMR (CDC13 ): ı = 1.15–1.52 (m, 18H, CH2 ), 1.78 (m, 2H, CH2 P), 3.51 (m, 2H, CH2 O), 4.67 (s, 3H, OH); 31 P NMR: ı = 24.15. HR-MS (Calcd. for C11 H25 O4 P: 252.1490): 251.1414 (C− ). 2.4. Synthesis and characterization of OEGBr initiator Oligomeric methoxy polyethylene glycol 2-bromoisobutyrate (OEGBr) was prepared by the following procedure reported by Jin et al. [42]. To a solution of methoxyoligo(ethylene glycol) (10.0 g, 28.6 mmol) and triethylamine (6.0 mL, 42.8 mmol) in 50.0 mL of dried CH2 Cl2 cooled in an ice-water bath was added dropwise 2bromoisobutyryl bromide (10.1 g, 42.8 mmol) diluted in 50.0 mL of dried CH2 Cl2 . A white precipitate, triethylamine hydrogenbromide salt, was formed immediately. The mixture was magnetically stirred for 2 h at 0 ◦ C and then carried over night at room
Fig. 1. Schematic figure of the grafting process.
temperature for a complete conversion. The triethylamine hydrogenbromide salt was filtered off and washed with 10.0 mL of CH2 Cl2 three times. The yellow solution of the crude product was washed with 100.0 mL of 1.0 M NaHCO3 solution three times, followed with saturated brine and deionized water three times, respectively. The solution was dried over MgSO4 . The CH2 Cl2 was evaporated under reduced pressure, and the OEGBr was obtained. Yield: 72.0% (20.5 g) with respect to methoxyoligo(ethylene glycol). 1 H NMR (CDCl3 , 400 MHz): ı = 4.27 (t, 2H), 3.75 (t, 2H), 3.62 (m, 24H), 3.56 (t, 2H), 3.37 (s, 3H), 1.90 (s, 6H). 2.5. Grafting of poly-MPC on TiO2 film surface Poly-MPC was grafted on TiO2 film surface according to the procedures described below. The principle is presented in Fig. 1. TiO2 film was cut into 1 cm × 1 cm samples and ultrasonically cleaned with acetone, ethanol and hot distilled water. The samples were dried under vacuum. TiO2 films were dipped in a dried THF solution of HUPA (10.0 mM) for 6 h at ambient temperature and pressure; films were then removed from solution and warmed in an oven under vacuum at 120 ◦ C for 20 h. Samples were then cooled, dipped in a dried THF solution of HUPA again, operated repeatedly for five times. After the reaction, the films were rinsed with THF, acetone, ethanol under ultrasound and dried under vacuum. TiOHUPA was obtained. The TiO-HUPA films were placed in three-necked flat bottom flask cooled in an ice-water bath, and 40.0 mL of anhydrous THF and triethylamine (4.8 mL, 20.0 mmol) was added, then 2bromoisobutyryl bromide (4.7 g, 20.0 mmol) was added after 1 h. The reaction was carried over night. After the reaction, triethylamine hydrogenbromide salt and solvent were removed. TiO-BUP was obtained and rinsed in acetone, ethanol, deionized water under ultrasound and dried under vacuum. Poly-MPC was grafted on TiO-BUP by surface-initiated ATRP [43,44]. In a typical ATRP grafting polymerization reaction, Cu(I)Br (11.5 mg, 0.08 mmol), bpy (25.0 mg, 0.16 mmol), and MPC (2.4 g, 8 mmol) were added to a 25.0 mL two-neck pear-shape flask containing a magnetic stir bar. The flask was evacuated and purged with argon (four cycles). Methanol (15.0 mL bubbled for 1 h with argon before use) was then added to the flask via syringe. The resulting dark brown liquid was stirred under argon for 10 min and then cooled to −20 ◦ C. OEGBr initiator (20.05 mg, 0.04 mmol) in deaerated methanol was added to the mixture via syringe. The reaction mixture was homogenized by agitation for 2 min and then transferred to the three-necked flat bottom flask containing initiator-treated TiO-BUP films. Polymerization was carried at
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Fig. 4. DR-FTIR spectra of samples. Fig. 2. XRD patterns of the deposited TiO2 film (A, anatase; R, rutile).
room temperature for 48 h to allow complete monomer conversion. The polymerization was terminated by exposing to air, causing the dark brown reaction solution to turn blue. The TiO-MPC films were removed from the solution, cleaned ultrasonically with methanol and deionized water to remove any unattached poly-MPC, rinsed again with methanol under ultrasound and dried under vacuum. 2.6. Characterization Samples for XPS, FTIR analyses were prepared in size of 1 cm × 1 cm and 0.8 cm × 0.8 cm for contact angle analysis. The crystalline phases of the film were identified by X-ray diffraction ˚ at 45 kV/35 mA instrument using Cu K␣ radiation ( = 1.5406 A) on Philips X’Pert diffractometer in 2 range of 20–70◦ , in steps of 0.03◦ and with a counting time of 0.5 s per step. XPS analysis was performed on a KRATOS XSAM800 X-ray photoelectron spectrometer using a Mg K␣ (h = 1253.6 eV) line excitation source. The pressure in the chamber was below 2 × 10−9 Torr before the data were taken. The binding energy scale was referenced by setting Cls peak at 284.6 eV. The FTIR spectra were obtained using NICOLET 5700 infrared spectroscopy instrument with the diffuse reflectance mode for modified films. The contact angle was measured using a DSA100 Drop Shape Analyzer. At least 6 contact angles on different areas were measured and averaged. The 1 H NMR spectra were recorded in deuteriochloroform (CDCl3 ) with tetramethylsilane (TMS) as the internal standard at
ambient temperature on an INOVA-400 (Varian America) spectrometer. ESI-MS was recorded on Bruker Daltonics Bio TOF mass spectrometer. 2.7. Platelet adhesion Fresh human whole blood was centrifuged at 1500 rpm for 15 min to separate the blood corpuscles, and the resulting platelet rich plasma (PRP) was used for the platelet adhesion experiment. After incubation in PRP for 1 h, samples were rinsed with phosphate buffered solution (PBS) and reserved for analysis via SEM. These samples were treated by 2% glutaraldehyde for 2 h, and were dehydrated and dealcoholed followed with drying by critical point dryer (BAL-TEC CPD 030). The platelets attached surface was gold deposited and examined by SEM (Philips Quanta 200). 3. Results and discussion 3.1. Poly-MPC chemically grated on TiO2 films surface It has been reported that the crystalline TiO2 films possess better biocompatibility than amorphous TiO2 films, and the anatase and rutile are the main types of TiO2 for biomedical applications [36,45]. Fig. 2 shows the XRD patterns of the prepared TiO2 film. The diffraction peaks corresponded to anatase as referred to JCPDS file 21-1272 and 21-1276.
Fig. 3. SEM images of the films: (a) unmodified TiO2 film, (b) TiO-MPC.
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Fig. 5. XPS survey of the surface of samples.
The TiO2 film was grafted with 11-hydroxyundecylphosphonic acid assembling in which the phosphonic acid groups anchored on the TiO2 surface [46,47]. The presence of hydroxyl groups on the surface plays an important role in covalently grafting poly-MPC onto the TiO2 -HUPA film surface by surface-initiated atom-transfer radical polymerization through chemical methods. Fig. 3 shows SEM of unmodified TiO2 film and MPC grafted TiO2 film. The surface of TiO2 film was smooth before modification (Fig. 3a). The surface of TiO-MPC film had white substances due to grafting poly-MPC attached (Fig. 3b). But poly-MPC did not cover the surface uniformly and completely, indicating that the reaction of HUPA with TiO2 film was inhomogenous. FTIR, XPS and contact angle measurements were carried out to check if poly-MPC had been grafted on the TiO2 film surface. Fig. 4 shows FTIR spectra of films before and after HUPA assembling. It can be clearly seen that in the TiO2 films spectrum after HUPA assembling, there were new absorptions between 3000 cm−1 and 2500 cm−1 compared with those of TiO2 . In the spectrum of TiO2 -MPC, it can be seen that the peak at 2921.9 cm−1 and 2851.3 cm−1 were characteristic adsorption peaks of –CH2 –, the peak at 1735.5 cm−1 was ascribed to C O, the peaks at 1458.4 cm−1 was ascribed to P O, the peak at 1289.8 cm−1 was ascribed to P–O–CH2 , the peaks at 1054.2 cm−1 was ascribed to P–O, the peak at 989.2 cm−1 was ascribed to C–N. This fact indicates that ploy-MPC was grafted successfully on the surfaces of TiO2 films by chemical methods. Fig. 5a shows the XPS survey result for TiO-HUPA and TiO-MPC. It shows that phosphorus was introduced in HUPA self-assembling and MPC grafting processes, simultaneously nitrogen was introduced in MPC grafting process as new elements on the surface of TiO2 film. In the XPS full spectrum of TiO-MPC surface, the peak of bromine element was too weak to determine (it is not shown in the figure).
Fig. 5b shows the element high-resolution results of XPS survey of the TiO-MPC surface. The peak of 133.7 eV represents the binding energy of P2p . The peaks of 399.2 and 402.3 eV represent the binding energy of N1s , the peak of 402.3 eV was introduced by the –N+ (CH3 )3 group of poly-MPC, and the peak of 399.2 eV was introduced by N2 adsorbed on the TiO-MPC surface from the air. The peaks of 529.9, 531.7 and 532.9 eV represent the binding energy of O1s , which were introduced by TiO2 film and chemical substances grafted on the surface. XPS analysis indicates that the poly-MPC was grafted on the TiO2 film surface. The water contact angle of TiO2 film was 55.4◦ , while the contact angle of TiO2 -HUPA film increased to 75.5◦ , which was due to contributed mainly by the hydrophobicity of the long aliphatic carbochain of HUPA, though HUPA contains the hydrophilic hydroxyl end group. And the contact angle decreased dramatically to 61.3◦ after poly-MPC was grafted on the surface. It was because that polyMPC contains a large number of hydrophilic phosphorylcholine groups, and increased the hydrophilicity of the surface.
3.2. Platelet adhesion SEM of adhered platelets on the sample surface after 1 h incubation is shown in Fig. 6. Although the platelet rich plasma was equal for all samples, the distribution of the platelets on the different samples showed obvious differences. On the poly-MPC grafted TiO2 film surface, adhered platelets were significantly decreased and there is no obvious aggregation and pseudopodium compared with that of pure TiO2 film, or different, frequently used materials for bloodcontacting devices, such as stainless steel and pyrolytic carbon surface, where platelets showed significant adhesion and pseudopod formation. The adhered platelets showed that poly-MPC grafted TiO2 film surface was effective to inhibit platelet adhesion.
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Fig. 6. SEM of adhered platelets on the sample surface after 1 h: (a) TiO-MPC, (b) unmodified TiO2 film, (c) stainless steel, (d) pyrolytic carbon.
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