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Heparin-grafted poly(tetrafluoroethylene-co-hexafluoropropylene) film with highly effective blood compatibility via an esterification reaction
Surface & Coatings Technology 228 (2013) S126–S130
Contents lists available at SciVerse ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Heparin-grafted poly(tetrafluoroethylene-co-hexafluoropropylene) film with highly effective blood compatibility via an esterification reaction Qiang Gao, Yashao Chen ⁎, Yanlin Wei, Xudong Wang, Yanling Luo Key Laboratory of Applied Surface and Colloid Chemistry (Shaanxi Normal University), Ministry of Education, School of Chemistry & Chemical Engineering, Xi'an 710062, China
a r t i c l e
i n f o
Available online 10 July 2012 Keywords: Plasma treatment UV-induced graft polymerization Heparin Esterification reaction Blood compatibility
a b s t r a c t The aim of this work was to modify poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surface in order to improve its hydrophilicity and blood compatibility. Acrylic acid (AAc) was introduced onto Ar plasma-pretreated FEP surface by UV-induced grafted copolymerization. Subsequently, heparin (Hp) was also immobilized covalently with the carboxylic group of PAAc grafted onto the FEP surface via an esterification reaction. The chemical composition and the morphological change of the modified films were characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electronic microscopy (SEM), atomic force microscopy (AFM) and water contact angel measurements. The root–mean–square average surface roughness (Ra) changed from 5.82 nm (FEP) to 13.60 nm (FEP-PAAc) and 11.74 nm (FEP-PAAc-Hp). Water contact angles changed from 115.0o (FEP) to 32.4 o (FEP-PAAc) and 44.8 o (FEP-PAAc-Hp). The blood compatibility of the modified films was evaluated by platelet adhesion experiments. All results suggested that the FEP-PAAc-Hp surface presented excellent antithrombotic properties and good hydrophilicity. Therefore, this method will provide a potential and effective solution for the surface modification of medical polymer materials. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It is known that biocompatibility plays an important role in implanted biomaterials. A truly biocompatible biomaterial should perform its function without causing undue host response or resulting adverse clinical reaction [1]. In general, biocompatibility of implant material mainly depends on its surface properties, for which reason it is necessary to modify the surface to improve the biocompatibility of biomaterials. Surface modification including physical, chemical modification and bio-molecule immobilization is considered as a promising approach to incorporate polymer materials onto a wide range of hydrophobic substrates for improved biocompatibility [2–5]. In our previous work, surface grafting modification is an important way to enhance the biocompatibility without changing the bulk structure of the polymer [6–8]. As we known, Hp is a critically clinical anticoagulant for minimizing thrombus formation on artificial surfaces since it is a kind of sulfated polysaccharide that contains sulfonic, sulfoamino, and carboxyl groups. Hence, to immobilize Hp molecules onto the material surface is the most popular technique for minimizing the thrombogenicity of materials. In this regard, the heparinized surfaces have anticoagulant properties that prolong blood clotting time when they are coated on blood devices or containers [9]. Recently, surface
⁎ Corresponding author. Tel.: +86 29 81530795; fax: +86 29 81530727. E-mail address: [email protected] (Y. Chen). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2012.07.015
heparinization is regarded as an effective way to improve the blood compatibility of hydrophobic polymeric film [10,11]. FEP has been widely used as biomaterial due to its excellent chemical resistance, non-toxicity, and chemical stability [12]. However, the blood compatibility of FEP film as blood-contacting material is not applicable due to its inert and hydrophobic surface. In general, in order to improve the blood compatibility and ensure the long-term effectiveness of the biomedical devices, surface modification has often been performed before used in biomedical fields. In the present work, surface modification of Ar plasma-pretreated FEP film was carried out by UV-induced graft copolymerization with water-soluble monomer of AAc to create carboxyl functional groups on FEP surfaces, followed by esterification reactions, and reactions with Hp to create anticoagulation surfaces. In-vitro blood compatibility of the film was evaluated by platelet adhesion studies. The process for functionalization of FEP film with Hp consisted of several steps and was illustrated in Fig. 1. 2. Experimental section 2.1. Materials FEP films having a thickness of about 0.1 mm and a density of 2.15 g/cm3 were purchased from Goodfellow Ltd., of Cambridge, UK, followed by washing with acetone in an ultrasonic washer, and dried at room temperature under vacuum conditions before use. Acrylic acid (AAc), heparin sodium (Hp), and methylene chloride (MC) were
Q. Gao et al. / Surface & Coatings Technology 228 (2013) S126–S130
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Fig. 1. Schematic diagram of surface reactions on FEP film: step A, pretreated by Ar plasma and immersed in AAc solution; step B, UV-induced graft copolymerization; step C, Hp reaction.
purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. 4-dimethylaminopyridine (DMAP) and N,N-dicyclohexyl carbodiimide (DCC) were purchased from Sigma-Aldrich, America. Argon (Ar, purity: 99.99%) was employed as process gas for plasma treatment of FEP.
placed into a 100 mL beaker with 25 mL of MC, then stirred and cooled in an ice-water bath. DCC (1.2 mmol) was added, and the mixture was continuously stirred for 5 h. Upon removal, all samples were washed respectively in MC and distilled water sequentially for 3 h, dried at 35 °C under vacuum conditions for 24 h, and then analyzed.
2.2. Graft polymerization of AAc onto FEP surface: FEP-PAAc 2.4. Characterization FEP films were pretreated by Ar plasma at a voltage of 6.0 kV for 5 min in a PECVD-500 plasma apparatus (Beijing Technol Science Co., LTD, Beijing, China) at a gas flow of 60 sccm and a pressure of 50 Pa. Subsequently, the films were exposed to air for 20 min, leading to surface oxidation and formation of peroxide and hydroperoxide species [13]. The air-exposed FEP films were immersed in an aqueous solution containing 6% (v/v) AAc for 4 h (Fig. 1, step a). In brief, an UV illumination system equipped with a high-pressure mercury lamps (1000 W, λ = 365 nm) was employed, and then exposed to UV irradiation for 1 h under nitrogen atmosphere (Fig. 1, step b) [14,15]. Finally, the films were washed with distilled water and stirred for 12 h at 50 °C to remove homo-polymer and residual monomer, then maintained under vacuum for at least 24 h to remove the water. 2.3. Immobilization of Hp onto FEP surface: FEP-PAAc-Hp To modify FEP-PAAc surfaces with Hp, we utilized an esterification reaction using DMAP catalyst and DCC coupling agent (Fig. 1, step c) [16–18]. FEP-PAAc films, Hp (5 mg), and DMAP (0.2 mmol) were
Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were performed with an EQUI NOX55 FT-IR spectrometer with an ATR unit (Bruker Co., Germany). Each spectrum was obtained by cumulating 64 scans with a resolution of 4 cm −1. Surface composition was determined by X-ray photoelectron spectra (XPS) on an ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV) (ThermoElectron Co., America). Spectra were recorded at a 45° take-off angle with 20 eV pass energy. The changes of surface roughness and morphology were observed by scanning electron microscopy (SEM; Quanta 200 Philips-FEI Co., Netherlands) and atomic force microscopy (AFM; WET-SPM9500 J3, SHIMADZU Co., Japan) in the tapping mode. Static contact angle was measured by the sessile drop method with a video-based contact angle measuring device (OCA20, Dataphysisc Co., Germany). Each contact angle value reported here was an average of at least five measurements. 2.5. Platelets adhesion test A platelet adhesion test was used to evaluate the adhesion behavior and activation of the platelets on the samples. Fresh human whole blood (20 mL) was taken from a healthy volunteer and anticoagulated with 0.109 M solution of sodium citrate at a dilution ratio of 9:1 (blood/sodium citrate solution). Then platelet-rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1200 rpm for 10–12 min at room temperature. After that, the samples (1.0 cm × 1.0 cm) were placed in individual wells of a 24-well tissue culture plate, and each well was dropped into with 50 μL PRP for 1 h at room temperature. After the samples were carefully rinsed twice with PBS (pH, 7.2) to remove non-firmly adsorbed platelets, they were immersed into 2.0 wt.% glutaraldehyde solution for 1 h, Table 1 XPS atomic composition data for FEP films.
Fig. 2. ATR-FTIR spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
Samples
C (at.%)
F (at.%)
O (at.%)
N (at.%)
S (at.%)
Pristine FEP FEP-PAAc FEP-PAAc-Hp
21.21 66.73 64.01
78.79 0.69 8.86
0 32.58 23.29
0 0 2.41
0 0 1.43
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Fig. 3. XPS spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
and washed with triple-distilled water several times. Then, they were immersed into 30, 50, 70, 90, 100% (v/v) ethanol/water for 20 min at each step and dried in air. Finally, the samples were sputter-coated with gold prior to observation with SEM [19,20]. 3. Results and discussion ATR-FTIR analysis, shown in Fig. 2, indicates the presence of specific functional groups on the modified surface. Fig. 2a shows strong bands at 1150–1250 cm −1 in the spectra of the pristine FEP are characteristic for the stretching vibration of CF2 and CF3 present in the structure of the fluorinated films, and a sharp band at around 980 cm −1 is due to the stretching vibration of CF in the CF3 group [21,22]. Fig. 2b shows a strong new peak at 1750 cm −1 is corresponding to C_O, and the broad band from 2750 to 3300 cm −1 is assigned to the \OH stretch characteristic of carboxyl groups, verifying successful grafting of AAc to FEP [23]. Fig. 2c shows a reduced \OH peak because of the esterification reaction between the \COOH groups on substrate and \OH groups of Hp. In addition, IR spectra of FEP-PAAc-Hp displays the new absorption bands near 1050 and 1650 cm −1, which are characteristic adsorption peaks of sulfonated group (\SO3 stretching) and amino group (N\H bending) of conjugated Hp. At the same time, there are three absorptions at 845 cm −1, 860 cm −1, and 950 cm −1, which can be attributed to the characteristic absorption peak in Hp [16,20,24,25]. These results indicated that Hp is successfully immobilized onto the FEP-PAAc surface.
The chemical composition of the FEP surfaces at various stages of surface modification was determined by XPS (see Table 1). In Table 1, F (at.%) of the pristine FEP with grafting of AAc decrease from 78.79 to 0.69, that's because of the thickness of the grafted PAAc polymer layer is more than the probing depth of the XPS technique [13]. However, after the Hp covalent bonding to the FEP-PAAc film surface, F (at.%) of the FEP-PAAc-Hp film increases from 0.69 to 8.86 at.%. That's because part of the PAAc chain is loose due to the effect of solvent, this process lead to the pristine FEP film surface exposure, therefore, F (at.%) of the FEP-PAAc-Hp film was increased. Fig. 3a shows a typical XPS survey spectra for the pristine FEP surface, the typical signals for carbon (C1s at 293 eV) and fluorine (F1s at 691 eV) are clearly detected. After treatment of the FEP surfaces with AAc, the typical signals attributed to carbon, fluorine, and additional signals assigned to O1s at 534 eV, which indicate that AAc has been grafted to the FEP surfaces (Fig. 3b). Moreover, the carbon and oxygen signals are noticeably stronger compared with the pristine FEP surface, due to the additional carbon and oxygen in the AAc. In addition, the fluorine signal is noticeably reduced for the formation of a cross-linked PAAc coating onto the film surface. Because the reactions of FEP-PAAc with Hp molecules are conducted using the esterification process (Fig. 3c), the characteristic signals attributed to carbon, fluorine, and oxygen are again detected. Moreover, the appearance of weak signals assigned to nitrogen (N1s at 401 eV) and sulfur (S2p at 199.1 eV) indicates that Hp is formed on the FEP-PAAc surface. Overall, the XPS results further confirm that the surface modification method utilized here is very effective. To get more information on the changes of the surface compositions, we collect high resolution XPS data for C1s and O1s. Fig. 4 shows the C1s and O1s core-level spectra of the pristine FEP, FEP-PAAc, and FEP-PAAc-Hp. As shown in Fig. 4a, the C1s core-level spectra of the pristine FEP film can be curve-fitted into three peak components with binding energies (BEs) at 289.7 eV, 293.2 eV, and 296.3 eV, attributed to the CF, the CF2, and the CF3 species, respectively [26]. It also reveals that there is no oxygen in the surface of the pristine FEP film. Fig. 4b shows the C1s and O1s core-level spectra of the FEP-PAAc. The C1s core-level spectra of the FEP-PAAc surface can be curve-fitted with three new peak components at 285.6 eV, 289.2 eV, and 292.5 eV, attributed to the C\C/C\H, C\O, O_C\O, respectively. The three fluorocarbon peak components in the C1s core-level spectra almost disappeared, which indicate that the thickness of the grafted PAAc polymer layer is more than the probing depth of the XPS technique [13]. The O1s core-level spectra consists of the C_O, and C\O peak components at 532.8 eV, and 534.2 eV, respectively. The results indicate that AAc is successfully grafted on the plasma-treated PEP film surface to form the functional groups (\COOH). After Hp immobilization onto the FEP-PAAc surface (see
Fig. 4. C1s and O1s core-level spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
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Fig. 5. SEM and contact angel images of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
Fig. 4c), the C1s core-level can be curve-fitted with four peak components at 285.5 eV, 286.0 eV, 287.1 eV, and 289.8 eV, attributed to the C\N, C\C/C\H, \C\O\, C_O/N\C_O, respectively. The C-N peak component is associated with the linkages in Hp itself. The O1s core-level spectra consists of the C_O, C\O\C, and C\O peak components at 532.5 eV, 533.2 eV, and 534.3 eV, respectively. Compared with Fig. 4b, the ratio of the C\OH has reduced significantly, that is expected due to the introduction of the ester. Furthermore, the additional peak C\O\C verified formation of an ester linkage between the \COOH of FEP-PAAc and the \OH of Hp molecules. The changes in surface morphology of the FEP surfaces, at various stages of surface modification, were studied by SEM and AFM. Representative SEM and contact angel images are shown in Fig. 5. As can be seen from Fig. 5a, the SEM images of the pristine FEP relatively
appeared featureless and very smooth. The pristine FEP is highly hydrophobic with a contact angle of 115.0°. In Fig. 5b, FEP-PAAc surface implies that the FEP surface was almost covered with a PAAc layer and the hydrophilicity is greatly enhanced with contact angle of 32.4°. This could be ascribed to the hydrophilic nature of grafted PAAc chains which are abundant in carboxyl groups [27]. In Fig. 5c, FEP-PAAc-Hp surface shows that there are some particles appeared and contact angle changed to 44.8° due to Hp molecules after the esterification reaction. Commonly, grafted PAAc layer causes roughening of the FEP surface, and the grafting of large molecules, that is, Hp, further changes the surface roughness. The morphological change of the surface can be observed by AFM (Fig. 6). Here different geometric features are demonstrated: the AFM images of the pristine FEP and the FEP-PAAc
Fig. 6. AFM images of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp; (d) is RMS roughness of the samples surface.
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Fig. 7. SEM images of platelet on (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
films are shown in Fig. 6a and b, respectively. The root-mean-square average surface roughness (Ra) of the pristine FEP film is in the order of about 5.82 nm (Fig. 6d). The Ra value increases to about 13.60 nm after the FEP surface has been graft-copolymerized with AAc. The increase in the Ra value is consistent with the presence of the surface grafted PAAc polymer on the FEP film. However, the roughness of the FEP-PAAc-Hp surface is reduced to 11.74 nm (Fig. 6c). Part of the Hp molecules are strong filled to the interspace of the FEP-PAAc film surface, which cause the roughness of the FEP-PAAc-Hp surface down, this indicates that the presence of Hp on the FEP-PAAc surface. It is well known that when a material contacts with blood, proteins instantaneously adsorb onto the surface and deform. Subsequently, platelets that adhere, are activated, and aggregated. The adhesion and aggregation of platelets play a significant role in thrombus formation [28]. Therefore, the extent of platelet adhesion and the morphology of the adhered platelets are considered to be early indicators of the thrombogenicity of biomaterials in contact with blood [29,30]. SEM images were used to evaluate the adhesion and morphology of platelets on various sample surfaces, the representative typical images of platelets adhesion behavior on these surfaces are shown in Fig. 7. As can be seen, platelets are adhered more and largely aggregated on the surface of pristine FEP (Fig. 7a). It is clear that the number of adhered platelets was significantly decreased for FEP-PAAc surface due to the hydrophilicity of PAAc layer (Fig. 7b). However, Fig. 7c shows that was no platelets adhesion on FEP-PAAc-Hp surface, thus, these heparinized surfaces can effectively improve the antithrombogenecity of the FEP films. That's because of the excellent hydrophilic and anticoagulant characteristics of the Hp molecules, also explained the good effect of modified FEP film surface by Hp binding. 4. Conclusions In this paper, a convenient approach of surface hydrophilization and heparinization for inert FEP film was developed. ATR-FTIR, XPS, AFM and SEM revealed that the FEP-PAAc film surface was successfully prepared by Ar plasma treatment and UV-induced graft polymerization with AAc, followed by the immobilization of Hp on the FEP-PAAc surface. The water contact angle indicated that the hydrophilicity improvement of the modified FEP surface by the grafting of PAAc and the bonding of Hp, respectively. Results of platelet adhesion experiments clearly indicated that the blood compatibility of the pristine FEP and FEP-PAAc surfaces were bad, the FEP-PAAc-Hp film surface exhibited excellent antithrombogenicity property. The results
presented in this study consistently demonstrated that covalent co-immobilization of Hp can simultaneously improve blood compatibility and hydrophilicity of medical polymer materials, which would be of potentially great significance for long-term implantation of biomedical devices in the human body. Acknowledgements This work has been supported by funds from the Fundamental Research Funds for the Central Universities (GK201004001) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1070). References [1] F. Williams, Biomaterials 29 (2008) 2941. [2] L. Li, C.Y. Tu, J.S. Huang, Y.L. Liu, K.R. Lee, J.Y. Lai, Surf. Coat. Technol. 201 (2006) 63. [3] Y. Iwasaki, K. Ishihara, Anal. Bioanal. Chem. 381 (2005) 534. [4] F.J. Xu, K.G. Neoh, E.T. Kang, Prog. Polym. Sci. 34 (2009) 719. [5] S.Y. Jiang, Z.Q. Cao, Adv. Mater. 22 (2010) 920. [6] G.W. Zhao, Y.S. Chen, X.L. Wang, Appl. Surf. Sci. 253 (2007) 4709. [7] L.H. Zhang, D. Wu, Y.S. Chen, Appl. Surf. Sci. 255 (2009) 6840. [8] Y.L. Wei, X.L. Wang, G.W. Zhao, J. Appl. Polym. Sci. 118 (2010) 943. [9] B.D. Ratner, A.D. Hoffman, F.D. Schoen, J.E. Lemons, Biomaterials Science, an Introduction to Materials in Medicine, 2nd ed. Elsevier, Amsterdam, 2004. [10] F.R. Gong, X.Y. Cheng, S.F. Wang, Y.C. Zhao, Acta Biomater. 6 (2010) 534. [11] G.C. Li, P. Yang, Y.Z. Liao, N. Huang, Biomacromolecules 12 (2011) 1155. [12] B. Coupe, M.E. Evangelista, R.M. Yeung, W. Chen, Langmuir 17 (2001) 1956. [13] K.L. Tan, L.L. Woon, H.K. Wong, Macromolecules 26 (1993) 2832. [14] H.P. Brack, C. Padeste, M. Slaski, S. Alkan, J. Am. Chem. Soc. 126 (2004) 1004. [15] E. Bormashenko, R. Pogreb, G. Whyman, Y. Bormashenko, R. Jager, T. Stein, A. Schechter, D. Aurbach, Langmuir 24 (2008) 5977. [16] K.S. Jee, H.D. Park, K.D. Park, Y.H. Kim, Biomacromolecules 5 (2004) 1877. [17] M. Shizuka, T.O. Schrader, M.L. Snapper, J. Org. Chem. 71 (2006) 1330. [18] N. Aumsuwan, S. Heinhorst, M.W. Urban, Biomacromolecules 8 (2007) 713. [19] K. Yao, X.D. Huang, X.J. Huang, Z.K. Xu, J. Biomed. Mater. Res. Part A 78A (2006) 684. [20] J.H. Jiang, L.P. Zhu, X.L. Li, Y.Y. Xu, B.K. Zhu, J. Membr. Sci. 364 (2010) 194. [21] G. Fei, J. Shin, S.A. Kang, B.S. KO, P.H. Kang, Y.S. Lee, Y.C. Nho, J. Polym. Sci., Part A: Polym. Chem. 48 (2010) 563. [22] M.M. Nasef, Polym. Int. 50 (2001) 338. [23] G. Lisbeth, C.T. Adrienne, T. Matt, Biomacromolecules 6 (2005) 2197. [24] Y.J. Kim, I.K. Kang, M.W. Huh, S.C. Yoon, Biomaterials 21 (2000) 121. [25] J.X. Liu, D.Z. Yang, Y.J. Cai, Chin. J. Mater. Res. 16 (2002) 529. [26] Y. Zhang, A.C.H. Huan, K.L. Tan, E.T. Kang, Nucl. Instrum. Methods B 168 (2000) 29. [27] Z.Y. Cheng, S.H. Teoh, Biomaterials 25 (2004) 1991. [28] L.J. Yu, X. Wang, X.H. Wang, X.H. Liu, Surf. Coat. Technol. 128–129 (2000) 484. [29] M. Tanaka, A. Mochizuki, N. Ishii, T. Motomura, T. Hatakeyama, Biomacromolecules 3 (2002) 36. [30] Q. Yang, Z.K. Xu, Z.W. Dai, J.L. Wang, Chem. Mater. 17 (2005) 3050.
Contents lists available at SciVerse ScienceDirect
Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
Heparin-grafted poly(tetrafluoroethylene-co-hexafluoropropylene) film with highly effective blood compatibility via an esterification reaction Qiang Gao, Yashao Chen ⁎, Yanlin Wei, Xudong Wang, Yanling Luo Key Laboratory of Applied Surface and Colloid Chemistry (Shaanxi Normal University), Ministry of Education, School of Chemistry & Chemical Engineering, Xi'an 710062, China
a r t i c l e
i n f o
Available online 10 July 2012 Keywords: Plasma treatment UV-induced graft polymerization Heparin Esterification reaction Blood compatibility
a b s t r a c t The aim of this work was to modify poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) film surface in order to improve its hydrophilicity and blood compatibility. Acrylic acid (AAc) was introduced onto Ar plasma-pretreated FEP surface by UV-induced grafted copolymerization. Subsequently, heparin (Hp) was also immobilized covalently with the carboxylic group of PAAc grafted onto the FEP surface via an esterification reaction. The chemical composition and the morphological change of the modified films were characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electronic microscopy (SEM), atomic force microscopy (AFM) and water contact angel measurements. The root–mean–square average surface roughness (Ra) changed from 5.82 nm (FEP) to 13.60 nm (FEP-PAAc) and 11.74 nm (FEP-PAAc-Hp). Water contact angles changed from 115.0o (FEP) to 32.4 o (FEP-PAAc) and 44.8 o (FEP-PAAc-Hp). The blood compatibility of the modified films was evaluated by platelet adhesion experiments. All results suggested that the FEP-PAAc-Hp surface presented excellent antithrombotic properties and good hydrophilicity. Therefore, this method will provide a potential and effective solution for the surface modification of medical polymer materials. © 2012 Elsevier B.V. All rights reserved.
1. Introduction It is known that biocompatibility plays an important role in implanted biomaterials. A truly biocompatible biomaterial should perform its function without causing undue host response or resulting adverse clinical reaction [1]. In general, biocompatibility of implant material mainly depends on its surface properties, for which reason it is necessary to modify the surface to improve the biocompatibility of biomaterials. Surface modification including physical, chemical modification and bio-molecule immobilization is considered as a promising approach to incorporate polymer materials onto a wide range of hydrophobic substrates for improved biocompatibility [2–5]. In our previous work, surface grafting modification is an important way to enhance the biocompatibility without changing the bulk structure of the polymer [6–8]. As we known, Hp is a critically clinical anticoagulant for minimizing thrombus formation on artificial surfaces since it is a kind of sulfated polysaccharide that contains sulfonic, sulfoamino, and carboxyl groups. Hence, to immobilize Hp molecules onto the material surface is the most popular technique for minimizing the thrombogenicity of materials. In this regard, the heparinized surfaces have anticoagulant properties that prolong blood clotting time when they are coated on blood devices or containers [9]. Recently, surface
⁎ Corresponding author. Tel.: +86 29 81530795; fax: +86 29 81530727. E-mail address: [email protected] (Y. Chen). 0257-8972/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2012.07.015
heparinization is regarded as an effective way to improve the blood compatibility of hydrophobic polymeric film [10,11]. FEP has been widely used as biomaterial due to its excellent chemical resistance, non-toxicity, and chemical stability [12]. However, the blood compatibility of FEP film as blood-contacting material is not applicable due to its inert and hydrophobic surface. In general, in order to improve the blood compatibility and ensure the long-term effectiveness of the biomedical devices, surface modification has often been performed before used in biomedical fields. In the present work, surface modification of Ar plasma-pretreated FEP film was carried out by UV-induced graft copolymerization with water-soluble monomer of AAc to create carboxyl functional groups on FEP surfaces, followed by esterification reactions, and reactions with Hp to create anticoagulation surfaces. In-vitro blood compatibility of the film was evaluated by platelet adhesion studies. The process for functionalization of FEP film with Hp consisted of several steps and was illustrated in Fig. 1. 2. Experimental section 2.1. Materials FEP films having a thickness of about 0.1 mm and a density of 2.15 g/cm3 were purchased from Goodfellow Ltd., of Cambridge, UK, followed by washing with acetone in an ultrasonic washer, and dried at room temperature under vacuum conditions before use. Acrylic acid (AAc), heparin sodium (Hp), and methylene chloride (MC) were
Q. Gao et al. / Surface & Coatings Technology 228 (2013) S126–S130
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Fig. 1. Schematic diagram of surface reactions on FEP film: step A, pretreated by Ar plasma and immersed in AAc solution; step B, UV-induced graft copolymerization; step C, Hp reaction.
purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. 4-dimethylaminopyridine (DMAP) and N,N-dicyclohexyl carbodiimide (DCC) were purchased from Sigma-Aldrich, America. Argon (Ar, purity: 99.99%) was employed as process gas for plasma treatment of FEP.
placed into a 100 mL beaker with 25 mL of MC, then stirred and cooled in an ice-water bath. DCC (1.2 mmol) was added, and the mixture was continuously stirred for 5 h. Upon removal, all samples were washed respectively in MC and distilled water sequentially for 3 h, dried at 35 °C under vacuum conditions for 24 h, and then analyzed.
2.2. Graft polymerization of AAc onto FEP surface: FEP-PAAc 2.4. Characterization FEP films were pretreated by Ar plasma at a voltage of 6.0 kV for 5 min in a PECVD-500 plasma apparatus (Beijing Technol Science Co., LTD, Beijing, China) at a gas flow of 60 sccm and a pressure of 50 Pa. Subsequently, the films were exposed to air for 20 min, leading to surface oxidation and formation of peroxide and hydroperoxide species [13]. The air-exposed FEP films were immersed in an aqueous solution containing 6% (v/v) AAc for 4 h (Fig. 1, step a). In brief, an UV illumination system equipped with a high-pressure mercury lamps (1000 W, λ = 365 nm) was employed, and then exposed to UV irradiation for 1 h under nitrogen atmosphere (Fig. 1, step b) [14,15]. Finally, the films were washed with distilled water and stirred for 12 h at 50 °C to remove homo-polymer and residual monomer, then maintained under vacuum for at least 24 h to remove the water. 2.3. Immobilization of Hp onto FEP surface: FEP-PAAc-Hp To modify FEP-PAAc surfaces with Hp, we utilized an esterification reaction using DMAP catalyst and DCC coupling agent (Fig. 1, step c) [16–18]. FEP-PAAc films, Hp (5 mg), and DMAP (0.2 mmol) were
Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were performed with an EQUI NOX55 FT-IR spectrometer with an ATR unit (Bruker Co., Germany). Each spectrum was obtained by cumulating 64 scans with a resolution of 4 cm −1. Surface composition was determined by X-ray photoelectron spectra (XPS) on an ESCALAB 250 spectrometer equipped with a monochromatic Al X-ray source (1486.6 eV) (ThermoElectron Co., America). Spectra were recorded at a 45° take-off angle with 20 eV pass energy. The changes of surface roughness and morphology were observed by scanning electron microscopy (SEM; Quanta 200 Philips-FEI Co., Netherlands) and atomic force microscopy (AFM; WET-SPM9500 J3, SHIMADZU Co., Japan) in the tapping mode. Static contact angle was measured by the sessile drop method with a video-based contact angle measuring device (OCA20, Dataphysisc Co., Germany). Each contact angle value reported here was an average of at least five measurements. 2.5. Platelets adhesion test A platelet adhesion test was used to evaluate the adhesion behavior and activation of the platelets on the samples. Fresh human whole blood (20 mL) was taken from a healthy volunteer and anticoagulated with 0.109 M solution of sodium citrate at a dilution ratio of 9:1 (blood/sodium citrate solution). Then platelet-rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1200 rpm for 10–12 min at room temperature. After that, the samples (1.0 cm × 1.0 cm) were placed in individual wells of a 24-well tissue culture plate, and each well was dropped into with 50 μL PRP for 1 h at room temperature. After the samples were carefully rinsed twice with PBS (pH, 7.2) to remove non-firmly adsorbed platelets, they were immersed into 2.0 wt.% glutaraldehyde solution for 1 h, Table 1 XPS atomic composition data for FEP films.
Fig. 2. ATR-FTIR spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
Samples
C (at.%)
F (at.%)
O (at.%)
N (at.%)
S (at.%)
Pristine FEP FEP-PAAc FEP-PAAc-Hp
21.21 66.73 64.01
78.79 0.69 8.86
0 32.58 23.29
0 0 2.41
0 0 1.43
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Fig. 3. XPS spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
and washed with triple-distilled water several times. Then, they were immersed into 30, 50, 70, 90, 100% (v/v) ethanol/water for 20 min at each step and dried in air. Finally, the samples were sputter-coated with gold prior to observation with SEM [19,20]. 3. Results and discussion ATR-FTIR analysis, shown in Fig. 2, indicates the presence of specific functional groups on the modified surface. Fig. 2a shows strong bands at 1150–1250 cm −1 in the spectra of the pristine FEP are characteristic for the stretching vibration of CF2 and CF3 present in the structure of the fluorinated films, and a sharp band at around 980 cm −1 is due to the stretching vibration of CF in the CF3 group [21,22]. Fig. 2b shows a strong new peak at 1750 cm −1 is corresponding to C_O, and the broad band from 2750 to 3300 cm −1 is assigned to the \OH stretch characteristic of carboxyl groups, verifying successful grafting of AAc to FEP [23]. Fig. 2c shows a reduced \OH peak because of the esterification reaction between the \COOH groups on substrate and \OH groups of Hp. In addition, IR spectra of FEP-PAAc-Hp displays the new absorption bands near 1050 and 1650 cm −1, which are characteristic adsorption peaks of sulfonated group (\SO3 stretching) and amino group (N\H bending) of conjugated Hp. At the same time, there are three absorptions at 845 cm −1, 860 cm −1, and 950 cm −1, which can be attributed to the characteristic absorption peak in Hp [16,20,24,25]. These results indicated that Hp is successfully immobilized onto the FEP-PAAc surface.
The chemical composition of the FEP surfaces at various stages of surface modification was determined by XPS (see Table 1). In Table 1, F (at.%) of the pristine FEP with grafting of AAc decrease from 78.79 to 0.69, that's because of the thickness of the grafted PAAc polymer layer is more than the probing depth of the XPS technique [13]. However, after the Hp covalent bonding to the FEP-PAAc film surface, F (at.%) of the FEP-PAAc-Hp film increases from 0.69 to 8.86 at.%. That's because part of the PAAc chain is loose due to the effect of solvent, this process lead to the pristine FEP film surface exposure, therefore, F (at.%) of the FEP-PAAc-Hp film was increased. Fig. 3a shows a typical XPS survey spectra for the pristine FEP surface, the typical signals for carbon (C1s at 293 eV) and fluorine (F1s at 691 eV) are clearly detected. After treatment of the FEP surfaces with AAc, the typical signals attributed to carbon, fluorine, and additional signals assigned to O1s at 534 eV, which indicate that AAc has been grafted to the FEP surfaces (Fig. 3b). Moreover, the carbon and oxygen signals are noticeably stronger compared with the pristine FEP surface, due to the additional carbon and oxygen in the AAc. In addition, the fluorine signal is noticeably reduced for the formation of a cross-linked PAAc coating onto the film surface. Because the reactions of FEP-PAAc with Hp molecules are conducted using the esterification process (Fig. 3c), the characteristic signals attributed to carbon, fluorine, and oxygen are again detected. Moreover, the appearance of weak signals assigned to nitrogen (N1s at 401 eV) and sulfur (S2p at 199.1 eV) indicates that Hp is formed on the FEP-PAAc surface. Overall, the XPS results further confirm that the surface modification method utilized here is very effective. To get more information on the changes of the surface compositions, we collect high resolution XPS data for C1s and O1s. Fig. 4 shows the C1s and O1s core-level spectra of the pristine FEP, FEP-PAAc, and FEP-PAAc-Hp. As shown in Fig. 4a, the C1s core-level spectra of the pristine FEP film can be curve-fitted into three peak components with binding energies (BEs) at 289.7 eV, 293.2 eV, and 296.3 eV, attributed to the CF, the CF2, and the CF3 species, respectively [26]. It also reveals that there is no oxygen in the surface of the pristine FEP film. Fig. 4b shows the C1s and O1s core-level spectra of the FEP-PAAc. The C1s core-level spectra of the FEP-PAAc surface can be curve-fitted with three new peak components at 285.6 eV, 289.2 eV, and 292.5 eV, attributed to the C\C/C\H, C\O, O_C\O, respectively. The three fluorocarbon peak components in the C1s core-level spectra almost disappeared, which indicate that the thickness of the grafted PAAc polymer layer is more than the probing depth of the XPS technique [13]. The O1s core-level spectra consists of the C_O, and C\O peak components at 532.8 eV, and 534.2 eV, respectively. The results indicate that AAc is successfully grafted on the plasma-treated PEP film surface to form the functional groups (\COOH). After Hp immobilization onto the FEP-PAAc surface (see
Fig. 4. C1s and O1s core-level spectra of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
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Fig. 5. SEM and contact angel images of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
Fig. 4c), the C1s core-level can be curve-fitted with four peak components at 285.5 eV, 286.0 eV, 287.1 eV, and 289.8 eV, attributed to the C\N, C\C/C\H, \C\O\, C_O/N\C_O, respectively. The C-N peak component is associated with the linkages in Hp itself. The O1s core-level spectra consists of the C_O, C\O\C, and C\O peak components at 532.5 eV, 533.2 eV, and 534.3 eV, respectively. Compared with Fig. 4b, the ratio of the C\OH has reduced significantly, that is expected due to the introduction of the ester. Furthermore, the additional peak C\O\C verified formation of an ester linkage between the \COOH of FEP-PAAc and the \OH of Hp molecules. The changes in surface morphology of the FEP surfaces, at various stages of surface modification, were studied by SEM and AFM. Representative SEM and contact angel images are shown in Fig. 5. As can be seen from Fig. 5a, the SEM images of the pristine FEP relatively
appeared featureless and very smooth. The pristine FEP is highly hydrophobic with a contact angle of 115.0°. In Fig. 5b, FEP-PAAc surface implies that the FEP surface was almost covered with a PAAc layer and the hydrophilicity is greatly enhanced with contact angle of 32.4°. This could be ascribed to the hydrophilic nature of grafted PAAc chains which are abundant in carboxyl groups [27]. In Fig. 5c, FEP-PAAc-Hp surface shows that there are some particles appeared and contact angle changed to 44.8° due to Hp molecules after the esterification reaction. Commonly, grafted PAAc layer causes roughening of the FEP surface, and the grafting of large molecules, that is, Hp, further changes the surface roughness. The morphological change of the surface can be observed by AFM (Fig. 6). Here different geometric features are demonstrated: the AFM images of the pristine FEP and the FEP-PAAc
Fig. 6. AFM images of (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp; (d) is RMS roughness of the samples surface.
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Fig. 7. SEM images of platelet on (a) pristine FEP, (b) FEP-PAAc, and (c) FEP-PAAc-Hp.
films are shown in Fig. 6a and b, respectively. The root-mean-square average surface roughness (Ra) of the pristine FEP film is in the order of about 5.82 nm (Fig. 6d). The Ra value increases to about 13.60 nm after the FEP surface has been graft-copolymerized with AAc. The increase in the Ra value is consistent with the presence of the surface grafted PAAc polymer on the FEP film. However, the roughness of the FEP-PAAc-Hp surface is reduced to 11.74 nm (Fig. 6c). Part of the Hp molecules are strong filled to the interspace of the FEP-PAAc film surface, which cause the roughness of the FEP-PAAc-Hp surface down, this indicates that the presence of Hp on the FEP-PAAc surface. It is well known that when a material contacts with blood, proteins instantaneously adsorb onto the surface and deform. Subsequently, platelets that adhere, are activated, and aggregated. The adhesion and aggregation of platelets play a significant role in thrombus formation [28]. Therefore, the extent of platelet adhesion and the morphology of the adhered platelets are considered to be early indicators of the thrombogenicity of biomaterials in contact with blood [29,30]. SEM images were used to evaluate the adhesion and morphology of platelets on various sample surfaces, the representative typical images of platelets adhesion behavior on these surfaces are shown in Fig. 7. As can be seen, platelets are adhered more and largely aggregated on the surface of pristine FEP (Fig. 7a). It is clear that the number of adhered platelets was significantly decreased for FEP-PAAc surface due to the hydrophilicity of PAAc layer (Fig. 7b). However, Fig. 7c shows that was no platelets adhesion on FEP-PAAc-Hp surface, thus, these heparinized surfaces can effectively improve the antithrombogenecity of the FEP films. That's because of the excellent hydrophilic and anticoagulant characteristics of the Hp molecules, also explained the good effect of modified FEP film surface by Hp binding. 4. Conclusions In this paper, a convenient approach of surface hydrophilization and heparinization for inert FEP film was developed. ATR-FTIR, XPS, AFM and SEM revealed that the FEP-PAAc film surface was successfully prepared by Ar plasma treatment and UV-induced graft polymerization with AAc, followed by the immobilization of Hp on the FEP-PAAc surface. The water contact angle indicated that the hydrophilicity improvement of the modified FEP surface by the grafting of PAAc and the bonding of Hp, respectively. Results of platelet adhesion experiments clearly indicated that the blood compatibility of the pristine FEP and FEP-PAAc surfaces were bad, the FEP-PAAc-Hp film surface exhibited excellent antithrombogenicity property. The results
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