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Surface sulfonation of silk fibroin film by plasma treatment and in vitro antithrombogenicity study
Materials Science and Engineering C 20 (2002) 199 – 202 www.elsevier.com/locate/msec
Surface sulfonation of silk fibroin film by plasma treatment and in vitro antithrombogenicity study Jinwei Gu, Xinlin Yang *, Hesun Zhu Research Center of Materials Science, Beijing Institute of Technology, Beijing, 100081, China
Abstract To improve the blood compatibility of Bombyx mori silk fibroin (SF) film, the film was modified by SO2 gas plasma treatment, or by a two-step process including NH3 gas plasma treatment and reaction with 1,3-propane sultone. XPS and ATR-FTIR were used to analyze the surface chemical elements. In vitro antithrombogenicity was determined by the method of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. Percents of sulfur element on the surfaces of both modified SF films were 4.03% and 3.30%, respectively, while that of the control film was only 0.32%. Moreover, the antithrombogencity of treated films was increased remarkably due to surface sulfonation. The results implied a potential use of sulfonated SF for blood-contacting biomaterials. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Silk fibroin (SF); Plasma; Antithrombogenicity
1. Introduction Silk fibroin (SF), a protein produced by silkworms, has attracted considerable attention recently as a biomaterial with good biocompatibility and mechanical properties [1]. Various studies on its applications in biological and biomedical fields have emerged such as enzyme immobilization, oxygen permeable membranes, drug permeable films and matrix for mammalian cell culture [2 –6]. As a biomaterial, the interaction between its surface and the biological environment must be considered. Especially, when a foreign material is exposed to blood, plasma proteins are absorbed onto the surface, followed by the activation of clotting factors or the adhesion and activation of platelets, and finally the formation of a thrombus [7]. Thus, the surface of a silk fibroin film should be modified to improve its blood compatibility when it is used as a blood-contacting material. Low-temperature plasma glow technique is an effective method for surface modification. Previous reports showed sulfur-contained groups can be incorporated onto the substrate by SO2 plasma and surface sulfonation of some polymer materials could reduce platelets deposition thereby improve their antithrombogenecity [8,9]. Besides of SO2 plasma, other methods have also been reported for surface sulfonation of the materials [10,11]. In the present study, *
Corresponding author. Fax: +86-10-68913584. E-mail address: [email protected] (X. Yang).
SO2 or NH3 gas plasma was used to make sulfonic groups covalently linked to the surface of SF films. Then the sulfonated film was tested for the antithrombogenicity by in vitro coagulation time tests, including tests of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). The results showed the antithrombogenecity of SF films could be significantly increased after surface sulfonation.
2. Experimental 2.1. Materials Bombyx mori silk was commercially from China Import & Export. Toluene, methyl alcohol (both at HPLC grade) and 1,3-propane sultone were products of Sigma. Kits for in vitro coagulation time (PT, APTT and TT) tests were purchased from Huashan Hospital of Shanghai Medical University. Human blood plasma was obtained from China Red Cross Association. 2.2. Preparation of SF films B. mori silk was boiled with 1%(W) Na2CO3 for about 1 h in order to remove the sericin protein. SF was extracted by a solution of calcium chloride/ethanol/distilled water (1:2:8 mole ratio) at 80 jC. After dialyzed against distilled water
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J. Gu et al. / Materials Science and Engineering C 20 (2002) 199–202
for 3 days, SF solution was filtered and its concentration was determined by an UV –VIS spectrometer. Then the solution was cast into a polystyrene plate and incubated at 50 jC for 24 h. SF film was formed after drying the plate in vacuum at room temperature (RT) for another 24 h. 2.3. Surface sulfonation of SF films by SO2 plasma The plasma reactor system designed by our lab consists of a glass chamber with a pair of stainless steel disc electrodes (Fig. 1). The upper electrode was connected to a 13.56-MHz radio frequency generator and the lower electrode was grounded. An automatic matching network was utilized to minimize the reflection power of the system. The SF film was placed on lower electrode and then the base gas pressure was pumped down to less than 1 Pa. The processing parameters of SO2 plasma treatment were set as follows: gas flow rate, 20 SCCM; working pressure, 20 Pa; net plasma power, 30 W; time, 5 min; temperature, RT. After the plasma treatment was done, the gas flow was continued for 5 min at the working pressure in order to quench reactive sites.
Table 1 Surface atomic relative ratios of C1s, O1s, N1s, and S2p measured by XPS analysis in variously treated SF films Treatment
C1s (%)
O1s (%)
N1s (%)
S2p (%)
N/C
O/C
S/C
Control SO2 plasma NH3 plasma Two-step process
68.65 66.61 66.47 67.12
19.43 21.29 19.69 18.86
11.60 8.07 13.45 10.71
0.32 4.03 0.39 3.30
0.085 0.045 0.112 0.126
0.287 0.466 0.230 0.273
0.003 0.182 0.006 0.048
2.6. In vitro coagulation time tests for SF films The antithrombogenicity of SF films was evaluated by in vitro coagulation time tests, including PT, APTT and TT [12]. Briefly, the tested film was incubated with healthy human blood plasma in a transparent plastic tube, and the reagents for each coagulation time test were added to the tube immediately. The clotting times were measured by a photo-optical clot detection instrument Coag-A-MateR-XM (Organon Teknika, USA).
2.4. Surface sulfonation of SF films by a two-step process The chemical grafting of sulphonyl group onto SF films was also performed by a two-step process, which was previously described by Lin et al. [11]. At first, the film was treated by NH3 plasma to produce amino groups on the surface, and then incubated for 24 h with a 10% (V/V) solution of 1,3-propane sultone in toluene (HPLC grade). The film was rinsed in anhydrous methyl alcohol (HPLC grade) and dried in a vacuum oven for 24 h at RT. 2.5. Surface characterization of SF films The ATR-FTIR spectra of SF films were recorded on an EQUINOX55 spectrometer (Bruker). XPS analysis of SF films was performed by an ESCA XSAM800 spectrometer (Kratos, UK) equipped with an Al Ka non-monochromatic X-ray source (E=1486.6 eV). The surface elemental compositions of C1s, O1s, N1s and S2p were measured and detailed scans of S2p and N1s were made in the present experiments.
Fig. 1. Schematic diagram of plasma glow discharge apparatus. 1. Reaction chamber; 2. RF shield; 3. Upper electrode; 4. Lower electrode (grounded); 5. RF power generator; 6. Matching network; 7. Pumping system; 8. Reactive gas flow system.
Fig. 2. XPS analysis results of surface S2p scan spectra of SF films treated by SO2 plasma (A) and by a two-step process (B).
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3. Results and discussion 3.1. XPS analysis of SF films XPS analysis revealed carbon (C), nitrogen (N), oxygen (O), and sulfur (S) atoms on the surface of untreated and plasma-treated SF films. The surface atomic relative percentages in various SF films were listed in Table 1. The surface percentages of C, O, N, and S of untreated SF film were 68.65%, 19.43%, 11.60% and 0.32%, respectively. The O/C ratio of the SO2 plasma-treated SF film was higher than that of the control, which indicated the strong oxidizing capability of SO2 plasma [9]. Less surface oxygen element was found on NH3 plasma-treated SF film than SO2 plasmatreated sample. Compared with untreated sample, SF film treated by SO2 plasma or by the two-step process had more abundant surface sulfur element, which showed some groups containing sulfur were introduced onto the surface of SF films. The surface S/C ratio after the two-step process was 0.048, much lower than 0.182 of that after treatment by SO2 plasma, implying the latter may be a more effective method for surface sulfonation of the silk fibroin material than the former.
Fig. 4. ATR-FTIR spectra of SF films treated by SO2 plasma (A), a two-step process (B) and untreated SF films (C).
Fig. 2 showed XPS analysis results of the surface S2p spectra of SF films by SO2 plasma modification and by the two-step process. There were two distinctive peaks near 169.1 and 164.2 eV within the S2p spectrum of SO2 plasma modified SF (Fig. 2A), while there was only one peak near 169.1 eV within that of SF after the two-step process (Fig. 2B). The higher energy peak may represent sulfur at a higher oxidation state, assigned to sulfur atoms bonded to two or three oxygen atoms such as sulfone, sulfonate or sulfonic acid. In contrast, the lower energy peak may be attributed to sulfur atoms linked to carbon or to one oxygen atom [9]. It was also found the former sulfur form was 70.89% of total sulfur atoms in SF treated by SO2 plasma, implying that sulfonate group was the main type of the sulfur-containing groups generated onto the surface of SF film by SO2 plasma. In addition, the existence of the only higher energy peak within S2p spectrum of SF after the twostep process indicated 1,3-propane sultone had been grafted chemically to the amino sites produced from NH3 plasma treatment. Fig. 3 showed XPS analysis results of the surface N1s spectra of untreated SF films and SF films treated by the two-step process. There were two major types of N atoms within the SF molecule: N atoms of NH2 and acylamide groups, whose binding energies are 398.9 and 399.7 eV, respectively (Fig. 3A). The percentage of N atom within NH2 group increased from 36.5% to 46.4% (Fig. 3A,B), showing the treatment of NH3 plasma is effective for the chemical grafting of NH2 group onto the surface of SF film. 3.2. ATR-FTIR analysis of SF films
Fig. 3. XPS analysis results of surface N1s scan spectra of untreated (A) and NH3 plasma-treated (B) SF films.
The ATR-FTIR spectra of untreated SF, SO2 plasmatreated SF and the two-step processed SF were shown in Fig. 4. Within three spectra, the peak in 3278 cm1 (not
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J. Gu et al. / Materials Science and Engineering C 20 (2002) 199–202
Table 2 In vitro coagulation times (PT, TT and APTT) of human blood plasma incubated with variously treated SF films. The data were mean values of those obtained from two repeated experiments Samples
PT (s)
TT (s)
Blood Blood Blood Blood Blood
10.5 10.3 11.9 12.1 18.0
14.8 32.3 15.5 31.3 > 90 >150 14.3 27.4 >90 >150
plasma plasma+untreated SF plasma+SF treated by SO2 plasma plasma+SF treated by NH3 plasma plasma+SF treated by the two-step process
APTT (s)
shown) represented the stretching vibration of NH and the peaks in 1645, 1518 and 1232 cm1 might derive from the stretching vibration of C¼O and CN, respectively. However, there was a peak located at 1120 –1180 cm1 within spectra A (peak 1141 cm1) and B (peak 1153 cm1) but not within that of C, which could be explained that the peak came from the stretching vibration of S¼O of sulfonate group. This peak is relatively low within both spectra probably due to the low concentration of surface sulfonate group in both kinds of SF films. 3.3. In vitro coagulation time tests of SF films The PT, APTT and TT tests were widely used for the clinical detection of the abnormality of blood plasma and for the primary screening of the anticoagulative chemicals [12]. They were recently applied in the evaluation of the in vitro antithrombogenicity of biomaterials [13,14]. The normal ranges of PT, APTT and TT for a healthy blood plasma were regarded to be 11F3, 28F10 and 16F5 s, respectively. When the blood plasma was incubated with the SF film untreated or treated by NH3 plasma alone, the corresponding data of PT, APTT and TT were 10.3, 31.3 and 15.5 s or 12.1, 27.4 and 14.3 s, indicating neither SF itself nor NH3 plasmatreated SF had few influence on the thrombogenicity of the blood plasma (Table 2). However, when the blood plasma was incubated with the sulfonated SF films, the APTT and TT became undetectable, because they both are over the
maximums of 150 and 90 s set to the instrument, respectively (Table 2). Therefore, it was thought that SO3 group on the surface of SF might play an important role in inhibiting the activities of some clotting factors of blood plasma involved in APTT and TT tests. In conclusion, this paper described two methods for the surface sulfonation of silk fibroin film, involving the use of plasma glow. In addition, the antithrombogenicity of silk fibroin could be significantly improved after surface sulfonation.
Acknowledgements This work was supported by ‘‘973 projects of China (No. G1999064705)’’ from Chinese Science and Technology Ministry.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
N. Minoura, M. Tsukada, M. Nagura, Biomaterials 11 (1990) 430. M. Demura, T. Asakura, Biotechnol. Bioeng. 33 (1989) 598. N. Minoura, M. Tsukada, M. Nagura, Polymer 31 (1990) 265. N. Minoura, S.I. Aiba, M. Higuchi, Y. Gotoh, M. Tsukada, Y. Imai, Biochem. Biophys. Res. Commun. 208 (1995) 511. K. Inouye, M. Kurokawa, S. Nishikawa, M. Tsukada, J. Biochem. Biophys. Methods 37 (1998) 159. J. Chen, N. Minoura, A. Tanioka, Polymer 35 (1994) 2853. J.S. Bae, E.J. Seo, I.K. Kang, Biomaterials 20 (1999) 529. T.G. Grasel, S.L. Cooper, J. Biomed. Mater. Res. 23 (1989) 1371. J.C. Lin, S.L. Cooper, Biomaterials 16 (1995) 1017. I.C. Kim, J.G. Chio, T.M. Tak, J. Appl. Polym. Sci. 74 (1999) 2046. J.C. Lin, T.M. Ko, S.L. Cooper, J. Colloid Interface Sci. 164 (1994) 99. F. Khan, L.M. Snyder, L. Pechet, J. Thromb. Thrombolysis 5 (1998) 83. I.K. Kang, O.H. Kwon, K.H. Byun, J. Mater. Sci. Mater. Med. 7 (1996) 135. Y.H. Sun, L.X. Feng, J. Appl. Polym. Sci. 74 (1999) 2826.
Surface sulfonation of silk fibroin film by plasma treatment and in vitro antithrombogenicity study Jinwei Gu, Xinlin Yang *, Hesun Zhu Research Center of Materials Science, Beijing Institute of Technology, Beijing, 100081, China
Abstract To improve the blood compatibility of Bombyx mori silk fibroin (SF) film, the film was modified by SO2 gas plasma treatment, or by a two-step process including NH3 gas plasma treatment and reaction with 1,3-propane sultone. XPS and ATR-FTIR were used to analyze the surface chemical elements. In vitro antithrombogenicity was determined by the method of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) tests. Percents of sulfur element on the surfaces of both modified SF films were 4.03% and 3.30%, respectively, while that of the control film was only 0.32%. Moreover, the antithrombogencity of treated films was increased remarkably due to surface sulfonation. The results implied a potential use of sulfonated SF for blood-contacting biomaterials. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Silk fibroin (SF); Plasma; Antithrombogenicity
1. Introduction Silk fibroin (SF), a protein produced by silkworms, has attracted considerable attention recently as a biomaterial with good biocompatibility and mechanical properties [1]. Various studies on its applications in biological and biomedical fields have emerged such as enzyme immobilization, oxygen permeable membranes, drug permeable films and matrix for mammalian cell culture [2 –6]. As a biomaterial, the interaction between its surface and the biological environment must be considered. Especially, when a foreign material is exposed to blood, plasma proteins are absorbed onto the surface, followed by the activation of clotting factors or the adhesion and activation of platelets, and finally the formation of a thrombus [7]. Thus, the surface of a silk fibroin film should be modified to improve its blood compatibility when it is used as a blood-contacting material. Low-temperature plasma glow technique is an effective method for surface modification. Previous reports showed sulfur-contained groups can be incorporated onto the substrate by SO2 plasma and surface sulfonation of some polymer materials could reduce platelets deposition thereby improve their antithrombogenecity [8,9]. Besides of SO2 plasma, other methods have also been reported for surface sulfonation of the materials [10,11]. In the present study, *
Corresponding author. Fax: +86-10-68913584. E-mail address: [email protected] (X. Yang).
SO2 or NH3 gas plasma was used to make sulfonic groups covalently linked to the surface of SF films. Then the sulfonated film was tested for the antithrombogenicity by in vitro coagulation time tests, including tests of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). The results showed the antithrombogenecity of SF films could be significantly increased after surface sulfonation.
2. Experimental 2.1. Materials Bombyx mori silk was commercially from China Import & Export. Toluene, methyl alcohol (both at HPLC grade) and 1,3-propane sultone were products of Sigma. Kits for in vitro coagulation time (PT, APTT and TT) tests were purchased from Huashan Hospital of Shanghai Medical University. Human blood plasma was obtained from China Red Cross Association. 2.2. Preparation of SF films B. mori silk was boiled with 1%(W) Na2CO3 for about 1 h in order to remove the sericin protein. SF was extracted by a solution of calcium chloride/ethanol/distilled water (1:2:8 mole ratio) at 80 jC. After dialyzed against distilled water
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J. Gu et al. / Materials Science and Engineering C 20 (2002) 199–202
for 3 days, SF solution was filtered and its concentration was determined by an UV –VIS spectrometer. Then the solution was cast into a polystyrene plate and incubated at 50 jC for 24 h. SF film was formed after drying the plate in vacuum at room temperature (RT) for another 24 h. 2.3. Surface sulfonation of SF films by SO2 plasma The plasma reactor system designed by our lab consists of a glass chamber with a pair of stainless steel disc electrodes (Fig. 1). The upper electrode was connected to a 13.56-MHz radio frequency generator and the lower electrode was grounded. An automatic matching network was utilized to minimize the reflection power of the system. The SF film was placed on lower electrode and then the base gas pressure was pumped down to less than 1 Pa. The processing parameters of SO2 plasma treatment were set as follows: gas flow rate, 20 SCCM; working pressure, 20 Pa; net plasma power, 30 W; time, 5 min; temperature, RT. After the plasma treatment was done, the gas flow was continued for 5 min at the working pressure in order to quench reactive sites.
Table 1 Surface atomic relative ratios of C1s, O1s, N1s, and S2p measured by XPS analysis in variously treated SF films Treatment
C1s (%)
O1s (%)
N1s (%)
S2p (%)
N/C
O/C
S/C
Control SO2 plasma NH3 plasma Two-step process
68.65 66.61 66.47 67.12
19.43 21.29 19.69 18.86
11.60 8.07 13.45 10.71
0.32 4.03 0.39 3.30
0.085 0.045 0.112 0.126
0.287 0.466 0.230 0.273
0.003 0.182 0.006 0.048
2.6. In vitro coagulation time tests for SF films The antithrombogenicity of SF films was evaluated by in vitro coagulation time tests, including PT, APTT and TT [12]. Briefly, the tested film was incubated with healthy human blood plasma in a transparent plastic tube, and the reagents for each coagulation time test were added to the tube immediately. The clotting times were measured by a photo-optical clot detection instrument Coag-A-MateR-XM (Organon Teknika, USA).
2.4. Surface sulfonation of SF films by a two-step process The chemical grafting of sulphonyl group onto SF films was also performed by a two-step process, which was previously described by Lin et al. [11]. At first, the film was treated by NH3 plasma to produce amino groups on the surface, and then incubated for 24 h with a 10% (V/V) solution of 1,3-propane sultone in toluene (HPLC grade). The film was rinsed in anhydrous methyl alcohol (HPLC grade) and dried in a vacuum oven for 24 h at RT. 2.5. Surface characterization of SF films The ATR-FTIR spectra of SF films were recorded on an EQUINOX55 spectrometer (Bruker). XPS analysis of SF films was performed by an ESCA XSAM800 spectrometer (Kratos, UK) equipped with an Al Ka non-monochromatic X-ray source (E=1486.6 eV). The surface elemental compositions of C1s, O1s, N1s and S2p were measured and detailed scans of S2p and N1s were made in the present experiments.
Fig. 1. Schematic diagram of plasma glow discharge apparatus. 1. Reaction chamber; 2. RF shield; 3. Upper electrode; 4. Lower electrode (grounded); 5. RF power generator; 6. Matching network; 7. Pumping system; 8. Reactive gas flow system.
Fig. 2. XPS analysis results of surface S2p scan spectra of SF films treated by SO2 plasma (A) and by a two-step process (B).
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3. Results and discussion 3.1. XPS analysis of SF films XPS analysis revealed carbon (C), nitrogen (N), oxygen (O), and sulfur (S) atoms on the surface of untreated and plasma-treated SF films. The surface atomic relative percentages in various SF films were listed in Table 1. The surface percentages of C, O, N, and S of untreated SF film were 68.65%, 19.43%, 11.60% and 0.32%, respectively. The O/C ratio of the SO2 plasma-treated SF film was higher than that of the control, which indicated the strong oxidizing capability of SO2 plasma [9]. Less surface oxygen element was found on NH3 plasma-treated SF film than SO2 plasmatreated sample. Compared with untreated sample, SF film treated by SO2 plasma or by the two-step process had more abundant surface sulfur element, which showed some groups containing sulfur were introduced onto the surface of SF films. The surface S/C ratio after the two-step process was 0.048, much lower than 0.182 of that after treatment by SO2 plasma, implying the latter may be a more effective method for surface sulfonation of the silk fibroin material than the former.
Fig. 4. ATR-FTIR spectra of SF films treated by SO2 plasma (A), a two-step process (B) and untreated SF films (C).
Fig. 2 showed XPS analysis results of the surface S2p spectra of SF films by SO2 plasma modification and by the two-step process. There were two distinctive peaks near 169.1 and 164.2 eV within the S2p spectrum of SO2 plasma modified SF (Fig. 2A), while there was only one peak near 169.1 eV within that of SF after the two-step process (Fig. 2B). The higher energy peak may represent sulfur at a higher oxidation state, assigned to sulfur atoms bonded to two or three oxygen atoms such as sulfone, sulfonate or sulfonic acid. In contrast, the lower energy peak may be attributed to sulfur atoms linked to carbon or to one oxygen atom [9]. It was also found the former sulfur form was 70.89% of total sulfur atoms in SF treated by SO2 plasma, implying that sulfonate group was the main type of the sulfur-containing groups generated onto the surface of SF film by SO2 plasma. In addition, the existence of the only higher energy peak within S2p spectrum of SF after the twostep process indicated 1,3-propane sultone had been grafted chemically to the amino sites produced from NH3 plasma treatment. Fig. 3 showed XPS analysis results of the surface N1s spectra of untreated SF films and SF films treated by the two-step process. There were two major types of N atoms within the SF molecule: N atoms of NH2 and acylamide groups, whose binding energies are 398.9 and 399.7 eV, respectively (Fig. 3A). The percentage of N atom within NH2 group increased from 36.5% to 46.4% (Fig. 3A,B), showing the treatment of NH3 plasma is effective for the chemical grafting of NH2 group onto the surface of SF film. 3.2. ATR-FTIR analysis of SF films
Fig. 3. XPS analysis results of surface N1s scan spectra of untreated (A) and NH3 plasma-treated (B) SF films.
The ATR-FTIR spectra of untreated SF, SO2 plasmatreated SF and the two-step processed SF were shown in Fig. 4. Within three spectra, the peak in 3278 cm1 (not
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J. Gu et al. / Materials Science and Engineering C 20 (2002) 199–202
Table 2 In vitro coagulation times (PT, TT and APTT) of human blood plasma incubated with variously treated SF films. The data were mean values of those obtained from two repeated experiments Samples
PT (s)
TT (s)
Blood Blood Blood Blood Blood
10.5 10.3 11.9 12.1 18.0
14.8 32.3 15.5 31.3 > 90 >150 14.3 27.4 >90 >150
plasma plasma+untreated SF plasma+SF treated by SO2 plasma plasma+SF treated by NH3 plasma plasma+SF treated by the two-step process
APTT (s)
shown) represented the stretching vibration of NH and the peaks in 1645, 1518 and 1232 cm1 might derive from the stretching vibration of C¼O and CN, respectively. However, there was a peak located at 1120 –1180 cm1 within spectra A (peak 1141 cm1) and B (peak 1153 cm1) but not within that of C, which could be explained that the peak came from the stretching vibration of S¼O of sulfonate group. This peak is relatively low within both spectra probably due to the low concentration of surface sulfonate group in both kinds of SF films. 3.3. In vitro coagulation time tests of SF films The PT, APTT and TT tests were widely used for the clinical detection of the abnormality of blood plasma and for the primary screening of the anticoagulative chemicals [12]. They were recently applied in the evaluation of the in vitro antithrombogenicity of biomaterials [13,14]. The normal ranges of PT, APTT and TT for a healthy blood plasma were regarded to be 11F3, 28F10 and 16F5 s, respectively. When the blood plasma was incubated with the SF film untreated or treated by NH3 plasma alone, the corresponding data of PT, APTT and TT were 10.3, 31.3 and 15.5 s or 12.1, 27.4 and 14.3 s, indicating neither SF itself nor NH3 plasmatreated SF had few influence on the thrombogenicity of the blood plasma (Table 2). However, when the blood plasma was incubated with the sulfonated SF films, the APTT and TT became undetectable, because they both are over the
maximums of 150 and 90 s set to the instrument, respectively (Table 2). Therefore, it was thought that SO3 group on the surface of SF might play an important role in inhibiting the activities of some clotting factors of blood plasma involved in APTT and TT tests. In conclusion, this paper described two methods for the surface sulfonation of silk fibroin film, involving the use of plasma glow. In addition, the antithrombogenicity of silk fibroin could be significantly improved after surface sulfonation.
Acknowledgements This work was supported by ‘‘973 projects of China (No. G1999064705)’’ from Chinese Science and Technology Ministry.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
N. Minoura, M. Tsukada, M. Nagura, Biomaterials 11 (1990) 430. M. Demura, T. Asakura, Biotechnol. Bioeng. 33 (1989) 598. N. Minoura, M. Tsukada, M. Nagura, Polymer 31 (1990) 265. N. Minoura, S.I. Aiba, M. Higuchi, Y. Gotoh, M. Tsukada, Y. Imai, Biochem. Biophys. Res. Commun. 208 (1995) 511. K. Inouye, M. Kurokawa, S. Nishikawa, M. Tsukada, J. Biochem. Biophys. Methods 37 (1998) 159. J. Chen, N. Minoura, A. Tanioka, Polymer 35 (1994) 2853. J.S. Bae, E.J. Seo, I.K. Kang, Biomaterials 20 (1999) 529. T.G. Grasel, S.L. Cooper, J. Biomed. Mater. Res. 23 (1989) 1371. J.C. Lin, S.L. Cooper, Biomaterials 16 (1995) 1017. I.C. Kim, J.G. Chio, T.M. Tak, J. Appl. Polym. Sci. 74 (1999) 2046. J.C. Lin, T.M. Ko, S.L. Cooper, J. Colloid Interface Sci. 164 (1994) 99. F. Khan, L.M. Snyder, L. Pechet, J. Thromb. Thrombolysis 5 (1998) 83. I.K. Kang, O.H. Kwon, K.H. Byun, J. Mater. Sci. Mater. Med. 7 (1996) 135. Y.H. Sun, L.X. Feng, J. Appl. Polym. Sci. 74 (1999) 2826.