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Analysis of cell-adhesion surface induced by ion-beam irradiation into biodegradable polymer
NIM B Beam Interactions with Materials & Atoms
Nuclear Instruments and Methods in Physics Research B 242 (2006) 51–54 www.elsevier.com/locate/nimb
Analysis of cell-adhesion surface induced by ion-beam irradiation into biodegradable polymer Tasuku Yotoriyama a,*, Aiko Nakao b, Yoshiaki Suzuki b, Takeyo Tsukamoto a, Masaya Iwaki b a
Graduate School, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan b Beam Application Team, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Available online 28 November 2005
Abstract Biodegradable polymers such as poly (L-lactic acid) (PLLA) are widely used to construct cell scaffolds for tissue engineering. We previously reported that cell attachment on a PLLA surface was improved by ion-beam irradiation. In this report, we attempted to characterize the cell-adhesion surface induced by ion-beam irradiation into PLLA by means of X-ray photoemission spectroscopy (XPS), Raman scattering measurement (Raman), and a fluorescence antibody technique. The substrates used were PLLA films; Ar+ ion-beam irradiation was performed at an energy of 50 keV with fluences of 1 · 1014 and 1 · 1015 ions/cm2. The results of XPS and Raman studies indicated the formation of new carbon structures and new functional groups by ion-beam irradiation. The protein adsorption was improved by Ar+ irradiation. We concluded that the cell-adhesion surface was induced as a consequence of production of new functional groups by ion-beam irradiation. 2005 Published by Elsevier B.V. PACS: 87.68.+z; 87.64.Gb; 81.65.Lp Keywords: Ion implantation; Biodegradable polymer; Cell adhesion; XPS
1. Introduction Aliphatic polyesters based on glycolic and lactic acids are the most investigated and advanced bioresorbable polymers used to make biomedical and pharmaceutical devices. Poly (L-lactic acid) (PLLA) is a biodegradable polymer that hydrolyzes into a low molecule when implanted in a body. The hydrolysis-generated reaction products are non-toxic to the body since they are broken down into carbon dioxide and water by the bodyÕs metabolism cycle. However, the lack of functional groups
* Corresponding author. Address: Beam Application Team, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: +81 48 467 9359; fax: +81 48 462 4623. E-mail address: [email protected] (T. Yotoriyama).
0168-583X/$ - see front matter 2005 Published by Elsevier B.V. doi:10.1016/j.nimb.2005.08.201
for covalent cell-recognition signal molecules to promote cell attachment has limited PLLAÕs further application in the field of tissue engineering. It is necessary to introduce some reactive groups on the chemically inert polymer surface [1]. We previously demonstrated that cell attachment on biodegradable polymer was improved by ion-beam irradiation [2]. This paper focuses to characterize the cell-adhesion surface induced by ion-beam irradiation into biodegradable polymer using X-ray photoemission spectroscopy (XPS) and Raman scattering measurement (Raman). Cell adhesion is mediated by the formation of an adsorbed interfacial protein layer coming from the complex serum medium [3,4]. There are several adhesion molecules in the serum, but we investigated fibronectin (FN) adsorption on an ion-beam irradiated surface using a fluorescence antibody technique.
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
2. Material and methods 2.1. Sample preparation The substrates used were 3 cm2, 200-lm-thick Poly-Llactic acid (PLLA) sheets (LACTY; Shimadzu, Japan). We performed Ar+ ion-beam irradiation into PLLA at an energy of 50 keV with fluences of 1 · 1014 and 1 · 1015 ions/cm2 at room temperature using a RIKEN 200 kV low-current implanter. The beam current was kept below 0.1 lA/cm2 to prevent heating the specimen, and the target chamber was maintained at a base pressure of 10 4 Pa during ion-beam irradiation. Non-irradiated PLLA sheets were used as controls in all investigations. 2.2. Fluorescence antibody technique Protein adsorption on an ion-irradiated surface was investigated using a fluorescence antibody technique. Fetal bovine serum (FBS, Sanko-Junyaku Co., Japan) was diluted with DulbeccoÕs phosphate-buffered saline solution (PBS, pH = 7.4) to a final concentration of 10%. Ion-irradiated PLLA sheets were cut into 1 cm2. Non-irradiated PLLA was used as the control. Samples were placed into a 24-well tissue culture plate and wetted with 10% FBS solution for 90 min at 37 C. The FBS solution was removed and samples were washed three times with PBS. Samples were fixed by ethanol for 10 min, and fixed surfaces were washed three times with PBS. Surfaces were blocked with 3% bovine albumin fraction V (BSA, GIBCO, NY) in PBS for 30 min. After washing twice with 10 mM glycine in PBS, we reacted the surfaces with a rabbit polyclonal anti-bovine FN antibody (LB1027, LSL Co., Ltd., Japan) at 1:500 dilution for 30 min at 37 C. The surfaces were washed three times with 0.1% BSA in PBS and then incubated for 30 min with 1:200 dilution of fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG antibody (55662, ICN Pharmaceuticals, Inc., US). Subsequently, the surfaces were again washed three times with 0.1% BSA in PBS. The FITC-labeled FN on the surfaces was observed using a fluorescent microscope (IX-70, Olympus, Japan) and Fluorescence Spectrophotometer (F-2500, HITACHI, Japan) with an excitation wavelength of 488 nm, and the intensity at the maximum emission wavelength of 530 nm was measured. 2.3. Surface characterization Chemical composition of the surface layer was determined by XPS (ESCALAB 250, Thermo-VG, England) employing monochromate Al Ka-X-ray source (1486.6 eV, 150 W). The overview spectra were taken between 0 and 1000 eV with an energy step of 1.0 eV, while the detailed spectra of the peaks of interest (O ls and C ls) were recorded with an energy step of 0.1 eV. Band deconvolution was performed to separate the multiple compo-
nents observed. The band shape used was a mixture of Gaussian and Lorentz functions (L/G = 0.3). Raman spectra were obtained with a Raman microspectrometer (LabRAM, Jobin–Yvon, France) at room temperature. A He–Ne laser (632.817 nm) was used as the excitation source. The spectral resolution was 1 cm 1. 3. Results and discussion 3.1. Fluorescence antibody technique Fig. 1 presents an immunofluorescence micrograph of an Ar+-irradiated PLLA surface at 50 keV with a fluence of 1 · 1014 ions/cm2 after adsorbed FN labeling with FITC. FN labeled with FITC was excited at 488 nm; the dark region is the non-irradiated area, and the bright region is the Ar+-irradiated area. This result indicated that FN adsorption on the irradiated surface increased relative to the non-irradiated surface. Fig. 2 depicts a bar diagram
Fig. 1. Immunofluorescence micrograph of Ar+-irradiated PLLA surface at an energy of 50 keV with a fluence of 1 · 1014 ions/cm2 after adsorbed FN labeling with FITC. The dark region is the non-irradiated area; the bright region is the Ar+-irradiated area. Bar = 100 lm.
Intensity ratio ( I (sample) / I (control))
52
2.5
2.0
1.5
1.0
0.5
0.0 Control
1×1014 ions/cm2
1×1015 ions/cm2
Fig. 2. Graph of fluorescence intensity of non-irradiated and Ar+irradiated surface of PLLA measured by fluorometer after adsorbed FN labeling with FITC.
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
53
Table 1 Composition (in at.%) of C ls and O ls peaks of XPS spectra C ls
O ls
COO (289 eV) C@O (288 eV) C–O (286.5 eV) C–C (285 eV) Amorphous carbon (284.3 eV) O–C (533.8 eV) O@C (532.6 eV)
Samples Original 1 · 1014 cm 1 · 1015 cm
0 7.5 2.7
2 2
18.2 5.7 4.3
17.6 11.6 10.5
28.9 31.4 29.5
of fluorescence intensity of the non-irradiated and Ar+irradiated surface measured by a spectrofluorometer after adsorbed FN labeling with FITC. Considering the change of surface morphology by Ar+ irradiation, the surface area was measured using the atomic force microscope and the fluorescence intensity was corrected using surface area value. The fluorescence intensity of the irradiated surfaces increased compared with the non-irradiated surface. FN adsorption was maximum for Ar+ irradiation at 50 keV with a fluence of 1 · 1014 ions/cm2. These results demonstrated that ion-beam irradiation increase FN adsorption on the modified surface. 3.2. Surface characterization Table 1 lists the chemical compositions of non-irradiated PLLA and Ar+-irradiated PLLA layers derived from analyses of the C 1s and O 1s peaks of the XPS spectra. The spectrum of the non-irradiated PLLA contains three peaks at 285, 286.5 and 288 eV; these are assigned to the
0 19.8 38.9
17.7 9.4 7.3
17.5 14.5 6.8
C–H group, C–O group and C@O group. After Ar+ irradiation with a fluence of 1 · 1014 ions/cm2, new components appeared at 284.3 and 289 eV; these were assigned to the amorphous carbon and COO group [5]. The C@O and COO groups were destroyed by Ar+ irradiation with a fluence of 1 · 1015 ions/cm2. The oxygen percentage was also decreased, and the amorphous carbon was consequently increased [6]. Fig. 3 illustrates the Raman spectra of non-irradiated PLLA (a), Ar+-irradiated PLLA at an energy of 50 keV with fluences of 1 · 1014 (b) and 1 · 1015 ions/cm2 (c). Raman spectra of the ion-irradiated PLLA at an energy of 50 keV with a fluence of 1 · 1015 ions/cm2 contained two new Raman active modes, a peak centered at 1600 cm 1 and a peak centered 1360 cm 1. The higher wave number peak is characteristic of polycrystalline graphite (G band) or amorphous carbon with graphitic bonding. The lower wave number peak seems to be in agreement with that observed in disordered graphite (D band) [7]. These results indicated that Ar+ irradiation at a fluence of 1 · 1015 ions/cm2 destroyed the original surface structure and produced an amorphous carbon phase.
Intensity (arb. unit)
4. Conclusion
(c)
(b)
(a)
2000
1500 Raman Shift
1000 (cm-1)
Fig. 3. Raman spectra of non-irradiated PLLA (a), Ar+-irradiated PLLA at an energy of 50 keV with fluences of 1 · 1014 (b) and 1 · 1015 ions/cm2 (c).
We investigated cell-adhesion surfaces induced by ionbeam irradiation into PLLA by means of XPS, Raman, and a fluorescence antibody technique. XPS and Raman studies demonstrated that the original surface structure of PLLA was destroyed by Ar+ irradiation at 50 keV with a fluence of 1 · 1014 ions/cm2, which subsequently induced the formation of new functional groups such as C@O and COO . Then C@O and COO groups were destroyed by Ar+ irradiation with a fluence of 1 · 1015 ions/cm2, which decreased the oxygen percentage and consequently increased amorphous carbon. The fluorescence antibody technique indicated that the amount of protein adsorption was relative to new radical and functional groups. Considering that the amount of protein adsorption was significantly reduced by Kr+ irradiation at 150 keV with a fluence of 1 · 1015 ions/cm2, new functional groups and radical are important factor for protein adsorption. We concluded that large increases in protein adsorption and subsequent cell adhesion were correlated with production of new functional groups by ion-beam irradiation.
54
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
References [1] Z. Ma, C. Gao, J. Ji, J. Shen, Eur. Polym. J. 38 (2002) 2279. [2] T. Yotoriyama, T. Tsukamoto, Y. Suzuki, M. Iwaki, Nucl. Instr. and Meth. B 206 (2003) 527. [3] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, Biomaterials 16 (1995) 1057.
[4] C. Satrio, C. Scifo, G. Marletta, Nucl. Instr. and Meth. B 166–167 (2000) 782. [5] E. Kiss, I. Bertoti, I. Vargha-Butler, J. Colloid Interface Sci. 245 (2002) 91. [6] A. Nakao, M. Kaibara, M. Iwaki, Y. Suzuki, M. Kusakabe, Appl. Surf. Sci. 100–101 (1996) 112. [7] G. Kister, G. Cassanas, M. Vert, Polymer 39 (1998) 267.
Nuclear Instruments and Methods in Physics Research B 242 (2006) 51–54 www.elsevier.com/locate/nimb
Analysis of cell-adhesion surface induced by ion-beam irradiation into biodegradable polymer Tasuku Yotoriyama a,*, Aiko Nakao b, Yoshiaki Suzuki b, Takeyo Tsukamoto a, Masaya Iwaki b a
Graduate School, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan b Beam Application Team, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Available online 28 November 2005
Abstract Biodegradable polymers such as poly (L-lactic acid) (PLLA) are widely used to construct cell scaffolds for tissue engineering. We previously reported that cell attachment on a PLLA surface was improved by ion-beam irradiation. In this report, we attempted to characterize the cell-adhesion surface induced by ion-beam irradiation into PLLA by means of X-ray photoemission spectroscopy (XPS), Raman scattering measurement (Raman), and a fluorescence antibody technique. The substrates used were PLLA films; Ar+ ion-beam irradiation was performed at an energy of 50 keV with fluences of 1 · 1014 and 1 · 1015 ions/cm2. The results of XPS and Raman studies indicated the formation of new carbon structures and new functional groups by ion-beam irradiation. The protein adsorption was improved by Ar+ irradiation. We concluded that the cell-adhesion surface was induced as a consequence of production of new functional groups by ion-beam irradiation. 2005 Published by Elsevier B.V. PACS: 87.68.+z; 87.64.Gb; 81.65.Lp Keywords: Ion implantation; Biodegradable polymer; Cell adhesion; XPS
1. Introduction Aliphatic polyesters based on glycolic and lactic acids are the most investigated and advanced bioresorbable polymers used to make biomedical and pharmaceutical devices. Poly (L-lactic acid) (PLLA) is a biodegradable polymer that hydrolyzes into a low molecule when implanted in a body. The hydrolysis-generated reaction products are non-toxic to the body since they are broken down into carbon dioxide and water by the bodyÕs metabolism cycle. However, the lack of functional groups
* Corresponding author. Address: Beam Application Team, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: +81 48 467 9359; fax: +81 48 462 4623. E-mail address: [email protected] (T. Yotoriyama).
0168-583X/$ - see front matter 2005 Published by Elsevier B.V. doi:10.1016/j.nimb.2005.08.201
for covalent cell-recognition signal molecules to promote cell attachment has limited PLLAÕs further application in the field of tissue engineering. It is necessary to introduce some reactive groups on the chemically inert polymer surface [1]. We previously demonstrated that cell attachment on biodegradable polymer was improved by ion-beam irradiation [2]. This paper focuses to characterize the cell-adhesion surface induced by ion-beam irradiation into biodegradable polymer using X-ray photoemission spectroscopy (XPS) and Raman scattering measurement (Raman). Cell adhesion is mediated by the formation of an adsorbed interfacial protein layer coming from the complex serum medium [3,4]. There are several adhesion molecules in the serum, but we investigated fibronectin (FN) adsorption on an ion-beam irradiated surface using a fluorescence antibody technique.
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
2. Material and methods 2.1. Sample preparation The substrates used were 3 cm2, 200-lm-thick Poly-Llactic acid (PLLA) sheets (LACTY; Shimadzu, Japan). We performed Ar+ ion-beam irradiation into PLLA at an energy of 50 keV with fluences of 1 · 1014 and 1 · 1015 ions/cm2 at room temperature using a RIKEN 200 kV low-current implanter. The beam current was kept below 0.1 lA/cm2 to prevent heating the specimen, and the target chamber was maintained at a base pressure of 10 4 Pa during ion-beam irradiation. Non-irradiated PLLA sheets were used as controls in all investigations. 2.2. Fluorescence antibody technique Protein adsorption on an ion-irradiated surface was investigated using a fluorescence antibody technique. Fetal bovine serum (FBS, Sanko-Junyaku Co., Japan) was diluted with DulbeccoÕs phosphate-buffered saline solution (PBS, pH = 7.4) to a final concentration of 10%. Ion-irradiated PLLA sheets were cut into 1 cm2. Non-irradiated PLLA was used as the control. Samples were placed into a 24-well tissue culture plate and wetted with 10% FBS solution for 90 min at 37 C. The FBS solution was removed and samples were washed three times with PBS. Samples were fixed by ethanol for 10 min, and fixed surfaces were washed three times with PBS. Surfaces were blocked with 3% bovine albumin fraction V (BSA, GIBCO, NY) in PBS for 30 min. After washing twice with 10 mM glycine in PBS, we reacted the surfaces with a rabbit polyclonal anti-bovine FN antibody (LB1027, LSL Co., Ltd., Japan) at 1:500 dilution for 30 min at 37 C. The surfaces were washed three times with 0.1% BSA in PBS and then incubated for 30 min with 1:200 dilution of fluorescein isothiocyanate (FITC)conjugated goat anti-rabbit IgG antibody (55662, ICN Pharmaceuticals, Inc., US). Subsequently, the surfaces were again washed three times with 0.1% BSA in PBS. The FITC-labeled FN on the surfaces was observed using a fluorescent microscope (IX-70, Olympus, Japan) and Fluorescence Spectrophotometer (F-2500, HITACHI, Japan) with an excitation wavelength of 488 nm, and the intensity at the maximum emission wavelength of 530 nm was measured. 2.3. Surface characterization Chemical composition of the surface layer was determined by XPS (ESCALAB 250, Thermo-VG, England) employing monochromate Al Ka-X-ray source (1486.6 eV, 150 W). The overview spectra were taken between 0 and 1000 eV with an energy step of 1.0 eV, while the detailed spectra of the peaks of interest (O ls and C ls) were recorded with an energy step of 0.1 eV. Band deconvolution was performed to separate the multiple compo-
nents observed. The band shape used was a mixture of Gaussian and Lorentz functions (L/G = 0.3). Raman spectra were obtained with a Raman microspectrometer (LabRAM, Jobin–Yvon, France) at room temperature. A He–Ne laser (632.817 nm) was used as the excitation source. The spectral resolution was 1 cm 1. 3. Results and discussion 3.1. Fluorescence antibody technique Fig. 1 presents an immunofluorescence micrograph of an Ar+-irradiated PLLA surface at 50 keV with a fluence of 1 · 1014 ions/cm2 after adsorbed FN labeling with FITC. FN labeled with FITC was excited at 488 nm; the dark region is the non-irradiated area, and the bright region is the Ar+-irradiated area. This result indicated that FN adsorption on the irradiated surface increased relative to the non-irradiated surface. Fig. 2 depicts a bar diagram
Fig. 1. Immunofluorescence micrograph of Ar+-irradiated PLLA surface at an energy of 50 keV with a fluence of 1 · 1014 ions/cm2 after adsorbed FN labeling with FITC. The dark region is the non-irradiated area; the bright region is the Ar+-irradiated area. Bar = 100 lm.
Intensity ratio ( I (sample) / I (control))
52
2.5
2.0
1.5
1.0
0.5
0.0 Control
1×1014 ions/cm2
1×1015 ions/cm2
Fig. 2. Graph of fluorescence intensity of non-irradiated and Ar+irradiated surface of PLLA measured by fluorometer after adsorbed FN labeling with FITC.
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
53
Table 1 Composition (in at.%) of C ls and O ls peaks of XPS spectra C ls
O ls
COO (289 eV) C@O (288 eV) C–O (286.5 eV) C–C (285 eV) Amorphous carbon (284.3 eV) O–C (533.8 eV) O@C (532.6 eV)
Samples Original 1 · 1014 cm 1 · 1015 cm
0 7.5 2.7
2 2
18.2 5.7 4.3
17.6 11.6 10.5
28.9 31.4 29.5
of fluorescence intensity of the non-irradiated and Ar+irradiated surface measured by a spectrofluorometer after adsorbed FN labeling with FITC. Considering the change of surface morphology by Ar+ irradiation, the surface area was measured using the atomic force microscope and the fluorescence intensity was corrected using surface area value. The fluorescence intensity of the irradiated surfaces increased compared with the non-irradiated surface. FN adsorption was maximum for Ar+ irradiation at 50 keV with a fluence of 1 · 1014 ions/cm2. These results demonstrated that ion-beam irradiation increase FN adsorption on the modified surface. 3.2. Surface characterization Table 1 lists the chemical compositions of non-irradiated PLLA and Ar+-irradiated PLLA layers derived from analyses of the C 1s and O 1s peaks of the XPS spectra. The spectrum of the non-irradiated PLLA contains three peaks at 285, 286.5 and 288 eV; these are assigned to the
0 19.8 38.9
17.7 9.4 7.3
17.5 14.5 6.8
C–H group, C–O group and C@O group. After Ar+ irradiation with a fluence of 1 · 1014 ions/cm2, new components appeared at 284.3 and 289 eV; these were assigned to the amorphous carbon and COO group [5]. The C@O and COO groups were destroyed by Ar+ irradiation with a fluence of 1 · 1015 ions/cm2. The oxygen percentage was also decreased, and the amorphous carbon was consequently increased [6]. Fig. 3 illustrates the Raman spectra of non-irradiated PLLA (a), Ar+-irradiated PLLA at an energy of 50 keV with fluences of 1 · 1014 (b) and 1 · 1015 ions/cm2 (c). Raman spectra of the ion-irradiated PLLA at an energy of 50 keV with a fluence of 1 · 1015 ions/cm2 contained two new Raman active modes, a peak centered at 1600 cm 1 and a peak centered 1360 cm 1. The higher wave number peak is characteristic of polycrystalline graphite (G band) or amorphous carbon with graphitic bonding. The lower wave number peak seems to be in agreement with that observed in disordered graphite (D band) [7]. These results indicated that Ar+ irradiation at a fluence of 1 · 1015 ions/cm2 destroyed the original surface structure and produced an amorphous carbon phase.
Intensity (arb. unit)
4. Conclusion
(c)
(b)
(a)
2000
1500 Raman Shift
1000 (cm-1)
Fig. 3. Raman spectra of non-irradiated PLLA (a), Ar+-irradiated PLLA at an energy of 50 keV with fluences of 1 · 1014 (b) and 1 · 1015 ions/cm2 (c).
We investigated cell-adhesion surfaces induced by ionbeam irradiation into PLLA by means of XPS, Raman, and a fluorescence antibody technique. XPS and Raman studies demonstrated that the original surface structure of PLLA was destroyed by Ar+ irradiation at 50 keV with a fluence of 1 · 1014 ions/cm2, which subsequently induced the formation of new functional groups such as C@O and COO . Then C@O and COO groups were destroyed by Ar+ irradiation with a fluence of 1 · 1015 ions/cm2, which decreased the oxygen percentage and consequently increased amorphous carbon. The fluorescence antibody technique indicated that the amount of protein adsorption was relative to new radical and functional groups. Considering that the amount of protein adsorption was significantly reduced by Kr+ irradiation at 150 keV with a fluence of 1 · 1015 ions/cm2, new functional groups and radical are important factor for protein adsorption. We concluded that large increases in protein adsorption and subsequent cell adhesion were correlated with production of new functional groups by ion-beam irradiation.
54
T. Yotoriyama et al. / Nucl. Instr. and Meth. in Phys. Res. B 242 (2006) 51–54
References [1] Z. Ma, C. Gao, J. Ji, J. Shen, Eur. Polym. J. 38 (2002) 2279. [2] T. Yotoriyama, T. Tsukamoto, Y. Suzuki, M. Iwaki, Nucl. Instr. and Meth. B 206 (2003) 527. [3] J.G. Steele, B.A. Dalton, G. Johnson, P.A. Underwood, Biomaterials 16 (1995) 1057.
[4] C. Satrio, C. Scifo, G. Marletta, Nucl. Instr. and Meth. B 166–167 (2000) 782. [5] E. Kiss, I. Bertoti, I. Vargha-Butler, J. Colloid Interface Sci. 245 (2002) 91. [6] A. Nakao, M. Kaibara, M. Iwaki, Y. Suzuki, M. Kusakabe, Appl. Surf. Sci. 100–101 (1996) 112. [7] G. Kister, G. Cassanas, M. Vert, Polymer 39 (1998) 267.