Langmuir 1999, 15, 1667-1672
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Surface Hydrogelation Using Photolysis of Dithiocarbamate or Xanthate: Hydrogelation, Surface Fixation, and Bioactive Substance Immobilization Y. Nakayama, M. Takatsuka, and T. Matsuda* Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan Received September 4, 1998. In Final Form: December 10, 1998 This article reports a novel technology for photoinduced surface hydrogelation with regional precision. The technology is based on the photochemistry of dithiocarbamate and xanthate groups, both of which are dissociated into highly reactive radical pairs upon ultraviolet (UV) light irradiation. Photoreactive hydrophilic polymers containing dithiocarbamate or xanthate groups were prepared by radical copolymerization of N,N-dimethylacrylamide with vinylbenzyl N,N-diethyldithiocarbamate or 2-(ethylxanthate)ethyl methacrylate, respectively. Upon UV light irradiation, the films of the photoreactive copolymers were converted to water-absorbable cross-linked gels. Higher derivatization of the photoreactive groups and longer irradiation times resulted in higher gel yields and reduced swellability. X-ray photoelectron spectroscopic (XPS) analyses and water contact angle measurements showed that upon UV light irradiation of the photoreactive copolymer cast on a poly(ethylene terephthalate) (PET) film, hydrogels were simultaneously formed and fixed onto the PET film. When a mixture of the photoreactive copolymer with a bioactive substance such as heparin and urokinase was used, prolonged whole-blood-coagulation times for heparin-immobilized surfaces and fibrinolysis for urokinase-immobilized surfaces were observed. A micropatterned hydrogelated surface was prepared with regional precision under UV light irradiation through a photomask. The photochemical surface technology developed here is expected to be useful for conferring potent antithrombogenicity on biomedical devices.
Introduction Since nonionic hydrogels suppress protein adsorption and cell adhesion,1-4 reliable blood compatibility of fabricated biomedical devices, such as blood pumps,5,6 catheters,7-11 and biosensors,12-17 may be achieved by hydrogel formation on blood-contacting surfaces, the usefulness of which can be enhanced by the immobilization of biologically active substances, such as heparin,18-22 * Corresponding author. Telephone: (+81) 6-6833-5012. Fax: (+81) 6-6872-7485. E-mail:
[email protected]. (1) Szycher, M. Biocompatible Polymers, Metals and Composites; Technomic Publ., Co., Inc.: Lancaster, PA, 1983. (2) Park, J. B. Biomaterials Science and Bioengineering, Plenum Press: New York and London, 1984. (3) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985; Vol. 1, p 1. (4) Curtis, A. S. G. The Cell Surface; Its Molecular Role in Morphogenesis; Academic Press: London, 1967. (5) Santin, M.; Motta, A.; Cannas, M. J. Biomed. Mater. Res. 1998, 40, 424. (6) Walker, A. S.; Blue, M. A.; Brandon, T. A.; Emmanual, J.; Guilbeau, E. J. ASAIO J. 1992, 38, M550. (7) Mitchel, J. F.; Shwedick, M.; Alberghini, T. A.; Knibbs, D.; McKay, R. G. Circulation 1994, 90, 1979. (8) Fram, D. B.; Aretz, T.; Azrin, M. A.; Mitchel, J. F.; Samady, H.; Gillam, L. D.; Sahatjian, R.; Waters, D.; McKay, R. G. J. Am. Coll. Cardiol. 1994, 23, 1570-1577. (9) Uyama, T.; Tadokoro, H.; Ikada, Y. Biomaterials 1991, 12, 71. (10) Cox, A. J. Biomaterials 1987, 8, 500. (11) Margules, G. S.; Hunter, C. M.; MacGregor, D. C. Med. Bio. Eng. Comput. 1983, 21, 1. (12) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829. (13) Nakayama, Y.; Zheng, Q.; Nishimura, J.; Matsuda, T. ASAIO J. 1995, 41, M418. (14) Vaidya, R.; Wilkins, E. Med. Eng. Phys. 1994, 16, 416. (15) Linke, B.; Kerner, W.; Kiwit, M.; Pishko, M.; Heller, A. Biosens. Bioelectron. 1994, 9, 151. (16) Nakayama, Y.; Matsuda, T. ASAIO J. 1992, 38, M421. (17) Brinkman, E.; van der Does, L.; Bantjes, A. Biomaterials 1991, 12, 63. (18) Falb, R. D. In Polymers in medicine and surgery; Kronenthal, R. L., Ed.; Plenum Press: New York, 1976; p 77. (19) Bruck, S. D. J. Biomed. Mater. Res. 1972, 6, 173.
urokinase,23-27 and prostaglandins,28-32 entrapped in or released from a hydrogel fixed on blood-contacting surfaces. Although various approaches to prepare hydrogels via chemical or physical cross-linking reactions have been developed,33-41 very few methods have been developed for hydrogel formation on substrate surfaces. (20) Ito, Y.; Sisido, M.; Imanishi, Y. J. Biomed. Mater. Res. 1986, 20, 1017. (21) Vulic, I.; Okano, T.; Kim, S. W. J. Polym. Sci., Polym. Chem. Ed. 1988, 26, 381. (22) Mori, Y.; Nagaoka, S.; Masubuchi, Y. Trans. Am. Soc. Artif. Organs 1988, 26, 381. (23) Liu, L. S.; Ito, Y.; Imanishi, Y. Biomaterials 1991, 12, 545. (24) Watanabe, S.; Shimizu, Y.; Teramatsu, T.; Murachi, T.; Hino, T. J. Biomed. Mater. Res. 1981, 15, 553. (25) Kusserow, B. K.; Larrow, R. W.; Nichols, J. E. Trans. Am. Soc. Artif. Intern. Organs 1973, 19, 8. (26) Senatore, F.; Bernath, F.; Meisner, K. J. Biomed. Mater. Res. 1986, 20, 177. (27) Oshiro, T.; Lin, M. C.; Kambayashi, J.; Mori, T. Methods Enzymol. 1988, 137, 529. (28) Greisler, H. P.; Klosak, J. J.; McGurrin, J. F.; Endean, E. D.; Ellinger, J.; Pozar, J. D.; Henderson, S. C.; Kim, D. U. J. Cardiovasc. Surg. 1990, 31, 640. (29) Kim, S. W.; Jacobs, H.; Lin, J. Y.; Nojori, C.; Okano, T. Ann. N.Y. Acad. Sci. 1987, 516, 116. (30) McRea, J. C.; Ebert, C. D.; Kim, S. W. Trans. Am. Soc. Artif. Intern. Organs 1981, 27, 511. (31) Akashi, M.; Takeda, S.; Miyazaki, T.; Yashima, E.; Miyauchi, N. J. Bioact. Compat. Polym. 1989, 4, 4. (32) Ebert, C. D.; Lee, E. S.; Kim, S. W. J. Biomed. Mater. Res. 1982, 16, 629. (33) Otterbritte, R. M., Huang, S. J., Park, K., Eds. Hydrogels and biodegradable polymers for bioapplications; ACS Symposium Series 627; American Chemical Society: Washington, DC, 1996. (34) Zekorn, T. D.; Horcher, A.; Mellert, J.; Siebers, U.; Altug, T.; Emre, A.; Hahn, H. J.; Federlin, K. Int. J. Art. Organs 1996, 19, 251. (35) Peppas, N. A.; Sahlin, J. J. Biomaterials 1992, 17, 1553. (36) Corkhill, P. H.; Hamilton, C. J.; Tighe, B. J. Biomaterials 1989, 10, 3. (37) Graham, N. B.; McNeill, M. E. Biomaterials 1984, 5, 27. (38) Ichimura, K.; Watanabe, S. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 1419. (39) Ichimura, K. J. Polym. Sci., Chem. Ed. 1984, 22, 2817. (40) Chujo, Y.; Sada, K.; Saegusa, T. Macromolecules 1990, 23, 2693.
10.1021/la981169h CCC: $18.00 © 1999 American Chemical Society Published on Web 02/06/1999
1668 Langmuir, Vol. 15, No. 5, 1999 Scheme 1. Photoinduced Radical Generating Reactions of Alkyl N,N-Diethyldithiocarbamate and Alkyl O-Ethylxanthate
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surface. Heparin- and urokinase-releasing hydrogelated surfaces and micropatterned hydrogelated surfaces are demonstrated in the latter part of this article. Materials and Methods
We have been involved in developing surface photochemical process technologies that enable the simultaneous formation and fixation of a hydrogel onto polymer surfaces of biomedical devices. Such technologies have been realized through the photochemistry of photodimerizable groups,16,42-45 such as cinnamate, coumarin, and thymine base, and/or photoinduced radical generating groups,14,46-48 such as phenyl azide and benzophenone groups. In this article, we report a novel photochemically driven surface hydrogelation technology using dithiocarbamate or xanthate as the radical generating group for improving the blood compatibility of fabricated devices. Dithiocarbamate and xanthate derivatives, which have been utilized as vulcanization accelerators for rubbers49-51 and also photopolymerization initiators52-56 in photolithography,57-59 are known to dissociate into highly reactive radical pairs upon ultraviolet (UV) light irradiation or heating (Scheme 1).55,59 The generated alkyl and alkylthiyl radicals trigger complex reactions including radical recombination, chain transfer, and addition reaction to radical polymerizable vinyl monomers. In the first part of this article, the preparation of photoreactive hydrophilic polymers having dithiocarbamate or xanthate is described. The recombination reaction between polymer radicals generated by the photoreactive groups of the polymers upon UV light irradiation produces hydrogels. In addition, the recombination reaction between the polymer radical and a substrate radical generated by chain transfer reaction from the polymer radical to the substrate may fix the hydrogel formed onto the substrate (41) Sawhney, A. S.; Pathak, C. P.; Hubbell, J. A. Macromolecules 1993, 26, 581. (42) Nakayama, Y.; Matsuda, T. J. Polym. Sci., Polym. Chem. Ed. 1993, 31, 977. (43) Nakao, H.; Matsuda, T.; Nakayama, Y.; Hara, Y.; Saishin, M. ASAIO J. 1993, 39, M257. (44) Chung, D. J.; Matsuda, T. Private communication. (45) Matsuda T.; Moghaddam M. J.; Miwa H.; Sakurai, K.; Iida, F. ASAIO J. 1992, 38, M154. (46) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1995, 29, 749. (47) Nakayama, Y.; Matsuda, T. ASAIO J. 1995, 41, M374. (48) Nakayama, Y.; Matsuda, T. J. Biomed. Mater. Res. (Appl. Biomater.), submitted for publication. (49) Koch, H. P. J. Chem. Soc. 1949, 401. (50) Ebich, Y. R.; Biba, A. D.; Blokh, G. A.; Kremlev, M. M. Kim. Tekhnol. (Kharkov) 1971, 21, 112. (51) Lawrence, J. P. U.S. Patent US 4017489, 1977. (52) Otsu, T.; Yoshida, M., Makromol. Chem., Rapid Commun. 1982, 3, 127. (53) Otsu, T.; Yoshida, M.; Tazaki, T. Makromol. Chem., Rapid Commun. 1982, 3, 133. (54) Otsu, T.; Yoshida, M. Polym. Bull. (Berlin) 1982, 7, 197. (55) Otsu, T.; Matsunaga, T.; Doi, T.; Matsumoto, A. Eur. Polym. J. 1995, 31, 67. (56) Nakayama, Y.; Matsuda, T. Macromolecules 1996, 29, 8622. (57) Okawara, M.; Yamashita, N.; Ishiyama, K.; Imoto, E. Kogyo Kagaku Zasshi 1963, 66, 1383. (58) Okawara, M.; Nakai, T.; Otsuji, Y.; Imoto, E. J. Org. Chem. 1965, 30, 2025. (59) Okawara, M.; Nakai, T.; Imoto, E. Kogyo Kagaku Zasshi 1965, 68, 582.
General Methods. All 1H NMR spectra were recorded in DMSO-d6 with a 270 MHz NMR spectrometer (JEOL, JNMJX-270, Tokyo, Japan) using tetramethylsilane (0 ppm) as the internal standard at room temperature. Gel permeation chromatographic (GPC) analyses in chloroform were carried out with a HLC-8020 instrument (Tosoh, Tokyo, Japan, at Osaka National Research Institute, Osaka, Japan) (column: Tosoh TSKgel G5000HXL, G4000HXL and G3000HXL). The columns were calibrated with narrow weight distribution poly(ethylene glycol) standards. X-ray photoelectron spectroscopy (XPS) was performed with an ESCA 750 (Shimadzu, Kyoto, Japan) using a magnesium anode (Mg KR radiation) at room temperature and 2 × 10-7 Torr (8 kV, 20 mA) at the takeoff angle of 90° connected to an ESCAPAC-760 data processor. Static contact angles with deionized water were measured with a contact angle meter (Kyowa Kaimen Kagaku Co., Ltd., Tokyo, Japan) at 25 °C. Atomic force microscopic (AFM) images were obtained on a NanoScope IIIa (Digital Instruments, Inc., Santa Barbara, CA) using a Si3N4 cantilever with a spring constant of 0.12 N/m. AFM images (400 × 400 pixels) were obtained using the “contact mode”. UV/vis absorption spectra were recorded using a JASCO U-best 30 spectrophotometer (Tokyo, Japan). Materials. Vinylbenzyl chloride (mixture of m and p isomers) was purchased from Tokyo Chemical Ind. Ltd. (Tokyo, Japan). 2,2′-Azobis(isobutyronitrile) (AIBN) was recrystallized twice from methanol. Heparin sodium salt (1.8 kDa) was purchased from Wako Pure Chem. Ind. Ltd. (Osaka, Japan). High-molecularweight human urokinase (54 kDa) with specific activity of 1.5 × 105 IU/mg was kindly supplied by Unichika Co. (Kyoto, Japan). Solvents and other reagents, all of which are of special reagent grade, were purchased from Wako and used after conventional purification. Synthesis of Vinylbenzyl N,N-Diethyldithiocarbamate (1). This compound was prepared according to a previously reported procedure.60 Briefly, 20 mL of an ethanolic solution of vinylbenzyl chloride (12.7 g, 0.083 mol) was added dropwise to an ethanolic solution (80 mL) of sodium N,N-diethyldithiocarbamate trihydrate (22.5 g, 0.1 mol) at 0 °C. After being stirred for 24 h at room temperature, the reaction mixture was poured into a large amount of water and extracted with diethyl ether. The organic phase was washed with water and dried with Na2SO4, filtered, and evaporated under vacuum. The residue was recrystallized three times from methanol; the yield of vinylbenzyl N,N-diethyldithiocarbamate (1) is 20.1 g (91.3%). Synthesis of Poly(N,N-dimethylacrylamide-co-vinylbenzyl N,N-diethyldithiocarbamate) (2). A typical procedure for preparation is as follows. A glass tube containing a mixture of N,N-dimethylacrylamide (DMAAm) (2.0 g, 20.2 mmol), vinylbenzyl N,N-diethyldithiocarbamate (0.30 g, 1.13 mmol), AIBN (molar ratio of [monomer]/[initiator] ) 152), and N,N-dimethylformamide (DMF) (7.6 mL) was sealed under reduced pressure after three freeze-pump-thaw cycles. After shaking of the sample at 60 °C for 24 h, the precipitate obtained by the addition of a large amount of diethyl ether was separated from the solution by filtration. Reprecipitation was carried out three times in a DMF-diethyl ether system. The last precipitate was dried under vacuum and stored in a dark desiccator. The yield of poly(N,Ndimethylacrylamide-co-vinylbenzyl N,N-diethyldithiocarbamate) (2) was 2.05 g (89%). The molecular weight was estimated by GPC analysis: Mn ) 1.08 × 104. The content of dithiocarbamate groups was 6.0 mol %, as determined by 1H NMR measurements. Synthesis of 2-(Ethylxanthate)ethyl Methacrylate (3).56 2-Bromoethyl methacrylate (10 g, 52 mmol) and ethylxanthatic acid potassium salt (8 g, 50 mmol) were dissolved in 30 mL of acetone. After being refluxed for 2 h, the reaction mixture was poured into a large amount of water and then extracted with diethyl ether. The organic phase was washed with water, dried (60) Otsu, T.; Yamashita, K.; Tsuda, K. Macromolecules 1986, 19, 287.
Photoinduced Surface Hydrogelation with Na2SO4, filtered, and evaporated under vacuum. The residue was distilled (bp 94-103 °C/0.3 mmHg); the yield of 2-(ethylxanthate)ethyl methacrylate (3) was 77%. 1H NMR (DMSO-d6): δ 6.03 (s, 1H, -CHdCH2), 5.68 (s, 1H, -CHdCH2), 4.65 (q, 2H, -CH2CH3, J ) 7.12 Hz), 3.46 (t, 2H, -OCH2CH2-, J ) 6.1 Hz), 3.44 (t, 2H, -SCH2CH2-, J ) 6.1 Hz), 1.88 (s, 3H), 1.37 (t, 3H, -CH2CH3, J ) 7.02 Hz). Synthesis of Poly[N,N-dimethylacrylamide-co-2-(ethylxanthate)ethyl methacrylate] (4). This copolymer was prepared by radical copolymerization of DMAAm with 2-(ethylxanthate)ethyl methacrylate (3) in DMF as for copolymer (2) (data in the text). Photogelation. A DMF solution (0.2 mL) of the photoreactive copolymer (2 or 4) (40 mg; Ws, weight of the solid content) was poured on a circular glass coverslip (diameter, 15 mm, Matsunami Glass Ind., Ltd., Osaka, Japan) and then dried for 3 h under heating at 80 °C in air. The resultant film was UV light-irradiated for a predetermined length of time. The irradiation was conducted using a 500 W xenon lamp (UXL-500D, USHIO, Tokyo, Japan) through a Pyrex cooler, and the wavelength of illumination (λ > 260 nm) was selected with the aid of cutoff filter (UV-27, Toshiba, Tokyo, Japan). The light intensity was measured with a photometer (1.2 mW/cm2, UVR-1, TOPCON, Tokyo, Japan). The disk-shaped gel formed was repeatedly rinsed with DMF, allowed to equilibrate with deionized water for 24 h at room temperature, and then weighed (Ww) after the excess water was carefully swabbed away. The gel was dried under vacuum for 24 h at room temperature and weighed (Wg). The gel yield was calculated as Wg/Ws × 100. The degree of swelling was calculated as (Ww Wg)/Wg. As the data of the gel yield and degree of swelling were reproducible [n ) 5, standard deviation (SD) < 5%], only the average values are reported. Surface Hydrogelation. A phosphate-buffered saline (PBS) solution of the photoreactive copolymers (2 or 4, 5 w/v %) with or without heparin or urokinase (0.5 w/v %) was coated onto a poly(ethylene terephthalate) (PET) film (Bellco Glass Inc., Vineland, NJ) and dried for 12 h at room temperature. The treated PET film was UV light-irradiated as described above, washed with PBS, and then dried. Two-Dimensional Micropatterning. Micropatterning of surface hydrogelation was carried out using a projection mask with a lattice pattern of 20 µm width (Hirai Co., Ltd., Osaka, Japan). A PET sheet (diameter, 14 mm; thickness, 200 µm; Wako), cast with the photoreactive copolymer (2) from its aqueous solution (5 w/v %), was allowed to come in tight contact with the mask. The treated PET sheet was UV light-irradiated as described above through the mask and then thoroughly washed with deionized water. Measurements of Heparin and Urokinase Release. The concentration of heparin released into a PBS solution from a hydrogel fixed onto a PET sheet at 37 °C was determined spectrophotometrically using the toluidine blue-metachromatic shift assay.61 The activity of urokinase released into a PBS solution from hydrogel at 37 °C was determined fluorometrically using the glutaryl-Gly-Arg-4-methylcoumarin amide as a synthetic substrate for urokinase.62 Fibrin Dissolution Experiment. Dissolution of fibrin by urokinase-immobilized hydrogels on a PET film was observed using conventional fibrin plate method.63 Blood Coagulation Experiment. The inner surface of a poly(propylene) (PP) tube was fixed with hydrogel with or without heparin. Human blood samples collected from healthy volunteers were each poured into the tubes. The Lee-White whole blood coagulation time was measured at 37 °C.64 Platelet Adhesion on Hydrogelated Surfaces. PET sheets coated with hydrogels with or without heparin were placed at the bottom of a 24-well tissue culture dish (Corning Lab. Sci. Co., (61) Smith, P. K.; Mallia, A. K.; Mermanson, G. T. Trans. Am. Soc. Artif. Intern. Organs 1980, 36, 466. (62) Morita, T.; Kato, H.; Iwanaga, S.; Takeda, K.; Kimura, T.; Sakakibara, S. J. Biochem. 1977, 82, 1495. (63) Kanai, I., Kanai, M., Eds. In Rinsho Kensahou Teiyou; Kanahara Press: Tokyo, 1978; Vol. VI, pp 105-106. (64) MacAulay, M. A.; Frisch, C. R.; Klionsky, B. L. Tech. Bull. Regist. Med. Technol. 1968, 38, 223.
Langmuir, Vol. 15, No. 5, 1999 1669 Chart 1. Chemical Structures of the Photoreactive Hydrophilic Copolymers: 2, Poly(N,N-dimethylacrylamide-co-vinylbenzyl N,N-diethyldithiocarbamate); 4, Poly[N,N-dimethylacrylamide-co-2-(ethylxanthate)ethyl methacrylate]
Corning, NY). After incubation for 60 min at 37 °C in a humid atmosphere of 95% air and 5% CO2 in 1 mL of platelet-rich plasma (PRP, 6.6 × 105 cells/mL), which was prepared by centrifugation of citrated human whole blood at 900 rpm for 15 min, the sheets were fixed in 3% glutaraldehyde, critical-point-dried, and sputtercoated with platinum. The number of platelets adhered to the surfaces was determined under a scanning electron microscope (SEM, S-4000, Hitachi, Tokyo, Japan).
Results and Discussion Preparation of Photoreactive Copolymer. Two kinds of photoreactive hydrophilic copolymers, 2 and 4, whose chemical structures are shown in Chart 1, were prepared by radical copolymerization of N,N-dimethylacrylamide (DMAAm) with vinylbenzyl N,N-diethyldithiocarbamate (1) or 2-(ethylxanthate)ethyl methacrylate (3), respectively. Table 1 summarizes the preparation conditions and the compositions of the photoreactive copolymers 2 and 4. The content of dithiocarbamate groups in copolymer 2 ranged from approximately 4 to 29 mol % and that of xanthate groups in copolymer 4 ranged from approximately 3 to 18 mol %. The copolymers with a lower content of the photoreactive groups were soluble in water. These water-soluble copolymers (runs 1, 2, 5, and 6 in Table 1) were used for surface hydrogelation. Photogelation and Swelling Properties. Upon UV light irradiation, films of dithiocarbamate- or xanthatederivatized photoreactive copolymers, which were prepared by casting on glass coverslips of their DMF solutions, were not dissolved in water and DMF but swelled in their solvents. Irradiation time-dependent UV absorption changes of the photoreactive copolymer films on a quartz crystal plate are shown in Figure 1. Upon UV light irradiation of the dithiocarbamate-derivatized copolymer (2; dithiocarbamate content, 28.6 mol %) for 5 min, the absorption peaks at about 280 and 250 nm, attributed to the characteristic peaks of N-CdS and SdC-S conjugated systems of the dithiocarbamate groups, respectively,62 disappeared, and an absorption peak at about 275 nm, probably due to decomposed derivatives of the dithiocarbamate groups, was newly detected (Figure 1a). This absorption peak disappeared upon washing with methanol, strongly suggesting that a low-molecularweight product derived from the dithiocarbamate groups was washed away. On the other hand, upon UV light irradiation of the xanthene-derivatized copolymer (4; xanthate content, 17.5 mol %), the absorption peak at 280 nm, which is attributed to the SdC-S conjugated system of xanthates,62 decreased slowly with irradiation time without the appearance of any new absorption peak (Figure 1b). At about 0.5-h-irradiation, the xanthate peak
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Table 1. Syntheses of Poly(N,N-dimethylacrylamide-co-vinylbenzyl N,N-diethyldithiocarbamate) (2) and Poly(N,N-dimethylacrylamide-co-2-(ethylxanthate)ethyl methacrylate) (4)a monomer run
no.
feed (mol %)
1 2 3 4 5 6 7 8
1 1 1 1 3 3 3 3
3.3 5.3 10.0 20.0 3.0 5.0 10.0 20.0
copolymer no.
conversion (%)
Mnb
photoreactive group contentc (mol %)
solubility in water
2 2 2 2 4 4 4 4
90 89 87 77 93 96 91 92
14 300 10 800 11 500 11 100 15 000 11 500 13 300 37 000
3.6 6.0 12.9 28.6 3.2 5.2 10.2 17.5
soluble soluble insoluble insoluble soluble soluble insoluble insoluble
a Total monomer concentration ) 300 g/L in N,N-dimethylformamide; initiator concentration ) [monomer]/[initiator] ) 100, 60 °C, 24 h in sealed tubes. b Determined by GPC measurements (calibrated with poly(ethylene glycol); eluent, chloroform). c Content of photoreactive groups determined by 1H NMR analyses.
Figure 1. Absorption spectral changes of (a) dithiocarbamatederivatized photoreactive copolymer (2; dithiocarbamate content, 28.6 mol %) in film and (b) xanthate-derivatized photoreactive copolymer (4; xanthate content, 17.5 mol %) in film as a function of UV light irradiation time with λ > 260 nm. Table 2. Photogelation of Photoreactive Copolymers and Swelling Properties photoreactive photoreactive group content irradiation gel yield degree of copolymer (mol %) timea (min) (%) swellingb 2 2 2 2 2 2 2 4 4
3.6 6.0 6.0 6.0 6.0 12.9 28.6 10.2 17.5
10 5 10 15 30 10 10 10 10
11.4 14.2 33.4 45.5 58.0 45.5 89.7 33.3 75.3
25.2 29.6 24.3 19.5 15.4 6.3 0.9 11.3 2.6
a Light intensity is 1.2 mW/cm2. b Grams of adsorbed H O per 2 gram of dry gel.
was almost absent. This indicates that the photodecomposition of xanthate groups was slower than that of the dithiocarbamate groups. Gel yields and degrees of swelling of the resultant gels were determined after equilibration with water (Table 2). Both increases in irradiation time and the content of the photoreactive groups in the copolymers tended to an increased gel yield but reduced water-swellability. Surface Hydrogelation. A schematic diagram of the sequences of the preparation method of a hydrogelated
Figure 2. A schematic diagram of the preparation of (A) a hydrogel-fixed surface and (B) a hydrogel-fixed surface immobilized with a bioactive substance, such as heparin and urokinase, and its overlayering with other hydrogel, where the lower hydrogel layer functions as a matrix layer to immobilize a bioactive substance and the upper hydrogel layer functions as a protective layer to regulate the release of an immobilized bioactive substance.
surface on a polymer film is illustrated in Figure 2A. At first, a photoreactive copolymer was coated onto a poly(ethylene terephthalate) (PET) film from its aqueous or DMF solution and then air-dried (step I in Figure 2A). Subsequently, the cast PET film was UV light-irradiated and then rinsed with water and methanol (step II in Figure 2A). The surface compositional characterization of the PET film coated with the dithiocarbamate-derivatized copolymer (2; dithiocarbamate content, 6.0 mol %) before and after UV light irradiation was carried out using XPS measurements. The O/C, N/C, and S/C elemental ratios, calculated from the respective peak areas of the C1S, O1S, N1S, and S1S signals, are summarized in Table 3. There was little difference in these elemental ratios between nontreated PET films and coated PET films subjected to washing without irradiation, indicating that the photoreactive copolymer on the surface was removed by washing. On the other hand, upon UV light irradiation, the O/C ratio decreased from 0.33 (PET) to 0.16 but the N/C and S/C ratios increased from zero (PET) to 0.16 and 0.01, respectively. These values were very close to the respective theoretical values for the photoreactive copolymer used (O/C ) 0.17, N/C ) 0.18, S/C ) 0.02). Little appreciable spectral change was observed after extensive washing with water and methanol. The irradiated PET
Photoinduced Surface Hydrogelation
Langmuir, Vol. 15, No. 5, 1999 1671
Table 3. Surface Chemical Compositions and Water Contact Angles of PET Films Coated with the Photoreactive PDMAAma before and after UV Light Irradiation elemental ratiob
c
N/C
contact angle (deg)c
sample
O/C
advancing
receding
nontreated PET coated PET without UV with UV
0.33 (0.40)d
0 (0)d
0 (0)d
S/C
64.3 ( 0.6
59.4 ( 1.3
0.31 0.16 (0.17)d
0.03 0.16 (0.18)d
0 0.01 (0.02)d
63.0 ( 2.5 18.6 ( 3.2
51.2 ( 2.8 24
a
Values compared with the case for glass tubes ) 1.0.
Table 5. Number of Platelets Adherent and Deformed on Poly(ethylene terephthalate) (PET) Films Coated with Hydrogel no. of platelets sample
adherent
deformed
PET hydrogelated surface heparin-immobilized hydrogelated surface
92.9 ( 12.4 5.3 ( 0.5 0.4 ( 0.3
15.9 ( 3.5 ∼0 ∼0
a Platelet number per 0.015 mm2. b 1 h after incubation for a density of 6.6 × 105/mL platelets.
layers were examined in a PBS solution (Figure 5). The urokinase release rate decreased as the thickness of the protective layer increased, indicating that the release rate was controlled to some extent by the thickness of the protective layer. An inner surface of a poly(propylene) tube was fixed with a thin hydrogel layer of a photoreactive copolymer (2; dithiocarbamate content, 6.0 mol %) with or without heparin, which was prepared as mentioned above (Figure 2B). The whole-blood-coagulation times for the nonheparinized or heparinized hydrogels were measured (Table 4). The coagulation time was prolonged when the surface of the tube was coated with the hydrogel. A more prolonged anticoagulant activity was obtained when heparin was immobilized into the hydrogel. Platelet adhesion behavior on a hydrogelated surface was examined by scanning electron microscopy (Table 5). Nontreated PET surfaces exhibited marked platelets adhesion and deformation upon incubation with human platelet-rich plasma for 1 h. Platelet adhesion was significantly reduced on the hydrogelated surface and especially so on a heparin-immobilized hydrogelated surface. Markedly reduced platelet deformation was observed on both heparinized and nonheparinized-hydrogel-fixed surfaces. Micropatterning of Surface Hydrogelation. Because photolysis of the dithiocarbamates occurs only on photoirradiated regions, a micropatterned hydrogelated surface can be formed using patterned projected light irradiation. A PET film coated with a photoreactive copolymer (2; dithiocarbamate content, 6.0 mol %) was UV light-irradiated through a photomask having a line pattern (width of line: 20 µm), which was in contact with
Figure 6. Atomic force microscopic observation of a regionally specific micropatterned hydrogelated surface which was prepared by UV light irradiation to the dithiocarbamate-derivatized photoreactive copolymer (2; dithiocarbamate content, 6.0 mol %), cast on a PET film, through the line-patterned photomask (line width: 20 µm): a, nontreated PET surface; b, hydrogel fixed regions.
the copolymer. The topographic image of the treated surface, recorded by scanning the surface with the sharp tip of an AFM, is shown in Figure 6. A regionally micropatterned surface was produced by fixation of a large densely gelled block (b in Figure 6) onto a relatively flat nontreated PET surface (a in Figure 6). The regional dimensions of the pattern obtained were the same as those of the photomask used. Conclusions Surface hydrogelation was achieved by casting of photoreactive copolymers, derivatized with photoreactive groups such as dithiocarbamate and xanthate, from their aqueous solution onto a polymer film and subsequent UV light irradiation. Control of the degree of cross-linking in the hydrogel, immobilization of bioactive substances, and overlayering of hydrogel to control the release rate of immobilized substances was feasible. In addition, this phototechnology may offer promise for realizing the desired biocompatibility on a given part of fabricated medical devices such as artificial organs, catheters, and biosensors. Acknowledgment. The authors thank the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) for financial support of this work under Grant No. 97-15. We are grateful to Atsuyoshi Nakayama for the GPC measurements. LA981169H
