Bioconjugate Chem. 1997, 8, 730−734
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Photoimmobilization of Sulfated Hyaluronic Acid for Antithrombogenicity Guoping Chen,† Yoshihiro Ito,*,†,‡ Yukio Imanishi,† Agnese Magnani,§ Stefania Lamponi,§ and Rolando Barbucci§ Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan, PRESTO, JST, Keihanna Plaza, Hikaridai 1-7, Seika-cho, Kyoto 612-02, Japan, and CRISMA, Siena University, Via E. Bastianini n. 12, 53100 Siena, Italy. Received April 2, 1997; Revised Manuscript Received July 2, 1997X
Anticoagulant polymer sulfated hyaluronic acid was patterned immobilized on a poly(ethylene terephthalate) (PET) film in a specific pattern by photolithography. Hyaluronic acid was sulfated by a sulfur trioxide-pyridine complex. The polymer was coupled with azidoaniline. The derivatized polymer was cast on a PET film from aqueous solution. After drying, the film was photoirradiated in the presence or absence of a photomask. The micropatterning was confirmed by staining with a dye, brilliant green. Since the anticoagulant polymer has negative charges, the cationic dye was adsorbed on the regions where the anticoagulant polymer was immobilized. Platelet adhesion was reduced on the sulfated hyaluronic acid-immobilized areas. The immobilized sulfated hyaluronic acid significantly reduced thrombus formation.
INTRODUCTION
One of the most widely employed approaches in the synthesis of blood compatible materials is heparinization, in which synthetic polymers are coated or immobilized with heparin. Heparin is a natural polyanionic polysaccharide carrying sulfonate groups (1, 2). Therefore, several investigations using sulfonated polymers have been performed (3-13). Recently, it has been found that sulfated hyaluronic acid has a heparin-like property (1417). In the present study, the anticoagulant polymer was immobilized on a poly(ethylene terephthalate) (PET) film and its interaction with blood was investigated. In order to immobilize the polymer on the film surface, the photoimmobilization method developed by Matsuda and Sugawara (18-21) was used. Because the method does not need the presence of functional groups on the matrix surface to connect the functional unit with the polymer, it has been applied for surface functionalization of conventional polymers (22, 23). Micropatterning of anticoagulant polysaccharide by the photolithography technique allowed visualization of interactions with blood in the regions with or without immobilized polysaccharide. MATERIALS AND METHODS
Materials. 4-Azidoaniline hydrochloride, 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (watersoluble carbodiimide, WSC), Triton X-100, and brilliant green were purchased from Wako Pure Chemical Ind. Ltd. (Osaka, Japan). The photolithographic mask was purchased from Nippon Filcon Co., Ltd. (Osaka, Japan). Poly(ethylene terephthalate) (PET) film (diameter, 13.5 mm) was purchased from Akita Sumitomo Bake Co. * Address correspondence to Yoshihiro Ito, Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan. Telephone: 81-75-753-5638. Fax: 81-75-753-4911. E-mail:
[email protected]. † Kyoto University. ‡ JST. § Siena University. X Abstract published in Advance ACS Abstracts, September 1, 1997.
S1043-1802(97)00049-9 CCC: $14.00
(Akita, Japan). Bovine serum albumin (BSA) was purchased from Intergen Co. (Purchase, NY). Propidium iodide was purchased from Sigma Co. (St. Louis, MO). Vectashield mounting medium for fluorescence was purchased from Vector Laboratories, Inc. (Burlingame, CA). Synthesis of the Photoreactive Polymer. The synthetic scheme of the photoreactive polymer is shown in Figure 1. Sulfated hyaluronic acid (SHyal) was prepared as previously reported (14-17). Briefly, a solution of tributylammonium hyaluronate in N,N-dimethylformamide (DMF) was mixed with a solution of the sulfur trioxide-pyridine complex in DMF under a nitrogen gas flow. The mixture was kept at 0 °C for 1 h. Then, double-distilled water and a 1 M NaOH solution were added. The sulfated hyaluronic acid was precipitated by an excess of ethanol. The product was separated by centrifugation (15 min at 1500 rpm) and dialyzed against double-distilled water in a dialysis tube with a cutoff MW of 12 000. The solution was then lyophilized. The purified product was referred to as SHyal. NMR analysis revealed that 87.5% of the OH groups were sulfated (14). SHyal (18.20 mg), 4-azidoaniline (7.75 mg), and WSC (10 mg) were dissolved in deionized water (20 mL), and the pH of the solution was adjusted to 7.0. The solution was stirred at 4 °C for 24 h. Then, the solution was ultrafiltered (Millipore MoleCut II, filtration off below a MW 10 000) and the residue was repeatedly washed with distilled water until the absence of 4-azidoaniline in the filtrate was confirmed by ultraviolet absorption at 257 nm. The purified solution was then lyophilized. The photoreactive SHyal conjugate is referred to as AzPhSHyal. The content of azidophenyl groups in the copolymer was measured by ultraviolet absorption. Photoimmobilization. The microprocessing procedure is shown in Figure 2. An aqueous solution of AzPhSHyal (1 mg/mL, 0.1 mL) was dropped onto a PET film and air-dried at room temperature. Subsequently, the film was irradiated with an ultraviolet lamp (Koala, 100 W) at a distance of 5 cm for 10 s in the presence or absence of a photomask. The film was washed with distilled water until the absence of AzPhSHyal in the wash was confirmed by ultraviolet spectroscopy. The © 1997 American Chemical Society
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Photoimmobilization of Sulfated Hyaluronic Acid
Figure 1. Preparation of azidophenylamino-derivatized sulfated hyaluronic acid.
Figure 3. UV spectra of 4-azioaniline (6.2 µg/mL), azidophenylamino-derivatized SHyal (34.0 µg/mL), and the original Shyal (34.0 µg/mL).
Figure 2. Photoimmobilization of azidophenylamino-derivatized sulfated hyaluronic acid. The photoimmobilization was carried out with or without a photomask.
PET film immobilized with AzPhSHyal in a specific pattern was immersed in an aqueous solution of brilliant green (1 mg/mL) for 5 min. After washing with distilled water, the stained film was observed using a microscope (Olympus Co., Tokyo, Japan). Anticoagulant Activity Assays. Human blood was collected by gravity through a 19-gauge scalp vein needle into polypropylene tubes containing 1 part of ACD solution for 9 parts of blood. The ACD solution contains anhydrous D-glucose (2.45 g), sodium citrate dihydrate (2.20 g), and citric acid monohydrate (0.08 g) in distilled water (100 mL). The citrated blood was used for the following measurements: recalcification coagulation time, platelet adhesion, and in vitro thrombus formation. The recalcification coagulation time was measured as follows. SHyal or AzPhSHyal (2.5 mg) was added in tubes containing 0.5 mL of citrated blood, and subsequently, 0.05 mL of 0.1 M CaCl2 was added. The tubes were gently mixed until the thrombus was formed. The time required for thrombus formation was measured. The platelet adhesion experiment was performed as follows. The citrated human blood was centrifuged at
1200 rpm for 20 min at 4 °C. The supernatant (0.2 mL), which is platelet-rich plasma, was added to the microprocessed PET film. After incubation at 37 °C for 2 h, the film was gently washed with distilled water and immersed in a phosphate-buffered saline (PBS) solution containing 3% paraformaldehyde for 10 min. The film was washed three times for 10 min each with PBS and treated with PBS containing 0.25% Triton X-100. After washing with PBS three times, the film was incubated in PBS containing 3% BSA at room temperature for 1 h. Subsequently, the film was incubated in PBS containing 0.1% BSA and 1 µg/mL propidium iodide at room temperature for 1 h. The film was washed three times for 10 min each with PBS, briefly rinsed with distilled water, and then mounted in Vectashield mounting medium. The film adhered with platelets was observed by a fluorescence microscope (Olympus). In vitro thrombus formation on the sample film was carried out as reported previously (24). The citrated blood (0.2 mL) was put to a sample film, and 0.02 mL of a 0.1 M CaCl2 solution was added. After incubation at 37 °C for a prescribed period, the film was gently washed with distilled water and immersed in a PBS solution containing 3% paraformaldehyde for 10 min. Then, the formed thrombus was weighed after drying. RESULTS
Synthesis and Activity of AzPhSHyal. Ultraviolet spectra of SHyal and AzPhSHyal are shown in Figure 3. In the spectrum of AzPhSHyal, an absorption at 268 nm
732 Bioconjugate Chem., Vol. 8, No. 5, 1997
Chen et al.
Table 1. Recalcification Coagulation Times of Sulfated Hyaluronic Acid sample
timea (min)
nothing sulfated hyaluronic acid azidophenylamino-derivatized sulfated hyaluronic acid
20 ( 3 >120 >120
a
n ) 5, ( standard deviation.
which is assignable to the azidophenyl group was observed. The absorption was somewhat red-shifted from the corresponding absorption of 4-azidoaniline. This shift could be due to electron delocalization of the azidophenyl group by the amido bond formation. Assuming that the molecular absorption coefficient of azidophenyl group at 268 nm was the same as that of 4-azidoaniline at 257 nm, the content of azidophenyl groups in the copolymer was 1.01 ( 0.04 mol/mol of disaccharide unit of sulfated hyaluronic acid. It is known that one carboxyl group exists in a disaccharide unit of SHyal. Under the present reaction conditions, the carboxyl groups were fully coupled with the azidophenylamino group. Table 1 shows the recalcification coagulation time of the two kinds of sulfated hyaluronic acids. The incorporation of the azidophenylamino group did not seem to affect the anticoagulant activity of SHyal. This result indicates that the availability of carboxylate groups, unlike that of heparin (25), is not indispensable for the anticoagulant activity of SHyal. In fact, in the case of SHyal, the anticoagulant activity is mainly based on its high density of negative charges as previously demonstrated (15). Since AzPhSHyal can elongate the coagulation time in the solution, the polymer was immobilized on a substrate. Micropattern Immobilization of AzPhSHyal. AzPhSHyal was photoimmobilized in the presence of a photomask. Upon UV irradiation, the azidophenyl group was easily photolyzed to generate highly reactive nitrene, which spontaneously formed covalent bonds with the neighboring hydrocarbon in the PET film surface. After being thoroughly washed with distilled water, the immobilized polymer was exactly in the pattern of the photomask as shown in Figure 4. Since the polymer was anionic, the cationic dye, brilliant green, selectively stained the areas of AzPhSHyal immobilization. Interaction with Blood. Platelet adhesion onto the micropattern of immobilized AzPhSHyal is shown in Figure 5. The adhered platelets were detected only in the regions without AzPhSHyal immobilization. It is visually shown that the micropatterning of sulfated hyaluronic acid reduced the interaction of the PET film with platelets. Thrombus formation on the PET film with AzPhSHyal photoimmobilized in the absence of the photomask was investigated, and the result is shown in Figure 6. On the AzPhSHyal-immobilized film, the thrombus was not formed in the initial 20 min. The amount of thrombus formed on the AzPhSHyal-immobilized PET film in 1 h was about 25% of that formed on the PET film without immobilization. We previously showed that no thrombus formation was found on the heparinized polyurethane over the course of 20 min by the same evaluation method (24). It was demonstrated that the antithrombogenic effect of immobilized sulfated hyaluronic acid corresponded to that of immobilized heparin. DISCUSSION
Hyaluronic acid is a linear polysaccharide and consists of repeating disaccharide units of β-glucuronic acid and
Figure 4. Micrographs of (a) an AzPhSHyal-immobilized film and (b) a photomask. AzPhSHyal was stained by brilliant green.
Figure 5. Fluorescence micrograph of platelets adhered onto a PET film micropattern-immobilized with AzPhSHyal. The adhered platelets were stained by fluorescent propidium iodide.
N-acetyl-β-D-glucosamine. By itself, hyaluronic acid shows a growth-promotion effect, and investigations were carried out to enhance wound-healing properties, for instance, by culturing human keraticocytes on sponges and membranes composed of the benzyl ester of hyaluronic acid (26). Inoue and Katakami (27) reported that the hyaluronic acid stimulated proliferation of epithelial cells more than fibronectin and that as the result corneal epithelial wound healing was promoted. Hamann et al. (28) demonstrated that hyaluronic acid promoted proliferation of undifferentiated progenitor cells through the
Photoimmobilization of Sulfated Hyaluronic Acid
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material surface is necessary to give biomaterials antithrombogenicity. Recently, various types of combinatorial approaches have been carried out for design of biomaterials (34, 35). The lithographic technique allowed visual evaluation of biological activities of the hybrid material under the same conditions as for nontreated material. The photoimmobilization or photolithographic method will be useful for design and synthesis of new biomaterials. ACKNOWLEDGMENT Figure 6. Time course of thrombus formation on a sulfated hyaluronic acid-immobilized PET film and a PET film without immobilization.
CD44 receptor on the surface of progenitor during eosinopoiesis in vitro. Recently, Rooney et al. (29) reviewed the role of hyaluronic acid in tumor neovascularization upon which tumor growth and metastasis are totally dependent. They suggested a model to account for hyaluronic acid of differing molecular mass being present, at different locations, within a single tumor and how this hyaluronic acid aids both general tumor growth and tumor metastasis (29). To endow the polymer with anticoagulant properties, hyaluronic acid was sulfated by Balazs et al. (30). The sulfated hyaluronic acid (SHyal) inactivated thrombin in a manner different from that of heparin. While heparin inhibits thrombin via antithrombin III, SHyal directly binds thrombin by specific electrostatic interactions (15). To make conventional polymers antithrombogenic, several methods, including coating and immobilization of the anticoagulant, have been developed. Covalent immobilization is thought to be suitable for attaining a long-term antithrombogenicity. However, the presence of functional groups such as carboxyl groups and amino groups is needed on the matrix surface as a scaffold for covalent immobilization. The photoimmobilization method used in the present investigation does not need such functional groups. The photochemical method was used by Erdtmann et al. (31) to immobilize heparin, dermatan sulfate, and dextran sulfate. Kuijpens et al. (32) also photoimmobilized a small molecule, theophylline, on medical-grade polyurethane for inhibition of surfaceinduced activation of blood platelets by the same method in which a 4-azidophenylamino group is involved. Although they did not use a lithographic technique and did not investigate the interaction of photoimmobilized material with whole blood, they found that the theophyllineimmobilized polyurethane reduced platelet adhesion by 1 order of magnitude. Considering no platelet adhesion on AzPhSHyal-immobilized regions, the immobilized AzPhSHyal was more effective than the immobilized theophylline. In this investigation, it was shown that the polymer having anticoagulant activity in the solution was photoimmobilized on a conventional polymeric material to make the material antithrombogenic. In the case of heparinization of the material, the heparinized surface generally reduced platelet adhesion, although the effect of heparin on platelet is unclear in solution (1). On the other hand, some anticoagulants did not effectively reduce thrombus formation after immobilization. For synthesis of blood compatible materials, it is necessary to choose adequate anticoagulants and immobilization methods for them. Well-known is the fact that the absence of platelets on the substrate surface following exposure to blood does not in itself indicate that the material possesses anticoagulant properties (33). However, the bioinertness of the
The authors thank Drs. T. Matsuda and Y. Nakayama at the National Cardiovascular Center Research Institute for their helpful suggestions on the photolithography technique. This work was partially supported by a Grantin-Aid for scientific research from the Ministry of Education, Science and Culture of Japan and partially supported by the Nissan Science Foundation. LITERATURE CITED (1) Ito, Y. (1987) Antithrobogenic heparin-bound polyurethanes. J. Biomater. Appl. 2, 235-265. (2) Ito, Y., Okuyama, T., Kashiwagi, T., and Imanishi, Y. (1994) Interaction of heparin with amphiphile assemblies and biocompatibility of the heparin complexes. J. Biomater. Sci., Polym. Ed. 6, 707-714. (3) Sederal, L. C., van der Does, L., van Doijl, J. F., Beugeling, T., and Bantjes, A. (1981) Anticoagulant activity of a synthetic heparinoid in relation to molecular weight and N-sulphate content. J. Biomed. Mater. Res. 15, 819-827. (4) Muzzarelli, R. A., Tanfani, F., Emanuelli, M., Pace, D. P., Chiurazzi, E., and Piani, M. (1984) Sulfated N-(carboxymethyl)-chitosans: novel blood anticoagulants. Carbohydr. Res. 126, 225-231. (5) Crassous, G., Harjanto, F., Mendjel, H., Sledz, J., and Schue F. (1985) A new symmetric membrane having blood compatibility. J. Membr. Sci. 22, 269-282. (6) Gebelein, C. G., and Murphy, D. (1987) The synthesis of some potentially blood compatible heparin-like polymeric materials. In Advances in Biomedical Polymers (C. G. Gebelein, Ed.) pp 277-284, Plenum Press, New York. (7) Ito, Y., Liu, L.-S., and Imanishi, Y. (1991) Interaction of poly(sodium vinyl sulfonate) and its surface graft with antithrombin III. J. Biomed. Mater. Res. 25, 99-105. (8) Ito, Y., Iguchi, Y., Kashiwagi, T., and Imanishi, Y. (1991) Synthesis and nonthrombogenicity of polyetherurethaneurea film grafted with poly(sodium vinyl sulfonate). J. Biomed. Mater. Res. 25, 1347-1361. (9) Ito, Y., Iguchi, Y., and Imanishi, Y. (1992) Synthesis and nonthrombogenicity of heparinoid polyurethanes. Biomaterials 13, 131-135. (10) Hergen, R. W., and Cooper, S. L. (1992) Improved materials for blood-contacting applications: blends of sulphonated and non-sulphonated polyurethanes. J. Mater. Sci.: Mater. Med. 3, 313-321. (11) Jozefonvicz, J., and Jozefowicz, M. (1994) Blood contacting polymers. In Polymeric Materials (S. Dumitriu, Ed.) pp 349366, Marcel Dekker, New York. (12) Lim, F., and Cooper, S. L. (1995) Effect of sulphonate incorporation on in vitro leucocyte adhesion to polyurethanes. Biomaterials 16, 457-466. (13) Han, D. K., Lee, N. Y., Park, K. D., Kim, Y. H., Cho, H. I., and Miu, B. G. (1995) Heparin like anticoaglunat activity of sulphonated poly(ethylene oxide) and sulphonated poly(ethylene oxide)-grafted polyurethane. Biomaterials 16, 467471. (14) Barbucci, R., Magnani, A., Casolaro, M., Marchettini, N., Rossi, C., and Bosco, M. (1995) Modification of hyaluronic acid by insertion of sulfate groups to obtain a heparin-like molecule. Part I. Characterization and behaviour in aqueous solution towards H+ and Cu2+ ions. Gazz. Chim. Ital. 125, 169-180. (15) Magnani, A., Albanese, A., Lamponi, S., and Barbucci, R. (1996) Blood-interaction performance of differently sulfated hyaluronic acids. Thromb. Res. 81, 383-395.
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