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Preparation and Biocompatibility of Novel UV-Curable Chitosan Derivatives† Eiko Renbutsu,‡ Masaru Hirose,‡ Yoshihiko Omura,§ Fumiaki Nakatsubo,| Yasuhiko Okamura,‡ Yoshiharu Okamoto,‡ Hiroyuki Saimoto,⊥ Yoshihiro Shigemasa,⊥ and Saburo Minami*,‡ Department of Clinical Veterinary Medicine, Tottori University, 4-101 Koyma-Minami, Tottori 680-8553, Japan, Omura Toryo Company, Limited, 3-87, Chiyomi, Tottori 680-0911, Japan, Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto University, 606-8502 Kyoto, Japan, and Department of Materials Science, Faculty of Engineering, Tottori University, 4-101 Koyma-Minami, Tottori 680-8552, Japan Received February 2, 2005; Revised Manuscript Received May 10, 2005
UV-curable chitosans (UVCC-7-10) were synthesized using less-toxic agents. The UVCC-7 was completely cured by UV spot irradiation for 4 s. The UVCC-7 was implanted into murine subcutaneous tissues, and the response to the implantation was observed by histological examination at 7 days after implantation. In the histological findings, the implant was surrounded by thin fibrous granulating tissue with no inflammatory cellular infiltration. Fibroblasts infiltrate between the cured implant. The novel synthesized UVCC-7 showed good biocompatibility. Polymers have long been utilized in various fields and now are indispensable materials. Chitin and chitosan, natural biopolymers that are the second-most abundant biopolysaccharides after cellulose, have potential applications in medicine as a result of their biocompatibility and useful biological reactivity. We have investigated the efficacy of chitin and chitosan in animal wound treatment, and we have examined their humeral and cellular activation both in vitro and in vivo.1 Chitosan, poly(2-amino-2-deoxy-β-D-glucose), is soluble in mild acid solution, such as acetic acid, and is more easily modified than chitin. Omura et al. have previously developed a nonelectrolysis plating method utilizing the chelating ability of chitosan amino groups and have applied the technique to paints.2 However, chitosan is difficult to dissolve in many ordinary organic solvents and due to the poor compatibility between chitosan and paint resins, much time is wasted in the drying process after painting. These shortcomings have been the biggest disadvantages in the industrial development of chitosan in medical applications. To overcome these disadvantages, we attempted to prepare UV-sensitive chitosan derivatives that can be cured in seconds or minutes by UV exposure and were successful in developing some UV-curable chitosan derivatives (UVCC 7-10). We then proceeded to characterize these derivatives as biomedical materials. There are few reports on the application of UV-curable chitosan. Based on the adsorbent behavior of palladium on * To whom correspondence should be addressed. E-mail: minami@ muses.tottori-u.ac.jp. Tel.: +81-857-31-5433. Fax: +81-857-31-5433. † This paper was presented at the III Iberoamerican Symposium on Chitin (III SIAQ), held in Crdoba, Spain, September 27-29, 2004. ‡ Dept. Clin.Vet. Med., Tottori University. § Omura Toryo Company, Limited. | Kyoto University. ⊥ Dept. Materials Sci., Tottori University.
Figure 1. Structures of photosensitive aldehydes.
UV-cured chitosan derivative films, a novel plating method was discovered in our recent studies and was applied to microelectronics.3-6 On the other hand, basic experiments using photo-cross-linkable chitosan hydrogels have been reported by Ishihara et al. for medical applications.7,8 We succeeded in developing UV-curable chitosans using less-toxic chemical agents. The side chains of UV-curable chitosan consist of vanillin or hydroxybenzaldehydes, both of which are natural resources present in plants. Vanillin is a familiar food additive with a sweet smell. Introduction of these side chains improved the solubility of chitosan. In the present paper, we report the preparation of less-toxic UV-
10.1021/bm0500796 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/16/2005
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Figure 2. Preparation of UV-curable chitosan derivatives from aldehydes 1, 2, 4, and 6.
Figure 4. Histological findings of the suture material implantation (group B). An absorbable synthesis suture material (polyglyconate, Maxon) is observed (arrow) surrounded by inflammatory granulating tissue.
Figure 3. Histological findings of the control (group A): a thick scab (a) was covered on the regenerated epidermis. The regenerated dermis has a random alignment of epidermal cells (b). A hyperemia of corium with moderate cellular (neutrophils) infiltration is observed (c).
curable chitosans and the biological responses to one of these derivatives in mouse subcutaneous tissues. N-Selective introduction of a UV-curable side chain to chitosan was accomplished by reductive N-alkylation with photosensitive aldehydes. As shown in Figure 1, we prepared six types of photosensitive aldehydes: 3-methoxy-4-(2-hydroxy-3-methacryloyloxypropoxy)benzaldehyde (1),9 3,4-bis(2-hydroxy-3methacryloyloxypropoxy)benzaldehyde (2), 3,5-bis(2-hydroxy3-methacryloyloxypropoxy)benzaldehyde (3), 3-methoxy-4-
methacryloyloxybenzaldehyde (4),10,11 3,4-dimethacryloyloxybenzaldehyde (5), and 3,5-dimethacryloyloxybenzaldehyde (6). Photosensitive functional groups (2-hydroxy-3-methacryroyloxypropoxy group) were introduced to vanillin or hydroxybenzaldehyde using glycidyl methacrylate to give aldehydes 1-3. Methacryloyloxy functions were then prepared by esterification using methacrylic acid and dicyclohexylcarbodiimide, to give aldehydes 4-6. Among these, aldehyde 1 derived from vanillin was the cheapest and was easily recovered in good yield by recrystallization. Aldehydes 3 and 5 were found to be unsuitable for preparing chitosan derivatives because of poor solubility and low yield, respectively. Details for the preparation of these products will be described in another report. In the present study, UVCC-7, shown in Figure 2, was prepared from chitosan (degree of deacetylation, 97%; molecular weight, 30 000-50 000; Dainichiseika Color & Chemicals Mfg. Co., Ltd.) and aldehyde 1 (0.8 mol equivalent relative to the glucosamine unit). N-Selective introduction of the side chain to the chitosan was accomplished by reductive N-alkylation, using NaBH3CN via Schiff base in a McIlvaine buffer (0.2 M acetic acid, 0.2 M sodium acetate) solution (pH 4.5). After neutralization, the product was collected by centrifugation and washed with ethanol and water. Excess water was removed by suction to give a wet
Communications
Figure 5. Histological findings of the cyanoacrylate implantation (group C). Cyanoacrylate implants (arrows) are observed surrounded by thick inflammatory granulating tissue. Numerous inflammatory cells migrated toward the implantations.
colorless amorphous compound containing about 31 wt % UVCC-7. This moist UVCC-7 was used as the implant. UVCC-7 was identified and characterized by 500 MHz 1H NMR spectra recorded on a JEOL ANM-GCP(V3) spectrometer, Fourier transform infrared (FT/IR) spectra on a Nicolet AVATAR 360 type spectrophotometer, and elemental analysis (J-SCIENCE HCN recorder MT-700 Type HCN). 1H NMR spectra of UVCC-7 in 2% CD3COOD showed typical peaks corresponding to the side chains at δ 1.86 (sCH3), 2.02 (sNHAc), 3.31 (H-2), 3.95 (-OCH3), 5.69, 6.16 (sCdCH2), and 7.06-7.12 (aromatic H), while no formyl peak (δ 9.86) corresponding to the starting aldehyde was observed. In FT/IR spectra, characteristic bands corresponding to the side chains were observed at 1716 cm-1 (CdO), 1637 (CdC), and 1517 (aromatic). The degree of UV-curable substitution was determined to be 0.8 by elemental analysis. To prepare an implant containing UVCC-7, wet UVCC-7 compound (3.2 g) was mixed with dimethylsulfoxide (DMSO; 16.8 g; to give 5 wt % UVCC-7; 11 wt % water; 84 wt % DMSO) and stirred vigorously for 1 day to give a clear viscous solution. Irgacure 1000 (200 µL; Ciba Specialty Chemicals) was then added to the mixture as a photoinitiator. Part of this solution (100 µL), the UVCC-7 mixture, was poured into the 96-well microplate. UV light irradiation was performed with a short arc metal-halogen lamp installed in
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Figure 6. Histological findings of the UVCC-7 implantation (group D). UV-cured UVCC-7 (arrows) is surrounded by thin fibrous granulating tissue with no inflammatory cellular infiltration. Fibroblasts infiltrate between each UVCC-7. UVCC-7 showed good biocompatibility compared to the other groups.
a UV spot irradiator (Eye Cure Light Spot UP150M, Eye Graphics) for 4 s at a distance of 10 mm from the solution. For the determination of the biocompatibility of UVCC7, histological experiments were performed using 12 ddy mice randomly divided into four groups (groups A-D). The experimental mice were 8-week-old females weighing 30 g. Three mice were provided for each treatment group. Group A underwent surgical intervention (skin and subcutaneous tissue dissection and suturing) without implantation. Group B was implanted with an absorbable polyglyconate (USP 3-0, Maxon) suture thread and was subjected to surgical intervention. Group C was implanted with commercial surgical adhesive, 2-ethylcyanoacrylate (Aron Alpha, Sankyo) and was subjected to surgical intervention. Group D was implanted with the UV-cured UVCC-7 mixture and was subjected to surgical intervention. The procedure for the animal experiments was approved by the Animal Research Committee in accordance with the guidelines for animal experimentation of the Faculty of Agriculture, Tottori University (Approval No. 04-44). Under general anesthesia by peritoneal administration (ip) of 50 mg/kg pentobarbital (Nembutal, Dai-Nippon Seiyaku, Osaka), the dorsal skin was dissected with a surgical scalpel parallel to the body and subcutaneous tissue was bluntly dissected using a small spatula to create space for implantation (group A underwent surgical intervention without
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Microscopic observation of group A is shown in Figure 3. Figure 4 shows group B. Figure 5 shows group C. Figure 6 shows group D. As shown in Figure 7, cross-linked UVCC-7 includes a biocompatible chitosan site. Thus, UV-cured UVCC-7 induced mild inflammation in mouse tissues when compared with the other commercial implants. The properties of UVCC-7 for medical applications will further be tested in subsequent investigations. References and Notes
Figure 7. Structure of UV-cured UVCC-7.
implantation). Each implant was inserted into the subcutaneous space, and the skin was closed with nylon sutures (USP 3-0, Suplyron). After 1 week, the surgically treated tissue of each mouse was collected and fixed with neutral 10% formaldehyde solution for histological examination. The fixed tissue was rinsed overnight with water and was dehydrated using a graded series of alcohol and 100% xylene. Dehydrated tissue was embedded in paraffin, and thin sections are produced using a microtome. Sections were stained by hematoxylin and eosin. Mice showed no abnormal findings with regard to the general condition during the experimental period.
(1) Minami, S.; Okamoto, Y.; Hamada, K.; Fukumoto, Y.; Shigemasa, Y. Chitin and Chitinase; Birkhauser Verlag Basel: Switzerland, 1999; pp 265-277. (2) Omura, Y.; Nakagawa, Y.; Murakami, T. AdVances in Chitin Science; Jacques Andre Pub.: Lyon, France, 1998; Vol. II, pp 902-907. (3) Omura, Y.; Renbutsu, E.; Saimoto, H.; Shigemasa, Y. Jpn. Kokai Tokkyo Koho, JP 02, 226503, 2002, 7 pp. (4) Renbutsu, E.; Omura, Y.; Saimoto, H.; Shigemasa, Y. Chitosan in Pharmacy and Chemistry; Atec Edizioni: Grottammare, Italy, 2003; pp 455-460. (5) Renbutsu, E.; Omura, Y.; Nakatsubo, F.; Okamoto, Y.; Minami, S.; Saimoto, H.; Shigemasa, Y. Chitin Chitosan Res. 2003, 9, 104. (6) Renbutsu, E.; Omura, Y.; Asaka, M.; Nakatsubo, F. Jpn. Kokai Tokkyo Koho, JP 00, 109501, 2000, 7 pp (Patent No. JP 2962717, 1999). (7) Ishihara, M.; Nakanishi, K.; Ono, K.; Sato, M.; Kikuchi, M.; Saito, Y.; Yura, H.; Matsui, T.; Hattori, H.; Uenoyama, M.; Kurita, A. Biomaterials 2002, 23, 833. (8) Ishihara, M. Trends Glycosci. Glycotechnol. 2002, 14, 331. (9) Ichimura, K.; Oohara, N. J. Polym. Sci., Part A: Polym. Chem. 1987, 25, 3063. (10) Brown, E.; Racois, A. Tetrahedron Lett. 1972, 50, 5077. (11) Imoto, M.; Maeda, T.; Ouchi, T. Chem. Lett. 1978, 153.
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