Biomacromolecules 2004, 5, 1110-1115
1110
Novel Biodegradable Polyphosphate Cross-Linker for Making Biocompatible Hydrogel Yasuhiko Iwasaki,*,† Chigusa Nakagawa,†,‡ Michiko Ohtomi,† Kazuhiko Ishihara,§ and Kazunari Akiyoshi† Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai; Chiyoda-ku, Tokyo 101-0062, Japan, Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama; Funabashi-shi, Chiba 274-8510, Japan, and Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo; Bunkyo-ku, Tokyo 113-8656, Japan Received January 14, 2004; Revised Manuscript Received March 5, 2004
To obtain a novel biodegradable cross-linker, polymerizable polyphosphate (PIOP) was synthesized by ringopening polymerization of 2-i-propyl-2-oxo-1,3,2-dioxaphospholane with 2-(2-oxo-1,3,2-dioxaphosphoroyloxyethyl methacrylate) (OPEMA). The number averaged molecular weight of the PIOP was 1.2 × 104, and the number of OPEMA units in one PIOP molecule was 2.2. Nonenzymatic degradation of the PIOP was evaluated in various pH aqueous media. The degree of hydrolysis was dependent on the pH; that is, it increased with an increase in the pH of the medium. At pH 11.0, the PIOP completely degraded in only 6 days. The poly[2-methacryloyloxyethyl phosphorylcholine (MPC)] cross-linked with the PIOP was prepared by radical polymerization. This polymer could form hydrogel, and the free water fraction in the hydrogel was high. The enzymatic activity of trypsin in contact with the hydrogel was similar to that in buffer solution. There is no adverse effect caused by the hydrogel to reduce the function of the trypsin. The cytotoxicity of poly(MPC) and degraded PIOP was evaluated using v79 cells, and it was not observed in either case. In conclusion, PIOP is a hydrolyzable polymer, which can be used as a cross-linker, and novel hydrogels having biodegradability and biocompatibility were prepared from poly(MPC) cross-linked with the PIOP. Introduction There has been a great deal of interest in polyphosphates, which are biodegradable through hydrolysis, and possibly enzymatic digestion of phosphate linkages under physiological conditions.1 These biodegradable polyphosphates appear interesting for biological and pharmaceutical applications because of their biocompatibility and structural similarities to the naturally occurring nucleic and teichoic acid. Recently, polyphosphates have been proposed for use in the field of tissue engineering as scaffolds and as gene carriers.2,3 A variety of synthetic routes for polyphosphates have been reported including ring-opening polymerization,4,5 polycondensation,6 and enzymatic polymerization.7 However, the functionalities of the polyphosphates in these reports are relatively poor, and almost all of the polymers have alkyl groups in their side chains. We hypothesized that a polyphosphate with polymerizable groups might be a novel biodegradable cross-linker for polymeric hydrogel for biomedical uses. Hydrogels are insoluble, cross-linked polymer networks that can absorb significant amounts of water.8,9 Also, hydrogels are as flexible as soft tissues, which minimizes * To whom correspondence should be addressed. E-mail: yasu.org@ tmd.ac.jp. Telephone: +81-3-5280-8026. Fax: +81-3-5280-8027. † Tokyo Medical and Dental University. ‡ Toho University. § The University of Tokyo.
their potential irritation to surrounding tissue. More recent trends in hydrogel research are macromolecular drug delivery and cell entrapment for tissue engineering.10,11 For these applications, biodegradability and biocompatibility of hydrogels are important. To achieve biocompatibility, we have been studying 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers synthesized as biomimetics to biomembrane structures.12 The MPC polymers exhibit a surface property that resists nonspecific protein adsorption and cell adhesion, i.e., “biofouling”.13 Biofouling reduces the functionality of a material and induces an unexpected bioreaction. MPC polymers are thus quite useful for surface modification of biomedical devices.14 Further, it has been shown that the activation and inflammatory response of cells in contact with MPC polymers are not induced.15-17 MPC polymers can have a hydrogel state in which they swell with water and might be suitable for forming complexes with biosubstances. In this study, we synthesized polymerizable polyphosphate (PIOP) as a novel biodegradable cross-linker and a biomimetic hydrogel consisting of poly(MPC) cross-linked with the PIOP. We also studied their biodegradability, protein interaction, and cytotoxicity. Experimental Section General Information. 2-Propanol, tetrahydrofurane (THF), and triethylamine (TEA) were purified by distillation. Poly-
10.1021/bm049961m CCC: $27.50 © 2004 American Chemical Society Published on Web 04/09/2004
Biomacromolecules, Vol. 5, No. 3, 2004 1111
Novel Biodegradable Polyphosphate Cross-Linker Table 1. Synthetic Results of PIOP
a
[OPEMA] (mol %)
[initiator] (mol %)
in feed
0.30
2.0 b
in copolymer
yield (%)
Mna (×103)
Mw/Mna
composition of OPEMA units in a PIOPb (OPEMA units/PIOP)
3.0
17
12.0
1.3
2.2
Determined by GPC. Determined by
1H
NMR.
Scheme 1. Synthetic Route of PIOP
Table 2. Hydration Degree of PCPG Abb.
MPC/OPEMA unit in PIOPa (mol %)
Heqb (wt %)
PCPG 1.0 PCPG 0.2 PCPG 0.1 PEPG 0.1
99.0/1.0 99.8/0.2 99.9/0.1 99.0/1.0
90.2 92.8 97.8 83.3
free water fractionc 0.77 (0.02)
0.56 (0.03)
a [Monomer] ) 1.0 mol/L, [AIBN] ) 5 mmol/L. b Hydration degree (Heq) ) (weight of water in polymer gel/weight of polymer gel saturated with water) × 100. c Determined by DSC measurement: Mean (S. D.).
(2-i-propyl-2-oxo-1,3,2-dioxaphospholane-co-2-(2-oxo-1,3,2dioxaphosphoroyloxyethyl methacrylate) (IPP-co-OPEMA)18) (PIOP) was synthesized by the method previously described.19 For a typical reaction, IPP (19.6 mmol) and OPEMA (0.4 mmol) were placed into a thoroughly dried 50-mL round-bottomed flask equipped with a three-way stopcock. After the mixture was stored under reduced pressure for 2 h, triisobutyl aluminum (15 µL) was added under an argon gas atmosphere. The polymerization was continued until the magnetic stirrer was stopped. Dry THF was then added to dilute the polymer. The polymer was purified by reprecipitation from diethyl ether. Scheme 1 and Table 1 show the chemical structure of PIOP and the synthetic results of the PIOP, respectively. MPC was synthesized by the method previously described.12 Poly(ethylene glycol) monomethacrylate (PEGMA) was kindly purchased from NOF. Co., Tokyo, Japan. Trypsin from porcine pancreas and N-R-benzoyl-L-arginine ethyl ester (BAEE) were purchased from Wako Pure Chemicals, Tokyo, Japan and Aldrich, St. Louis, USA, respectively. They were used without further purification. Preparation of Hydrogel Cross-Linked with the PIOP (PCPG). PIOP, MPC, and 2,2′-azobisisobutyronitrile (AIBN) were dissolved in ethanol, and argon gas was bubbled into the solution to eliminate oxygen (Table 2). The monomer solution was poured into a polyethylene dish under an argon gas atmosphere. A glass cover was placed on the polyethylene dish, which was then stored at 60 °C for 18 h. The polymerized gel was soaked in ethanol for 3 days to eliminate
unreacted monomers. Instead of MPC, PEGMA was used to make a reference-hydrogel (PEPG), as shown in Figure 3. Determination of Equilibrated Hydration Degree and Free Water Fraction of Hydrogels. The polymer gel was immersed in water to equilibrate at 25 °C, and the degree of hydration in the gel was determined from an increment in the weight. The equilibrated degree of hydration (Heq) of the polymer gel saturated with water was determined by the following equation: Heq (wt%) ) (weight of water in the polymer gel) × 100 (weight of polymer gel saturated with water) The water structure of PIOP was estimated by a differential scanning calorimeter (DSC; DSC-100, Seiko-I, Chiba, Japan).20 The DSC analysis was conducted between -50 and +50 °C at a heating rate of 5 °C/min. About 100 mg of gel was placed in an aluminum pan, and a given amount of water was added for DSC analysis. The pan was tightly sealed to prevent water evaporation and stored overnight before measurement. The endothermic peak of the hydrated gel of around 0 °C, which is attributed to the melting of frozen water, was compared with that of pure water, and the free water fraction was calculated. Hydrolysis Test. The hydrolysis of PIOP and PCPG was evaluated by soaking the polymers (0.10 g) in an aqueous media (25 mL) at 37 °C. The pH of the medium was adjusted at 4.0 (citric acid/NaH2PO4), 7.4 (phosphate buffer solution), or 11.0 (NaOH/NaH2PO4). After soaking the polymer in water for the given time period, the weight loss of the hydrogel and the molecular weight were measured and recorded with a balance and a gel-permeation chromatography [for measurement of Mn of PIOP and degradation products from PIOP: Tosoh GPC system with refractive index detector and double size-exclusion columns, Nacalai Tesque, 5GPC-60 and 5GPC-300 with a polystyrene (PSt, Tosoh standard sample) standard in THF; for measurement
1112
Biomacromolecules, Vol. 5, No. 3, 2004
of Mn of degradation products: Tosoh GPC system with refractive index detector and size-exclusion columns, Shodex, SB-804 HQ with a poly(ethylene glycol) (PEG, Tosoh standard sample) standard in distilled water containing 10 mM LiBr]. The chemical structure of the degradation products was confirmed by 1H and 31P NMR (R-500, JEOL, Tokyo, Japan) analyses. Five samples were prepared for each polymer to evaluate their hydrolysis. Effect of Hydrogel Environment on Enzyme Activity. The enzymatic activity of trypsin in contact with the hydrogel was measured using BAEE as a substrate according to the method reported by Erlanger.21 Briefly, freeze-dried hydrogels (0.05 g) were soaked in 0.05 M Tris-HCl buffer solution (pH 8.15) having 13.5 mg/mL of trypsin (0.5 mL for PCPG, 0.25 mL for PEPG due to the degree of hydration of the hydrogels). The hydrogels were immediately saturated and the enzyme solution was completely absorbed in the PCPG. After being stored for 1 or 3 h at 37 °C, the hydrogel was gently washed with Tris-HCl buffer solution and soaked in 2.0 mL of the buffer solution for 1 h. Then, the buffer solution containing the hydrogels was filtered and the solution was kept at 0 °C until measurement of trypsin activity. To determine the trypsin activity, the concentration of the trypsin was normalized to 175 µg/mL. The trypsin solution (0.2 mL) was added to 6.0 × 10-4 M BAEE/TrisHCl buffer solution. The extent of BAEE hydrolysis was determined by measurement of N-R-benzoyl-L-alginine at 253 nm. Apparent activity is defined as the relative release rate of N-R-benzoyl-L-alginine. The data are relative activity to that of native trypsin and described with averaged amount and standard deviation from five measurements for each sample. The statistic analysis was performed with student’s t-test. Cytotoxicity Test of Degradation Products. Chinese hamster fibroblasts (v79 cells) were purchased from RIKEN Cell Bank. The v79 cells were maintained in a culture medium (Eagle’s MEM; Nissui Pharmaceutical, Tokyo, Japan) containing 10% fetal bovine serum at 37 °C in a humidified atmosphere of air containing 5% CO2. The contents of the flasks used for cell maintenance were detached by trypsin treatment, and 50 cells in 1 mL of culture medium were seeded in each of the 24 wells. The cells were stored overnight at 37 °C in a CO2 incubator with 95% humidity to adhere on the wells’ surfaces. The given amount of poly(MPC) and degradation products from PIOP were then introduced into the culture media. Zinc diethyldithiocarbamate (ZDEC) and zinc dibutyldithiocarbamate (ZDBC) were used as probe cytotoxic compounds. The cells were cultured for 7 days in a CO2 incubator. The wells were rinsed with PBS and treated with 10% formaldehyde solution to fix the colony that formed on the surface. After being washed with water, the colony was stained with 10% Giemsa solution and the number of colonies were counted. Results and Discussion Table 1 shows the synthetic results of PIOP in this study. The molecular weight of PIOP was 1.2 × 104, and the molecular weight distribution of PIOP was 1.3. In addition,
Iwasaki et al.
Figure 1. Change in weight of PIOP caused by hydrolysis from soaking in various pH media at 37 °C. Circle, pH 4.0; square, pH 7.4; triangle, pH 11.0.
Figure 2. GPC spectra of PIOP and degraded PIOP soaked in PBS for 40 days.
the number of OPEMA units in each PIOP chain was 2.2 as determined by 1H NMR. The PIOP synthesized in this study was soluble in THF and CH3Cl, and in ethanol, but was insoluble in water. Aliphatic polyesters have been widely used as biodegradable polymers and are believed to be safe biocompatible polymers due to the nontoxicity of the degradation products. However, control of the degradability of the polyesters is difficult because of high crystallinity, hydrophobicity, and poor solubility.22 Figure 1 shows the changes in PIOP weights due to hydrolysis in various pH media for given periods. Under the acidic condition (pH 4.0), hydrolysis of the PIOP was slow. In contrast, the PIOP was completely degraded in only 6 days in the basic condition (pH 11.0). Penczek et al. reported the hydrolysis of poly(methyl ethylene phosphate) in the range from pH 1 to 12.23 In the basic condition, the rate of hydrolysis of the main chain (km) was dramatically faster (1.32 × 10-5 s-1 at pH 11.16) than that in the acidic condition (1.14 × 10-8 s-1 at pH 3.78). Moreover, the ratio of km to the rate of hydrolysis of side chain (ks) was nearly equal in the basic condition; ks is larger than km in the acidic condition. The GPC charts of PIOP and degraded PIOP are shown in Figure 2. From the elution profile of the PIOP, a single peak at 11-min was observed. The number averaged molecular weight of the degraded PIOP standardized with poly(styrene) was 1.0 × 102. On the 1H NMR spectrum of the degraded PIOP, the signals are due to the -CdCH2, i-propyl group, etc (Figure 3). To make hydrogels, MPC and PEGMA were used as monomers, as shown in Figure 4. Table 2 shows the degree of hydration and the free water fraction of hydrogels crosslinked with the PIOP. The Heq value was changed depending
Biomacromolecules, Vol. 5, No. 3, 2004 1113
Novel Biodegradable Polyphosphate Cross-Linker
Figure 6. Change in weight of PCPG caused by hydrolysis from soaking in various pH media at 37 °C.
Figure 3.
1H
NMR spectrum of hydrated PIOP.
Figure 4. Structures of MPC and PEGMA. Figure 7. GPC spectra of hydrated PCPG soaked in various pH media for 44 days.
Figure 5. Picture of PCPG1.0 saturated with water.
on the concentration of the PIOP. The free water fractions of PCPG and PEPG at Heq were 0.77 and 0.56, respectively. Figure 5 shows a picture of the transparent hydrogel, PCPG1.0 containing 1.0 mol % OPEMA unit of PIOP. Figure 6 shows the degradation profile of PCPG soaked in aqueous media with various pH values. In the acidic medium, the weight loss of the gel was observed as being less. Under physiological pH conditions, the weight of the PCPG decreased to 80% after soaking in the PBS for 44 days. In the basic condition, the gel almost degraded completely within 20 days. This behavior is similar to the hydrolysis of PIOP. Thus, the degradation of PCPG is due to hydrolysis of the PIOP. In the basic condition, the observed elute had a molecular weight of 1.0 × 105. Moreover, the NMR spectrum of the elute was similar to
Figure 8. Apparent enzymatic activity of trypsin in contact with hydrogel for 1 or 3 h.
Table 3. % Elution of GPC for PCPG Hydrolyzed at Various pH Mediaa
a
pH
I (1.0 × 105)
II (3.0 × 102)
4.0 7.4 11.0
0.2 6.1 97.0
99.8 93.9 3.0
( ): molecular weight.
that of the poly(MPC). It has been reported that the property of poly(MPC) is not influenced by pH conditions from 4.0 to 11.0.13 Figure 7 and Table 3 show the GPC profiles of degradation products from PCPG soaked in each medium for 44 days and the percent of elution of the degradation products. At
1114
Biomacromolecules, Vol. 5, No. 3, 2004
pH 4.0, elution was observed with a molecular weight of 3.0 × 102 (II in Figure 7). The fraction with high molecular weight (1.0 × 105) increased with an increase in pH. Figure 8 shows the apparent enzymatic activity of trypsin in contact with hydrogel. The activity of native trypsin was decreased to 0.55 for 1 h and 0.47 for 3 h incubation in the buffer solution due to autolysis. The activity of trypsin in contact with the PCPG was also decreased but was similar to that of trypsin in buffer solution. However, the activity of trypsin in contact with PEPG was significantly (P < 0.01) lower than that of the trypsin stored in the buffer solution or the PCPG. Although poly(ethylene glycol) (PEG) is believed to be a promising polymer for reducing the nonspecific interaction with biomolecules,24 the molecular weight and density of the PEG molecules might influence the degree of biocompatibility. The hydrogels synthesized in this study can absorb significant amounts of water due to the hydrophilic MPC and PEGMA units. We then focused on the water absorbed in the hydrogels to clarify the interaction between the hydrogel and biomolecules. Tsuruta considered the importance of the water structure on biomedical polymers.25 In his review article, he explained that polymers having a hydroxyl group such as poly(HEMA) can incorporate water molecules at the surface and form a network structure of water molecules. Protein interaction starts with protein trapping by a network structure of water molecules on the surface. The longer the contact of a protein on the surface, the greater the chance of the protein interacting in denaturation and irreversible adsorption. This is a highly probable explanation for the mechanism of protein interaction on hydrophilic polymers. The free water content in the adsorbed water in the PCPG was relatively high. Thus, PCPG may not interact with protein and would be a good container for biomolecules. We have reported on the free water fraction in hydrophilic polymers and protein adsorption on polymer surfaces.20 When the free water fraction in the polymer increased, the adsorption and denaturation of bovine serum albumin or bovine plasma fibrinogen reduced. Furthermore, the effect of water-soluble polymers on the water structure has been discussed by Kitano et al.26 They explained that water structure in a poly(MPC) aqueous solution is similar to natural water. Figure 9 shows the number of v79 cell colonies that formed after contact with degraded PIOP or poly(MPC). When the cells were in contact with control compounds such as, ZDEC and ZDBC, the number of colonies decreased and was completely reduced at 0.125 and 7.5 µg/mL, respectively. In contrast, no decrease in the number of colonies formed due to contact with degraded PIOP or poly(MPC) was observed. This result indicates that PCPG is quite safe material. The investigation of in vivo biocompatibility of the PCPG is on going. In conclusion, a novel biodegradable cross-linker, a polymerizable polyphosphate (PIOP) was synthesized. Hydrolysis of the PIOP was influenced by pH of media. Hydrogels were prepared by radical polymerization of the PIOP and hydrophilic methacrylates. Nonenzymatic degradation of the hydrogel also occurred due to hydrolysis of the
Iwasaki et al.
Figure 9. Number of v-79 cell colonies formed after contact with hydrated PCPG. Filled circle: ZDEC, Filled square: ZDBC, Open circle: degradation products from PIOP immersed in PBS for 5 days. Open square: degradation products from PIOP immersed in PBS for 44 days. Open triangle: poly(MPC), [Cells] ) 50 cells/well.
PIOP. Particularly, the hydrogel having phosphorylcholine groups (PCPG) did not reduce enzyme activity, and degradation products from PCPG were noncytotoxic. Newly designed hydrogels prepared with biomimetic polymers having biocompatibility and biodegradability may be suitable polymeric materials in biomedical application. Acknowledgment. The Cosmetology Research Foundation supported a part of this study. We gratefully acknowledge the valuable discussion provided by Dr. Atsushi Harada of Osaka Prefecture University and Dr. Kikuko Fukumoto of The University of Tokyo. References and Notes (1) Renier, M. L.; Kohn, D. H. J. Biomed. Mater. Res. 1997, 34, 95104. (2) Wan, A. C.; Mao, H. Q.; Wang, S.; Leong, K. W.; Ong, L. K.; Yu, H. Biomaterials 2001, 22, 1147-1156. (3) Wang, J.; Zhang, P. C.; Lu, H. F.; Ma, N.; Wang, S.; Mao, H. Q.; Leong, K. W. J. Controlled Release 2002, 83, 157-168. (4) Libiszowski, J.; Kaluzynski, K.; Penczek, S. J. Polym. Sci., Part A: Polym. Chem. 1978, 16, 1275-1283. (5) Pretula, J.; Kaluzynski, K.; Penczek, S. Macromolecules 1986, 19, 1797-1799. (6) Richards, M.; Dahiyat, B. I.; Arm, D. M.; Lin, S.; Leong, K. W. J. Polym. Sci., Part A: Polym. Chem. 1991, 29, 1157-1165. (7) Wen, J.; Zhuo, R. X. Macromol. Rapid Commun. 1998, 19, 641642. (8) Peppas, N.; Ed. Hydrogels in Medicine and Pharmacy 1-3; CRC Press: Boca Raton, FL, 1987. (9) Derossi, D.; Kajiwara, K.; Osada, Y.; Yamaguchi, A.; Eds. Polymer Gels-Fundamentals and Biomedical Applications; Plenum: New York, 1989. (10) Drury, J. L.; Mooney, D. J. Biomaterials 2003, 24, 4337-4352. (11) Jeong, B.; Kim, S. W.; Bae, Y. H. AdV. Drug DeliV. ReV. 2002, 17, 37-51 (12) Ishihara, K.; Ueda, T.; Nakabayashi, N. Polym. J. 1990, 22, 355360. (13) Ishihara, K.; Oshida, H.; Endo, Y.; Ueda. T.; Watanabe, A.; Nakabayashi, N. J. Biomed. Mater. Res. 1992, 26, 1543-1552. (14) Ishihara, K.; Hasegawa, T.; Watanabe, J.; Iwasaki, Y. Artif. Organs 2002, 26, 1014-1019. (15) Iwasaki, Y.; Mikami, A.; Kurita, K.; Yui, N., Ishihara, K.; Nakabayashi, N. J. Biomed. Mater. Res. 1997, 36, 508-515. (16) Iwasaki, Y.; Sawada, S.; Ishihara, K.; Khang, G.; Lee, H. B. Biomaterials 2002, 23, 3897-3903. (17) Sawada, S.; Sakaki, S.; Iwasaki, Y.; Nakabayashi, N.; Ishihara, K. J. Biomed. Mater. Res. 2003, 64A, 411-416. (18) Edmundson, R. S. Chem. Ind. (London) 1962, 1828. (19) Iwasaki, Y.; Komatsu, S.; Narita, T.; Akiyoshi, K.; Ishihara, K. Macromol. Biosci. 2003, 3, 238-242.
Novel Biodegradable Polyphosphate Cross-Linker (20) Ishihara, K.; Nomura, H.; Mihara, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi, N. J. Biomed. Mater. Res. 1998, 39, 323-30. (21) Erlanager, B.; Kokowsky, N.; Cohen, W. Arch. Biochem. Biophys. 1961, 95, 271-278. (22) Kimura, Y. In Biomedical applications of polymeric materials; Tsuruta, T., Hayashi, T., Kataoka, K., Ishihara, K., Kimura, Y., Eds.; CRC Press: Boca Raton, FL, 1993; p 163-190. (23) Baran, J.; Penczek, S. Macromolecules 1995, 28, 5167-5176.
Biomacromolecules, Vol. 5, No. 3, 2004 1115 (24) Harris, J. M.; Ed. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical applications; Plenum: New York, 1992. (25) Tsuruta, T. AdV. Polym. Sci. 1996, 126, 1-51. (26) Kitano, H.; Sudo, K., Ichikawa, K.; Ide, M., Ishihara, K. J. Phys. Chem. B 2000, 104, 11425-11429.
BM049961M
