Bioconjugate Chem. 2002, 13, 23−28
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Novel Functional Biodegradable Polymer: Synthesis and Anticoagulant Activity of Poly(γ-Glutamic Acid)sulfonate (γ-PGA-sulfonate) Michiya Matsusaki,† Takeshi Serizawa,† Akio Kishida,‡ Takeshi Endo,§ and Mitsuru Akashi*,† Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, I-21-40 Korimoto, Kagoshima 890-0065, Japan, Department of Biomedical Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan, and Faculty of Engineering, Yamagata University, 4-13-16 Johnan, Yonezawa 992-8510, Japan. Received January 29, 2001; Revised Manuscript Received August 15, 2001
γ-Poly(glutamic acid) (γ-PGA), which is produced by Bacillus subtilis, was sulfonated using 2-aminoethane-1-sufonic acid (taurine) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) to give sulfonated γ-PGA (γ-PGA-sulfonate). From 1H NMR spectroscopy and IR spectroscopy, it was confirmed that taurine was introduced to the side chain of γ-PGA via an amide linkage. By altering the synthetic conditions, it was possible to control the content of sulfonate in γ-PGA-sulfonate. Anticoagulant activity was investigated in order to evaluate the biological activity of γ-PGA-sulfonate by the Lee-White test. The clotting time was prolonged when the concentration of γ-PGA-sulfonate on the degree of sulfonation was increased. It becomes clear that γ-PGA-sulfonate is potentially useful for various medical applications, such as drug delivery, tissue engineering, and medical materials.
INTRODUCTION
Biodegradable polymers are used in a wide variety of applications, such as suture materials, bone fixation materials, environmental materials, and in drug delivery systems. Recently, further application of biodegradable polymers has been desired in many situations, such as a simuli-responsible drug delivery system and tissue engineering. However, it is difficult to prepare such functional biodegradable polymers because most biodegradable polymers have a simple chemical structure and lack reactive main or side chains. Some researchers have tried to prepare the functional biodegradable polymers. Ohya and co-workers have studied polydepsipeptides, which are copolymers of R-amino acids and R-hydroxy acids having a reactive side chain (1, 2). Mikos and coworkers have synthesized poly(propylene fumarate) (PPF), which is a reactive unsaturated functional biodegradable polymer (3, 4). These polymers have unique properties and are further improved in performances as compared with the conventional ones; however, there are so many aspects of the demands to biodegradable polymers. For tissue engineering applications, there are some special demands to biodegradable polymers (in many cases, to scaffolds). A typical one is the ability of incorporating growth factors. Others are the fabrication of a complex shape, degradation rate control, and stimuli responsibility. On the other hand, when biodegradable polymers are used in biomedical fields, the polymers should be metabolized in biological systems at a final stage. So, R-hydroxy acids, amino acids, and lipids are currently used. When one would synthesize novel func* To whom correspondence should be addressed. Phone: +81-99-285-8320. Fax: +81-99-255-1229. E-mail: akashi@ apc.kagoshima-u.ac.jp. † Kagoshima University. ‡ National Cardiovascular Center Research Institute. § Yamagata University.
Scheme 1. Synthesis of Poly(γ-glutamic acid Sulfonate) (γ-PGA-Sulfonate)
tional biodegradable polymers, one should use the same resources, which have functional groups or double bonds in their chemical structure. Poly(γ-glutamic acid) (γ-PGA) is bioproduct which is secreted by a Bacillus subtilis strain, B. subtilis F-2-01 (5), and is mass produced by fermentation. The molecular weight of γ-PGA is very high (100 000-1 000 000), and this is a unique character of γ-PGA. Some researchers have tried to use γ-PGA as a novel biodegradable polymer by modifying the γ-PGA side chain. Kubota et al. have reported alkaline hydrolysis of γ-PGA (6) and esterification of the carboxyl group for the purpose of providing better solubility in organic solvents (7) or for the preparation of fiber (8). Kunioka et al. have studied the crosslinking reaction of γ-PGA by γ irradiation (9, 10). Hara has reported the preparation of γ-PGA hydrogel for applications as an environmental material (11). We have also reported the polyion complex formation between γ-PGA and chitosan (12) and polymer drug (13). For further applications, we tried to prepare the sulfonated γ-PGA (γ-PGA-sulfonate). Generally, it is well-known that heparin has many biological activities, such as anticoagulant activity, stabilizing activity of growth factors, antiviral activity, and
10.1021/bc010008d CCC: $22.00 © 2002 American Chemical Society Published on Web 12/20/2001
24 Bioconjugate Chem., Vol. 13, No. 1, 2002
plasma clearing activity. Various heparinoids were synthesized, and their biological activities were studied. Sulfated naturally occurring polysaccharides have been studied, such as dextran, chitin, and chitosan (14-17). Synthetic polymers, such as poly(vinyl sulfonate) and polysaccharide derivatives having sulfamide and Osulfonic acid groups (18, 19), are the examples. The heparinoid, having antiviral activity or cell growth stimulation activity, are also the examples (20-23). In this study, the sulfonic group was introduced into γ-PGA by using 2-aminoethane-1-sulfonic acid (taurine) in order to obtain sulfonated γ-PGA (γ-PGA-sulfonate). γ-PGA-sulfonate was inspired from the chemical structure of heparin, which is well-known to have anticoagulant activity and biodegradability. Biodegradable γ-PGAsulfonate could be termed “a bioinspired material” and is expected to have heparin-like biological activities. Taurine is one of the most abundant amino acids in animal tissues and is described as an essential amino acid for human and other mammalian species. Taurine has been reported to have several putative roles, functioning as a neurotransmitter, a neuromodulator, and a neurogrowth factor and functioning in membrane stabilization. Studies of sulfonation by conjugation with taurine have been reported in amino acids and oligopeptides (24, 25). Sulfonation of γ-PGA using taurine is a new trial in the meaning of the modification of γ-PGA in aqueous media. Because of the high molecular weight and poor solubility to aqueous media, the chemical modification of γ-PGA was done in organic media. In this study, the preparation of γ-PGA aqueous solutions and esterification reactions were studied (in detail). The anticoagulant activity of γ-PGA-sufonated was also investigated.
Matsusaki et al. Table 1. Preparation of γ-PGA-sulfonatea
runs
γ-PGA (mmol)
taurine (mmol)
WSC (mmol)
yield (%)
sulfonate contentb (%)
1 2 3 4 5 6 7 8
5 5 5 5 5 5 5 5
5 5 5 0.5 2.5 5 10 50
2.5 5 10 5 5 5 5 5
58 61 64 53 61 61 56 63
25 51 81 13 37 51 54 48
a Reaction time was 24 h at room temperature. b The sulfate content against total carboxyl groups was estimated by a 1H NMR spectrum.
were clotted. In vitro clotting time was evaluated using the following equations:
Lee-White clotting time ) time which sample tube was clotted time which reference tube was clotted Hydrolysis Testing of γ-PGA and γ-PGA-Sulfonate. γ-PGA (MW ) 350 000) and γ-PGA-sulfonate (10% and 90%, MW ) 150 000) were dissolved in a 0.2 M phosphate buffer (pH ) 7.4), and the concentration of samples was prepared at 0.1 wt %. Samples were stored at 80 °C until several hydrolysis times, molecular weights, and molecular weight distributions were measured by gel permeation chromatography (GPC) (TSK-GEL G5000PW and G4000PWxL column; flow rate was 1.0 mL/min), calibrated with pullulan (Shodex STANDARD P-82) standards. RESULTS AND DISCUSSION
MATERIALS AND METHODS
Materials. γ-PGA (MW 1 230 000, MW/Mn 1.20) was kindly donated by Meiji Seika Kaisya, Ltd. (Tokyo, Japan) and was used without further purification. Sodium hydrogen carbonate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC), taurine, and sodium dextran sulfate (DS) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Heparin sodium salt, polyvinyl sulfuric acid potassium salt (PVS), and calcium chloride were purchased from Nacalai Tesque (Kyoto, Japan). Poly(p-styrenesulfonic acid sodium salt) (PSS) was purchased from Tokyo Kasei (Tokyo, Japan). Synthesis of γ-PGA-Sulfonate. Adequate amounts of sodium hydrogen carbonate, γ-PGA, WSC, and taurine were dissolved in 30 mL of pure water under magnetic stirring for 30 min at 0 ∼ 4 °C. The reaction solution was maintained for 24 h at ambient temperature. The reaction solution was dialyzed using a Spectra/Pore membrane (wt off molecular weight of 50 000) for 3 days. γ-PGA-sulfonate was obtained by freeze-drying for 3 days. The reaction conditions are shown in Table 1. Anticoagulant Activity Test (Lee-White Test). The Lee-White test was carried out as follows. One milliliter of human whole blood, which was collected with a 21-gauge needle, was added to tubes each containing 1 mg/mL of each sample solution (sample tubes) and to an empty tube (reference tube). These tubes were allowed to stand for 3 min at 37 °C. After 3 min, the reference tube was tilted, and if the reference tube was not clotted, the tube was tilted every 30 s until clotting occurred. When the reference tube had clotted, the sample tubes were tiled every 30 s until the contents of these tubes
Synthesis of γ-PGA-Sulfonate. Figure 1 shows the H NMR spectra of γ-PGA, taurine, and γ-PGA-sulfonate (the synthetic condition is no. 2 in Table 1). All of the peaks corresponding to γ-PGA and the CH2 of taurine were observed after the reaction. The peak of a CH2 next to a nitrogen atom was shifted from 3.4 ppm in taurine to 3.6 ppm in γ-PGA-sulfonate, indicating that taurine was introduced into γ-PGA by an amide linkage. Figure 2 shows the FT-IR spectra of γ-PGA and γ-PGAsulfonate. The peaks at 1100-1200 and 1350 cm-1 for γ-PGA-sulfonate show the asymmetric and symmetric stretching vibration modes of S (dO2), respectively. These peaks were not observed for γ-PGA. The peaks at 1600 cm-1 of amide I and 1650 cm-1 of amide II were similarly observed for γ-PGA and γ-PGA-sulfonate. It was also confirmed that a carboxyl group of the side chain of γ-PGA was converted to an amide group, because a peak at 1750 cm-1 which corresponds to carbonyl vibration mode of carboxyl groups in γ-PGA was reduced in γ-PGAsulfonate. These results indicated that γ-PGA-sulfonate was prepared by the conjugation of taurine with γ-PGA. Sawa et al. has reported the molecular structure analysis of γ-PGA by IR spectra and by optical rotatory dispersion curves (26). Their report indicated that H-RPGA (COOH type) was an R-helix structure, H-γ-PGA (COOH type) was a parallel β structure, and Na-γ-PGA (COONa type) was a random structure. In this study, Naγ-PGA (COONa type) was used, so γ-PGA-sulfonate was thought to be a random structure. In Figure 2, when the FT-IR spectrum of γ-PGA is compared with that of γ-PGA-sulfonate, the reduction of a carboxyl group peak and the increase of sulfone group peaks were observed. Other changes of spectra were not observed. Accordingly, 1
Novel Functional Biodegradable Polymers
Figure 1.
1H
Bioconjugate Chem., Vol. 13, No. 1, 2002 25
NMR spectra of (A) γ-PGA, (B) taurine, and (C) γ-PGA-sulfonate in D2O.
Figure 3. Effect of concentration on anticoagulant activity (n ) 3).
Figure 2. FT-IR spectra of γ-PGA and γ-PGA-sulfonate.
the molecular structure of γ-PGA-sulfonate is confirmed to be a random structure. γ-PGA-sulfonate is thought to have an extended structure, because of ion repulsion by a sulfonic group. It is expected that the solubility of γ-PGA-sulfonate is different from that of γ-PGA by sulfonic group. The solubility of γ-PGA was compared with that of γ-PGAsulfonate. γ-PGA was dissolved in a pH > 7.4 aqueous solution. However, it was only dissolved in DMF, NMP, and DMSO and was not dissolved in other organic solvents. The solubility of γ-PGA-sulfonate was better than that of γ-PGA. For example, γ-PGA-sulfonate was dissolved in all pH’s for aqueous solutions, and it had a good solubility in organic solvents (DMF, NMP, and so forth). This result confirmed that the properties of γ-PGAsulfonate were different from those of γ-PGA. Control of Sulfonate Content. The amount of taurine and WSC in feed was varied in order to control the sulfonate content. The yield and sulfonate content in the γ-PGA-sulfonate are shown in Table 1. The sulfonate content increased with increasing WSC in feed, resulting in a maximum content of 81%. On the other hand, when the amount of taurine in feed was also increased in the
presence of a constant concentration at WSC (5 mmol), the sulfonate content was not increased to more than approximately 50%. Yields were around 60% under all preparing conditions. These results indicated that, in order to control sulfonate content, changing the WSC amount is most valuable. Anticoagulant Activity Test (Lee-White Test). It is expected that γ-PGA-sulfonate has various biological activities. In this study, anticoagulant activity was investigated as a typical bioactive evaluation of γ-PGAsulfonate. Anticoagulant activity was evaluated by the Lee-White test. Figure 3 shows the dependence of the clotting time on the γ-PGA-sulfonate concentration. The clotting time increased with an increase in the γ-PGAsulfonate concentration. This result indicates that the anticoagulant activity of γ-PGA-sulfonate was concentration-dependent. Figure 4 shows the dependence of the anticoagulant activity on the degree of sulfonation. The clotting time of all γ-PGA-sulfonate samples were prolonged when they were compared to those of γ-PGA. This result indicates that γ-PGA-sulfonate obtained anticoagulant activity as a result of introducing taurine. Moreover, the clotting time increased with an increase in the degree of sulfonation. Figure 5 compares the anticoagulant activity of γ-PGAsulfonate, heparin, dextran sulfate (DS), poly(vinyl sul-
26 Bioconjugate Chem., Vol. 13, No. 1, 2002
Matsusaki et al.
Figure 4. Effect of sulfonate content on anticoagulant activity (n ) 3). Solution concentration was 1 mg mL-1.
Figure 6. Degradation behavior of (A) γ-PGA and (B) 10% γ-PGA-sulfonate.
Figure 5. Comparison of anticoagulant activity between γ-PGA-sulfonate, heparin, dextran sulfate (DS), poly(vinyl sulfuric acid) (PVS), and poly(styrene sulfonic acid) (PSS) (n ) 3). Solution concentration was 1 mg mL-1.
furic acid) (PVS) and poly(styrene sulfonic acid) (PSS). The concentration of each solution was 1 mg mL-1, and heparin was used at a concentration of 1 IU mL-1 (approximately 6 µg/mL). The anticoagulant activity of γ-PGA-sulfonate was not high as compared with heparin or DS. However, it was possible to realize the anticoagulant activity on γ-PGA by the introduction of taurine. γ-PGA-sulfonate can be prepared on a large scale, suggesting that the sulfonation of γ-PGA has significant implications. In this study, the anticoagulant activity of γ-PGAsulfonate was evaluated by the Lee-White test using human whole blood. It was indicated that γ-PGA-sulfonate has higher anticoagulant activity than synthetic sulfated polymers, such as PSS and PVS. However, it is difficult to clarify the mechanism of anticoagulant activity by the Lee-White method. We have reported that not only the interaction with antithrombin III but also activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT) tests recognize the mechanism of anticoagulant activity of poly(glucosyloxyethyl methacrylate)sulfate [poly(GEMA)-sulfate] (27, 28). The results described that the antioagulant activity mechanism of poly(GEMA) sulfate was different from heparin. The mechanism of anticoagulant activity of γ-PGA-sulfonate will be evaluated in the future. Hydrolysis Testing of γ-PGA and γ-PGA-Sulfonate. The hydrolysis of γ-PGA has been reported in detail. Kubota et al. have investigated the alkaline hydrolysis of γ-PGA for the purpose of obtaining various molecular weights of γ-PGA (6). It was possible to obtain the expected molecular weight of γ-PGA by controling the NaOH concentration. Kunioka et al. have also reported the effect of temperature on the hydrolysis of γ-PGA hydrogel prepared by γ irradiation. The rate of hydrolysis increased with an increase in hydrolysis temperature. On
the basis of these observations, it is expected that γ-PGAsulfonate is biodegradable polymer. In this study, the hydrolysis of γ-PGA and γ-PGA-sulfonate (10 and 90%) was evaluated in a phosphate buffer (pH ) 7.4) at 80 °C. After 6, 12, 24, 48 h, the hydrolysis reaction was stopped by cooling at 4 °C, and the molecular weight of each sample was measured by GPC. Figure 6 shows the GPC profiles of the hydrolysis of γ-PGA and γ-PGAsulfonate (10%). High molecular weight polymers and oligomers coexisted without any intermediate molecular weight polymers. The ratio of high molecular weight polymers against its oligomer (molecular weight of about 2000) increased with an increase in time, indicating the hydrolysis of the polymers. Ninety percent of the γ-PGAsulfonate showed a similar tendency (data not shown). Figure 7 indicated the existence ratio of high molecular weight polymer and oligomer estimated by GPC. The change of existence ratio of oligomer γ-PGA and γ-PGAsulfonate has a similar tendency; however, the existence ratio of oligomer γ-PGA-sulfonate was more rapidly changed as compared to that of γ-PGA. It was confirmed that the hydrolysis ratio of γ-PGA-sulfonate was more rapid than that of γ-PGA. Further, the hydrolysis ratio of 90% γ-PGA-sulfonate was faster than that of 10% γ-PGA-sulfonate, indicating that the introduction of sulfonic groups to γ-PGA accelerate the polymer hydrolysis. The hydrolysis mechanisms of γ-PGA and γ-PGAsulfonate were suggested as follows. γ-PGA-sulfonate was thought to be more extended than γ-PGA, because of electrostatic repulsion of sulfonic groups. Accordingly, it was presumed that water molecules can readily attach the polymers, resulting that the hydrolysis of γ-PGAsulfonate was easier and faster than that of γ-PGA. The hydrolysis ratio of 90% γ-PGA-sulfonate was faster than that of 10% γ-PGA-sulfonate, because 90% γ-PGA-sulfonate has many sulfonic groups. In addition, the pH of the neighborhood of the polymer seemed to be lowered by the introduction of a sulfonic group, resulting that the acid hydrolysis of the polymers was accelerated. For these reasons, the hydrolysis rate of γ-PGA-sulfonate was dependent on the degree of sulfonation. The hydrolysis of γ-PGA and γ-PGA-sulfonate had high molecular weight polymers and oligomers without any intermediate molecular weight polymers. It was thought that when hydrolysis was started, hydrolysis of γ-PGA
Novel Functional Biodegradable Polymers
Bioconjugate Chem., Vol. 13, No. 1, 2002 27 ACKNOWLEDGMENT
This study was partially supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science. LITERATURE CITED
Figure 7. Existence ratio of (O) high molecular weight polymers and (4) oligomers was estimated by GPC: (A) γ-PGA and (B) 10% γ-PGA-sulfonate.
and γ-PGA-sulfonate seemed to rapidly proceed to the oligomer level. So, it was presumed that Figure 6 was only confirming the high molecular weight polymer and low molecular weight polymer (oligomer) peaks. This result was not clear in this study. Accordingly, further study of the hydrolysis mechanism of γ-PGA and γ-PGAsulfonate will be done in the future. It was confirmed that γ-PGA and γ-PGA-sulfonate had degradability. In this study, a hydrolysis test was carried out at 80 °C as an acceleration test. It is expected that γ-PGA-sulfonate will be hydrolyzed in vivo. γ-PGA is a component of Japanese traditional food Natto; in other words, γ-PGA is edible natural product. In the meaning of toxicity, γ-PGA is very safe; however, the biocompatibility of γ-PGA is now investigated. Taurine exists in animal tissues, and is an essential amino acid for mammalian species. So, the toxicity of taurine is thought to be very low. It is expected that γ-PGAsulfonate is a nontoxic material. The toxicity test of γ-PGA-sulfonate will be done in the future. CONCLUSION
γ-PGA-sulfonate was prepared by the reaction of taurine with γ-PGA. Results indicated that the sulfonate content could be controlled by changing the WSC amount. γ-PGA-sulfonate was found to have anticoagulant activity. Anticoagulant activity was dependent on the concentration and sulfonate content of γ-PGA-sulfonate. γ-PGAsulfonate has the potential to combine with cytokines, such as basic fibroblast growth factor, and to form polyion complexes with cationic biodegradable polymers. γ-PGAsulfonate is expected to have applications in a wide variety of areas, such as drug delivery systems, environmental materials, tissue engineering, and medical materials.
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BC010008D
