Novel Functional Biodegradable Polymer II: Fibroblast Growth Factor

Basic fibroblast growth factor (FGF-2) mitogenic activities of sulfonated poly(γ-glutamic acid) (γ-PGA-S) were investigated with chlorate-treated L9...
0 downloads 0 Views 463KB Size
Download PDF
Biomacromolecules 2005, 6, 400-407

400

Novel Functional Biodegradable Polymer II: Fibroblast Growth Factor-2 Activities of Poly(γ-glutamic acid)-sulfonate Michiya Matsusaki,† Takeshi Serizawa,‡ Akio Kishida,§ and Mitsuru Akashi*,† Department of Molecular Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita 565-0871, Japan, Research Center of Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, and Department of Biomedical Engineering, Advanced Biomedical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita 565-8565, Japan Received August 25, 2004; Revised Manuscript Received October 21, 2004

Basic fibroblast growth factor (FGF-2) mitogenic activities of sulfonated poly(γ-glutamic acid) (γ-PGA-S) were investigated with chlorate-treated L929 fibroblast culture tests. When 72% of the carboxyl groups in γ-PGA were sulfonated (γ-PGA-S72), cell numbers reached a maximum. The activity of γ-PGA-S72 was higher than that of γ-PGA and synthetic heparinoids and was almost comparable to that of heparin. Cytotoxicity of γ-PGA-S72 was not observed, regardless of the degree of sulfonation. FGF-2-protective effects of γ-PGA-S72 against acid and thermal inactivation were also evaluated, and γ-PGA-S72 showed higher FGF-2-protective effects in comparison to nonsulfonated γ-PGA. The steric structures of various sulfonated γ-PGA-Ss were analyzed by molecular modeling (molecular orbital method (MOPAC)) and indicated that γ-PGA-Ss are helical in vacuo. Results from MOPAC and the molecular mechanics method (MM2) demonstrated that electrostatic interactions can take place between sulfonic and carboxyl groups of γ-PGA-S and basic amino acid residues in FGF-2. γ-PGA-S72 can interact with FGF-2 strongly. Introduction Ever since the concept of tissue engineering was proposed in 1993,1 many trials have been performed to develop nextgeneration medical technologies for regenerating tissues and organs. Three key factors are required for tissue engineering: cells, scaffolds, and growth factors. Typical cell sources have included bone marrow, progenitor, and various stem cells; these cell types have been extensively studied in terms of development, differentiation, and proliferation.2-5 Scaffolds are also important, because they provide cell growth and differentiation environments. Collagen, poly(lactic acid) and poly(glycolic acid) have been widely used as tissue engineering matrices. Recently, novel approaches to functional scaffold preparation have extensively investigated. Mikos et al. prepared poly(propylene fumarate) as novel orthopedic composites,6,7 and Hubbell et al. synthesized cell adhesive fibrin matrices.8 We also reported novel biodegradable polymers composed of hydroxycinnamic acid and D,Llactic acid.9 However, it is difficult to control cellular functions by solely manipulating the scaffold or matrix. Therefore, the application of cytokines is thought to be essential for tissue engineering. Various cytokines, such as platelet-derived growth factor,10 hepatocyte growth factor,11 and basic fibroblast growth factor (FGF-2),12 have been used to enhance tissue regeneration. However, it is difficult to * To whom correspondence should be addressed. Tel.: +81-6-68797356. Fax: +81-6-6879-7359. E-mail: [email protected]. † Osaka University. ‡ The University of Tokyo. § National Cardiovascular Center Research Institute.

directly apply cytokines to tissue-engineering processes because of their sensitivity to denaturation by external environmental factors such as acidic- or basic-pHs and high temperatures. Therefore, we suggest a method for the preparation and application of polymers that, can stably interact with cytokines, which is crucial for the effective application of cytokines. FGF-2 belongs to a group of growth factors that have mitogenic, neurotrophic and angiogenic activities, and is widely used in tissue engineering.13-15 FGF-2 is unstable protein, but, heparin and heparan sulfate proteoglycans (HSPG) that have FGF-2 activity, keep up the stability of FGF-2 in the human body. FGF-2 was reported to stably interact with heparin or HSPG by electrostatic interaction.16-19 Gospodarpwocz et al. reported heparin, HSPG, and synthetic heparinoids have protective effect of FGFs.20,21 In interactions between sulfated polysaccharides and FGFs, sulfate groups appeared to predominantly bind to basic amino acid residues in FGFs. Polyanions such as dextran sulfate,22 nucleotides,23 sulfated β-cyclodextrin,24 glycosaminoglycnas (GAG),25 and synthetic (1 f 6)-R-D-mannopyranan sulfate26 having sulfate groups strongly interact with FGFs and mimic heparin effects on the biological activities of FGFs.27 These days, however, Kunou et al. reported that carboxyl groups in polysaccharides complement sulfate groups to significantly contribute to interactions between sulfated polysaccharide and FGFs.28 Since carboxyl groups in acidic polysaccharides have negative charges under physiological environments, it is plausible that carboxyl groups are important for interactions with FGFs.

10.1021/bm049492o CCC: $30.25 © 2005 American Chemical Society Published on Web 12/18/2004

Novel Functional Biodegradable Polymer II

In a previous study, we reported the design, synthesis, and anticoagulant activities of sulfonated poly(γ-glutamic acid) (γ-PGA-S) which was expected to have biological FGF-2 activities.29 γ-PGA is a naturally occurring polymer secreted by a Bacillus subtilis strain30 and potentially provides a resource for environmental and biodegradable materials.31-35 Sulfonate contents of γ-PGA-S were easily controlled (090%) by changing concentration of condensation agent in feed; sulfonation control is significant as an alteration in the sulfate content for conventional heparinoids is difficult due to the low chemical reactivity of the saccharic structure. We maintain that γ-PGA-S provides a suitable balance of sulfonic and carboxyl groups for interaction with FGF-2 due to the high chemical reactivity of γ-PGA. γ-PGA-S may be useful for tissue engineering because anticoagulant activities are much lower than those of heparin and heparinoids (e.g., dextran sulfate). Heparin and heparinoids sometimes give rise to side effects such as bleeding, which are dependent on the circumstances, as heparin and heparinoids possess high anticoagulant activities. Coagulant activity is important for implant materials except cardiovascular implant materials, because adhesive cells such as fibroblasts adhere onto blood clots, which form on/in implanted materials. In this study, we investigated interaction between FGF-2 and γ-PGA-S. We also evaluated FGF-2 protective effects on γ-PGA-S against acidic and thermal inactivation experiments in detail. Materials and Methods Materials. γ-PGA (Mw 310 000, Mw/Mn 1.20) was kindly donated by Meiji Seika Kaisya, Ltd. (Tokyo, Japan) and used without further purification. Sodium chlorate, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (WSC), sodium dextran sulfate (DS; Mw is 500 000) and 2-aminoethanesulfonic acid (taurine) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used as received. Recombinant human FGF-2 was purchased from Sigma Aldrich (St. Louis, MO). Poly(p-styrenesulfonic acid sodium salt) (PSS; Mw is 200 000) was purchased from Tokyo Kasei (Tokyo, Japan) and used as received. Heparin sodium salt (Mw is 20 000), poly(vinyl sulfuric acid potassium salt) (PVS; Mw is 500 000), and other chemicals were purchased from Nacalai Tesque (Kyoto, Japan) and used without further purification. Preparation of γ-PGA-Ss. γ-PGA-Ss were synthesized according to our former study.29 Briefly, adequate amounts of sodium hydrogen carbonate, γ-PGA, WSC, and taurine were dissolved in 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 (cutoff molecular weight of 50 000) for 3 days. The γ-PGA-S was obtained by freeze-drying the solution for 3 days. Fibroblast Cell Proliferation Assay. Chlorate-treated L929 fibroblasts that had suppressed biosynthesis of heparan sulfate proteoglycans (HSPG)36 were used in order to analyze the FGF-2 activity of γ-PGA-Ss, heparin heparinoids. The medium for the chlorate treatments was prepared as follows. Eagle’s minimal essential medium (MEM) was supplemented

Biomacromolecules, Vol. 6, No. 1, 2005 401

with 50 mM cystine, 30 mM sodium chlorate, 80 mM sodium chloride, L-glutamine, sodium hydrogen carbonate, and 10% fetal calf serum (FCS) (Sigma, St. Louis, MO) to form medium A. The cells were incubated for 48 h at 37 °C with medium A. The medium was then exchanged with Eagle’s MEM (10% FCS) and the cells were seeded in 48-well multiplate at 1∼104 cells/well. The cells were allowed to adhere overnight at 37 °C, and were washed twice with medium A supplemented with 0.1% BSA (medium B). After that FGF-2 (10 ng/mL) and various samples (10 µg/mL) were added and incubated in serum-free culture medium for 96 h at 37 °C. The number of cells was counted by using WST-1 (Dojindo, Kumamoto, Japan).37 FGF-2-protective Effects on γ-PGA-S against Acidic Inactivation Experiment. The method used is that of Shibata21 with minor modifications. The FGF-2 solution (10 µg/mL) was diluted to 20-fold with phosphate buffered saline (PBS) with or without γ-PGA-S and the other polymers. The mixture was adjusted to pH 7.4, 4.0, and 2.0 with HCl. After 30, 60, and 120 min of incubation, the solutions were further diluted 25-fold with Eagle’s MEM containing 10% FCS, and then these solutions added to chlorate-treated L929 fibroblasts which had been preincubated 24-well multiplates at 1∼104 cells/well for 24 h at 37 °C. The final concentration of FGF-2 in the medium was 10 ng/mL, whereas the concentration of all other polymers was 10 µg/mL. After 72 h, the number of cells was counted. The FGF-2 protective activity was evaluated as relative cell proliferation activity using the following formula: Percent cell number ) (cell number in treated FGF-2 and polymer treated groups)/(cell number in the control ∼100). The control (100%) consists of cells incubated with FGF-2 and polymers under normal conditions (37 °C, 5% CO2, pH ) 7.4). FGF-2-protective Effects on γ-PGA-S against Thermal Inactivation Experiment. The FGF-2 solution (10 µg/mL) was diluted to 20-fold with PBS, with or without γ-PGA-S, and the other polymers. The mixtures were then maintained at 37, 60, and 90 °C, respectively. After 30, 60, and 120 min of incubation, the solutions were further diluted 25-fold with Eagle’s MEM (10% FCS). These solutions were added to chlorate-treated L929 fibroblasts, which had been preincubated into 24-well multiplates at 1∼104 cells/well for 24 h at 37 °C. The final concentration of FGF-2 in the medium was 10 ng/mL, whereas the concentration of the sample polymers was 10 µg/mL. After 72 h, the number of cells was counted, and the percent cell number was calculated by the equation mentioned above. Molecular Modeling. Molecular modeling was performed using BioMedCAChe6.0 software (Fujitsu, Inc., Chiba, Japan). In our attempt to find stable and low-energy conformations of various sulfonated γ-PGA-Ss (six residues), the initial structure was subjected to energy minimization and molecular orbital method (MOPAC; MOZYME method). Structural data of the FGF-2 and heparin derivative complex was taken from the Protein Data Bank (entry code 1BFC). Heparin derivative hexasaccharide and H2O were removed from the complex. The energy minimized various sulfonated γ-PGA-Ss were manually docked to the heparin binding site in FGF-2 by molecular mechanics method (MM2).

402

Biomacromolecules, Vol. 6, No. 1, 2005

Figure 1. Relationship between L929 fibroblast cell number and sulfonate content of γ-PGA-S by chlorate-treated L929 fibroblasts proliferation in serum-free culture medium with FGF-2 (10 ng/mL) and γ-PGA-Ss (10 µg/mL) for 96 h (n ) 3). The number of cells was counted by using WST-1 reagent.37 The chlorate treatment is showed briefly as follows. The cells were incubated for 48 h at 37 °C with medium A (see materials and methods). The medium was then exchanged with 10% FCS Eagle’s MEM and the cells were seeded in 48-well multiplate at 1∼104 cells/well. The cells were allowed to adhere overnight at 37 °C, and were washed twice with medium B (see materials and methods). *Statistically significant difference (p < 0.05) using a two-sample t test for each comparison. NS ) no significant difference.

Matsusaki et al.

Results and Discussion

Figure 2. Comparison of FGF-2 activity of γ-PGA-S72 to that of various heparinoids were evaluated by chlorate-treated L929 fibroblasts proliferation in serum-free culture medium with FGF-2 (10 ng/ mL) and polymers (10 µg/mL) for 96 h (n ) 3). The number of cells was counted by using WST-1 reagent.37 The chlorate treatment is showed briefly as follows. The cells were incubated for 48 h at 37 °C with medium A (see materials and methods). The medium was then exchanged with 10% FCS Eagle’s MEM and the cells were seeded in 48-well multiplate at 1∼104 cells/well. The cells were allowed to adhere overnight at 37 °C, and were washed twice with medium B (see materials and methods). *Statistically significant difference (p < 0.05) using a two-sample t test for each comparison. NS ) no significant difference.

FGF-2 Activity of γ-PGA-Ss. Figure 1 shows the relationship between L929 fibroblast cell number and sulfonate content of γ-PGA-S produced by chlorate-treated L929 fibroblasts proliferation in serum-free culture medium with FGF-2 (10 ng/mL) and γ-PGA-Ss (10 µg/mL) for 96 h. Since chlorate treatment suppresses endogenous biosynthesis of HSPG in these cells, chlorate-treated L929 fibroblasts cannot proliferate in serum-free culture medium by themselves. If added polymers interact with FGF-2 (like HSPG), chlorate-treated L929 fibroblasts can proliferate in serum-free culture medium. The effect of each polymer on FGF-2 activity could be clearly evaluated by chlorate treatment.38 The cell number increased with increasing γ-PGA-S sulfonate content and the addition of 72% sulfonated γ-PGA-S (γ-PGA-S72) showed the highest cell number particularly in all of the γ-PGA-Ss. When the sulfonate content exceeded 72%, the cell number decreased with increasing sulfonate content. These results would indicate γ-PGA-S72 can stably interact with FGF-2 and the γ-PGA-S72-FGF-2 complex binds to the FGF receptor on cellular surfaces. In comparison with other γ-PGA-Ss, γ-PGA-S72 may have well-balanced steric and electrostatic structures that interact with FGF-2. Figure 2 shows a comparison of FGF-2 activity of γ-PGAS72 to that of various heparinoids. The addition of γ-PGAS72 induced higher cell numbers than those of other polymers and describes that γ-PGA-S72 has higher activity than heparin and other heparinoids. This result also demonstrates a significant point of γ-PGA-S72 because many

researchers have reported FGF-2 activities of conventional heparinoids without saccharic structures, which were lower than those of saccharic heparinoids.25 In general, saccharic heparinoids sulfate content cannot be easily controlled because of the low chemical reactive properties of the saccharic structure. However, the sulfonate content of γ-PGA-S is easily controlled because of inherent flexibility and high-chemical reactivities of polypeptide main-chains.29 A balance between sulfonic and carboxyl groups in γ-PGAS72 can be appropriate for interaction with FGF-2. The high molecular weight (Mw is 310 000) may also contribute to the high FGF-2 activity of γ-PGA-S72. However, FGF-2 activities of DS (Mw is 500 000), PSS (Mw is 200 000), and PVS (500 000) were lower than that of γ-PGA-S72 despite the higher molecular weight. Kunou et al. reported that carboxyl groups in polysaccharides complement sulfate groups to interaction between sulfated polysaccharide and FGFs.28 Faham et al. reported carboxyl group of iduronic acid residues in heparin interact with R121 (at a high affinity binding site) and K136 (at a low affinity binding site) in FGF-2.19 The interaction between polyanion and FGF-2 requires not only a sulfonic group but also a carboxyl group. Therefore, a balance of sulfonic and carboxyl groups should be important to foster an interaction with FGF-2. The physicochemical studies on the interaction with FGF-2 and γ-PGA-Ss such as dissociation constant (Kd) are currently being performed in order to elucidate more clearly the interaction between γ-PGA-S72 and FGF-2, and we will report it in the next paper.

Novel Functional Biodegradable Polymer II

Biomacromolecules, Vol. 6, No. 1, 2005 403

Figure 3. Comparison of the FGF-2-protective effect of heparin, γ-PGA, and γ-PGA-S72 under acidic conditions (pH ) 7.4 (O), 4.0 (4), and 2.0 (0)). The polymer concentrations were 10 µg/mL, and the FGF-2 concentration was 10 ng/mL. The number of cells was counted by using WST-1 reagent.37 The chlorate treatment is showed briefly as follows. The cells were incubated for 48 h at 37 °C with medium A (see materials and methods). The medium was then exchanged with 10% FCS Eagle’s MEM and the cells were seeded in 48-well multiplate at 1∼104 cells/ well. The cells were allowed to adhere overnight at 37 °C and were washed twice with medium B (see materials and methods). The controls consisted of chlorate-treated L929 fibroblasts incubated with FGF-2 and polymers without any treatment (n ) 3) *Statistically significant difference (p < 0.05) using a two-sample t test for each comparison with (a) under same condition.

FGF-2-protective Effects on γ-PGA-S against Acidic and Thermal Inactivation Experiments. Heparin and heparinoids were reported to have FGFs protective effects against acidic- and thermal- inactivation and enzymatic degradation.20,21 Like heparin, γ-PGA-S72 is expected to have FGF-2-protective effects; thus, we investigated the γ-PGA-S72 protective effects against acidic and thermal inactivation with chlorate-treated L929 cells proliferation tests. FGF-2 and various polymers were incubated in acidic and thermal culture media for predetermined times, and mixed solutions were added to chlorate-treated L929 culture media. If FGF-2 remains active during acidic or thermal treatments, chlorate-treated L929 cells can proliferate. The decrease in cell number indicates FGF-2 inactivation by the treatment. Figure 3 shows a comparison of acidic condition FGF-2-protective effects for heparin, γ-PGA, and γ-PGAS72. From Figure 3a, it is clear that FGF-2 itself is unstable even at pH ) 7.4 and 37 °C, as FGF-2 activation was only 68.3%. Furthermore, 77% FGF-2 was inactivated by incubation at pH ) 4.0 and 2.0 for 120 min. When γ-PGA was added to the culture medium (Figure 3b), chlorate-treated L929 cellular proliferation rose to 80% of that calculated

for the positive control. Cell growth promotion may not only be due to FGF-2-protective effects, but also due to γ-PGA cell-protective effects. Similar results have been previously reported; for example, water-soluble polysaccharides could protect chlorate-treated L929 cells and enhance proliferation activities.21,38 When the environmental pH was lowered, the proliferation of chlorate-treated L929 cells was drastically reduced even in the presence of γ-PGA. From this result, it is suggested that the FGF-2-protective effect of γ-PGA from acidic condition is relatively low. Interestingly, γ-PGA-S72 maintains high cellular proliferation comparable to heparin under all of the tested pH conditions. Cell numbers for heparin and γ-PGA-S72 were greater than those of γ-PGA over the entire pH-range and indicate that both heparin and γ-PGA-S72 can protect FGF-2 under low pH. Figure 4 shows results from FGF-2-protective effects under high-temperature conditions. In all cases, the cell number decreased with increasing treatment times and temperatures. Under the most severe condition (90 °C for 120 min), FGF-2 activity was drastically reduced when not protected by polymer and protected by γ-PGA; however, in the presence of heparin, 42% FGF-2 activity remained. In the presence

404

Biomacromolecules, Vol. 6, No. 1, 2005

Matsusaki et al.

Figure 4. Comparison of the FGF-2-protective effect of heparin, γ-PGA, and γ-PGA-S72 under various temperature conditions (37 °C (O), 60 °C (4), and 90 °C (0)). The polymer concentrations were 10 µg/mL, and the FGF-2 concentration was 10 ng/mL. The number of cells was counted by using WST-1 reagent.37 The chlorate treatment is showed briefly as follows. The cells were incubated for 48 h at 37 °C with medium A (see materials and methods). The medium was then exchanged with 10% FCS Eagle’s MEM and the cells were seeded in 48-well multiplate at 1∼104 cells/well. The cells were allowed to adhere overnight at 37 °C and were washed twice with medium B (see materials and methods). The controls consisted of chlorate-treated L929 fibroblasts incubated with FGF-2 and polymers but without any treatment (n ) 3) *Statistically significant difference (p < 0.05) using a two-sample t test for each comparison with (a) under same condition.

of γ-PGA-S72, the remaining FGF-2 activity was 30%, which was slightly lower than that of heparin. Shibata et al. reported FGF-2-protective effects of DS and other polysaccharides.21 In their study, the FGF-2-protective effect of DS at pH ) 2.0 for 120 min was 13%. The activity of chondroitin sulfate (CS) was 40%, and fucus fucoidan was 42% at pH ) 2.0 for 120 min. In comparison, the FGF-2protective effect of γ-PGA-S72 was 43% at pH ) 2.0 for 120 min. This result indicates that the FGF-2-protective effect of γ-PGA-S72 was greater than that of other heparinoids. Based on this comparison, sulfonation is thought to be necessary for FGF-2 protective effects.24,26 Coltrini et al. reported the capacity of different GAGs to protect FGF-2 from proteolysis.25 The protect effect from proteolysis decreases in the following order: heparin > heparan sulfate > dermatan sulfate ) chondroitin sulfates A and C > hyaluronic acid. They reported the protective effect might be caused by the degree of sulfation and the backbone structure of GAG modulate. In the case of γ-PGAS72, it showed high FGF-2 protective effect comparable to heparin, although the backbone structure is different. We

would like to suggest a balance of sulfonic acid and carboxyl groups is also important to enhance an interaction with FGFs. It is a noteworthy phenomenon that γ-PGA-S72 can protect FGF-2 under low pH. FGF-2 is sometimes used at wound sites, where severe inflammatory reactions occur and the surrounding pH is lowered. Wound site inflammatory reactions last 6 to 24 h. Therefore, γ-PGA-S72 is expected to protect FGF-2 during inflammatory reactions. On the other hand, the protective activity of γ-PGA-S72 against heat treatment supports a substrate protective mechanism during chemical reactions at high temperatures and during storage under normal temperatures. Both FGF-2 stabilizing activities of γ-PGA-S72 are useful from the point of view of reducing the necessary amount of FGF-2. This is an important issue in regards to functional materials that will be used for tissue engineering. The methodology for the incorporation of γ-PGA-S into tissue engineering scaffolds has been established. In a previous study, we reported ultrathin film formation of γ-PGA and chitosan.39 Moreover, the preparation and biological evaluation of γ-PGA-S hydrogels was

Novel Functional Biodegradable Polymer II

Biomacromolecules, Vol. 6, No. 1, 2005 405

Figure 5. Simulated structure of heparin and γ-PGA (six residues).

Figure 6. Simulated structure of various sulfonated γ-PGA-Ss.

studied.40 Therefore, γ-PGA-S is thought to be useful in a wide variety of areas, including biomedical applications. Molecular Modeling. It was confirmed that γ-PGA-S had high FGF-2 activities and FGF-2-protective effects by cell culture tests. However, a detailed reason or mechanism could not be elucidated from cell culture tests. Accordingly, we considered the detailed interaction between γ-PGA-S and FGF-2 by molecular modeling. A heparin-FGF-2 binding site and heparin-derived hexasaccharide complex has been described by X-ray crystallography.19 Simulated structures of heparin, γ-PGA, and various sulfated γ-PGA-Ss (six residues) are shown in Figures 5 and 6. Initial structures were subjected to energy minimization and molecular orbital

method (MOPAC; MOZYME method). Heparin was helical and its carboxyl and sulfate groups were arranged outside the helix. γ-PGA and γ-PGA-Ss were also helical because of electrostatic reflection between carboxylic and sulfonic acids. Figure 7 shows a simulated structure of γ-PGA-S71 (six residues) and FGF-2 complex based on the crystal structure.19 Energy minimized γ-PGA-S71 is manually docked to the heparin-binding site of FGF-2. To fit γ-PGAS71 on the binding site, various orientations of γ-PGA-S71 were examined. The binding site of FGF-2 to γ-PGA-S71 was the same as that shown for heparin in the literature.19 Carboxylic and sulfonic acids of γ-PGA-S71 could interact with basic amino residues of FGF-2. Sulfonic acid groups

406

Biomacromolecules, Vol. 6, No. 1, 2005

Matsusaki et al.

Figure 7. Supposed binding site of γ-PGA-S71 and FGF-2 constituted by molecular modeling using BioMedCAChe6.0. The higher binding sites were red, whereas the lower binding sites were blue.

were assigned in the vicinity of basic amino residues that were located at the high affinity FGF-2 binding site. It has been reported that carboxyl groups of iduronic acid residues in heparin hexasaccharide can interact with R121 (at a high affinity binding site) and K136 (at a low affinity binding site) in FGF-2.19 In the case of γ-PGA-S71, carbonyl groups of amide bonds could interact with R121 and K136 in FGF-2 (Figure 7). These results suggest that γ-PGA-S71 was able to interact with FGF-2 in heparin-like manner. Kunou et al. reported a sulfated colominic acid with well FGF-2 activity and basic amino acid residues interaction with FGF-2 (except K136) by molecular modeling.28 The simulated structure of other γ-PGA-Ss (S29, S57 and S100) and FGF-2 complex were also calculated. To fit γ-PGA-Ss on the binding site, various orientations of γ-PGA-Ss were examined, however, in the case of γ-PGA-S29 and S57, there is no sulfonic acid groups which were assigned in the vicinity of basic amino residues that were located at the high affinity FGF-2 binding site. In the case of γ-PGA-S100, carbonyl groups of amide bonds could not interact with R121 and K136 in FGF-2 (data not shown). These results of molecular modeling indicated that γ-PGA-S29, -S57, and -S100 cannot interact strongly with FGF-2 because of lacking in balance of carboxylic and sulfonic acids. This reason supports cell culture results that cell numbers at γ-PGA-S10, -S29, and -S91 were lower than that of γ-PGA-S72 (Figure 1). In the case of γ-PGA-S10, -S29, and -S91, γ-PGA-Ss cannot interact strongly with FGF-2 and may not accelerate so much the mitogenic activities of FGF-2.The molecular modeling results support cell culture test results, and indicate that γ-PGA-S sulfonated to 70% could form a stable FGF-2 complex. Conclusion It was confirmed that γ-PGA-S72 had high FGF-2 activities, which was comparable to that of heparin. This is

a significant point of γ-PGA-S72 because many researchers have reported that FGF-2 activities of conventional heparinoids without saccharic structures were lower than that of saccharic heparinoids. Furthermore, results of molecular modeling supported that γ-PGA-S71 could interact with FGF-2 in a similar manner as heparin does. These results suggested that a novel molecular-design indicator, which was suggested in previous report29 and this paper, is useful for a novel molecular-design method of novel tissue engineering material. γ-PGA is a biodegradable polymer and can be applied in various forms such as a solid resin, sheet, or hydrogel. Therefore, the fairly high biological activities of γ-PGA-S suggest that we could prepare various novel materials for cell culture and tissue engineering materials. Acknowledgment. This study was partially supported by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science. This study was also supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. The authors are grateful to Assistant Professor Dr. T. Kaneko and Lecturer Dr. T. Kida of Osaka University and Professor Dr. Y. Nakatsuji of Osaka Institute of Technology University for their advice and suggestions. References and Notes (1) Langer, R.; Vacanti, J. P. Science 1993, 260, 920-925. (2) Iwaguro, H.; Yamaguchi, J.; Kalka, C.; Murasawa, S.; Masuda, H.; Hayashi, S.; Silver, M.; Li, T.; Isner, J. M.; Asahara, T. Circulation 2002, 105, 732-738. (3) Isner, J. M.; Kalka, C.; Kawamoto, A.; Asahara, T. Ann. N. Y. Acad. Sci. 2002, 953, 75-84. (4) Kawasaki, H.; Mizuseki, K.; Nishikawa, S.; Kaneko, S.; Kuwana, Y.; Nakanishi, S.; Nishikawa, S. I.; Sasai, Y. Neuron 2000, 28, 3140. (5) Matsui, M.; Mizuseki, K.; Nakatani, J.; Nakanishi, S.; Sasai, Y. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5291-5296. (6) He, S.; Timmer, M. D.; Yaszemski, M. J.; Yasko, A. W.; Engel, P. S.; Mikos, A. G. Polymer 2000, 42, 1251-1260.

Novel Functional Biodegradable Polymer II (7) He, S.; Yaszemski, M. J.; Yasko, A. W.; Engel, P. S.; Mikos, A. G. Biomaterials 2000, 21, 2389-2394. (8) Schense, J. C.; Bloch, J.; Aebischer, P.; Hubbell, J. A. Nat. Biotechnol. 2000, 18, 415-419. (9) Matsusaki, M.; Kishida, A.; Stainton, N.; Ansell, C. W. G.; Akashi, M. J. Appl. Polym. Sci. 2001, 82, 2357-2364. (10) Richardson, T. P.; Peters, M. C.; Ennett, A. B.; Mooney, D. J. Nat. Biotechnol. 2001, 19, 1029-1034. (11) Atabey, N.; Gao, Y.; Yao, Z. J.; Breckenridge, D.; Soon, L.; Soriano, J. V.; Burke, T. R., Jr.; Bottaro, D. P. J. Biol. Chem. 2001, 276, 14308-14314. (12) Kawai, K.; Suzuki, S.; Tabata, Y.; Ikada, Y.; Nishimura, Y. Biomaterials 2000, 21, 489-499. (13) Van, W. P. B.; Plantinga, J. A.; Wissink, M. J.; Beernink, R.; Poot, A. A.; Engbers, G. H.; Beugeling, T.; Van, A. W. G.; Feijen, J.; Van, L. M. J. J. Biomed. Mater. Res. 2001, 55, 368-378. (14) Sakiyama, E. S. E.; Hubbell, J. A. J. Controlled Release 2000, 65, 389-402. (15) Tabata, Y.; Miyao, M.; Inamoto, T.; Ishii, T.; Hirano, Y.; Yamaoki, Y.; Ikada, Y. Tissue Eng. 2000, 6, 279-289. (16) Spivak-Kroizman, T.; Lemmon, M. A.; Dikic, I.; Ladbury, J. E.; Pinchasi, D.; Huang, J.; Jaye, M.; Crumley, G.; Schlessinger, J.; Lax, I. Cell 1994, 79, 1015-1024. (17) Pantoliano, M. W.; Horlick, R. A.; Springer, B. A.; Dyk, D. E. V.; Tobery, T.; Wetmore, D. R.; Lear, J. D.; Nahapetian, A. T.; Bradley, J. D.; Sisk, W. P. Biochemistry 1994, 33, 10229-10248. (18) Plotnikov, A. N.; Schlessinger, J.; Hubbard, S. R.; Mohammadi, M. Cell 1999, 98, 641-650. (19) Faham, S.; Hileman, R. E.; Fromm, J. R.; Linhardt, R. J.; Rees, D. C. Science 1996, 271, 1116-1120. (20) Gospodarowicz, D.; Cheng, J. J. Cell Biol. 1986, 128, 475-484. (21) Shibata, H.; Takagi, I. K.; Nagaoka, M.; Hashimoto, S.; Aiyama, R.; Iha, M.; Ueyama, S.; Yokokura, T. BioFactors 2000, 11, 235245. (22) Kajio, T.; Kawahara, K.; Kato, K. FEBS 1992, 306, 243-246. (23) Chavan, A. J.; Haley, B. E. Biochemistry 1994, 33, 7193-7202. (24) Burke, C. J.; Volkin, D. B.; March, H.; Middaugh, R. Biochemistry 1993, 32, 6419-6426.

Biomacromolecules, Vol. 6, No. 1, 2005 407 (25) Coltrini, D.; Rusnati, M.; Zoppetti, G.; Oreste, P.; Isacchi, A.; Caccia, P.; Bergonzoni, L.; Presta, M. Eur. J. Biochem. 1993, 214, 51-58. (26) Kunou, M.; Hatanaka, K. Carbohydr. Res. 1995, 28, 107-112. (27) Liekens, S.; Leali, D.; Neyts, J.; Esnouf, R.; Rusnati, M.; Dell’Era, P.; Maudgal, P. C.; Clercq, E. D.; Presta, M. Mol. Pharm. 1999, 56, 204-213. (28) Kunou, M.; Koizumi, M.; Shimizu, K.; Kawase, M.; Hatanaka, K. Biomacromolecules 2000, 1, 451-458. (29) Matsusaki, M.; Serizawa, T.; Kishida, A.; Endo, T.; Akashi, M. Bioconjugate Chem. 2002, 13, 23-28. (30) Kubota, H.; Matsunobu, T.; Uotani, K.; Takebe, H.; Satoh, A.; Tanaka, T.; Taniguchi, M. Biosci. Biotechnol. Biochem. 1993, 57, 1212-1217. (31) Kubota, H.; Nambu, Y.; Endo, T. J. Polym. Sci., Part A: Polym. Chem. 1995, 33, 85-88. (32) Serizawa, T.; Goto, H.; Kishida, A.; Endo, T.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. 1999, 36, 801-804. (33) Tachaboonyakiat, W.; Serizawa, T.; Endo, T.; Akashi, M. Polym. J. 2000, 32, 481-485. (34) Tachibana, Y.; Kurisawa, M.; Uyama H.; Kobayashi S. Biomacromolecules 2003, 4, 1132-1134. (35) Matsusaki, M.; Hiwatari, K.; Higashi, M.; Kaneko, T.; Akashi, M. Chem. Lett. 2004, 33, 398-399. (36) Keller, K. M.; Brauer, P. R.; Keller, J. M. Biochemistry 1989, 28, 8100-8107. (37) Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K. Biol. Pharm. Bill. 1996, 19, 1518-1520. (38) Taguchi, T.; Kishida, A.; Sakamoto, N.; Akashi, M. J. Biomed. Mater. Res. 1998, 41, 386-391. (39) Tachaboonyakiat, W.; Serizawa, T.; Endo, T.; Akashi, M. Polym. J. 2000, 32, 481-485. (40) Matsusaki, M.; Serizawa, T.; Kishida, A.; Akashi M. J. Biomed. Mater. Res., submitted.

BM049492O