Thiol-Modified Chitosan Sulfate Nanoparticles for Protection and

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Bioconjugate Chem. 2010, 21, 28–38

Thiol-Modified Chitosan Sulfate Nanoparticles for Protection and Release of Basic Fibroblast Growth Factor Yi-Cheng Ho,‡ Shao-Jung Wu,§ Fwu-Long Mi,*,†,‡,⊥ Ya-Lin Chiu,| Shu-Huei Yu,# Nilendu Panda,| and Hsing-Wen Sung*,†,| Department of Biotechnology, Nano Materials R&D Center, and Department of Polymer Materials, Vanung University, Chung-Li, Taiwan, ROC, Department of Chemical Engineering, MingChi University of Technology, Taipei, Taiwan, ROC, and Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC. Received May 8, 2009; Revised Manuscript Received November 20, 2009

A series of chitosan (CS) derivatives, the 6-O-carboxymethylchitosan (6-O-CC), 2-N sulfated 6-O-carboxymethylchitosan (N-SOCC) and the 2-N and 3,6-O sulfated 6-O-carboxymethyl chitosan (N,O-SOCC) were synthesized in this study. The chemical structures and the degrees of substituted carboxymethyl and sulfate groups of the synthesized compounds were respectively determined by FT-IR spectra and elemental analysis. N,O-SOCC displayed the highest protective efficiency for basic fibroblast growth factor (bFGF) as examined by the L929 fibroblast culture test and docking simulation. N,O-SOCC-4-thio-butylamidine (TBA) conjugates prepared by modification of N,O-SOCC with 2-iminothiolane were in situ cross-linkable. The degrees of thiol substitution of the 2-iminothiolane modified N,O-SOCC polymers were determined to be in the ranges of 45.9 ( 3.7 and 415.6 ( 12.5 µmol SH/g SOCC by quantifying the amount of thiol groups on the thiolated polymers with Ellman’s reagent. The 2-iminothiolane modified N,O-SOCC and CS complex could be used for preparing nanoparticles by a polyelectrolyte self-assembly method, and the release of bFGF from the nanoparticles was successfully controlled. L929 fibroblast culture tests showed that the thiol modified N,O-SOCC/CS nanoparticles could effectively protect bFGF from inactivation over a 120 h period. The results of this study suggest that the thiol modified N,O-SOCC/ CS nanoparticles may be useful as novel materials for specific delivery of bFGF with mitogenic activity.

INTRODUCTION Heparin and heparan sulfate (HS) are members of the glycosaminoglycan family possessing higher negative charge density than other known biological macromolecules. Heparin/ HS is able to establish ionic interactions with a variety of proteins including enzymes, extracellular-matrix proteins, various cytokines, and members of the family of fibroblast growth factors (FGFs), epidermal growth factor (EGF), and the transforming growth factor (TGF) (1, 2). Such interactions especially control specified polysaccharide-protein interactions and possess biological potential in relation to cell growth, wound healing, and tissue regeneration, as well as thrombin inhibition (3). Basic fibroblast growth factor (bFGF) is a heparin/HS-binding growth factor and has shown therapeutic potential to enhance angiogenesis, soft tissue formation, re-epithelialization, and collagen maturation, as well as to accelerate bone fracture healing (4, 5). Heparin/HS can stabilize an active conformation of bFGF and enhance interaction with specific cellular receptors (6, 7). On the basis of these reasons, many studies have reported the development of heparin-functionalized materials for the purpose of delivering bFGF to enhance tissue * Correspondence to Fwu-Long Mi, PhD, Professor, Department of Biotechnology, Vanung University, Chung-Li, Taiwan 320, ROC, Fax: 886-3-4333063, E-mail: [email protected]. Dr. H.W. Sung: [email protected]. † The contributions by the two collaborating parties are equal. ‡ Department of Biotechnology, Vanung University. § MingChi University of Technology. | National Tsing Hua University. ⊥ Nano Materials R&D Center, Vanung University. # Department of Polymer Materials, Vanung University.

regeneration (8-10). Besides, preparations of conjugates of bFGF and polymer-based gene delivery vectors became potential targets for cancer gene therapy because fibroblast growth factor receptors (FGFRs) are highly expressed on a variety of human cancer cells (11, 12). In recent years, significant advances in the modification of polysacchsarides with specific structures or synthesis of polymers with pendent saccharides offer one biomimetic strategy to obtain heparin-like materials. For example, acrylamide with alkene-derivatized mono- and disaccharides can be polymerized to yield glycopolymers with biologically active, sulfated Nacetyl-D-glucosamines on the side chains (13). Chemical modifications of biological macromolecules such as dextran, colominic acid, poly(γ-glutamic acid), and alginate with sulfate or sulfonate groups have been shown to bind various growth factors and protect them from inactivation (14, 15). Chitosan (CS) and its derivatives have been used for the purpose of biomedical applications, such as gene delivery (16). A series of chitosan derivatives, the 6-O-carboxymethylchitosan (6-O-CC), 2-N sulfated 6-O-carboxymethylchitosan (N-SOCC) and the 2-N and 3,6-O sulfated 6-O-carboxymethylchitosan (N,O-SOCC) were synthesized in this work. The structures of 6-O-CC, N-SOCC, and N,O-SOCC were characterized by FTIR spectra analysis. The studies of L929 cell proliferation and docking simulation were used to investigate the growth factor protective effects of these chitosan derivatives with carboxymethyl and sulfate groups substituted in different position. 2-Iminothiolane as a prethiomer was reacted with N,O-SOCC to prepare the thiol-modified N,O-SOCC polymers, and the degrees of thiolation were determined by a colorimetric assay using Ellman’s reagent. The 2-iminothiolane modified N,OSOCC polymers displayed in situ cross-linking properties due to the oxidation of substituted thiol groups. Basic fibroblast

10.1021/bc900208t  2010 American Chemical Society Published on Web 12/14/2009

Thiol-Modified Chitosan Sulfate Nanoparticles

growth factor (bFGF) were readily immobilized in the 2-iminothiolane modified N,O-SOCC/chitosan (CS) nanoparticles, and the immobilized bFGF could be released in a sustained manner with mitogenic activities. The novel materials prepared from the thiol-modified N,O-SOCC polymer may be useful to deliver bFGF for biomedical applications.

EXPERIMENTAL PROCEDURES Materials. Monochloroacetic acid, isopropyl alcohol, and sodium carbonate were purchased from ACROS Chemical Co. (USA). Chitin powder was acquired from Tokyo Chemical Industry Co., Ltd. Oleum (H2SO4 · SO3), dithiothreitol (DTT), and trimethylamine sulfur trioxide were purchased from SigmaAldrich Chemical Co., Ltd. (USA). 2-Iminothiolane was from Aldrich Chemical Co. (Milwaukee, WI). CS (MW 60 kDa) with a degree of deacetylation of approximately 85% was acquired from Koyo Chemical Co. Ltd. (Japan). Recombinant human bFGF and enzyme-linked immunosorption assay (ELISA) kit were obtained from R&D Systems (Minneapolis, MN, USA). Synthesis of 6-O-CChitin and 6-O-CC. 6-O-CChitin was prepared according to the method described in the previous literature with some modification (17). 8.16 g of chitin powder was suspended in 40 mL sodium hydroxide solution (0.4 g/mL) including 0.2% sodium dodecyl sulfate (SDS) at 4 °C, and the slurry was kept in a freezer at -20 °C overnight. The frozen alkali-chitin was suspended in 200 mL isopropyl alcohol at room temperature, and 11.34 g of monochloroacetic acid was added in portions with mechanical stirring until the reaction mixture was neutralized. The product was filtered and washed with ethanol. The residue was extracted with water, and the water extract was slowly added to acetone to precipitate the crude product. The crude product was extensively dialyzed against deionized water and finally freeze-dried to obtain 6-O-CChitin. 9.65 g of 6-O-CChitin was placed in 40 mL NaOH/isopropyl alcohol aqueous solution (0.4 g/mL) and refluxed for 3 h under purging with nitrogen. The gel-like product was dissolved in water and precipitated by slowly adding acetone to the aqueous solution. The precipitation was dissolved in water and lyophilized after thorough dialysis against deionized water to remove the salt. The final product was 6-O-carboxymethylchitosan (6O-CC). Synthesis of N-Sulfated 6-O-CC. 6-O-CC was N-sulfated according to the method developed by Holme and Perlin (18). 6-O-CC (0.23 g) was suspended in 60 mL of an aqueous solution of sodium carbonate (0.33 g), stirred at 65 °C, and trimethylamine sulfur trioxide complex (0.62 g) was added. The reaction was carried out for 20 h. The media became clear and homogeneous at the end of the reaction. At the determined reaction time, the mixture was cooled to room temperature, extensively dialyzed against deionized water, and finally freeze-dried to obtain N-sulfated 6-O-CC (N-SOCC). Synthesis of N,O-Sulfated 6-O-CC. N-SOCC was further sulfated for the preparation of N,O-Sulfated 6-O-CC (N,OSOCC), according to the method developed by Vikhoreva et al. (19). Sulfation of N-SOCC was conducted in a reaction vessel by adding of small portions of oleum to a dimethylformamide (DMF) excess (0.53 g oleum and 2.12 g DMF per 0.29 g N-SOCC). The mixture was stirred vigorously at 5 °C, and then N-SOCC was added to the mixture for sulfation reaction. By stirring for 2 h, the reaction mixture was cooled to 20 °C, and a solid fraction was separated by filtration through a glass filter. Crude N,O-SOCC was precipitated from solution in 5-fold acetone volume and washed with acetone twice, then dissolved in distilled water with simultaneous neutralization with a 20% NaOH solution. Sodium salts of N,O-SOCC were extensively dialyzed against deionized water, and finally freeze-dried to obtain purified N,O-SOCC. Degrees of carboxymethylation and

Bioconjugate Chem., Vol. 21, No. 1, 2010 29

sulfation of the chitosan derivatives were determined by elemental analysis. The schematic procedure for the synthesis of 6-O-CChitin, 6-OCC, N-SOCC, and N,O-SOCC were shown in Figure 1. Characterization of the Synthesized Compounds. The synthesized 6-O-CChitin, 6-OCC, N-SOCC, and N,O-SOCC products were characterized by FT-IR (Perkin-Elmer Spectrum RX1 FT-IR System, Buckinghamshire, England). The substitutions of carboxymethyl groups and sulfated groups were determined by elemental analysis (Elementar vario EL 0) of the content ratios of C, H, O, N, and S in the synthesized 6-OCChitin, 6-OCC, N-SOCC, and N,O-SOCC polymers. Molecular weights of chitin and its derivatives were measured by gel permeation chromatography (GPC), which incorporated a TSK G3000-PW column and a RI 150 refractive index detector with 0.1 M aq NaCl eluent flowing at 1.0 mL/min at 30 °C (20). Protection of Mitogenic Activity of bFGF Using 6-OCC, N-SOCC, and N,O-SOCC Polymers. The mitogenic activity of the bFGF protected by the synthesized polymers, under acidic condition, was assessed in vitro by determining its ability to stimulate the proliferation of L929 fibroblast cells cultured in serum-free Eagle’s MEM medium, according to the method reported by Akashi et al. (21). The bFGF solution (5 µg/mL) was dissolved in phosphate buffered saline (PBS) containing the synthesized 6-OCC, N-SOCC, and N,O-SOCC polymers. The mixture was adjusted to pH 4.0 with HCl. After 30-120 min of incubation, the solutions were diluted with Eagle’s MEM containing 10% FCS, and then these solutions added to chloratetreated L929 fibroblasts which had been preincubated 24-well multiplates at 1 × 104 cells/well for 24 h at 37 °C. The final concentration of bFGF in the medium was 250 ng/mL, whereas the concentration of all synthesized polymers was 10 µg/mL. Cell viability is determined in percent by comparison to control cell cultures uisg a MTT cell viability assay kit (Biotium, Inc. USA). The bFGF protective activity was evaluated as relative cell proliferation respectively in polymer treated groups, at pH 4.0, as compared with the control group (in PBS, pH 7.4). Percent cell number ) (cell number in treated bFGF and polymer treated groups)/(cell number in the control). The control (100%) consists of cells incubated with bFGF and polymers under normal conditions (37 °C, 5% CO2, pH )7.4). Docking Simulation. Molecular modeling was performed with the program AutoDock using parameters adapted from the AMBER force field. The dimer units of 6-OCC, N-SOCC, and N,O-SOCC polymers and heparin were used for molecular docking in Sybyl mol2 format. A heparin-binding site of bFGF was obtained from X-ray crystallography as reported in the literature (22). The structural data of bFGF were obtained from the Protein Data Bank (entry code 1BFC). The flexible torsions of ligands were calculated with AutoTors, which docked a flexible ligand into a rigid receptor. To carry out docking simulations, a grid box was defined to enclose the heparin binding site with dimensions of 60 × 60 × 60 Å3 and a grid spacing of 0.375 Å. Docking was performed using the Lamarckian genetic algorithm (LGA) and Solis and Wets algorithm, where the number of GA is 10, the population size is 50, the maximum number of energy evaluations is 250 000, and the size of local search space to sample is 1.0 will perform at most 300 interactions. The simulation results were shown by figures displaying atomistic pictures of the molecules generated using the molecular graphic program UCSF Chimera. Thiolation of N,O-SOCC. Thiolated N,O-SOCC was prepared by reacting 2-iminothiolane with amino groups bestows SH groups to N,O-SOCC (23). Initially, 500 mg of N,O-SOCC was dissolved in 50 mL of DI water and different amounts of 2-iminothiolane (200-600 mg) was added with 1 mL of 2% DTT. The pH value was adjusted, respectively, with 0.1 M

30 Bioconjugate Chem., Vol. 21, No. 1, 2010

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Figure 1. Schematic illustration of the procedures for synthesis of 6-O-CC, N-SOCC, and N,O-SOCC. SDS, sodium dodecyl sulfate; IPA, isopropanol; Oleum, fuming sulfuric acid; DMF, dimethylformamide.

NaOH and HCl to pH 5.0 or 7.0. The coupling reaction was allowed to proceed for 24 h at room temperature under continuous stirring. For purification, the resulting polymer conjugate was dialyzed against 5 mM HCl containing 1% NaCl, against 5 mM HCl and finally against 1 mM HCl to obtain a final pH of 3. Thereafter, the reaction mixtures were dialyzed to remove unreacted 2-iminothiolane at 10 °C and obtain the new kind of thiolated polymer, the 2-iminothiolane modified N,O-SOCC. Determination of the Thiol Group Content. The degree of modification was determined by quantifying the thiolated N,OSOCC polymers with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) (24, 25). DTNB working reagent was prepared by add 50 µL of the DTNB solution and 100 µL Tris solution into 840 µL deionized water. First, 5 mg of thiolated N,O-SOCC polymers was dissolved in 2.5 mL of deionized water. Afterward, 10 µL sample solution was mixed with 990 µL of DTNB reagent and incubated 5 min at 37 °C. The absorbance values of the samples were immediately measured at 415 nm using PowerWave X340 (Bio-TEK Instrument, Inc., Winooski, VT, USA) plate reader. The amount of thiol moieties was calculated from an standard curve obtained by the 2-iminothiolane modified N,O-SOCC solutions with increasing amount of cysteine. Thiol Oxidation. The 2-iminothiolane modified N,O-SOCC was then dissolved in 100 mM acetate buffer pH 5.0, 100 mM phosphate buffer pH 6.0, or PBS (phosphate buffered saline) buffer pH 7.4, in a final concentration of 0.5% (w/v) at 4 °C. After dissolution, the samples were incubated at 37 °C under permanent shaking. At predetermined time points, aliquots of 500 µL were withdrawn and 50 µL of 1 M HCl was added in order to quench any further reaction. The amount of remaining thiol groups was determined with Ellman’s reagent as described above.

Preparation of Polyelectrolyte Complex Nanoparticles. The 2-iminothiolane modified N,O-SOCC/CS nanoparticles were prepared using a polyelectrolyte complex method under magnetic stirring at room temperature. In brief, an aqueous 2-iminothiolane modified N,O-SOCC (1.0 mg/mL, 2 mL, pH 7.4) was added by flush mixing with a pipet tip into aqueous CS at various known concentrations (0.15, 0.30, 0.60, 0.90, or 1.20 mg/mL, 8 mL, pH 6.0). The polyelectrolyte complex nanoparticles were collected by centrifugation at 12 000 rpm for 20 min. Supernatants were discarded, and nanoparticles were resuspended in DI water for further studies. The mean particle sizes and zeta potential values of nanoparticles were measured using a Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK). bFGF Entrapment. For preparing the bFGF-loaded nanoparticles, 1 mL of bFGF stock solution (1 µg/mL) was added to the dissolved N,O-SOCC or 2-iminothiolane modified N,OSOCC solutions (2.0 mg/mL, 1 mL) with continuous stirring at 4 °C. After dissolution of bFGF, the mixed N,O-SOCC or 2-iminothiolane modified N,O-SOCC solution was respectively added into an aqueous CS (1.2 mg/mL, 8 mL) as described before. Afterward, the bFGF-loaded nanoparticles were collected by centrifugation and the amounts of bFGF in the supernatants were analyzed to determine the free bFGF content. The bFGF loading efficiency was calculated by dividing the weight of the loaded bFGF (subtract free bFGF from total amount of bFGF) by the total amount of bFGF (26). The amounts of bFGF were measured by enzyme-linked immunosorption assay (ELISA) kit (Human bFGF Duoset; R&D Systems, Minneapolis, MN, USA). ELISA plates (NUNC; Polylabo, Strasbourg, France) were coated with capture monoclonal antibodies and blocked with 1% BSA (w/v) for 1 h. After adding the appropriately diluted samples to the ELISA plates,



Figure 2. FT-IR spectra of (A) chitin, 6-O-CChitin, and 6-O-CC; (B) 6-O-CC, N-SOCC, and N,O-SOCC; and (C) N-SOCC, and N,O-SOCC.

released bFGF was detected using antihuman bFGF polyclonal antibodies. Then, streptavidin-conjugated horseradish peroxidase was added to the plates. The enzyme substrate (tetramethylbenzidine and peroxide) was added and incubated for color development for 20 min. The enzyme reaction was stopped by adding an acidic solution. The absorbance of the samples was read at 450 nm using PowerWave X340 (Bio-TEK Instrument, Inc., Winooski, VT, USA) plate reader. All of the experiments were repeated six times. Long-Term Release of Protected bFGF for Cell Proliferation. The release profiles of bFGF from the 2-iminothiolane modified N,O-SOCC/CS nanoparticles were determined to study the effects of the in situ cross-linkable thiolated N,O-SOCC on long-term release of protected bFGF. The samples were incubated at 37 °C under continuous agitation, in 5 mL of PBS (pH 7.4) or PBS containing 5.0 mM glutathione (GSH) (27). At various time points, the release medium was collected and centrifuged at 12 000 rpm for 20 min, and the supernatant was withdrawn and fresh buffer was replenished. The amounts of bFGF in the supernatants were determined with the above-mentioned method of ELISA assay. All of the experiments were repeated four times. Long-term protection of the mitogenic activity of the growth factor was assessed by proliferation of L929 fibroblasts cultured in serum-free Eagle’s MEM medium with 250 ng/mL of bFGF either in solution or equivalent weight bFGF in nanoparticles. Cell cultured in serum-free Eagle’s MEM medium without adding bFGF was used as a control. The cell proliferation was measured using a hemocytometer at different time points over a period of 5 days post-treatment.

13

NMR spectra of 6-O-CC,

Statistical Analysis. Statistical analysis for the determination of differences in the measured properties between groups was accomplished using one-way analysis of variance and determination of confidence intervals, performed with a computer statistical program (Statistical Analysis System, v 6.08, SAS Institute Inc., Cary, NC). All data are presented as a mean value with its standard deviation indicated (mean ( SD). Differences were considered to be statistically significant when the p values were less than 0.05.

RESULTS AND DISCUSSION Synthesis and Characterization of 6-O-CC, N-SOCC, and N,O-SOCC. 6-O-CC was synthesized from its original compoundschitin. Figure 2A showed the FT-IR spectrum of chitin, 6-O-CChitin and 6-O-CC polymers. The characteristic absorption at 1580 cm-1 and 1620-1660 cm-1 was respectively assigned to the amide I (NsH) and amide II (CdO) stretch of chitin (18), while the characteristic absorption at 1710-1715 cm-1 was assigned to the carboxylic acid groups on 6-OCChitin. After deacetylation of 6-OCChitin in alkali solution, the carboxylic acid groups were converted into carboxylate ions (∼1650 cm-1), which were partially overlapped with the amine groups (∼1560 cm-1) on 6-O-CC. Figure 2B showed the FT-IR spectrum of 6-O-CC, N-SOCC, and N,O-SOCC polymers. N-SOCC demonstrated a significant decrease of NsH absorption bands around 1560 cm-1 as compared with 6-O-CC. The characteristic absorption bands in the area of 1160-1260 cm-1 (OdSdO asymmetric stretch) indicated the presence of sulfamate groups (N-sulfation) (18). N-SOCC was further sulfated with oleum, leading to the final


Ho et al.

Table 1. Substitution Degrees of 6-O-CChitin, 6-O-CC, N-SOCC, and N,O-SOCC Chitin 6-O-CChitin 6-O-CC N-SOCC N,O-SOCC

DDAa

DCb

DSc

Mwd

s s 0.92 0.92 0.92

s 0.46 0.46 0.46 0.46

s s s 0.51 1.13

s 1.54 × 105 1.38 × 105 8.75 × 104 5.73 × 104

a

DDA means the degree of deacetylation. b DC means the degree of carboxymethylation. c DS means the degree of sulfation. d Mw means the weight-average molecular weights determined by GPC.

product, N,O-sulfated 6-O-CC (N,O-SOCC) with nonselective sulfate groups at the 2-N, 3-O, and 6-O positions (19). The additional characteristic band of N,O-SOCC observed at 800 cm-1 representing the CsOsS stretch, suggesting the formation of sulfate ester linkage via the O-sulfation reactions with hydroxyl groups (20). Figure 2C showed the 13C NMR spectra of 6-O-CC, NSOCC, and N,O-SOCC polymers of chitosan and carboxymethylchitosan. In the spectrum of 6-O-CC, the signals observed at 176.5 and at 23.6 ppm were assigned to the carbonyl carbon of COCH3 and the methyl carbon (CH3), respectively. The signal at 96.7 ppm was assigned to carbon C-1 of 6-O-CC, and those signals at 56.3, 70.1, 75.9, 74.3, and 60.2 ppm were assigned to carbons C-2, C-3, C-4, C-5, and C-6, respectively. The signal observed at 179.5 ppm is assigned to the carbonyl carbons of carboxymethyl groups (28). The spectrum of N-SOCC showed split signals at 58.1 ppm corresponding to N-sulfation, though it was not completely sulfated because the unsulfated signal of C-2 could still be observed (C2+C2′) (18). The 13C NMR spectrum of N,O-SOCC indicated that in additional to the N-sulfation, the C-6 and C-3 hydroxyl groups were both partly sulfated (O-sulfation) because of the existence of sulfated and unsulfated signals (C6+C6′+C6′′ and C3+C3′) (19, 20). The degrees of carboxymethylation and sulfation estimated from elemental analysis, and the molecular weights determined by GPC were shown in Table 1. bFGF Protective Efficiency and Docuking Simulation. Figure 3A shows the effect of the substituted carboxymethyl and sulfate groups of the synthesized 6-O-CC, N-SOCC, and

N,O-SOCC on the retention of bFGF activity. At pH 4.0, the L929 cells proliferated well in serum-free medium containing bFGF and N-SOCC or N,O-SOCC. Dextran sulfate and (1f6)R-D-mannopyranan sulfate having sulfate groups have been reported to interact with FGFs and mimic heparin effects on the biological activities of FGFs (29, 30). Interestingly, N,OSOCC showed higher bFGF-protective effect compared with its N-SOCC counterpart under the tested pH condition (p < 0.05), suggested that the interaction between the sulfated polymer and bFGF requires not only N-sulfate groups, but also the O-sulfate groups. In contrast, 6-O-CC without the sulfate groups could not significantly protect bFGF, and the bFGFinduced cell growth in serum-free Eagle’s MEM medium was not observed. It is noted that binding of bFGF and heparin is mediated by ionic interaction between both N- and O-sulfate groups of heparin molecules and certain lysine (R) and arginine (K) cations in bFGF (31). In the study of docking simulation, four docking ligand molecules, respectively, correlating with the dimer units of 6-O-CC, N-SOCC, N,O-SOCC, and heparin were designed (Figure 4A), and their docking energies were computed (Table 2). The N,O-SOCC ligand with the lowest docked energy structure showed sulfate-mediated interactions between bFGF and the ligand involving 2-N-sulfate, 3-O-sulfate, and 6-Osulfate groups of the ligand and R121, K126, and K136 residues of bFGF (Figure 4B). The interactions between K120, K126 residues and the N,O-SOCC ligand are also mediated via 6-Ocarboxymethyl groups (Figure 4B). The sulfate groups appeared to predominantly bind to basic amino acid residues, while the carboxymethyl groups complement sulfate groups to contribute in stabilizing the binding structure (14). N,O-SOCC substituted with 6-O-carboxymethyl groups and 2-N, 3-O, and 6-O sulfate groups may have well-balanced steric and electrostatic structures that can stably interact with bFGF to protect the growth factor against acidic inactivation. The results of docking simulation and cell proliferation reveal that N,O-SOCC shows the lowest computed docking energy and is the most effective ligand in the retention of bFGF activity (Table 2). Although the N,O-SOCC ligand might have higher affinity to bFGF than heparin, N,O-SOCC is still less effective in the retention of bFGF activity for cell proliferation, as

Figure 3. Comparison of the bFGF-protective effect of 6-O-CC, N-SOCC, N,O-SOCC, and heparin polymers under acidic condition (bFGF in pH 4.0 medium, 30-120 min of incubation). Chlorate-treated L929 fibroblasts proliferated in serum-free culture medium with bFGF (250 ng/mL) and polymers (10 µg/mL) for 96 h.



Figure 4. Molecular modeling of docking simulation. (A) Docking conformations of 6-O-CC, N-SOCC, N,O-SOCC, and heparin docking ligands after binding to the heparin-binding site of bFGF. (B) Interaction of 6-O-CC, N-SOCC, N,O-SOCC, and heparin with the amino acids residues at the heparin-binding site of bFGF.

compared with its heparin counterpart (Figure 4A). This will be attributed to heparin not only demonstrating high affinity to FGF family growth factors but also binding to its receptor that facilitates FGF-receptor dimerization and activation (32).

Synthesis and Characterization of Thiolated N,O-SOCC. In situ gelling systems based on chitosan derivatives have been developed for glucose-sensitive, pulsatile delivery of insulin (33). Thiol-functionalized polymeric micelles or nanoparticles


Ho et al.

Table 2. Docked Energy of 6-O-CC, N-SOCC, N,O-SOCC, Thiolated N,O-SOCC, and Heparin 6-O-CC N-SOCC N,O-SOCC Thiolated N,O-SOCCe Heparin

Table 3. Substituted Thiol Moieties on the 2-Iminothiolane Modified N,O-SOCC Polymers

Fa

INTMb

INTNc

Dkd

-5.02 -5.60 -5.29 -4.24 -4.63

-5.02 -5.60 -5.29 -4.24 -4.63

0.62 0.77 -1.19 0.56 -0.52

-4.40 -4.83 -6.48 -3.69 -5.15

a

F means the free energy (kcal/mol). b INTM means the intermolecular energy (kcal/mol). c INTN means the internal energy (kcal/mol). d DK means the docked energy (kcal/mol). e Thiolated N,O-SOCC means the 2-iminothiolane modified N,O-SOCC.

have also been investigated for molecular recognition, tumor immunotargeting, or intracellular DNA delivery in response to glutathione (28, 34, 35). In this work, N,O-SOCC was selected for thiolation because the correlated ligand has the lowest docking energy. The thiolated N,O-SOCC polymer prepared by modification of N,O-SOCC with 2-iminothiolane was in situ cross-linkable. Apart from a sulfhydryl group, a cationic moiety is also introduced in the form of an amidine substructure resulting in a N,O-SOCC-4-thio-butylamidine (TBA) conjugate (23). The schematic procedure for the synthesis of 2-iminothiolane modified N-SOCC and the formation of its disulfide compound is shown in Figure 5. The modification of N,O-SOCC with 2-iminothiolane can be performed under inert condition to avoid the oxidation of thiol groups. The low reactivity of 2-iminothiolane in acid has also been reported (23). Therefore, we synthesized the thiolated N,O-SOCC in acidic (pH 5) and neutral (pH 7) conditions, and the reactants were purged with nitrogen. The amount of covalently attached thiol groups on the thiolated polymers was shown in Table 3. The docking ligand molecule correlates with the 2-iminothiolane modified N,O-SOCC displayed a higher docking energy as compared with its original N,O-SOCC counterpart, revealing a lower thiolated N,O-SOCC affinity of bFGF (Table 2). The steric hindrance of the conjugated thiomer (TBA) resulted in the shift of 2-N, 3-O, and 6-O sulfate groups to the region far from the heparin-binding residues, such as R121, K126, and

polymer N,O-SOCC

2-iminothiolane (g)

pH

conjugated thiol groups (µmol/g polymer)

500 mg 500 mg 500 mg 500 mg 500 mg 500 mg

200 mg 400 mg 600 mg 200 mg 400 mg 600 mg

5.0 5.0 5.0 7.0 7.0 7.0

45.2 ( 3.7 67.5 ( 7.9 298.4 ( 9.8 97.3 ( 7.1 258.4 ( 8.6 415.6 ( 12.5

K136 (Figure 6A). This simulated result suggested that bFGF protective efficiency of the thiolated N,O-SOCC might be decreased with the increase of the substituted thiomer degree. Therefore, we selected a 2-iminothiolane modified N-SOCC product with mediate thiol substitution [258.4 ( 8.6 (µmol/g polymer)] for the following studies. Thiol Oxidation of 2-Iminothiolane Modified N,O-SOCC. At pH 7.4, in the presence of oxygen, the thiol groups on the 2-iminothiolane modified N,O-SOCC can be easily oxided to disulfide bonds (Figure 6B), forming an in situ cross-linked network structure. Depending on the pH value, the thiol groups of 2-iminothiolane modified N,O-SOCC were oxidized, thereby forming inter- as well as intramolecular disulfide bonds. At pH 5.0, the thiol groups of 2-iminothiolane modified N,O-SOCC remained stable toward oxidation, whereas at pH 6.0, a decrease of 56% in the thiol moieties was observed, within 6 h of oxidation in air. Raising the pH value from 6.0 to 7.4 led to a higher concentration of negative thiolate anions, representing the reactive form for oxidation, finally resulting in a significant decrease of thiol moieties due to the formation of disulfide bonds (23, 25). These results revealed that thiolated N,O-SOCC could be used as an in situ cross-linkable polymer to entrap peptide or protein drugs for specific drug delivery purpose. Characterization of 2-Iminothiolane Modified N,O-SOCC/ CS Nanoparticles. 2-Iminothiolane modified N,O-SOCC and CS complex could be used for preparing nanoparticles by a polyelectrolyte self-assembly method. The particle sizes and the zeta potential values of the thiol-modified N,O-SOCC/CS

Figure 5. Schematic procedures for the synthesis of 2-iminothiolane modified N,O-SOCC (A) and the formation of its disulfide compound (B).



Figure 6. (A) Docking conformation of the 2-iminothiolane modified N,O-SOCC ligand after binding to the heparin binding site of bFGF and the interaction of the 2-iminothiolane modified N,O-SOCC with the amino acid residues of bFGF. (B) Decrease in thiol groups of 1.0% (w/v) 2-iminothiolane modified N,O-SOCC at pH values 5.0, 6.0, and 7.4. The thiol content of the 2-iminothiolane modified N,O-SOCC was 258.4 ( 8.6 µmol/g polymer.

nanoparticles, prepared at varying concentrations of the thiolated N,O-SOCC and CS, were determined, and the results are shown in Table 4. It was found that the particle size values of the prepared nanoparticles were mainly determined by the relative amount of the local concentration of the 2-iminothiolane modified N,O-SOCC in the added solution to the surrounding concentration of CS in the sink solution. At a fixed concentration of CS, an increase in the CS concentration allowed the thiolated N,O-SOCC molecules interacting with more CS molecules and thus formed a larger-sized nanoparticle. The formed nanoparticles had CS exposed on the surfaces and thus had a positively charged zeta potential. In contrast, as the amount of local 2-iminothiolane modified N,O-SOCC molecules sufficiently exceeded that of surrounding CS molecules, the formed nanoparticles had the thiolated N,OSOCC exposed on the surfaces and thus had a high negative charge of zeta potential. Therefore, the particle size and the zeta potential value of the prepared thiolated N,O-SOCC/CS nano-

Table 4. Effects of Various Weight Ratios (or charge ratios) of Chitosan to 2-Iminothiolane Modified N,O-SOCC on Mean Particle Size and Zeta Potential of Nanoparticles 2-iminothiolane N,O-SOCC/CS (w/w)

2-iminothiolane N,O-SOCC/CS charge ratio

1/0.6 1/1.2 1/2.4 1/3.6 1/4.8

1.00/0.48 1.00/0.95 1.00/1.90 1.00/2.86 1.00/3.81

mean particle size (nm)

zeta potential (mV)

218.4 ( 8.6

-21.8

c

242.6 ( 6.9 262.8 ( 3.4 289.9 ( 4.5

c

20.3 28.1 32.8

a Prepared in deionized water (pH 6.0, n ) 5). b Ratio of positively charged -NH3+ groups on CS to negatively charged -COO- and -SO3- groups on 2-iminothiolane modified N,O-SOCC. c Precipitation of aggregates was observed after 1 h.

particles can be controlled by their constituted compositions. The nanoparticles shelled with 2-iminothiolane modified N,OSOCC were unstable, while those shelled with CS remained


Ho et al. Table 5. Variation of Mean Particle Size of Thiolated N,O-SOCC/ CS Nanoparticles in GSH-Containing PBS Solution time (h)

pH 6.0 (in DI water)

pH 7.4 (in PBS)

pH 7.4 (in PBS/GSH medium)

0 24 48 72 96 120

289.9 ( 4.5 293.7 ( 7.8 298.2 ( 5.9 301.6 ( 6.7 299.1 ( 8.5 298.9 ( 6.9

289.9 ( 4.5 307.4 ( 7.3 309.5 ( 5.8 307.6 ( 7.1 308.4 ( 6.5 309.1 ( 7.7

289.9 ( 4.5 425.4 ( 7.3 447.8 ( 8.6 462.1 ( 6.8 471.5 ( 7.9 480.6 ( 8.4a

a

Figure 7. TEM micrographies of the 2-iminothiolane modified N,OSOCC/CS nanoparticles shelled with (A) CS (1.0 mg/mL thiolated N,OSOCC: 1.2 mg/mL CS) and (B) 2-iminothiolane modified N,O-SOCC (1.0 mg/mL thiolated N,O-SOCC: 0.30 mg/mL CS).

intact in PBS buffer. Even a minute amount of free thiolated N,O-SOCC in the medium could react with the thiol groups shelled on nanoparticles to form disulfides and finally resulted in the aggregation of nanoparticles. The results obtained by the TEM examination showed that the morphology of the prepared nanoparticles shelled with CS (1.0 mg/mL thiolated N,O-SOCC: 1.2 mg/mL CS) was spherical in shape with a smooth surface, while the nanoparticles shelled with 2-iminothiolane modified N,O-SOCC (1.0 mg/mL thiolated N,O-SOCC: 0.30 mg/mL CS) were aggregated due to the oxidation of surface thiol groups to disulfide bonds between the nanoparticles (Figure 7). Therefore, the CS shelled nanoparticles (1.0 mg/mL thiolated N,O-SOCC: 1.2 mg/mL CS) was selected for the following studies of bFGF entrapment and release. In body fluids and extracellular matrixes, a relatively low concentration of GSH (approximately 2-20 µM) could be found. In contrast, inside cells the concentration of GSH is 0.5-10 mM. The variation of particle size in simulated physiological condition (pH 7.4 with 10 mM glutathione) was determined to study the stability of nanoparticles. As shown in

The concentration of GSH is 10 mM.

Table 5, the mean particle size of GSH-treated nanoparticles gradually increased, reaching about 480 nm within 120 h. However, without GSH treatment, the nanoparticles in PBS solution showed negligible size variation within the same period of time. This result suggested that the cleavage of disulfide linkage in the presence of GSH led to swelling of the nanoparticles. bFGF Entrapment and Release. Chitosan and its derivativebased nanoparticles have been previously investigated for deliveryofbioactivereagents,includingproteinsandgenes(36-38). In this study, the thiolated N,O-SOCC, as an in situ crosslinkable polymer, has the advantage of entrapping bFGF without leading to the decrease in bioactivity of the peptide drug. The encapsulation of bFGF does not seem to significantly change the size and zeta potential of the nanoparticles. We have found that nanoparticles prepared only by CS and N,O-SOCC (the N,O-SOCC/CS nanoparticles), without modification with thiol groups, were very unstable and disintegrated rapidly in PBS buffer due to the balance of electrostatic charges by phosphate salts. Owing to the formation of covalently disulfide bonds, the 2-iminothiolane modified N,O-SOCC/CS nanoparticles with a positive surface charge (1.0 mg/mL thiolated N,O-SOCC: 1.2 mg/mL CS) could be used for bFGF entrapment and were kept stable in PBS buffer. The bFGF loading efficiency for N,OSOCC/CS and 2-iminothiolane modified N,O-SOCC/CS nanoparticles were 61.4% ( 5.8% and 54.9% ( 3.6%, respectively. Figure 8A shows the release profile of bFGF from the nanoparticles of N,O-SOCC/CS and 2-iminothiolane modified N,O-SOCC/CS nanoparticles in PBS buffer and the buffer containing glutathione. The N,O-SOCC/CS nanoparticles were unstable in PBS and could disintegrate rapidly to release the growth factor quickly. In contrast, the 2-iminothiolane modified N,O-SOCC introduced the disulfide bonds could assembled with CS into stable nanoparticles, coinciding with a slower release rate. The thiolated N,O-SOCC/CS nanoparticles shelled with CS suggested that bFGF-binding N,O-SOCC could be incoporated in the core of nanoparticles. Glutathione can reduce the covalent disulfide bonds to thiols leading to a quicker release of the growth factor from the nanoparticles. Cell Proliferation. The mitogenic activity of bFGF released from the 2-iminothiolane modified N,O-SOCC/CS nanoparticles after 5 days of incubation was evaluated by L929 cell culture in serum-free Eagle’s MEM medium (Figure 8B). The cell number of L929 fibroblasts does not seem to increase in the serum-free basal medium (control), indicating no additional cell growth in the medium without bFGF. The addition of bFGF in a free form to the basal medium stimulated the growth of L929 fibroblasts at day 2, and the growth increase of cells is significant as compared with that of the control group. The cells in the medium added with the bFGF-loaded, N,O-SOCC/CS nanoparticles showed increased cell proliferation comparable to the group of cells cultured with a free-form bFGF, because the nanoparticles disintegrate rapidly to release the growth factor quickly. Conversely, cells treated with bFGF-loaded, thiol modified N,O-SOCC/CS nanoparticles demonstrated a sustained significant increase in the cell proliferation activity (p < 0.05).



by encapsulating the factors in micro- or nanoparticles before being incorporated with hydrogels or 3-D scaffolds (40). The results of this study suggest that the thiol modified N,O-SOCC/ CS nanoparticles may be useful as novel materials for tissue engineering and might yield interesting future work.

CONCLUSIONS In conclusion, we have synthesized a heparin-like chitosan derivative, N,O-sulfated, 6-O-carboxymethylchitosan (N,OSOCC). N,O-SOCC demonstrated high affinity to bFGF and could protect mitogenic activity of bFGF as investigated by cell proliferation and docking simulation. Thiol modified N,O-SOCC was assembled with CS to form thiolated N,O-SOCC/CS nanoparticles. The thiolated N,O-SOCC/CS nanoparticles enhance the bFGF-stimulated cell growth through protecting the released growth factor from inactivation. It may be useful as a new biomaterial for the controlled release of bFGF for tissueengineering applications.

ACKNOWLEDGMENT The financial support for this research was provided by the National Science Council (NSC 95-2120-M-007-010 and NSC 96-2221-E-238-010), Taiwan, R.O.C.

LITERATURE CITED

Figure 8. (A) Release profiles of bFGF from N,O-SOCC/CS and 2-iminothiolane modified (thiolated) N,O-SOCC/CS nanoparticles at 37 °C in 10 mL of PBS (pH 7.4) or PBS containing 5.0 mM glutathione (GSH). The thiol content of the 2-iminothiolane modified N,O-SOCC was 258.4 ( 8.6 µmol/g polymer. (B) The growth kinetics of L929 fibroblasts cultured in serum-free culture medium (basal medium), basal medium with the addition of bFGF in free form, and the N,O-SOCC/ CS and 2-iminothiolane modified (thiolated) N,O-SOCC/CS nanoparticles loaded with bFGF.

This result indicates that only the thiol modified N,O-SOCC/ CS nanoparticle is able to protect bFGF from inactivation over the long term. Due to the protective effect of the nanoparticles, mitogenic activity of bFGF could be retained for a long time, without evident denaturation. The released growth factor could effectively bind to the cellular bFGF receptor for cell growth. It was encouraging to find that the nanoparticles could be easily mixed with several biological components such as alginate, chitosan, collagen, gelatin, and hyaluronic acid to be reshaped into nanoparticle-containing hydrogels or 3-D scaffolds. As reported in the literature, the therapy of ischemic disease and “in advance angiogenesis” for cell transplantation are two important objectives of angiogenesis in tissue engineering. In tissue repairing applications, therapeutic strategies of injecting bFGF-containing nanoparticles to stimulate arteriogenesis in animal models suffering from occlusive vascular disease received significant benefit from improved targeting, less invasiveness, better growth-factor stability, and more sustained growth-factor release (39). Other studies reported that prolonged retention of activity of the growth factors and controlled release of the signal proteins at the site of action with reduced burst effect became possible

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