A Glycopolymer Chaperone for Fibroblast Growth Factor-2 - American

Jan 3, 2004 - motes the interaction of FGF-2 with its receptor, fibroblast growth factor receptor-1 (FGFR-1), as well as the dimer- ization of both FG...
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Bioconjugate Chem. 2004, 15, 145−151

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A Glycopolymer Chaperone for Fibroblast Growth Factor-2 Ran Guan,† Xue-Long Sun,† Sijian Hou,† Peiyi Wu,† and Elliot L. Chaikof*,†,‡ Departments of Surgery and Biomedical Engineering, Emory University School of Medicine, Atlanta, Georgia 30322, and School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332. Received August 5, 2003; Revised Manuscript Received November 19, 2003

Mono- and disaccharide-containing glycopolymers were synthesized by cyanoxyl-mediated polymerization of acrylamide with acrylate-derivatized mono- and disaccharides. We demonstrate that a glycopolymer bearing pendant, fully sulfated lactose units effectively replaces heparin and heparan sulfate as a molecular chaperone for fibroblast growth factor-2 (FGF-2). Specifically, a compound was identified that protects FGF-2 from proteolytic, acid, and heat-induced degradation, while selectively promoting growth factor and receptor dimerization. Significantly, the capacity of this heparin-mimic to promote an FGF-2 specific proliferative cell response was confirmed and suggests potential applications for this compound and related derivatives in areas related to therapeutic angiogenesis.

INTRODUCTION

Heparan sulfates (HS) influence regenerative responses by acting as cofactors for a variety of cytokines (1, 2). For example, heparan sulfates bind fibroblast growth factors (e.g. FGF-1, FGF-2, FGF-7), heparin binding-epidermal growth factor (HB-EGF), plateletderived growth factor (PDGF), and vascular endothelial growth factor (VEGF), all of which promote endothelial and/or smooth muscle cell motility and proliferation (35). Significantly, the interaction of heparin-binding cytokines and growth factors with heparan sulfates serves to protect these molecules from heat, pH, and proteaserelated degradation mechanisms, establish high concentration storage sites and may, at times, assist in their appropriate presentation to specific cell-signaling receptors by promoting oligomerization or by altering cytokine or receptor conformation (6-8). For example, FGF-2 sequestration, dimerization, and stimulation of cell proliferation is dramatically reduced in extracellular matrix produced from chlorate treated cells that are unable to produce fully sulfated glycosaminoglycans (GAG) (9). The diverse biological properties of heparin and heparan sulfate combined with the inherent difficulty associated with the synthesis of these chemically complex natural products have motivated efforts to generate simpler structural mimics as component biomaterials for a variety of applications. In the past five years, significant advances in the synthesis of polymers with pendent saccharides, also known as “glycopolymers”, offer one such biomimetic strategy (10-13). For example, vinyl-, acrylate-, or norbornene-containing saccharide derivatives can be polymerized to yield glycopolymers with biologically active hydrophilic saccharides in the side chains. Moreover, the backbone structure may be altered to modulate both polymer biostability and processability. Significantly, the carbohydrate density of the polymer chain, which appears to affect both ligand binding * Address correspondence to Elliot L. Chaikof, M.D., Ph.D. 1639 Pierce Dr., Rm. 5105, Emory University, Atlanta, GA 30322. Phone (404) 727-8413. Fax: (404) 727-3660. E-mail: [email protected]. † Emory University School of Medicine. ‡ Georgia Institute of Technology.

affinity, as well as polymer physiochemical properties, can be controlled through the choice of polymerizable saccharide, associated comonomers, and dictated chain size. Overall, the potential to stably link bioactive oligosaccharide ligands is an important feature of the glycopolymer approach and presents an opportunity to create GAG mimicking structures that facilitate optimized sequestration and controlled local release of bioactive protein species. In prior reports, we have examined the applicability of cyanoxyl (•OCtN)-mediated free-radical polymerization in the synthesis of model glycopolymers from unprotected alkene- and acrylate-derivatized sulfated and unsulfated glycomonomers consisting of a variety of mono- and disaccharide species (14-17). Significantly, we observed that this polymerization technique can be conducted in aqueous solution, is tolerant of a broad range of functional groups (OH, NH2, COOH, and OSO3-), yields low-polydispersity polymers (PDI < 1.50) with high saccharide content, and can be applied to the synthesis of block and graft copolymers. We have used this approach to generate glycopolymers with pendant monosaccharides, as well as HS- and non-HS-related disaccharides. In this report, we demonstrate that this strategy can yield a novel HS-mimetic that acts as an effective macromolecular chaperone for a pro-angiogenic protein growth factor (FGF-2), sequestering and protecting FGF-2 from proteolytic, acid, or heat-induced degradation. In addition, this glycopolymer selectively promotes the interaction of FGF-2 with its receptor, fibroblast growth factor receptor-1 (FGFR-1), as well as the dimerization of both FGF-2 and the FGF-2/FGFR-1 complex. Significantly, all of these features are necessary for receptor activation. The capacity of this glycopolymer to uniquely promote a FGF-2-specific proliferative cell response is confirmed. MATERIALS AND METHODS

Materials. All solvents and reagents for monomer or polymer synthesis were purchased from commercial sources and were used as received, unless otherwise noted. Deionized water with a resistivity of 18 MΩ‚cm was used as a solvent in all polymerization reactions. FGF-2 (human recombinant, 17.2 kDa) was obtained

10.1021/bc034138t CCC: $27.50 © 2004 American Chemical Society Published on Web 01/03/2004

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from Pepro Tech Inc (Rocky Hill, NJ), 125I-FGF-2 was purchased from NEN (Boston, MA). Silver stain reagent was obtained from BioRad (Hercules, MA) and disuccinimidyl suberate (DSS) from Pierce (Rockford, IL). RPMI1640 media, penicillin-streptomycin, L-glutamine, and G418 were all obtained from Mediatech Inc. (Herndon, VA) and bovine calf serum from Hyclone (Logan, UT). Sodium heparin from porcine intestinal mucosa (MW between 12 000 and 20 000, lot 30K0572), heparin sulfate (MW ∼7000, lot 41K1130), and sodium dextran sulfate (MW 5000, 10 000, and 500 000) were obtained from Sigma (St. Louis, MO). Sucrose octasulfate potassium salt was aquired from Toronto Research Chemicals Inc. (Toronto, Canada), and β-mercaptoethanol, protein A sepharose, and [methyl-3H]-thymidine (5 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). FGFR-1 antibody raised against the carboxy terminus of FGFR-1 of human origin (identical to the corresponding mouse sequence) was purchased from Santa Cruz. General Synthetic Methods. Thin-layer chromatography (TLC) was performed on Whatman silica gel aluminum-backed plates of 250 µm thickness on which spots were visualized with UV light or by charring the plate after dipping in 10% H2SO4 in methanol. Mass spectra (MS/FAB) were obtained at an ionizing voltage of 70 eV. 1H NMR spectra were recorded at room temperature with a Varian INOVA 400 spectrometer (magnetic field strengths of 400 and 100 MHz for 1H and 13C NMR analyses, respectively). The size-exclusion chromatography (SEC) equipment comprised a Waters model 510 HPLC pump, a Waters Ultrahydrogel 250 column, and a Wyatt Technology Optilab 903 refractometer. The eluent consisted of a 0.1 mol/L NaNO3 deionized water solution containing 0.05 wt % sodium azide at a flow rate of 0.7 mL/min. The actual molar masses of the glycopolymer samples were determined from the response of the Dawn EOS (Wyatt Technology) multiangle laser light-scattering (LLS) detector that was connected to the outlet of the SEC apparatus. 2-O-Acryoylethoxyl-2-acetylamino-3,4,6-trisulfoxyβ-D-glucopyranoside (4) and 2-N-Acryoylaminoethoxyl 4-O-(2,3,4,6-Tetrasulfoxy-β-D-galatopyranosyl)-2,3,6-trisufoxy-β-D-glucopyranoside (5) were synthesized as previously reported (14, 16). Homopolymerization of Acrylamide-Derived Glycomonomers Initiated by ClC6H4NtN+BF4-/ NaOCN. In a three-neck flask, 3 mg (2.45 × 10-5 mol) of p-chloroaniline was reacted with 7 mg of HBF4 (48 wt % aqueous solution, 3.67 × 10-5 mol) in 2 mL of water at 0 °C and under an Ar atmosphere. The diazonium salt ClC6H4N+tNBF4- was generated by adding 2 mg (2.93 × 10-5 mol) of NaNO2 to the reaction medium. After 30 min, a degassed mixture of 390 mg (1.22 × 10-3 mol) of glycomonomer 5 (or 4) and 2 mg (2.45 × 10-5 mol) of sodium cyanate (NaOCN) dissolved in 0.5 mL of water was introduced into the flask containing the arenediazonium salt. The polymerization solution was then heated to 50 °C for 16 h. By varying the [M]/[I] ratio and reaction times resulting glycopolymers 7a and 7b (or 6a and 6b) were isolated by precipitation in a 10-fold excess of cold methanol and dried to yield white cotton woollike materials. Copolymerization of Acylamide-Derived Glycomonomers Initiated by ClC6H4NtN+BF4-/NaOCN. In a three-neck flask, 8 mg (6.03 × 10-5 mol) of pchloroaniline was reacted with 17 mg of HBF4 (48 wt % aqueous solution, 9.04 × 10-5 mol) in 2 mL of water at 0 °C and under an Ar atmosphere. The diazonium salt

Guan et al.

ClC6H4N+tNBF4- was then generated by adding 5 mg (7.2 × 10-5 mol) of sodium nitrite (NaNO2) to the reaction medium. After 30 min, a degassed mixture of 225 mg (6.03 ×10-4 mol) of glycomonomer 5 (or 4), 171 mg (2.41 × 10-3 mol) of acrylamide, and 4 mg (6.03 × 10-5 mol) of NaOCN dissolved in 1 mL of water were introduced into the flask containing the diazonium salt. The polymerization solution was then heated to 50 °C. The statistical copolymers 7c and 7d (or 6c-e) formed after 16 h of reaction were isolated by precipitation in a 10-fold excess of cold methanol and dried. The conversions were determined by weight of the resultant glycopolymer. Cell Culture. BaF3-FR1C-11 mouse lymphoma cells were kindly provided by Dr. D. Ornitz (Washington University, St. Louis, MO) and were cultured in RPMI 1640 media supplemented with 10% bovine calf serum, 10% WEHI-3 conditioned medium, 100 u/mL penicillin, 100 µg/mL streptomycin, 4 mM L-glutamine, 0.0035% β-mercaptoethanol, and 600 µg/mL G418. FR1C-11 cells were derived from the BaF3 line and have been transfected with a mouse cDNA encoding the 3Ig domain form of FGFR1-IIIc (18). Proliferation Assay. BaF3-FR1C-11 mouse lymphoma cells were washed twice with RPMI 1640 media lacking IL-3 and plated at a concentration of 2.2 × 104 cells/well in a 96-well plate in the presence of 3 ng/mL FGF-2 and increasing concentrations of heparin or various test compounds in a total volume of 150 µL of media (RPMI 1640-10% fetal calf serum without IL-3). After a 36 h incubation period, 1 µCi of 3H-thymidine was added to each well in a volume of 50 µL of RPMI media (without FGF or IL3). Cells were incubated for an additional 5 h and harvested onto glass fiber filters with a TOMTEC cell harvester (Tomtec Co., Orange, CT). Incorporated 3Hthymidine was determined by liquid scintillation counting. Each compound concentration was tested in triplicate. Dimerization of FGF-2 and FGFR-1. Compoundinduced FGF-2 dimerization was assessed, as detailed elsewhere (19). Briefly, 125I-FGF-2 (20 ng/mL) was incubated for 1 h at room temperature in the presence and absence of heparin (25 µg/mL) or various concentrations of test glycopolymer in 20 µL buffer containing 150 mM NaCl and 25 mM Hepes, pH 7.5. DSS was then added to a final concentration of 0.3 mM, and the mixture was incubated for an additional 20 min. The cross-linking reaction was quenched with 10 mM ethanolamine-HCl, pH 8.0, for 20 min and diluted with 2 × SDS-PAGE loading buffer. The protein was analyzed on a 15% SDSPAGE gel, and cross-linked FGF-2 was visualized by autoradiography on XAR film (Eastman Kodak Co., Rochester, NY). For FGFR cross-linking studies, 106 cells were washed with binding buffer (RPMI 1640), 1 mg/mL of bovine serum albumin (BSA), and 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, pH 7.5) and incubated with 3 ng/mL 125I-FGF-2 in the absence or presence of either heparin or HS-mimetic compound at 4 °C for 5 h. After three washes with ice cold PBS with 1 mg/mL of BSA, cells were resuspended and incubated in 250 µL of cross-linking solution (PBS, 0.2 mM DSS, 1 mg BSA/mL) for 45 min at room temperature. The reaction was quenched with 500 µL of TN buffer (50 mM tris, pH 7.4, 150 mM NaCl), washed twice with TN, and solubilized in RIPA buffer (PBS, 0.5% deoxycholic acid, 1% Triton X-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg aprotinin per mL). After 10 min on ice, the nuclei were removed by centrifugation and the supernatants were analyzed by electrophoresis through

A Glycopolymer Chaperone for FGF-2 Scheme 1. Cyanoxyl-Mediated Free-Radical Copolymerization of Sulfated Glycomonomer with Acrylamide

Bioconjugate Chem., Vol. 15, No. 1, 2004 147 Table 1. Sulfated Monosaccharide-Based Glycopolymers monomer ratio polymer glucosamine GM/AM composition content Mn Mw/Mn glyco(mol) (mol) (wt %)a (g/mol) SEC polymer 1/0 1/0 1/1 1/1 1/1 1/4

1/0 1/0 1/9 1/12 1/4 1/9

100 100 47 40 66 42

9900 20 800 5400 21 700 48 000 115 600

1.13 1.49 1.15 1.20 1.45 1.57

6a (SM1) 6b (SM2) 6c (SM3) 6d (SM4) 6e (SM5) 6f (SM6)

a Mass content of monosaccharide was determined by 1H NMR analysis.

Table 2. Sulfated Disaccharide-Based Glycopolymers

a 6% SDS-PAGE gel. The cross-linked FGF-2-FGFR was visualized by autoradiography on XAR film (Eastman Kodak Co., Rochester, NY) (20). Proteolytic Digestion of FGF-2. Trypsin-mediated digestion of FGF-2 in the presence of test compound was evaluated as described by Coltrini et al. (21). Briefly, 1-µg aliquots of the growth factor were incubated at 37 °C for 5 min in 5 mM Tris/HCl pH 7.6, in the presence of increasing concentrations of the test compound. This was followed by the addition of 100 ng of trypsin with incubation at 37 °C for 3 h. Samples were then supplemented with SDS-PAGE reducing sample buffer, heated to 100 °C for 5 min, and then electrophoresed on a SDSPAGE (15%) gel. The amount of undigested protein in a given lane was determined by densitometric analysis of silver-stained gels (21, 22). Acid and Heat Treatment of FGF-2. FGF-2 (100 µg/ mL) in 5 mM Tris (pH 7.6) was combined with heparin or test compound and then incubated for 2 h at room temperature in the presence of trifluoroacetic acid (TFA) diluted in water. TFA at a final concentration of 0.05, 0.1, 0.25, 0.5,1, 2.5, and 5% corresponds to a pH of 3.4, 2.37, 1.5, 1.08, 0.4, 0.2, and 0, respectively, which was unaffected by the addition of heparin, test compound, or FGF-2. The reaction mixture was further diluted as indicated with RPMI 1640-10% fetal calf serum. The final pH was neutral, as indicated by a phenol red indicator (23). To define the capacity of test compound to protect FGF-2 from heat inactivation, FGF-2 was incubated with heparin or test compound and then heated at 65 °C for 5 min. In both assays, residual FGF-2 activity was determined by measuring the proliferation of BaF3-FR1C-11 cells in response to 1 µL of reaction mixture. RESULTS AND DISCUSSION

Synthesis of Sulfated Glycopolymers. Polyacrylamide-based neoglycoconjugates have been used as diagnostic reagents, such as in the inhibition of hemagglutination by pathogens (24) or as solid-phase coatings in enzyme-linked immunosorbent assays (ELISA) (25, 26). Our prior investigations have confirmed that cyanoxyl (OCN)-mediated free-radical polymerization of acryl-derivatized glycomonomers is a convenient tool to produce water-soluble glycopolymers. In this reaction, cyanoxyl radicals were generated by an electron-transfer reaction between cyanate anions (-OCtN), from a NaOCN aqueous solution, and p-chlorobenzene-diazonium salts (ClC6H4NtN+BF4-), that were previously prepared in situ through a diazotization reaction of p-chloroaniline in water (Scheme 1). In addition to cyanoxyl persistent radicals, aryl-type active radicals were simultaneously produced, and only the latter species is capable of

monomer ratio GM/AM (mol)

polymer composition (mol)

lactose content (wt %)a

Mn (g/mol)

Mw/Mn SEC

glycopolymer

1/0 1/0 1/1 1/1

1/0 1/0 1/10 1/11

100 100 57 56

7500 114 000 9300 33 400

1.19 1.62 1.46 1.47

7a (SL5) 7b (SL7) 7c (SL3) 7d (SL4)

a Mass content of disaccharide was determined by 1H NMR analysis.

initiating chain growth. As summarized in Tables 1 and 2, cyanoxyl-mediated homopolymerization of sulfated N-acetylglucosamine glycomonomer 4 or lactose heptasulfate monomer 5 generated the expected sulfated glycopolymers 6 or 7 in good conversion yield (60-80%) and with relatively low polydispersity (1.13 < Mw/Mn < 1.62), respectively. Polymers of varying molecular weight were prepared by altering either monomer conversion or the initial ratio of monomer to initiator concentrations ([M]o/[I]o). Similarly, copolymers were prepared by copolymerization of sulfated glycomonomer 4 or 5 with acrylamide in low-polydispersity (1.15 < Mw/Mn < 1.57). The resultant glycopolymers were characterized by NMR spectroscopy as well as by SEC coupled with both refractive index and multiangle laser-scattered detectors. Glycopolymers Bearing Lactose Heptasulfate Dissacharides Stimulate FGF-2-Mediated Cell Proliferation. The capacity of selected glycopolymers to potentiate FGF-2 mitogenic activity was measured by [3H]-thymidine incorporation into BaF3-FR1C-11 cells, which express the FGF receptor, FGFR-1, but not cell surface heparan sulfate. In the absence of exogenous heparin or heparan sulfate, FGF-2 is incapable of eliciting cell proliferation. Cells were treated with 3 ng/mL of FGF-2 and increasing concentrations of indicated oligosacharides. Test compounds include a series of glycopolymers ranging in molecular weight, pendant group type, and density, as well as corresponding monomers, sucrose octasulfate (SOS), heparin, and heparan sulfate (Figure 1). The synthesis of sulfated glycopolymers did not ensure the generation of compounds that mimicked heparin’s ability to stimulate FGF-2-dependent cell proliferation. For example, glycopolymers 6 (SM1-SM6) carrying fully sulfated NAcGlc pendant groups were ineffective in promoting FGF-2-dependent cell proliferation (Figure 1A). However, glycopolymers 7 (SL3, SL4, SL5, and SL7) bearing sulfated lactose residues were able to elicit a range of proliferative activity depending upon the structural features of the polymer (Figure 1B-D). Of note, a high level of bioactivity was demonstrated by a glycopolymer designated SL3 (MW 9300, PDI 1.46) that was a copolymer of sulfated lactose monomers (GM) and acrylamide (AM) (molar ratio AM:GM 1:10, lactose

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Figure 1. Mitogenic activity of natural and synthetic carbohydrate-based compounds. Activation of FGF mitogenic activity as measured by [3H]-thymidine incorporation into BaF3-FR1C-11 cells treated with 3 ng/mL FGF-2 and increasing concentrations of the indicated compound. (A) Mitogenic activity of glycopolymers (SM1-SM6) in the presence of FGF-2. (B) Comparison of mitogenic activity of glycopolymers (SL3-SL7) with SOS and heparin. (C) Comparison of mitogenic activity of glycopolymer SL3 with SL3 monomer, SOS, and heparan sulfate. (D) Comparison of mitogenic activity of glycopolymer SL3 with dextran sulfate 5000, 10 000, and 500 000. (E) Mitogenic activity of synthetic glycopolymers in the presence of FGF-1. Activation of FGF-1 mitogenic activity as measured by [3H]-thymidine incorporation into BaF3-FR1C-11 cells treated with 4 ng/mL FGF-1 and increasing concentrations of the indicated compound.

content 57 wt %). SL3-mediated proliferative responses to FGF-2 exceeded those observed with heparan sulfate, as well as SOS and free SL3 monomer 5. Of interest, the SL3 effect is selective in that it was not capable of initiating a proliferative response to FGF-1 (Figure 1E). FGF-2 and FGFR-1 Dimerization Is Mediated by the Mitogenic Glycopolymer SL3. Characteristically, FGF-2 is sequestered in the ECM in a dimerized form only when matrix-producing cells produced fully sulfated GAGs. Significantly, dimerization of both FGF-2 and FGFR-1 are essential for an optimized biological response. To examine the capacity of SL3 to mediate FGF-2 dimerization, 20 ng/mL of 125I-FGF-2 was incubated with either heparin or SL3 for 1 h at room temperature. After

disuccinimidyl suberate cross-linking, the samples were resolved by electrophoresis and visualized by autoradiography (Figure 2). SL3 was as effective as heparin in mediating FGF-2 dimerization. Likewise, SL3-mediated FGFR-1 dimerization was investigated by incubating BaF3-FGFR-1C-11 cells with 125I-labeled FGF-2 in the presence or absence of either heparin or SL3. Cells were treated with disuccinimidyl suberate to cross-link FGF-2 to its receptor. Cross-linked proteins were electrophoresed on an SDS-6% polyacrylamide gel and detected by autoradiography (Figure 3). SL3 was as effective as heparin in mediating FGFR-1 dimerization. Glycopolymer SL3 Protects FGF-2 from Protein Degradation Induced by Trypsin, Acidic Condi-

A Glycopolymer Chaperone for FGF-2

Bioconjugate Chem., Vol. 15, No. 1, 2004 149

125I-

Figure 2. Dimerization of FGF-2 in the presence of SL3. FGF-2 20 (ng/mL) was incubated with either heparin or SL3 for 1 h at room temperature. After disuccinimidyl suberate crosslinking, samples were resolved by electrophoresis and visualized by autoradiography.

Figure 3. Cross-linking of 125I-FGF-2 to FGFR-1. BaF3-FGFR1C-11 cells were incubated with 125I -labled FGF-2 in the presence or absence of either heparin or SL3 and subsequently treated with disuccinimidyl suberate to cross-link FGF to the FGFR-1 receptor. Cross-linked proteins were electrophoresed on an SDS-6% polyacrylamide gel and detected by autoradiography.

tions, and Heat. It has been suggested that once soluble proteins, such as FGF-2, are released into the ECM, their sequestration by heparan sulfate proteoglycans is essential for preservation of bioactivity. In principle, complexation with HS chains should protect heparin-binding proteins from heat-, pH-, and protease-related degradation mechanisms. To assess protection from proteolysis, FGF-2 (1 µg) was incubated with trypsin for 3 h at 37 °C in the presence or absence of 25 µg/mL of heparin or decreasing concentration of glycopolymer SL3. Samples were then analyzed by SDS-PAGE and undigested FGF-2 was estimated by densitometry (Figure 4). SL3 was as effective as heparin in protecting FGF-2 from proteolytic degradation. Protection from acidic conditions was determined by incubation of FGF-2 with or without heparin or glycopolymer SL3 (25 µg/mL) in varying concentrations of trifluoroacetic acid (0.05 to 5%), corresponding to a pH range of 0 to 3.4. After 2 h incubation at room temperature, samples were diluted 20-fold with RPMI 1640 and incubated with heparin deficient cells (BaF3-FGFR-1) expressing the FGF receptor (FGFR-1). 3H-Thymidine incorporation was determined as an indicator of retained FGF-2 activity (Figure 5). Preservation of biological activity upon heat-treatment of FGF-2 was measured by incubation of FGF-2 at 65 °C for 5 min with or without heparin or glycopolymer SL3 (25 µg/mL). Samples were diluted in RPMI 1640 and incubated with BaF3-FGFR-1 cells, and 3H-thymidine incorporation was determined (Figure 6). Both heparin and the heparinmimetic glycopolymer SL3 provided substantial protection of FGF-2 from trypsin, heat, and acidic conditions.

Figure 4. SL3 protects FGF-2 from trypsin digestion. Aliquots (1 µg) of recombinant FGF-2 were incubated at 37 °C with trypsin in the absence or presence of 25 µg/mL heparin or decreasing concentration of SL3. Samples were then analyzed by SDS-PAGE and visualized by silver staining. Undigested FGF-2 in each lane of the gel was estimated by densitometry.

Figure 5. SL3 protects FGF-2 on exposure to acidic conditions. A stock solution of FGF-2 (100 µg/mL in 5 mM tris, pH 7.6) in the presence or absence of either heparin or SL3 was diluted 10-fold with TFA ranging in concentration from 0.05 to 5%. After 2 h incubation at room temperature, the samples were further diluted 20-fold with RPMI 1640-10% FCS without IL-3 (final FGF-2 concentration 0.5 µg/mL) and added to BaF3-FGFR-1C11 cells. Heparin or SL3 was added to those test samples that were incubated in TFA in the absence of either compound. The final FGF-2 concentration was 3.3 ng/mL, while that of heparin or SL3 was 25 µg/mL. After 36 h, 1 µCi of [3H]-thymidine was added to each sample. The cells were incubated for an additional 5 h and incorporated thymidine determined by liquid scintillation counting.

Despite recent advances in achieving sustained local delivery of protein growth factors through the use of synthetic or native macromolecules as controlled delivery vehicles, significant limitations remain in achieving optimal control over the degree of growth factor sequestration and maximum preservation of growth factor activity during and after release. For example, while the addition of MgOH to polylactide/glycolide (PLGA) microspheres have led to improvements in protein stabilization during material processing and polymer degradation, the risk of protein inactivation due to proteolytic, thermal, or other mechanisms that are operative at the depot site in the body remains (27-30). All of this necessitates the delivery of increased quantities of growth factor, with significant added cost, to compensate for its reduced bioavailability. Moreover, variability in the nature, extent, and magnitude of those physiochemical processes, which may degrade or denature protein growth factors, further complicates the goal of predictable uniform drug delivery.

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Mass Spectrometry Centers for their facilities. They are also indebted to Dr. Suri Iyer for kind technical assistance. LITERATURE CITED

Figure 6. SL3 protects FGF-2 from thermal denaturation. A stock solution of FGF-2 (100 µg/mL, in 5.0 mM tris, pH 7.6) was diluted 10-fold in PBS with or without SL3 or heparin and heat inactivated. An aliquot was added to BaF3-FGFR-1C-11 cells such that the final FGF-2 concentration was 3.3 ng/mL, while that of heparin or SL3 was 25 µg/mL. After 36 h, 1 µCi of [3H]thymidine was added to each sample. The cells were incubated for an additional 5 h and incorporated thymidine determined by liquid scintillation counting.

In this report, we postulate that heparan sulfate proteoglycans, which act as reservoirs for heparin-binding growth factors and cytokines, establishes a useful biomimetic paradigm for the generation of biomaterials for the controlled delivery of proangiogenic proteins or other growth factors. The diverse biological properties of heparin and heparan sulfate combined with the inherent difficulty associated with the synthesis of these chemically complex natural products have motivated efforts to generate simpler structural mimics as component biomaterials for a variety of applications. For example, Cooper et al. (31, 32), and Ito et al. (33, 34) and Onishi (35) have all produced sulfonate-functionalized polymers with promising anticoagulant-like properties. Of particular interest have been the efforts of Jozenfowicz and colleagues who have prepared dextrans derivitized with carboxymethyl, carboxymethylbenzamide, and carboxymethylbenzylamide sulfonate functional groups (36-39). The biological features of these polysaccharides are related to the number and distribution of chemical groups randomly attached along the dextran chain. Indeed, several of these polymers exhibit anticoagulant and anticomplement properties, and in some cases mimic heparin’s inhibitory effect on smooth muscle cell growth (9, 40-41). Other variants potentiate the activities of FGF-1 and FGF-2 and appear to stimulate the healing responses of a variety of tissue types in vitro and in vivo (38, 39). In this report, we demonstrate that a glycopolymer bearing pendant fully sulfated lactose units effectively replaces heparin and heparan sulfate as a molecular chaperone for FGF-2. A compound was identified that protects FGF-2 from proteolytic, acid, and heatinduced degradation, while selectively promoting its dimerization and interaction with its receptor. Significantly, the capacity of this heparin-mimic to promote an FGF-2 specific proliferative cell response suggests potential applications in areas related to therapeutic angiogenesis, including wound healing, myocardial or lower extremity revascularization, as well as in tissue engineering. ACKNOWLEDGMENT

This work was supported by grants from the NIH. The authors acknowledge the Emory University NMR and

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