Bioconjugate Chem. 2001, 12, 1085−1088
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Hyaluronic Acid-N-hydroxysuccinimide: A Useful Intermediate for Bioconjugation Yi Luo and Glenn D. Prestwich* Department of Medicinal Chemistry, The University of Utah, Salt Lake City, Utah 84112-5820. Received May 9, 2001; Revised Manuscript Received September 20, 2001
Hyaluronic acid (HA) is an abundant nonsulfated glycosaminoglycan component of synovial fluid and the extracellular matrix. HA is an important building block for biocompatible and biointeractive materials with applications in drug delivery, tissue engineering, wound repair, and viscosupplementation. Herein we describe the synthesis and characterization of HA-N-succinimide, an activated ester of the glucuronic acid moiety. This HA-active ester intermediate is a precursor for fluorescent probes, drug-polymer conjugates, and cross-linked hydrogels. As a demonstration, we used HA-NHS to prepare HA-BODIPY by coupling with the hydrazide derivative of the fluor. Intracellular uptake of HA-BODIPY into human ovarian cancer cells, which overexpress cell-surface HA receptors, was visualized using confocal microscopy.
Hyaluronic acid (hyaluronan, HA), a (β-1,4)-linked acid and (β-1,3) N-acetyl-D-glucosamine polysaccharide (Figure 1), is a nonsulfated glycosaminoglycan (GAG) found in the extracellular matrix (ECM). HA plays an important role in the structure and organization of ECM, including the maintenance of extracellular space, the transport of ion solutes and nutrients, and the preservation of tissue hydration. HA concentrations increase whenever rapid tissue proliferation, regeneration, and repair occur. HA also binds specifically to proteins in ECM, on cell-surface receptors, and within the cell cytosol. These protein-ligand interactions are important in stabilizing the cartilage ECM (1, 2), regulating cell adhesion and motility (3, 4), mediating proliferation and differentiation (5), and the action of growth factors (6). HA has also been implicated in morphogenesis and embryonic development (7), in cancer (8-14), in modulating inflammation (12), in stimulating angiogenesis and healing (15), and as a protective coating (16). HA and its chemically modified derivatives (17) have emerged as versatile building blocks for novel biomaterials (18). Chemical modification of HA has employed the carboxyl group, the hydroxyl groups, the reducing end, and in some cases partially N-deacetylated materials. The attempted carbodiimide-mediated coupling of primary amines to HA resulted in negligible coupling due to the protonation of the required nucleophilic nitrogen at the reaction pH. The major product was the N-acylurea adduct. Recently, a modified method for carbodiimide/ active ester-mediated coupling of polyfunctional amines to HA was developed in which HA was reacted with 30fold excess of the amine component at pH 6.8 in the presence of EDCI and 1-hydroxybenzotriazole (HOBt) with DMSO/H2O (1:1) as solvent (19). Antitumor agents have been coupled to HA to provide polymeric conjugates that deliver chemotherapeutics to target tissues. For D-glucuronic
* Address correspondence to this author: Professor Glenn D. Prestwich, The University of Utah, Department of Medicinal Chemistry, 30 South 2000 East, Room 201, Salt Lake City, UT 84112-5820, Phone: 801 585-9051; Fax: 801 585-9053, E-mail:
[email protected].
example, adriamycin (20), mitomycin C, and epirubicin (21) were coupled to HA using carbodiimide chemistry, and butyric acid-HA was synthesized by reaction of butyric anhydride with HA (22). In this technical note, we describe a convenient route to the HA-N-succinimide active ester (Figure 1). Ionexchange (23) and lyophilization afforded the HA tetrabutylammonium salt (HA-TBA). Next, 2 mol equiv (based on the calculated number of glucuronic acid residues present) of purified N-hydroxysuccinimido diphenyl phosphate (SDPP), mp 89-90 °C (24), were added to a solution of 2 wt % HA-TBA in DMSO. The reaction was stirred for either 2 h or overnight at room temperature, and the product was precipitated by addition of 3 volumes of hexane followed by excess acetone. The residue was dissolved in 2 mL of H2O and purified on a Sephadex G-25 (20-80 µm) (Sigma, St. Louis, MO) gel filtration column. In our experience, SDPP activation gave superior yields of HA-N-succinimide active ester (HA-NHS) in less time and under milder conditions than those reported previously (19, 23, 25-28). The degree of substitution was calculated by integration of the succinimide ester methylenes (1H NMR, broad singlet at δ ) 2.62 ppm, 4H) relative to the methyl of the acetamido moiety of the GlcNAc residues resonance (δ ) 1.95 ppm, 3H) (25). The degrees of substitution were 50 mol % and 80 mol % with the reaction time of 2 and 12 h, respectively. In some cases, triethylamine facilitated the carboxyl group activation reaction by SDPP (24). In the present example, however, use of triethylamine as a cosolvent led to ion exchange and isolation of the HAtriethylamine salt instead of HA-NHS. The HA-NHS active ester is a versatile precursor for bioconjugation with reporter and effector molecules. BODIPY-FL hydrazide (Molecular Probes, Inc., Portland, OR) was used to demonstrate this versatility. Thus, 20 mg of HA-NHS (50 mol %) was dissolved in 2 mL of H2O while stirring at room temperature; 1 mg of BODIPY-FL hydrazide in 0.5 mL of DMSO was then added with 3 drops of 0.1 N Na2CO3 buffer (pH 8.0). The reaction was stirred at room temperature for 3 days in the dark. Next, 2-aminoethanol (100 µL) was added to
10.1021/bc015513p CCC: $20.00 © 2001 American Chemical Society Published on Web 10/20/2001
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Figure 1. Synthesis of HA-NHS active ester and HA-BODIPY probe: (i) SDPP, DMSO, rt; (ii) DMSO/H2O, rt.
consume any unreacted active ester. Solvents were removed by lyophilization, and the residue was dissolved in 1 mL of H2O and purified by gel filtration. The purity of HA-BODIPY was measured at 210 nm (HA absorption) and 502 nm (BODIPY absorption) using analytical gel permeation chromatography (GPC) (Figure 2a). The low molecular weight HA-NHS has high efficiency of BODIPY coupling than the high molecular weight chains. The degree of substitution of BODIPY determined spectrophotometrically was 4.5 wt %. Interestingly, the HABODIPY conjugate had a 5-nm blue shifted absorbance with excitation at 488 nm as compared to the BODIPY hydrazide reagent (Figure 2b). In addition, a broadening of the fluorescence emission was observed (Figure 2c). It is possible that π-stacked or hydrophobically interacting aggregates could be formed in aqueous solution when a hydrophobic fluorophore is conjugated to a water-soluble polymer, thus altering the absorbance and emission of the fluorophore. (29). At the low modifications employed, this does not appear to be a problem, as a soluble, homogeneous derivative was obtained. This is consistent with previous work with HA-taxol (30) and HA-FITCtaxol derivatives (31). HA-BODIPY was readily internalized by human ovarian cancer cells (SK OV-3), which overexpresses the HA receptor CD44 on the cell surface. Thus, SK OV-3 cells were incubated in a cell culture flask, harvested by trypsinization, and transferred into an 8-well cell culture slide. Cells (20,000) were seeded in each well of the slide and cultured for 48 h. The culture medium was replaced with medium containing HA-BODIPY probe (100 µg/mL HA). Cells were cultured with the conjugates for various lengths of time. Unbound probe was removed by washing the cell layer three times with Dulbecco’s phosphatebuffered saline (DPBS) (Sigma, St. Louis, MO). Cells were fixed with 3% paraformaldehyde for 10 min at room temperature and washed again with DPBS. Internalization of BODIPY-HA was visualized by fluorescence confocal microscopy (Figure 3). Initially, BODIPY-HA can be seen on the cell membranes, and with time it is gradually endocytosed and accumulates in the cytoplasm. Thus, HA and probe-modified HA bind to the cell surface via interactions with CD44 or RHAMM, and a portion subsequently undergoes endocytosis (4, 30, 31). Internalization of [3H]-labeled HA previously revealed that intracellular degradation of HA occurs within a low pH environment (32), such as that of the lysosome. In the
Figure 2. (a) GPC profile of HA-BODIPY detected at 210 and 502 nm. GPC was carried out on the following system: Waters 515 HPLC pump, Waters 410 differential refractometer, Waters 486 tunable absorbance detector, Waters Ultrahydrogel 250 and 2000 columns (7.8 mm ID × 30 cm) (Milford, MA). The GPC eluent was 150 mM phosphate buffer/MeOH ) 80:20 (v/v), pH 6.5, and the flow rate was 0.5 mL/min. (b) UV/vis spectra of BODIPY and HA-BODIPY. (c) Fluorescence spectra of BODIPY and HA-BODIPY.
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Figure 3. Time course internalization of HA-BODIPY by SK OV-3 cells. Cells were examined using an inverted microscope (Nikon) and a Bio-Rad (Hercules, CA) MRC 1024 laser scanning confocal microscope. A coverslip was mounted on a microscope slide containing fixed cells with ProLong Antifade Kit (Molecular Probes, Eugene, OR) as the mounting medium. Cell images were collected by using a 60 × oil immersion objective, and no postacquisition enhancement of images were performed. Image acquisition used the BHS block of filters (excitation 488 nm and emission through a 522 nm 32 band-pass filter). Fluorescence images were scaled to 256 Gy levels.
present study, the uptake and degradation of HA in cellular compartments resulted in the release of fluorescent molecules into the cytoplasm. In conclusion, a new method was developed to prepare an HA-active ester derivative. This HA-active ester intermediate will find numerous applications in biomedical research, including the preparation of fluorescent probes, drug-polymer conjugates, and cross-linked hydrogels. ACKNOWLEDGMENT
Financial support for this work was provided by Department of Army (DAMD 17-9A-1-8254) and by the Huntsman Cancer Foundation at The University of Utah (UUtah). We are grateful to Joseph C. Shope and Dr. Daryll B. DeWald of Department of Biology of Utah State University for assistance with confocal microscopy. We thank Dr. L. Y.-W. Bourguignon of University of Miami Medical School for providing SK-OV-3 cells. We thank Clear Solutions Biotech, Inc. (Stony Brook, NY) for providing HA and the Center for Cell Signaling (UUtah), supported by the Utah Centers of Excellence Program, for cell culture facilities. LITERATURE CITED (1) Dowthwaite, G. P., Edwards, J. C. W., and Pitsillides, A. A. (1998) An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development. J. Histochem. Cytochem. 46, 641-651.
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