July/August 2002
Published by the American Chemical Society
Volume 3, Number 4
© Copyright 2002 by the American Chemical Society
Communications Thermosensitive Lanthanide Complexes of Hyaluronan Koen P. Vercruysse,†,‡ Hao Li,† Yi Luo,§ and Glenn D. Prestwich* Department of Medicinal Chemistry, The University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108 Received February 28, 2002; Revised Manuscript Received April 18, 2002
Addition of trivalent lanthanide salts to solutions of hyaluronan (HA), a naturally occurring anionic polysaccharide, resulted in an unexpected reverse-temperature phase transition (RTPT); i.e., the complexes were soluble when cooled but precipitated when warmed. A unique lower critical solution transition temperature (LCST) was observed for each lanthanide in the order (increasing LCST) Eu3+ < Nd3+< Ce3+ < Gd3+ < La3+ < Tb3+ < Dy3+ < Yb3+ < Lu3+ for high molecular weight HA. Moreover, the LCST values increased as the molecular weight of the HA decreased from 1730 to 190 kDa. The precipitationdissolution behavior was fully reversible in a cyclical fashion; a lag in redissolution was observed as the temperature was lowered, and longer incubation times at each temperature minimized this lag. The RTPT behavior could be ablated by titration with 8 kDa HA to produce complexes soluble at ambient temperature. This is the first description of RTPT behavior in a biologically important glycosaminoglycan. Introduction Hyaluronan (HA), a polysaccharide of alternating Dglucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) units (Figure 1), is the only nonsulfated glycosaminoglycan (GAG) found in the extracellular matrix.1 HA possesses extensive lubrication, water absorption, and water retention capabilities2,3 and influences cell adhesion, growth, and migration through interactions with cell surface receptors.4-6 HA also serves as a signaling molecule in cell motility, inflammation, wound healing, and cancer metastasis.7 With such unique physicochemical properties and biological functions, HA has become an attractive building block for new biocompatible and biointeractive materials.8 On the basis of its unique rheological properties, HA is * To whom all correspondence should be addressed: Phone: 801 5859051. Fax: 801 585-9053. E-mail:
[email protected]. † These authors contributed equally to this work. ‡ Current address: Department of Chemistry, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN 37209-1561. § Current address: Vertex Pharmaceuticals Inc., 130 Waverly St., Cambridge, MA 02139.
currently being used clinically for drug delivery,9 in viscosupplementation and viscosurgery,10 for the treatment of arthropathies by intra-articular injection, and to promote wound healing.11 Chemically modified and cross-linked HA has been used for wound healing,12 reducing postsurgical adhesions,13 and drug delivery.14-16 Many studies describe the interaction of HA with polypeptides, metal ions, and other molecules.17 For example, INTERGEL (LifeCore, Inc.), a hydrogel formed by the chelation of HA with ferric hydroxide, has been developed for prevention of postsurgical adhesions.18 Complexes of HA with copper, zinc, calcium, and barium have also been described, in particular with regard to the effects of divalent cations on HA degradation by hyaluronidase.19 However, the interactions of HA with trivalent lanthanide cations remain largely unexplored. In view of the importance of lanthanide complexes for fluorescence assay development, magnetic resonance imaging, and targeted radionuclide delivery, we embarked on the study of the interaction of HA with selected lanthanide trichloride salts. We now report an unprecedented HA-lanthanide interaction producing complexes that exhibit
10.1021/bm020026d CCC: $22.00 © 2002 American Chemical Society Published on Web 05/22/2002
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Communications
Figure 1. Chemical structure showing the repeating disaccharide unit of hyaluronan.
reverse-temperature phase transition (RTPT) behavior at element-specific lower critical solution transition temperatures (LCSTs). Experimental Section Lanthanide and Polymer Survey. All biochemical reagents were purchased from Sigma Chemical Company (St. Louis, MO) and used without further purification. Lanthanide chloride salts were obtained from Aldrich (Milwaukee, WI) and were of 99.99% purity. Aqueous 3 mg/mL solutions (0.5 mL) of the sodium salts of 1200 kDa HA and other anionic polysaccharides (chondroitin sulfate A (CS-A), chondroitin sulfate C (CS-C), heparin, carboxymethylcellulose (CMC), and pectin) were mixed with 0.15 mL of 100 mM aqueous EuCl3 or TbCl3 at ambient temperature. Cold HA-Eu3+ or HA-Tb3+ solutions were warmed in a controlled temperature bath, and precipitation reoccurred for the HA-Eu3+ and HA-Tb3+ complexes. Next, 1200 kDa HA (2.7 mg/mL) was mixed with 10 mM aqueous solution of the chloride salts of La3+, Ce3+, Nd3+, Eu3+, Gd3+, Tb3+, Dy3+, Yb3+, and Lu3+. Each mixture was then cooled in an ice bath and transferred to a quartz cuvette thermostated at 1.5 °C. The RTPT of each HA-Ln3+ complex was monitored by measuring the turbidity of the mixture at 650 nm on a HP 8453 UV-Vis spectrophotometer as the temperature was increased in 1 °C increments with a dwell time of 5-10 min at each temperature. Finally, Ln3+ solutions were mixed with 236 kDa HA (2.7 mg/mL), and LCST temperatures were determined as before. LCST Dependence on HA Size, Lanthanide, and Dwell Time. Four representative lanthanides were chosen for detailed studies: Gd3+, Tb3+, Dy3+, and Lu3+. As before, 1.8 mL of a 3.0 mg/mL solution of sodium HA at each of three sizes (1730, 824, and 190 kDa, as determined by calibrated gel permeation chromatography (GPC)) was mixed with 200 µL of 100 mM Ln3+ stock solution in the cold room, and then incubated at 5 °C below the LCST anticipated from the preliminary study. Each of the four lanthanide stock solutions had a pH of 5.80, compared to pH 5.65 for the Nanopure water employed. The homogeneous mixture was stirred, and the turbidity was measured at 650 nm as the temperature was increased in 1 °C increments with a dwell time of 10 min at each temperature. The reversibility and dwell-time dependence were examined using the HA-Dy3+ complex in the thermostated cuvette as described above. Dwell times of 5, 10, and 20 min were employed at each temperature setting.
Competitive Displacement by 8 kDa HA. HA (500 mg) was dissolved in 30 mL of PBS buffer and treated with 100 mg of solid bovine testicular hyaluronidase (300 units/mg, Sigma). During the incubation for 60 h at 37 °C, two batches of 100 mg of hyaluronidase were added. The solution was boiled for 3 min to stop the reaction and separated using a Sephadex G-25 column. The fractions were lyophilized, and the fraction used in the competitive displacement experiments was determined by matrix-assisted laser dessorption ionization mass spectrometry to be a mixture of HA oligosaccharides of 21-23 disaccharide units, with an average molecular weight of 8460 Da. RTPT was measured for seven solutions containing 0, 20, 33, 50, 66, 80, and 100 wt % of the 8 kDa HA oligosaccharide mixture relative to the 1730 kDa HA component. Results and Discussion In an effort to develop a fluorescence-based hyaluronidase assay, HA sodium salt (1200 kDa, 3 mg/mL) was mixed with Eu3+ or Tb3+ (final concentration, 5.5 mM) in water at ambient temperature. Precipitation was observed in all mixtures, accompanied by a loss in viscosity of the solutions, indicating HA precipitation in the form of a HA-Ln3+ complex. When stored at 3 ( 1 °C, the complexes of HA and Eu3+ or Tb3+ redissolved and the viscosity of the solutions increased correspondingly. Mixtures of CS-A, CSC, CMC, or pectin with 5.5 mM Eu3+ or Tb3+ also resulted in precipitation at ambient temperature, but none of these precipitates dissolved at 4 °C. Solutions of heparin with Eu3+ or Tb3+ remained homogeneous and without precipitation at all temperatures studied (data not shown). Soluble complexes of Eu3+ and Tb3+ have been widely used to study the ion binding properties of polyelectrolyte hydrogels and anionic polymers.20-22 When the HA-Eu3+ or HA-Tb3+ mixtures were warmed in a controlled temperature bath, precipitation reoccurred with increasing temperature, first for the HA-Eu3+ mixture and then for the HA-Tb3+ mixture. This precipitation-dissolution phenomenon was reversible over repeated cycles of warming and cooling. Alternating between dissolution and precipitation was accompanied by transitions between clear and turbid solutions. The temperature-dependent behavior of HA-Eu3+ or HA-Tb3+ complexes led us to investigate the interactions of HA with a larger range of trivalent lanthanides. Thus, aqueous solutions of the chloride salts of each of eight lanthanides (La3+, Ce3+, Nd3+, Eu3+, Gd3+, Tb3+, Dy3+,
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Communications Table 1. LCSTs for Aqueous HA-Ln3+ Complexesa LCST (°C) element lanthanum cerium neodymium europium gadolinium terbium dysprosium ytterbium lutetium
lanthanide salt 57LaCl
3
58CeCl
3
60NdCl
3
63EuCl
3
64GdCl 65TbCl
3
3
66DyCl
3 70YbCl 3 71LuCl 3
HA (1200 kDa)
HA (236 kDa)
