Synthesis of methacrylated hyaluronic acid with tailored degree of substitution

Abstract

The aim of this work was to develop a new method to derivatize hyaluronic acid (HyA) with polymerizable methacrylate residues with precise control over the substitution degree. The synthesis of methacrylated HyA (HyA-MA) was performed in dimethyl sulfoxide (DMSO) using glycidyl methacrylate (GMA) and 4-(N,N-dimethylamino)pyridine as a catalyst. HyA was rendered soluble in DMSO by exchanging the Na⁺ ions by the more lipophilic tetrabutylammonium ions. HyA-MA with a fully controlled degree of substitution (DS, defined as the number of methacrylate groups per 100 disaccharide units), ranging from 5 to 30, was obtained at 50 °C after 48 h. Hydrogels were obtained upon radical polymerization of aqueous solutions of HyA-MA using potassium peroxodisulfate (KPS) as initiator and N,N,N′,N′-tetramethylethylenediamine (TEMED) as catalyst. Almost complete methacrylate conversion (95%) was achieved for hydrogels obtained by polymerization of HyA-MA with a degree of substitution of 15. At lower DS (DS 8.5 and 5) the methacrylate conversion was 82% and 68%, respectively. Rheological characterization showed that with increasing DS the storage modulus of these HyA-MA hydrogels increased. Swelling experiments showed that HyA-MA gels with a DS of 15 or above were dimensionally stable, whereas HyA-MA gels with DS 5 and DS 8.5 swelled 1.6 and 1.4 times their initial weight, respectively. In conclusion, this paper shows that the DS of HyA-MA can be tailored by the reaction conditions and that consequently HyA-MA hydrogels with different characteristics can be prepared.

Introduction

Hyaluronic acid (HyA) is an endogenous polysaccharide, i.e. present in the vitreous body, synovial fluids and the extracellular matrix, which consists of repeating disaccharide units composed of β(1–4) linked N-acetyl-d-glucosamine and β(1–3) linked d-glucuronic acid [1]. Its biocompatibility, biodegradability and immunoneutrality make HyA an attractive polymer for biomedical and pharmaceutical applications. Currently, HyA is applied to treat joint diseases such as in osteoarthritis and in eye surgery as replacement fluid and is under investigation for drug delivery and tissue engineering applications [2], [3], [4]. For these applications besides unmodified HyA derivatized HyA is also used. Modification of HyA can be performed as hydroxyl and carboxy groups can be used for chemical derivatization [5], [6] and many chemical modifications of HyA have been described in literature [1]. One chemical modification concerns the derivatization of HyA with polymerizable methacrylate groups. This crosslinkable HyA can be used to form hydrogels for drug delivery and tissue engineering purposes [4], [5], [7], [8]. The synthesis of methacrylated HyA (HyA-MA) is performed in an aqueous environment with an excess of methacrylic anhydride with respect to the hydroxyl groups of HyA [3], [7], [9]. The major drawback of this synthesis lies in the aqueous basic (pH 8) reaction conditions: methacrylic anhydride can react with water to yield methacrylic acid and additionally the covalently linked methacrylic ester can be hydrolyzed during the reaction [10]. This makes it difficult to control the degree of substitution. Hence, there is need for a method in which the DS of methacrylated HyA can accurately be controlled as was achieved in the past for the modification of polysaccharides [11], [12], [13] allowing the preparation of hydrogels with tailored properties.

This paper reports on the synthesis of methacrylate derivatized HyA with precise control over the substitution degree in a suitable aprotic solvent by substitution of the parent polysaccharide with glycidyl methacrylate. Additionally, the characteristics of the hydrogels obtained with glycidyl methacrylate derivatized HyA are studied.