Determination Of Modification Degree In BDDE-modified Hyaluronic Acid Hydrogel

Hyaluronic acid (HA) is a natural unbranched polysaccharide with a repeating disaccharidic unit composed of β-D-glucuronic acid (GlcA) and β-D-N-acetylglucosamine (GlcNAc) linked by (1â†'4) and (1â†'3) glycosidic bonds. HA has excellent biocompatibility, high water retention capacity and viscoelasticity, which makes it suitable for various medical and cosmetic applications, like ophthalmic surgery adhesion agents, joint cavity supplements, surgery adhesions blocking agent and dermal filler. HA-based hydrogel is widely used as dermal filler during cosmetic surgery by physician.Natural HA can be easily degraded by hyaluronidase (HAase) in vivo. To improve the mechanical properties and prolong its duration in vivo, natural HA is chemical modified by cross-linkers to form HA hydrogel. The degree of modification is one very important characteristic parameter, which can effect on the rheological property, anti-enzymatic ability and swelling property of HA hydrogel in chemical structural foundation. Determination of modification degree will help us to understand different hydrogels based on its chemical structure.In this work, three crosslinking parameters (degree of total modification, t-MOD; degree of cross-link modification, c-MOD; degree of pendent modification, p-MOD) are defined and determined by quantification of the modified fragments in hydrogel digestion by size exclusion chromatography combined with mass spectrometry (SEC-MS).1. Preparation of hydrogelBDDE (1,4-butanediol diglycidyl ether)-modified HA hydrogels (with different addition of BDDE) were prepared. The mechanical and swelling properties of different HA hydrogels were studied. The morphology of HA hydrogel was characterized by scanning electron microscope (SEM).2. Analysis of enzymatic products of HA hydrogelA novel hyaluronidase HAase-B produced by Bacillus sp. A50 is used to digest HA hydrogel. HAase-B can digest HA into unsaturated disaccharide (â–³DiHA) completely. The digestion of hydrogel were analyzed by SEC using a Superdex Peptide 10/300 GL column. All fractions were collected and freeze-dried.1H-NMR analysis of each fraction was done on a Bruker 400MHz instrument. MS analysis provided accurate molecular weight of fractions. As a result, five modified fragments (HA oligosaccharides linked to BDDE residues) were found in the digestion.2. Determination of three crosslinking parametersThe t-MOD is equal to the total area percentage of modified fractions in SEC chromatogram, c-MOD and p-MOD are determined by relative quantification of five cross-link and pendent modified fragments in TIC of SEC-MS.3. Analysis of the HA hydrogel products in the marketSome HA hydrogel products in the market were analyzed by our method and differences between them were found.In this work, we report on a strategy to precisely identify and quantify the modified fragments in hydrogel digestion and determine three crosslinking parameters. This method is applicative for any BDDE-modified HA hydrogel products in the market, the differences between three parameters indicates various crosslinking modification strategies and diverse properties of hydrogel product.