Hyaluronic acid auto-crosslinked polymer (ACP): Reaction and particle size monitoring, polymer characterization and hyaluronidase stability

Mauro PAVAN, Fidia Farmaceutici

Background
Hyaluronic Acid (HA) is a non-sulphated glycosaminoglycan composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine; despite its high molecular weight, HA is soluble in water and is not resistant to enzymatic degradation, thus hindering its wider application as a biomedical material. Auto-Crosslinked polysaccharide (ACP) gel of HA is a fully biocompatible hydrogel, which has improved viscoelastic properties and prolonged in vivo residence time, compared to HA, for use in various biomedical applications. Crosslinking is achieved through the activation of HA carboxyl groups by 2-chloro-1-methylpyridinium iodide (CMPI) and subsequent nucleophilic attack by the hydroxyl groups of HA. The ester-type bond may involve the chain of hyaluronic acid itself (intramolecular) as well as other chains (intermolecular), and rheological properties of the product are strongly dependent on the intra/inter-molecular cross-linking ratio. Furthermore, many variables play a determinant role on both physical and structural features rendering the esterification conditions hard to be controlled if defined cross-linked hydrogels performances have to be attained.

Method
In this study, many ACP gels have been obtained by HA cross-linking with various CMPI equivalent. The chemical reaction has been optimized, the solvent choice, the initial reagents concentration and corresponding equivalent ratios have been assessed during the esterification reaction. The cross-linking reaction has been monitored by rheological measurements, during the CMPI addition in the reaction mixture. The products have been precipitated in ethanol as fine white powder, dried in vacuum and re-hydrated in water, sterilized by heat and characterized by rheological and particle size analysis. The crosslinking densities were also observed through ESEM analysis. Furthermore, GPC analysis, in vitro hyaluronidase degradation test and esterification degree determination have been performed out. All these parameters have been correlated with CMPI amount added during the synthesis.

Conclusion
A process development approach has been carried out studying the effect of CMPI concentration on physical-chemical properties of intermediates and final ACP products. Correlations between CMPI concentration and rheological properties, esterification degree and hydro-soluble content have been shown. Process reproducibility and robustness have been achieved, as a result of a scale-up phase obtaining a product with specific properties. Finally, a comparative hyaluronidase degradation assay revealed that the ACPs exhibited a higher resistance at low elastic modulus, compared to other more chemically resistant crosslinked HAs.

 
Back to  TS.VII.F