Linolenic acid grafted hyaluronan: Process development, structural characterization, biological assessing, and stability studies
Abstract
In this study, hyaluronan (HA) was grafted with alpha-linolenic acid (alpha LNA) by benzoyl mixed anhydrides methodology, which allowed the derivatization of HA under mild reaction conditions. The reaction was optimized and transferred from laboratory to semi-scale production.
The derivative revealed an unexpected cytotoxicity after oven drying and storage at 40 degrees C. For this reason, the storage conditions of sodium linolenyl hyaluronate (alpha LNA-HA) were optimized in order to preserve the beneficial effect of the derivative.
Oven, spray dried and lyophilized samples were prepared and stored at -20 degrees C, 4 degrees C and 25 degrees C up to 6 months. A comprehensive material characterization including stability study of the derivative, as well as evaluation of possible changes on chemical structure and presence of peroxidation products were studied by Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS), thermogravimetric analysis (TGA) and complemented with assessment of in vitro viability on mouse fibroblasts NIH-3T3.
The most stable ANA-HA derivative was obtained after spray drying and storage at ambient temperature under inert atmosphere. The choice of inert atmosphere is recommended to suppress oxidation of aLNA supporting the positive influence of the derivative on cell viability.
The encapsulation of hydrophobic drugs of ANA-HA were also demonstrated.