Chitosan nanoparticles production: optimization of physical parameters, biochemical characterization, and stability upon storage

Ionic gelation is among the simplest processes for the development of chitosan nanoparticles reported so far in the literature. Its one‑shot synthesis process in conjunction with the mild reaction conditions required are among the main causes for its success. In this work, we sought to optimize a set of physical parameters associated with the ionic gelation process at two different pH values. Following that, the NPs’ freeze‑drying and long‑term storage stability were assayed, and their biocompatibility with HaCat cells was evaluated. The results show that NPs were more homogenously produced at pH 5, and that at this pH value, it was possible to obtain a set of optimum production conditions. Furthermore, of the assayed parameters, TPP addition time and overall reaction time were the parameters which had a significant impact on the produced NPs. Nanoparticle freeze‑drying led to particle aggregation, and, of the cryoprotectants, assayed mannitol at 10% (w/v) presented the best performance, as the NPs were stable to freeze‑drying and maintained their size and charge in the long‑term stability assay. Lastly, the chitosan NPs presented no toxicity towards the HaCat cell line.