Chitosan acetylation degree influences the physical properties of polysaccharide nanoparticles: implication for the innate immune cells response.

The aim of the present contribution is twofold, as it reports: i) on the role played by chitosan acetylation degree for the stability of nanoparticles formed with hyaluronan (NPs); ii) on the effect of the interaction of such NPs with immune cells. Chitosans with similar viscosity-average molecular weight, (M_v ) ̅, (i.e. 200 000) and different fraction of acetylated units (FA), together with a low molecular weight hyaluronan were chosen for developing a selected library of formulations via electrostatic complex coacervation. Resulting NPs were analyzed in terms of size, polydispersity, surface charge and stability in physiological-mimicked media by Dynamic Light Scattering (DLS). Only medium acetylated chitosan (FA = 0.16) guaranteed the stability of nanoparticles. To explore the effect of NPs interaction with immune cells, the release of pro-inflammatory cytokines and reactive oxygen species (ROS) production by human macrophages and neutrophils, respectively, were evaluated. Strikingly, a structure-function relationship emerged, showing that NPs made of chitosans with FA = 0.02, 0.25, 0.46 and 0.63 manifested a pro-inflammatory activity, linked to the instability of the system. Conversely, NPs made of chitosan with FA = 0.16 neither modified the functional response of macrophages nor that of neutrophils. Of note, such NPs were found to possess additional properties potentially advantageous in applications as means of delivery of therapeutics to target inflamed sites, namely: (i) they are devoid of cytotoxic effects, (ii) they avoid engulfment during the early stage of interaction with macrophages and, (iii) they are muco-adhesive, thereby providing for site-specificity and long-residence effects.