New perspectives to counteract hypoxia in chronic wounds: a comparative evaluation of physico-chemistry, biocompatibility, and efficacy of oxygen-loaded nanodroplets shelled with different chitosan species and derivatives.

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs) - filled with oxygen-binding 2H,3H-decafluoropentane and shelled with changeable polysaccharides - were proposed as promising tools to counteract hypoxia by releasing clinically relevant oxygen doses in a time-sustained manner. Here, different chitosan species/derivatives [medium or low weight (MW or LW), glycol- (G), and methylglycol- (MG) chitosan] were compared as candidate biomaterials for shell manufacturing to optimise OLND physico-chemical characteristics, biocompatibility, and efficacy. All OLND formulations displayed spherical morphology, cationic surfaces, ≤ 500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability (with G chitosan-shelled OLNDs being the less stable), good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. MW, LW, and G chitosan were generally not toxic to human keratinocytes (HaCaT cell line), whereas MG chitosan reduced cell viability by 50%. Based on all these results, only LW and MW chitosan-shelled OLND formulations were comparatively analysed in the subsequent biological experiments. Both LW and MW chitosan-shelled OLNDs effectively improved migration of hypoxic keratinocytes. However, LW chitosan-shelled OLNDs appeared significantly less toxic to HaCaT cells than MW chitosan-shelled OLNDs. Therefore, LW chitosan emerges as the best candidate biomaterial to be employed for future OLND manufacturing.