Comparative evaluation of different chitosan species and derivatives as candidate biomaterials for oxygen-loaded nanodroplet formulations to treat chronic wounds.

INTRODUCTION Persistent hypoxia is a main clinical feature of chronic wounds. Recently, our group developed and patented a new platform of oxygen nanocarriers (oxygen-loaded nanodroplets, OLNDs) to release clinically relevant oxygen doses in a time-sustained manner1,2 and counteract the effects of hypoxia on several wound-related cell types (endothelium, keratinocytes, monocytes).3,4,5 ONLDs display a core-shell structure, with a specific oxygen-binding fluorocarbon (2H,3H-decafluoropentane) in the inner core and changeable polysaccharides (i.e. dextran or chitosan) in the outer shell. Here, different chitosan species/derivatives [medium or low weight (MW or LW), glycol- (G), and methylglycol- (MG) chitosan] were compared as candidate biomaterials for OLND manufacturing in order to optimise their physico-chemical characteristics, biocompatibility, and efficacy. EXPERIMENTAL STUDY OLND or control formulations (oxygen-free nanodroplets, OFNDs; oxygen-saturated solution, OSS) were prepared by following a purposely tuned multi-step protocol as previously described.1,2 Formulations were characterised for morphology by optical microscopy and for physico-chemical parameters by light scattering. In vitro oxygen release was monitored over time by using an oxymeter. Biocompatibility with human keratinocytes, endothelium, monocytes, and fibroblasts (HaCaT, HMEC-1, THP-1, and HDF cell lines, respectively) was assessed by LDH and MTT assays. In vitro cell abilities to promote wound healing were checked through scratch assay. All biological analyses were performed upon cell incubation with formulations (5, 10 or 20% v/v) for 24 h, either in normoxia (20% O2) or hypoxia (1% O2). RESULTS AND DISCUSSION Spherical OLNDs shelled with different chitosan molecules displayed cationic surfaces and ≤ 500 nm average diameters, with LW chitosan-shelled OLNDs being the smallest ones (see Table 1), as well as high stability and good oxygen encapsulation efficiency. All OLND formulations showed prolonged oxygen release kinetics, opposite to OFNDs (no release) or OSS (instant release). However, according to results from preliminary investigation on biocompatibility for each biomaterial, MG chitosan strongly reduced HaCaT cell viability by 50%, whereas MW, LW, and G chitosan were not toxic. Based on these results, only LW and MW chitosan-shelled OLND/OFND formulations were comparatively analysed in the subsequent biological experiments. LW chitosan-shelled OFNDs/OLNDs appeared significantly less toxic than MW chitosan-shelled OFNDs/OLNDs and OSS on HaCaT, HMEC-1, THP-1, and HDF cells. Nevertheless, both LW and MW chitosan-shelled OLNDs effectively improved wound healing abilities of hypoxic keratinocytes, opposite to OFNDs and OSS. CONCLUSION Among the four chitosan species/derivatives tested here, LW chitosan emerges as the biomaterial to be preferred for future OLND manufacturing and testing.