Chitosan oxygen-loaded nanobubbles counteract hypoxia dysregulation of MMP/TIMP balances in human keratinocytes: new perspectives for chronic wound healing.

In several pathological conditions, including diabetic foot and bedsores, efficient wound healing is hampered by hypoxia, altering the release from surrounding cells of molecules involved in matrix turn-over (matrix metalloproteinases, MMPs; tissue inhibitors of metalloproteinases, TIMPs). Interestingly, impaired chronic wounds might be effectively targeted by new oxygenating drugs such as O2-Loaded Nanobubbles (OLNs). In the present work, the effects of hypoxia on MMP/TIMP secretion from human keratinocytes (HaCaT cell line) were investigated, and the therapeutic potential of chitosan-shelled decafluoropentane-containing OLNs was evaluated. Normoxic keratinocytes released basal MMP-2, MMP-9, TIMP-1 and TIMP-2 protein levels. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9 and TIMP-2 without affecting TIMP-1 release. Chitosan OLNs, characterized by spherical morphology, diameters of 700 nm and cationic surfaces, were avidly internalized by keratinocytes, not displaying cell cytotoxicity and not affecting cell viability. After internalization, OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring normoxic balances. Neither O2-saturated solution (OSS) nor O2-free nanobubbles (OFNs) did mimic OLN abilities, suggesting that they were specifically dependent on time-protracted O2 diffusion from OLN core. Collectively, these data show that chitosan OLNs can effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Thus topical administration of exogenous O2, properly encapsulated in nanobubble formulations, could be a promising approach to promote healing processes in chronic wounds.