Nanotechnology and wound healing: chitosan-shelled/decafluoropentane-containing oxygen-loaded nanobubbles counteract hypoxia-dysregulated MMP/TIMP balances in human keratinocytes.

Introduction. In some pathological conditions (diabetic foot, bedsores), efficient wound healing is hampered by hypoxia, altering the release of molecules associated with matrix turn-over (matrix metalloproteinases, MMPs; tissue inhibitors of metalloproteinases, TIMPs) from surrounding cells, including keratinocytes. Provided that cellular phenotypes in such environments are better understood, impaired chronic wounds could be targeted by new effective oxygenating drugs, such as O2-Loaded Nanobubbles (OLNs). Here, hypoxia effects on human keratinocyte MMP/TIMP secretion were investigated, and the potential therapeutic role of chitosan-shelled decafluoropentane-containing OLNs was evaluated. Methods. HaCaT cells were incubated in serum-free medium for 24 h both in normoxia and hypoxia, in the absence/presence of OLNs, O2-free nanobubbles (OFNs) or O2-saturated solution (OSS). Thereafter, citotoxicity was evaluated by measuring lactate dehydrogenase activity; cell viability was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; MMP-2, MMP-9, TIMP-1, and TIMP-2 protein levels in cell supernatants were measured by enzyme-linked immunosorbent assay and gelatin zimography. Results. OLNs and OSS did not display significant cytotoxicity on keratinocytes, and did not affect cell viability, whereas OFNs slightly did. In normoxia, cells released basal MMP-2, MMP-9, TIMP-1 and TIMP-2 levels. Hypoxia strongly impaired MMP/TIMP balances, by reducing MMP-2, MMP-9, and TIMP-2 without affecting TIMP-1 release. OLNs abrogated all hypoxia effects, restoring normoxic MMP/TIMP levels. Neither OSS nor OFNs did mimic OLN effects, which appeared specifically dependent on time-sustained O2 diffusion from OLN core. Conclusion. Collectively, these data support the hypothesis that topical administration of exogenous O2, properly encapsulated in nanobubble formulations, might promote healing of hypoxia-associated chronic wounds. Acknowledgements. Work supported by funding from Ateneo-Compagnia di San Paolo (ORTO11CE8R 2011).