Introduction. Hypoxia can hamper efficient wound healing by altering the release of matrix turn-over-associated molecules (matrix metalloproteinases, MMPs; tissue inhibitors of metalloproteinases, TIMPs) from surrounding cells, including immune cells. Provided cellular phenotypes of such environments are better understood, impaired chronic wounds could be targeted by new effective oxygenating drugs, including O2-Loaded Nanobubbles (OLNs). Here, hypoxia effects on human monocyte MMP/TIMP secretion, along with potential therapeutic role of dextran-shelled decafluoropentane-containing OLNs, were investigated. Methods. Human monocytes isolated from peripheral blood 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; cell supernatant MMP-2, MMP-9, TIMP-1, and TIMP-2 protein levels were measured by enzyme-linked immunosorbent assay and gelatin zimography. Results. OLNs, OFNs and OSS did not display significant cytotoxicity, and did not affect cell viability. In normoxia, monocytes released basal levels of MMP-9, TIMP-1 and TIMP-2, but not MMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-9, enhancing TIMP-1, and not affecting TIMP-2 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 show that non-toxic dextran-shelled/decafluoropentane-containing OLNs counteract hypoxia effects on MMP/TIMP secretion from immune cells, supporting that the administration of exogenous O2, properly encapsulated in nanobubble formulations, might promote healing of chronic wounds. Acknowledgements. Work supported by funding from Ateneo-Compagnia di San Paolo (ORTO11CE8R 2011).
Nanotechnology and wound healing: dextran-shelled/decafluoropentane-containing oxygen-loaded nanobubbles counteract hypoxia-dysregulated MMP/TIMP balances in human monocytes.
GULINO, GIULIA ROSSANA;KHADJAVI, AMINA;GIRIBALDI, Giuliana;GUIOT, Caterina;PRATO, Mauro
2013-01-01
Abstract
Introduction. Hypoxia can hamper efficient wound healing by altering the release of matrix turn-over-associated molecules (matrix metalloproteinases, MMPs; tissue inhibitors of metalloproteinases, TIMPs) from surrounding cells, including immune cells. Provided cellular phenotypes of such environments are better understood, impaired chronic wounds could be targeted by new effective oxygenating drugs, including O2-Loaded Nanobubbles (OLNs). Here, hypoxia effects on human monocyte MMP/TIMP secretion, along with potential therapeutic role of dextran-shelled decafluoropentane-containing OLNs, were investigated. Methods. Human monocytes isolated from peripheral blood 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; cell supernatant MMP-2, MMP-9, TIMP-1, and TIMP-2 protein levels were measured by enzyme-linked immunosorbent assay and gelatin zimography. Results. OLNs, OFNs and OSS did not display significant cytotoxicity, and did not affect cell viability. In normoxia, monocytes released basal levels of MMP-9, TIMP-1 and TIMP-2, but not MMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-9, enhancing TIMP-1, and not affecting TIMP-2 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 show that non-toxic dextran-shelled/decafluoropentane-containing OLNs counteract hypoxia effects on MMP/TIMP secretion from immune cells, supporting that the administration of exogenous O2, properly encapsulated in nanobubble formulations, might promote healing of chronic wounds. Acknowledgements. Work supported by funding from Ateneo-Compagnia di San Paolo (ORTO11CE8R 2011).
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