Oxygen-loaded chitosan/perfluoropentane nanobubbles as promising antimicrobial devices to promote wound healing.

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Even more so, CWs are often worsened by microbial (either bacterial or fungal) infections. Intriguingly, nanobubbles (NBs) - displaying a peculiar structure based on fluorocarbons (perfluoropentane) in the inner core and polysaccharydes (dextran or chitosan) in the outer shell - have proven effective in delivering oxygen to hypoxic tissues. Moreover, antimicrobial properties have been largely reported for chitosan. For this reasons, in the present work chitosan/perfluoropentane nanobubbles were challenged for their anti-fungal and anti-bacterial properties against Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA), as well as biocompatibility with human skin cells and ability to promote wound healing processes. C. albicans or MRSA growth was monitored upon incubation with/without oxygen-loaded or oxygen-free NBs (OLNBs or OFNBs) at different times (2, 3, 4, 6, and 24 h). According to cell counts, both OLNBs and OFNBs significantly inhibited the growth of C. albicans up to 24 h; on the contrary, they exerted a bacteriostatic activity against MRSA only at earlier time-points (up to 6 h). Further investigation by confocal microscopy (performed either after 3 or 24 h of incubation) revealed that OLNBs and OFNBs were avidly internalized by C. albicans, but not by S. aureus (which was only associated with NB adhesion to the bacterial wall), possibly explaining the different cytostatic effects of NBs on either microbial species. In parallel, human keratinocytes (HaCaT cell line) were incubated with/without OLNBs or OFNBs for 24 h either in normoxia (20% O2) or hypoxia (1% O2). Analysis by confocal microscopy clearly showed nanobubble internalization by HaCaT cells. Complementary lactate dehydrogenase (LDH), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and adenosine triphosphate (ATP) assays demonstrated that OLNBs were not toxic to human keratinocytes, whereas OFNBs slightly affected their viability, thereby suggesting a protective role for oxygen molecule within NBs. Finally, results from scratch assay displayed a significant hypoxia-dependent inhibition of keratinocyte migratory ability, an effect that was fully reversed by OLNBs, but not OFNBs. Based on these data, chitosan-shelled and perfluoropentane-cored OLNBs appear as innovative, promising, non-toxic, and cost-effective anti-microbial devices able to promote repair processes in infected CWs