Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes

Abstract

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans-infected CWs.

Keywords: Candida albicans; Chronic wounds (CWs); Keratinocytes; Methicillin-resistant Staphylococcus aureus (MRSA); Nanobubbles (NBs).

Graphical abstract

Fig. 1

NB morphology and structure. OLNBs were checked for morphology by optical microscopy and TEM. Results are shown as representative images from ten different preparations. Panel A. OLNB image by optical microscopy. Magnification: 630 × . Panel B. OLNB image by TEM. Magnification: 52000 × .

Fig. 2

Effects of hypoxia and NBs on human keratinocyte viability, health, and metabolism. HaCaT cells (1.6 × 10⁵ cells/mL for MTT studies, 5 × 10⁵ cells/mL for LDH studies, and 5 × 10⁵ cells/mL for ATP studies) were left untreated or treated with OLNBs and OFNBs for 24 h in normoxia (20 % O₂, black columns) or hypoxia (1% O₂, white columns). After collection of cell supernatants and lysates, cell viability percentage was measured through MTT assay (panel A), cytotoxicity percentage through LDH assay (panel B), and ATP production through ATP assay (panel C). Results are shown as means + SEM from three independent experiments. Data were also evaluated for significance by ANOVA: * vs normoxic control cells: p < 0.05; ° vs hypoxic control cells: p < 0.05.

Fig. 3

OLNB internalization by human keratinocytes. HaCaT cells (6 × 10⁴ cells/mL) were left untreated or treated with FITC-labeled OLNBs for 24 h in normoxia (20 % O₂). After PI staining, cells were checked by confocal microscopy. Results are shown as representative images from three independent experiments. Panels a-c (red, green, merged): control cells; panels d-f (red, green, merged): OLNB-treated cells. Red: cell nuclei after PI staining. Green: FITC-labeled OLNBs. Magnification: 60×.

Fig. 4

Effects of hypoxia and OLNBs on the migration and wound healing abilities of human keratinocytes. HaCaT cells (3 × 10⁵ cells/mL) were seeded in two confluent monolayers, divided by a scratch of 500 μm, and incubated for 16 h in normoxia (20 % O₂) or hypoxia (1% O₂) with/without 10 % v/v OLNBs as well as OFNBs or OSS. Thereafter, scratch lengths were photographed and measured. Panel A: representative images. Panel B: means + SEM of scratch lengths. Results are from three independent experiments performed in triplicates. Data were also evaluated for significance by ANOVA: * vs normoxic untreated cells: p < 0.01; ° vs hypoxic untreated cells: p < 0.01.

Fig. 5

NB adhesion to MRSA bacterial wall. MRSA (10⁹ CFUs/mL) were left alone or incubated with 10 % v/v FITC-labeled OLNBs/OFNBs for 24 h. After staining bacteria with PI, confocal fluorescent images were taken using FITC and TRITC filters. Data are shown as representative images from three independent experiments. Red: PI. Green: FITC. Magnification: 100 × .

Fig. 6

Antibacterial activity of chitosan and chitosan-shelled NBs on MRSA. MRSA (10⁴ CFUs/mL) were incubated alone or with 10 % v/v OSS, free MW chitosan solution (0.139 % m/v), OFNB suspension, or OLNB suspension in sterile conditions at 37 °C and their growth was monitored for 2, 3, 4, 6 and 24 h. At each incubation time, the samples were spread on TSA agar medium to determine the CFUs/mL. Results are shown as means ± SEM from three independent experiments and expressed as Log CFUs/mL. Data were evaluated for significance by ANOVA: vs controls: 3-4 h, ** p < 0.01; 6 h, *** p = 0.0001.

Fig. 7

NB internalization by C. albicans.C. albicans (10⁸ CFUs/mL) were left alone or incubated with 10 % v/v FITC-labeled OLNBs or OFNBs for 3 h. After staining yeasts with PI, confocal fluorescent images were taken using FITC and TRITC filters. Data are shown as representative images from three independent experiments. Red: PI. Green: FITC. Magnification: 100 × .

Fig. 8

Antifungal activity of free chitosan and chitosan-shelled NBs on C. albicans.C. albicans (10⁵ CFUs/mL) were incubated alone or with 10 % v/v OSS, free MW chitosan (0.139 % m/v) solution, OFNB suspension, or OLNB suspension in sterile conditions at 37 °C and their growth was monitored for 2, 3, 4, 6 and 24 h. At each incubation time, the samples were spread on SAB agar medium to determine the CFUs/mL. Results are shown as means ± SEM from three independent experiments and expressed as Log CFUs/mL. Data were evaluated for significance by ANOVA: vs controls: 2 h, * p < 0.05; 3-6 h, *** p = 0.0001; 4 h, **** p < 0.0001; 24 h, ** p < 0.01.