Table 1.
Composition of OLND, OFND, OLNB, OFNB and OSS formulations.
Fig 1.
Schematic structure of nanodroplet and nanobubble formulations.
The oxygen nanocarriers described in the present work display a core-shell structure. As core fluorocarbon, DFP was employed for OLNDs, whereas PFP was used for OLNBs. Dextran was chosen as polysaccharidic shell molecule for both nanocarriers. In selected experiments, OLNDs were functionalized by conjugation with FITC. All nanocarrier solutions were prepared either in liquid (water or PBS) or gel (2% HEC) formulations.
Table 2.
Physical-chemical characterization of OLNDs, OFNDs, OLNBs, OFNBs and OSS.
Fig 2.
OLND and OLNB morphology, size distribution, and shell shear modulus.
OLNDs and OLNBs were checked for morphology by optical microscopy or by TEM, for size distribution by light scattering, and for shell shear modulus by rheometry. Results are shown as representative images from ten different preparations. Panel A. OLND image by optical microscopy. Magnification: 60X. Panel B. OLND image by TEM. Magnification: 21000X. Panel C. OLND size distribution. Panel D. OLND flow curve. Panel E. OLNB image by optical microscopy. Magnification: 60X. Panel F. OLNB image by TEM. Magnification: 21000X. Panel G. OLNB size distribution. Panel H. OLNB flow curve.
Fig 3.
OLND internalization by HaCaT cell line.
Human keratinocytes (106 cells /2 ml Panserin medium) were left untreated (upper panels) or treated with 200 l FITC-conjugated OLND PBS formulation (lower panels) for 24 h in normoxia (20% O2). After DAPI staining, cells were checked by confocal microscopy. Results are shown as representative images from three independent experiments. Left panels: cell nuclei after DAPI staining, in blue. Central panels: FITC-conjugated OLNDs, in green). Right panels: merged images. Magnification: 63X.
Table 3.
Refractive indexes of OLND and control formulations.
Fig 4.
OLND are not cytotoxic and improve viability of human keratinocytes in vitro.
Human keratinocytes (106 cells/2 ml Panserin 601 medium) were left untreated or treated with different doses (100–400 l) of OLND PBS formulation for 24 h in normoxia (20% O2; white-squared curves, both panels) or hypoxia (1% O2; black-squared curves, both panels). Thereafter, OLND cytotoxicity (Panel A) was measured through LDH assay, whereas cell viability (Panel B) was measured through MTT assay. Results are shown as means ± SEM from three independent experiments. Data were also evaluated for significance by ANOVA. Panel A. Versus normoxic untreated cells: p not significant. Panel B. Versus normoxic untreated cells: * p < 0.01; ** p < 0.001; *** p < 0.0001.
Fig 5.
In vitro oxygen release from OLND liquid and gel formulation and US-triggered sonophoresis through skin membranes.
Panels A-B. Oxygen release without US. OLND, OLNB and OSS water (A) and 2% HEC gel (B) formulations were monitored up to 6 h through an oxymeter for oxygen delivery by diffusion. Results are shown as a representative image from three independent experiments. Panels C-D. Oxygen release with US. US abilities to induce sonophoresis and oxygen release from OLND and control water (C) or 2% HEC gel (D) formulations were evaluated up to 135 min. Changes in oxygen levels in the hypoxic chamber between each time interval (0–45 min; 45–90 min; and 90–135 min) are indicated as ΔO2. Results are shown as means ± SD from three independent experiments. Data were also evaluated for significance by ANOVA. Versus OLND formulation: p < 0.001.
Fig 6.
Topical treatment with OLND gel formulation effectively enhances oxy-Hb levels in vivo.
The shaved hind limbs of nine anesthetized mice were monitored by photoacoustics for oxy-Hb and deoxy-Hb levels before (0 min, upper row), during (0–10 min, central row) and after (10 min, lower row) topical treatment with OSS (first column), OLND (second column) and OFND (third column) gel formulations. White/red pixels: oxy-Hb; blue pixels: deoxy-Hb. Data are shown as representative images from three independent experiments (three mice per experiment) with similar results.
Fig 7.
Topical treatment with US-activated OLNDs effectively enhances tcpO2 in vivo.
The shaved abdomens of eight anesthetized mice were topically treated with OLND gel formulation and sonicated (f = 1 MHz; P = 5 W; t = 30 sec). Before and after treatment, tcpO2 was monitored through transcutaneous oxymetry. Panel A. Short-term time-course (0–15 min) tcpO2 monitoring of three mice before and after treatment with OLND gel formulation. Data are shown as means ± SD. Results were also analyzed for statistical significance by Student’s t test. Versus untreated mice: p < 0.01. B. Long-term end-point (1 h) tcpO2 measurement of five mice (m1-m5) before and after treatment with OLND gel formulation. Data are shown individually per each mouse.