Acetone solution

Poly(ε-caprolactone) (PCL) nanocapsules loaded with oregano and thyme EOs were prepared according to the procedure described by Granata et al. [28 (link)] using the interfacial deposition of the preformed polymer method. Briefly, an acetone solution (80 mL) (Sigma-Aldrich/Merck, Darmstadt, Germany) containing EO (1.0 g), PCL (320 mg), and sorbitan monostearate (112 mg) (Sigma-Aldrich/Merck) was dropped under stirring at 25 °C into a polysorbate 80 aqueous solution (275 mL in 160 mL) (Sigma-Aldrich/Merck). The removal of the organic solvent provided an EO-NC suspension. The NCs without essential oils were prepared in the same conditions. The physicochemical parameters of the nanocapsules such as particle size, polydispersity, and zeta potential, were determined by light scattering measurements. The encapsulation efficiency and loading capacity values were obtained by UV–Vis spectroscopy. The exact composition of the commercial thyme and oregano EOs (provided by Flora s.r.l., Lorenzana, Italy, and Esperia S.p.A, Milan, Italy) were determined by GC-FID and GC-MS analysis. The values allowing the characterization of the nanocapsules and EOs were already reported [10 (link)] and are briefly shown in Table 5 and Table 6.

Tomato roots were fixed in 2.5% glutaraldehyde solution (EMS) in phosphate buffer (PB 0.1 M; pH 7.4) for 1 h at room temperature and subsequently fixed in a fresh mixture of osmium tetroxide (1%, EMS) with 1.5% potassium ferrocyanide (Sigma) in PB buffer for 1 h. The samples were then washed twice in distilled water and dehydrated in acetone solution (Sigma) in a concentration gradient (30% for 40 min, 50% for 40 min, 70% for 40 min and 100% for 1 h three times). This was followed by infiltration in LR White resin (EMS) in a concentration gradient (33% LR White in acetone for 6 h, 66% LR White in acetone for 6 h and 100% LR White for 12 h two times) and finally polymerized for 48 h at 60 °C in an oven in atmospheric nitrogen. Ultrathin sections (50 nm) were cut transversely at 2, 5 and 8 mm from the root tip, the middle of the root and 1 mm below the hypocotyl–root junction using a Leica Ultracut UC7 (Leica Mikrosysteme), picked up on a copper slot grid 2 × 1 mm (EMS) and coated with a polystyrene film (Sigma). Micrographs and panoramic images were taken with an FEI transmission electron microscope (FEI CM100) at an acceleration voltage of 80 kV with a TVIPS TemCamF416 digital camera (TVIPS) using the software EM-MENU (v.4.0) (TVIPS). Panoramic images were aligned with the software IMOD (v.4.11)^{73 (link)}.

Electron microscopy was performed at the Electron Microscopy Facility of the School of Biology and Medicine. Zebrafish skeletal muscle were dissected immediately after euthanasia and pre-fixed in PB 0.1M/2.5% glutaraldehyde/PFA4% solution at room temperature for 30min. 1mm cross-sectional sections were cut with a razor blade and fixation was prolonged until 2h. The samples were washed in PB three times (5min distilled water) and dehydrated in acetone solution (Sigma). Careful positioning was performed with binoculars on Aclar film (EMS, Hatfield, PA, United States) inside a Gene Frame (ThermoFischer Scientific) in 65μl of Epon resin and finally polymerized for 48h at 60°C in oven. Sagittal ultrathin sections of 50nm were cut on a Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) and picked up on a copper slot grid 2x1mm (EMS) coated with a polystyrene film (Sigma). Sections were post stained with 4% uranyl acetate (Sigma). Micrographs were taken with a transmission electron microscope Philips CM100 (ThermoFisher Scientific) with a TVIPS TemCam-F416 digital camera (TVIPS GmbH, Gauting, Germany).