Fluorescent NPs were formed by means of electrochemical anodization of a low-resistivity grade (< 1 Ω) bulk SiC polycrystalline wafer (NPs) [6
]. The etching process took place for 2.5 h at a current density of 25 mA/cm2
using a 1:1 HF (50%)/ethanol electrolyte. After the etching, a highly porous network constituted by interconnected SiC nanocrystals was formed. The ultraporous layer was washed several times with deionized water then naturally dried in ambient air, removed from the wafer and ground to powder. To obtain colloidal suspensions of NPs, the powder was dispersed in 1:1 deionized water/ethanol mixture or Dulbecco's Modified Eagle's Medium (DMEM) buffer for mammalian cell investigations, and then large NP crystallites were removed by centrifugation at 10,000 g for 5 min. These suspensions contained only small-sized (< 3 nm) and uniformly dispersed NPs. Typical TEM images, dynamic light scattering size distributions and zeta-potential measurement of which had already been shown in the study by Botsoa et al. [6
The inner layer of a fresh thin onion epidermis was peeled off. The onion peels were neatly spread in the center of a glass slide in a drop of water. The glass slide was then mounted on a mobile holder situated above a colloidal suspension (2 mL, surface area 4 cm2) containing the NP suspension (1 g/L). Height origin was fixed at the suspension surface. At a fixed height value, the onion epidermis samples were exposed to the suspension vapors for different periods of time varying from 3 to 17 h. Depending on exposure time, evaporated volume was changing from 0.5 to 1.5 mL. All experiments were carried out at a temperature of 21°C, humidity of 60% and vapor pressure of 1,018 hPa, without air mixing. Speed of convection flow was equal to 0 m/s. The manipulations were performed under two different conditions: (a) in open air and (b) in confined air with the whole system being covered by a cap. After a certain exposure time, the onion peels were intensively rinsed in deionized water to remove the NPs deposited onto the epidermis surface and unincorporated inside the epidermal cells. Control manipulations were carried out with the use of a pure suspension without the NPs.
Suspension containing NPs was also added to the 3 T3-L1 fibroblasts (American Type Culture Collection, Manassas, VA, USA). The cells were grown on glass substrates in DMEM supplemented with 10% newborn calf serum, 4 mM glutamine, 4 nM insulin (Actrapid Human Torrent Pharmaceuticals Ltd., Ahmedabad, India; Novo Nordisk A/S, Bagsvaerd, Denmark), 10 mM HEPES, 25 μg sodium ascorbate, 100 IU penicillin, 100 μg streptomycin and 0.25 mg/L amphotericin B at 37°C in a water-saturated atmosphere with 5% CO2 in a Heraeus incubator (BB16; Thermo Scientific, Brebières, France). The cells have been incubated for 15 h in the presence of NPs (with concentration of 0.8 g/L in suspension in DMEM). Some 3 T3-L1 samples were cultured in DMEM without NPs but, in air contact, were allowed with wells containing NP suspensions. After a certain incubation time, cells were rinsed twice in pure ethanol to eliminate NPs from the environment and to fix cells.
Finally, the samples were observed under a fluorescence microscope (Leica DMI 4000B, Leica Microsystèmes SAS, NANTERRE, France) using the following filter combination: UV/violet excitation band at 2.92 to 3.5 eV with an observation spectral range of < 2.64 eV. Leica EC 3 camera (Leica Camera Sarl, Saint Jorioz, France) was used.
For each image, luminosity per cell was calculated by summing intensities (in a range from 0 to 255) of each pixel in the picture and dividing this result by the number of fluorescent cells in the picture. The MathWorks Matlab R2009b and Simulink software (Torrance, CA, USA) were used for image analyses in order to quantify luminosity per one cell.