2.2. Methods

For the production of the inner oil phase, a mixture of d-α-tocopherol, Miglyol 812 and soybean lecithin was firstly prepared by stirring it for 5 min at 30 °C until a homogeneous solution has been obtained. The oil phase was dispersed in a water surfactant solution composed of Polysorbate 80 (Tween®80) 1.5% (m/V) under high shear homogenization (Ultra-Turrax®, T25, IKA, Germany) for 1 min to obtain a coarse emulsion. This emulsion was then processed in the high pressure homogenizer (EmulsiFlex®-C3, Avestin), in the continuous mode at 1000 bar and at room temperature.

The mean droplet size was determined by photon correlation spectroscopy (PCS) with a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). PCS yields the mean droplet size (z-Ave) and the polydispersity index (PI) as a measure of the width of the size distribution. For the determination of the surface electrical charge, the zeta potential (ZP) was measured in the samples previously diluted with double-distilled water adjusted to a conductivity of 50 μS/cm with a solution of 0.9% (m/V) NaCl. The pH was measured using a microprocessor-based pH and temperature bench meters (Hanna Instruments, Romania) with the glass electrode HI 1330. The pH of the nanoemulsions was in the range of 5.5–6.0 and the field strength was 20 V/cm, and the ZP analyzed by laser Doppler anemometry using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK).

The rheological analysis of the developed nanoemulsions was performed using the rheometer Rheo Stress RS 100 (Haake Instruments Karlsruhe, Germany) equipped with a cone-and-plate test geometry (plate diameter 20 mm, cone angle 4°). A volume of 2.5 mL of each nanoemulsion was tested at a controlled temperature of 22 ± 0.1 °C under shear rate control conditions within the range 1–50 s⁻¹. Rheograms of apparent viscosity were recorded against shear rate, and the data were fitted to Power Law (or Ostwald) model, according to the following equation: τ = K.γ^n-1, where τ stands for the shear stress (Pa), γ for the shear rate (s⁻¹) and K is the consistency index parameter that gives an idea of the viscosity of the fluid. The flow behavior index is represented by n (n < 1, Pseudoplastic behavior; n = 1, Newtonian behavior; n > 1, Dilatant behavior).

To determine the surface tension, a volume of 20 ml of sample was placed in a thermostatically controlled glass at 22 ± 0.1 °C, and the measurements were done in a Krüss Interfacial Tensiometer K10 PST (Krüss, Germany) equipped with a platinum ring. A Semi-Micro Osmometer K-7400 (Knauer, Berlin, Germany) was used to determine the osmolarity by placing a volume of 150 μL of sample in a glass capillary. For both analyses, measurements were carried out in triplicate.

Caco-2 cells, a human colorectal adenocarcinoma cell line, obtained from Cell Line Services, AG (Germany), were used as cell model to perform the cytotoxicity assay. Caco-2 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (V/V) fetal bovine serum (FBS), antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) and 1 mM l-glutamine in a controlled humidity atmosphere of 5% CO₂/95% air, at 37 °C, as previously described (Andreani et al., 2014, Severino et al., 2014). The cytotoxicity of NEs was evaluated applying the Alamar blue (AB) reduction method, for the comparison of the proliferation rate and viability of non-exposed Caco-2 cells (control, 0 μg/mL) with exposed Caco-2 cells, to appropriate NEs’ concentrations during pre-determined time intervals. Briefly, cells were detached from the culture flaks with trypsin, counted and seeded into 96-well microplates at a density of 5 × 10⁴ cells/mL (100 μL/well). NEs, diluted in FBS-free culture media to various concentrations, from 25 to 400 μg/mL (25, 50, 100, 200, 400 μg/mL), were added to the cells, 24 h after seeding and after removal of culture medium. Microplates were placed in the incubator, and cells were exposed for 48 h. After exposure, the media containing the NEs (and the control) was removed and replaced by FBS-free medium supplemented with 10% (V/V) of AB. The absorbance readings occurred about 4 h after AB addition, at 570 and 620 nm using a Multiskan EX microplate reader (MTX Labsystems, USA). The percentage of AB reduction was calculated as previously described by us (Andreani et al., 2014). For the statistical analysis of data, a one-way analysis of variance (ANOVA) test was applied. Results correspond to the mean of three independent experiments (n = 3) ± SD. Cell viability results were normalized to the control (untreated cells) and are expressed as % of control. A p-value < 0.05 was considered statistically significant.