*2.1. Materials*

Hyaluronic acid sodium salt (HA, MW = 1.01–1.8 MDa) was purchased from Lifecore Biomedical Co. (Chaska, MN, USA). Resveratrol (RSV) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Tween® 80 (Polysorbate 80) was purchased from Uniqema (Everberg, Belgium). Compritol® ATO C-888 was purchased from Gattefossé (Nanterre, France) and Miglyol 812® from Sasol Chemical Industries Ltd. (Homburg, Germany). Poloxamer® 188 (Pluronic F68, Kolliphor® P188) was obtained

from BASF (Ludwigshafen, Germany). All other reagents and solvents were analytical grade and used exactly as received. Ultra-purified water was obtained from Milli-Q ® Plus system, home supplied.

#### *2.2. Preparation of RSV-Loaded NLC*

RSV-loaded NLC were prepared through the high shear homogenization method associated with sonication [19,20]. The lipid phase was composed of 255 mg Compritol ® 888 ATO (C-888), 45 mg Miglyol 812 ®, and 10 mg RSV, and the aqueous phase was composed of 150 mg of Poloxamer ®, 188 (P188), and 75 mg of Tween ® 80 (Tw 80) in 12.5 mL bidistilled water were heated at 85 ◦C, separately. The aqueous phase was poured into the lipid phase and stirred with Ultra Turrax homogenizer Ystral GmbH X10/25 (Dottingen, Germany), followed by sonication for 15 min at 70% intensity (14 watts) using a Sonics and Materials Vibra-Cell ™ CV18 (Newtown, CT, USA), according to Table 1. The particles were dispersed in 12.5 mL of bidistilled water and kept at −20 ◦C for 10 min. Blank (i.e. non-loaded NLC) was prepared in a similar way, without the RSV. The formulations were stored at 4 ◦C.

**Table 1.** Preparation of resveratrol-loaded nanostructured lipid carriers (NLC). Factorial design, providing the lower (−1), upper (+1), and (0) central point level values for each variable.


#### *2.3. 22 Factorial Design*

The influence of the shear intensity and homogenization time on the NLC properties was evaluated using a 22 factorial design with triplicate runs of the central point to estimate the experimental error, composed of 2 variables, which were set at 2-levels each (Table 2). The mean particle size and polydispersity index (PDI) were the dependent variables. The design required a total of 7 experiments. Each factor, the lower and higher values of the lower and upper levels, was represented by a (−1) and a (+1), and the central point was represented by (0), as summarized in Table 1. The data were analyzed using STATISTICA 7.0.

**Table 2.** A 22 full factorial experimental design layout. The formulation codes of NLC with resveratrol are named as NLC-RSV(number of the experiment) and the one without resveratrol as NLC(number of the experiment).


#### *2.4. Characterization of RSV-Loaded NLC*

#### 2.4.1. Particle Size and Polydispersity Index

The particle size (PS) and polydispersity index (PDI) of NLC were measured at 25 ◦C using photon correlation spectroscopy (PCS) (dynamic light scattering, DLS, Zetasizer Nano NS, Malvern Instruments, Malvern, Worcs, UK). The measurements were carried out using a He–Ne laser at 633 nm and 4.0 mW power, with a back-scattering detection angle of 173◦ after dilution of formulations with ultra-purified

water. The average hydrodynamic diameter was recorded based on the observed diameters weighted by the number size distribution. The polydispersity index (PDI) was also calculated from cumulative analysis of the measured DLS intensity autocorrelation function (a dimensionless number that ranges from 0 to 1). PS and PDI of NLC were determined in triplicate. For each measurement, the NLC was diluted in Milli-Q ® water to an appropriate concentration to avoid multiple scattering.
