SLN production method

SLN were produced according to the method previously described.6,7 The waxes used were Witepsol H15 (Sasol, Hamburg, Germany) and Carnauba wax yellow number 1 (Sigma-Aldrich, St Louis, MO, USA). Surfactant polysorbate (Tween 80) and RA were purchased from Sigma-Aldrich. Briefly, SLN were prepared by hot-melt ultrasonication, loading the RA and herbal aqueous extracts at the final concentration of 0.15 mg RA/mL. The lipid at 0.5% (w/v) was warmed to a temperature of 5°C above the melting point (36°C for Witepsol and 86°C for Carnauba), then the melted matrix was added with RA or herbal extracts and subjected to ultrasonication (VCX 130, Sonics & Materials, Newtown, CT, USA) for 1 minute at 70% of intensity. Finally, the aqueous surfactant solution of polysorbate 80 was added to obtain a final concentration of 2% (v/v) and mixed for a few seconds, for completely homogenization of the O/W emulsion. The resulting fluid solutions were left to cool at room temperature (20°C). The SLN emulsions were stored at 5°C for 28 days until further use. When dried SLN were used, the emulsions were freeze-dried upon production using a vacuum freeze drier (Model FT33), under a vacuum pressure of 100 mTorr; the temperature in the freezing chamber was −46°C, and the temperature in the sample chamber was 15°C.