3.3. Production of Lipid Nanoparticles

Lipid nanoparticle dispersions were produced by applying a modified emulsification-sonication method based on a multiple emulsion (W/O/W) [19]. The ingredients of the lipid phase were introduced to a glass beaker: (i) Softisan^® 100–4.0; 4.5; 5.0 wt%; (ii) glycerol—37.5 wt%; (iii) CTAB—0.5 wt%, and heated to 50 °C. Concurrently, a small amount of Milli-Q^® Plus water was added to another beaker and also heated to 50 °C. Subsequently, the aqueous phase thus prepared was combined with the lipid phase, upon vigorous stirring the emerging emulsion using a high-speed Ultra-Turrax^® T25 Digital Homogenizer (Ystral GmbH D-7801, Ballrechten-Dottingen, Germany) at a speed of 24,000 rpm for approximately 5 min. Next, the obtained W/O emulsion was homogenized using a VC130 Ultrasonic Processor (Sonics, Newtown, CT, USA) for 30 s at 40% amplitude. Concurrently, an aqueous solution of the surfactant Tween^® 80 (0.5; 1.0; 1.5 wt%) was prepared by dispersing it in Milli-Q^® Plus water and heating in order to obtain a homogeneous dispersion. In the next step, a small amount of the cooled aqueous surfactant solution was added to the previously prepared W/O emulsion. The resulting mixture was sheared (homogenized) using an ultrasonic homogenizer for 90 s at an amplitude of 40%. After that, the remainder of the aqueous surfactant solution was added to the obtained dispersion and stirred with a magnetic stirrer at 300–350 rpm until the cooling-down of the system (approx. 15 min). The procedure of incorporating aucubin and catalpol (each of the glycosides separately) into the lipid nanoparticles consisted in adding appropriate amounts of the active compounds (0.1 wt% of aucubin and 0.09 wt% of catalpol) to the aqueous phase of the inner W/O emulsion.