*2.4. Preparation of Nanoemulsions*

The nanoemulsion region was identified from the phase diagrams depicted in Figures 3–5. Excess EFV was placed into a test tube containing the nanoemulsion preconcentrate that was one of five different surfactant mixtures and 10% *m*/*m* flaxseed oil. Of the five surfactant mixtures evaluated three were identified from each phase diagram and two were numeric optimization solutions extracted using the D-optimal design. The nanoemulsion mixtures were stirred at 100 rpm at laboratory room temperature under air-conditioner at 22 ± 2 ◦C with the aid of an FMH-STR magnetic stirrer (Lasec®, Cape Town, South Africa) for 48 h. The saturated nanoemulsions were centrifuged at 3000 rpm for 15 min using a model HN-SII IEC centrifuge (Thermo Scientific, Waltham, MA, USA) to separate excess

drug from the nanoemulsion. Aliquots (500 µL) of the supernatant were dispersed in 50 mL water for droplet size and ZP measurements. A further 500-µL aliquot of the supernatant was mixed with 50 mL of a 3:2 ratio ethanol: water solution, prior to quantitation of EFV. *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 7 of 22

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 7 of 22

**Figure 3.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S1 (0.5:1:1 *m/m* Ethanol: Tween® 80: Span® 20). **Figure 3.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S1 (0.5:1:1 *m*/*m* Ethanol: Tween® 80: Span® 20). **Figure 3.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S1 (0.5:1:1 *m/m* Ethanol: Tween® 80: Span® 20).

**Figure 4.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S2 (1:1:1 *m/m* Ethanol: Tween® 80: Span® 20). **Figure 4.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S2 (1:1:1 *m/m* Ethanol: Tween® 80: Span® 20). **Figure 4.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S2 (1:1:1 *m*/*m* Ethanol: Tween® 80: Span® 20).

Ethanol: Tween® 80: Span® 20).

Ethanol: Tween® 80: Span® 20).

**Figure 5.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S3 (1.5:1:1 *m/m*

**Figure 5.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S3 (1.5:1:1 *m/m*

Tween® 80: Span® 20).

Ethanol: Tween® 80: Span® 20).

**Figure 3.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S1 (0.5:1:1 *m/m*

**Figure 5.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S3 (1.5:1:1 *m/m* Ethanol: Tween® 80: Span® 20). **Figure 5.** Pseudo-ternary phase diagram of flaxseed oil with surfactant-mixture S3 (1.5:1:1 *m*/*m* Ethanol: Tween® 80: Span® 20).
