*3.3. Determination of Particle Size, Polydispersity Index (PDI), and Zeta Potential (ZP)*

The NLC mean particle size (Z-Ave) and PDI were measured by dynamic light scattering (DLS) technique using a Malvern nanozetasizer (Malvern, UK). A refractive index of 1.46 and an absorption index of 0.001 were used for the lipids, while a refractive index of 1.330 was used for the solvent (water). In addition, the NLC electrical surface charge was assessed by laser doppler electrophoresis by means of ZP measurements using the same apparatus. The dispersions were diluted with ultrapure water and the ZP was calculated using the Helmholtz–Smoluchowski equation and run on the system software. The temperature was set at 25 ± 1 ◦C. Each sample was analyzed in five replicates (*n* = 5) and the results were reported as the mean ± standard deviation (SD).

To confirm the absence of microparticles, particle size was measured by laser diffraction using a Malvern Mastersizer 3000E (Malvern, UK). The used particle refractive index was 1.4, the absorption index was 0.001, and the water dispersant refractive was 1.33, with Mi's theory being applied. The particle size was assessed by the values of the volume distribution (D50 and D90), indicating the percentage of particles with a diameter size equal or lower to the given values. The results were reported as the mean ± SD of five replicates (*n* = 5).
