2.4. Particle Size, Polydispersity Index, Zeta Potential Analysis, and NLC Particle Concentration

Hydrodynamic diameter and polydispersity index (PdI) were determined by dynamic light scattering (DLS). The zeta potential was measured by Doppler laser microelectrophoresis. Each sample was measured three times, each measurement corresponds to the average of 11 determinations in aqueous medium (viscosity = 0.08872 cp, medium refractive index = 1.330, and sample refractive index = 1.333) with a wavelength of 633 nm with a detection angle of 173° and equilibration time of 120 s. in a Malvern Zetasizer Nano ZS equipment (Malvern Panalytical Ltd., Malvern, UK). To compare the obtained size and to determine the NCL particle concentration, Nanoparticle Tracking Analysis (NTA) was performed using a NanoSight NS300 (Malvern Panalytical, Malvern, UK) equipment. For the measurements, all samples were diluted 1:10,000 with HPLC water. The diluted samples were injected with sterile syringes into the sample chamber equipped with a 532 nm diode laser (green). All samples were measured in a single shutter and gain mode for 90 s with manual shutter, gain, brightness, and threshold adjustments at room temperature. Three measurements of each newly injected sample were performed. To determine the D10, D50, and D90 as diameters size distributions, the mean size and SD values obtained by the NTA software were based on the arithmetic values calculated with all particles analyzed.