Z326k centrifuge

All chemical reagents, standards and solvents were purchased from Sigma Aldrich (Johannesburg, South Africa). All chemical reagents used were analytical grade.
All centrifugation was performed using a Hermle Z326k centrifuge (Hermle Labortechnik GmbH, Wehingen, Germany). All spectrophotometric determinations were performed using a microplate reader (BMG LABTECH GmbH, SpectroStar Nano, Ortenberg, Germany). Disease and decay-free African pumpkin leaves (20 kg) were selectively harvested twice according to maturity stage after 95 days of propagation from the vegetable garden belonging to the Zithobeni community (Bronkhorstspruit, South Africa). The leaves were cleaned by washing in running tap water and were transported to the laboratory at 10 °C within 2 h in cooler boxes and were stored at −80 °C for 48 h prior to cooking.

In a 1000 mL round-bottom flask, 6 mL TEOS, 300 mL EtOH, 22 mL H₂O, and 18.5 mL NH₄OH were added. This reaction mixture was stirred at room temperature at 1000 rpm. A second mixture of 36 mL TEOS and 150 mL EtOH was added drop-wise via a separatory funnel over about 2 h. The reaction mixture was left for 20 h at room temperature. At the end, the reaction mixture was neutralized with 12 mL of HCl. The silica nanoparticles were washed by dispersion in fresh solution, which was followed by centrifugation. The particles were washed in this way three times with EtOH and three times with water. The separation was done by centrifugation for 4 min, at 5500 rpm, in a Hermle Z 326K centrifuge (HERMLE Labortechnik GmbH, Wehingen, Germany) at 0 °C. At the end, the particles were dispersed in EtOH. The nanoparticle diameter was determined with a Verios G4 UC scanning electron microscope (SEM) (Thermo Fischer Scientific Inc., Eindhoven, The Netherlands) equipped with an energy dispersive spectrometer (EDS), EDAX Octane Elite (EDAX LLC/AMETEK, Mahwah, NJ, USA); see Figure S1 in the Supplementary Material. The zeta potential ζ of the pristine silica nanoparticles was determined with a Zetasizer NanoZS (Malvern Panalytical Ltd, Malvern, UK) to be ζ = −49.7 ± 1.4 mV, and the diameter D = 500 ± 7 nm by SEM; see Table S1 in the Supplementary Material.

The solubility of CIP.HCl was determined in nanoemulsion components as follows: OA and IPM as oil phases; TW-20 and Labrasol^® as surfactants; PG, PEG 400, and ethanol as cosurfactants; and 5% acetic acid and water as aqueous phases, as described in [49 (link)]. Briefly, an excess amount of CIP.HCl was dissolved in 3 mL of tested samples placed in 10 mL screwed-on test tubes. Samples were mixed using a vortex mixer and placed in a water bath at 37 °C for 24 h. Then samples were centrifuged at 3500× g for 15 min (Hermle Z326K centrifuge, Wehingen, Germany), and the supernatants were appropriately diluted with 0.1 N HCl. The concentration of CIP.HCl was assessed by UV-Vis spectrophotometry (Varian, Cary UV/Vis spectrophotometer) at a wavelength (λ_max) of 277 nm, using a standard calibration curve of CIP.HCl prepared in 0.1 N HCl.