*2.14. Uptake Study*

Caco-2 cells were seeded onto confocal dishes (NuncTM 4-well Rectangular Dishes, Thermo Fisher Scientific, Waltham, MA, USA) at a density of 10<sup>5</sup> cells/cm<sup>2</sup> and were cultured for 7 days until 100% confluence was reached. The experiment was initiated by the addition of 0.5 mL of a test sample. In order to mimic the intestinal environment, the test samples were prepared in 10-mM MES-HBSS buffer (pH 6.5, 0.05% *w/v* BSA). The uptake of OND was evaluated by testing the following samples: OND solution, nonloaded SEDDS dispersed in OND solution, DDAB-OND in SEDDS and DOTAP-OND in SEDDS dispersed in MES-HBSS buffer. The experiment was carried out for both Citrem and Standard SEDDS, and SEDDS were dispersed at 1:1000 (*w*/*w*). The cells were incubated with the samples for 2 h at 37 ◦C and 5% CO<sup>2</sup> without shaking. Fifteen minutes before the termination of the incubation, cell nuclei were stained by the addition of 10 µL of Hoechst 33342 (1 mg/mL). At the same timepoint, 10 µL of propidium iodide (PI) (0.1 mg/mL) was added to detect dead cells.

Immediately after incubation, cells were washed twice with prewarmed Opti-MEM® medium (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired with the Nikon Ti-E (Nikon, Minato, Japan) epifluorescence microscope equipped with a cooled scientific CMOS camera (AndorZyla 5.5; Andor Technology, Belfast, UK) and LED fluorescence source (CoolLED pE-300; CoolLED, Andover, UK). DAPI, FITC and Cy3 filter sets were used for image Hoechst, FAM-labeled OND and PI, respectively. Three representative images of each sample were taken using NIS Elements AR 4.20 software (Laboratory Imaging, Prague, Czech Republic).
