*2.12. Caco-2 Cell Monolayer Transport Study*

Caco-2 cells, a model of human enterocyte epithelial cells, were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were seeded on T12 filter inserts (pore size 0.4 mm, 1.13 cm<sup>2</sup> growth area, Corning, Sigma–Aldrich, Copenhagen, Denmark) at a final density of 1 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/cm<sup>2</sup> . The cells were cultured for 20–23 days before the transport study was conducted.

Transport experiments were conducted on a horizontal shaker at 37 ◦C using a pH gradient mimicking physiological conditions. A volume of 500 µL of 10-mM MES-HBSS buffer (pH 6.5, 0.05% *w/v* BSA) was used on the apical side and 1000 µL of 10-mM HEPES-HBSS buffer (pH 7.4, 0.05% *w/v* BSA) on the basolateral one for 120 min. The experiment was initiated by applying 500 µL of a formulation on the apical side. The following formulations were tested: OND solution, DDAB-OND in the SEDDS, DOTAP-OND in the SEDDS, DDAB in the SEDDS dispersed in OND solution, DOTAP in the SEDDS in dispersed OND solution and nonloaded blank SEDDS dispersed in OND solution for both the Citrem and Standard SEDDS. The SEDDS were dispersed 1:100 (*w*/*w*) shortly before the experiment in a MES-HBSS-based buffer preheated to 37 ◦C. All formulations finally contained 1 nmol/mL of OND.

Transepithelial electrical resistance (TEER) was monitored with the EVOM Epithelial Voltohmmeter (World Precision Instruments, East Lyme, CT, USA). Before and after the

experiment, the cell culture cup Endohm chamber was used at 25 ◦C in MES-HBSS and HEPES-HBSS applied apically and basolaterally, respectively. In addition, TEER values were also monitored using chopstick electrodes throughout the experiment at 30, 60, 90 and 120 min at 37 ◦C.

Samples of 100 µL were taken from the basolateral side at predetermined time points (15, 30, 45, 60, 90 and 120 min) and replaced with the same volume of fresh prewarmed HEPES-HBSS buffer. The withdrawn samples were analyzed for fluorescently labeled OND by measuring fluorescence excitation/emission wavelengths at 495/520 nm in a microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). The apparent permeability coefficient (Papp), and transported OND accumulated basolaterally across the Caco-2 monolayer were calculated using the following Equations (3) and (4).

$$P\_{\rm app} = \mathbf{J}/\mathbf{C}\_0 = \mathbf{Q}/(\mathbf{C}\_0 \times \mathbf{A})\tag{3}$$

$$\text{transported OND} = \left[ \text{Q} / (0.5 \times \text{C}\_0) \right] \times 100\text{\%} \tag{4}$$

where J indicates the flux over a steady-state time period (pmol/s/cm), and C<sup>0</sup> stands for the initial concentration of OND in the apical compartment (pmol/mL). Q is the accumulated mass of OND transported across the monolayer on the basolateral side (pmol) of the area A (cm<sup>2</sup> ) of membrane filters.
