4.8. Cellular Transport of AmB-Soluplus® Micelles Across Caco-2 Cell Monolayer

Caco-2 cells were seeded in a Transwell plate (Corning^®, polyester membrane insert, 12 mm with a pore size of 0.4 μm) at densities of 1 × 10⁵ cells/well. The cells were cultivated for 14 to 21 days to allow them to reach the required confluent monolayers. The culture media were changed every 2 days until experiments were conducted. The integrity of the Caco-2 cell monolayers was confirmed by measuring the transepithelial electrical resistance (TEER) with an EVOM2 (World Precision Instruments, Sarasota, FL, USA) before use. However, only cell monolayers with TEER values more than 500 Ω.cm² were used for transport studies. At the commencement of transport studies, the cellular monolayers were washed 3 times with warmed HBSS and allowed to equilibrate in the incubator for 30 min. Then, AmB-PM in HBSS, 0.4 mL, (equivalent to 15 μg/mL of AmB) was added to the apical chamber, while pre-warmed HBSS, 1.2 mL was added into each basolateral chamber. After incubation for 30, 60, 120, 180, and 240 min, 0.2 mL of basolateral medium was withdrawn and replaced with pre-warmed HBSS. Drug solutions in the collected samples were evaporated using the GeneVac EZ-2 evaporation system (GeneVac Ltd., Ipswich, UK) to increase drug concentrations. Collected samples were reconstituted with the mobile phase and centrifuged at 12,000 rpm for 10 min before HPLC analysis.

The transport of the drug from the apical to the basolateral chamber was represented as the amount of AmB transported versus time. In addition, the AmB apparent permeability coefficient (P_app) was calculated according to the following equation:

where dQ/dt is the slope of the cumulative drug permeated versus time curve (μg/h). A is the diffusion area (1.12 cm²), C₀ is the initial concentration of AmB in the apical chamber.

At the end of each incubation time, samples were removed from the apical and basolateral chambers, and the cells were washed three times with warmed HBSS. All wells were filled with 0.5 and 1.5 mL standard growth media into apical and basolateral chambers, respectively, then incubated for 24 h. Finally, the integrity of the cell monolayer was determined by the TEER measurement.