2.5. Permeability Study

Caco-2 cells were seeded at 1 × 10⁵ cells/well on Transwell inserts and cultured for 21 days. Prior to the permeability study, culture medium was replaced with HBSS; cells were equilibrated in this for around 40 minutes. FD4-loaded hydrogel (5% w/v AP) was diluted to 250 mg/mL (gel content) in HBSS and applied to the cells at this concentration. Basolateral solution was sampled (100 μL volumes) periodically for two h and FD4 was quantified by fluorescence. A parallel experiment was conducted with free FD4 application to Caco-2 monolayers at equivalent concentration. Experiments were performed in triplicates.