Ntervals. Cells were cultivated for at least 2 passages before seeding onto transwell filter supports to stabilize the cell phenotype 12].Validation of Transwell System using FITC-Insulin and Sulforhodamine B TransportUse of transwell system in determining transport of active molecules across Caco-2 monolayer was validated by using two fluorescent molecules, FITC-insulin and sulforhodamine-B, which would delineate the efficacy of system for both macromolecular and small molecular weight pharmaceutical moieties. Briefly, cells were pre-conditioned with basal seeding medium for 30 minutes before starting the experiment at 37uC. FITC-insulin andProtein Permeation across Caco-2 Monolayerssulforhodamine-B were loaded onto the individual Caco-2 monolayer filter supports at various concentrations (0.05, 0.15, 0.3, and 0.6 mg/well) dissolved in 500 ml of basal seeding medium. The basolateral chamber consisted 1400 ml of the same growth medium as per manufacturer’s protocol. The plates were incubated for 18204824 data ranged from 8.261.861026 cm/s to 10.561.861026 cm/s for the loading studied here (Table 1). Cumulative transport of FITC-insulin at the end of 5 hours for FITC-insulin ranged from 4.161.1 (0.15 mg loading) to 5.961.0 (0.6 mg loading) (Fig. 1b; Table 1). Transport of sulforhodamine-B also exhibited similar trends as FITC-insulin. Once again, a low percentage of applied sulforhodamine-B permeated through Caco-2 monolayer. The cumulative apical-to-basolateral transport of 0.00260.0008 mg, 0.00460.0007 mg, 0.00960.001 mg, and 0.0160.002 mg was observed at apical loadings of 0.05, 0.15, 0.3, and 0.6 mg/well at the end of 5 hours (r2 = 0.977; Fig. 2a). The cumulative transport ranged between 2.260.4 (0.6 mg loading) to 2.960.4 (0.3 mg loading) (Fig. 2b). At the same time, a consistent Papp was also ob.Ntervals. Cells were cultivated for at least 2 passages before seeding onto transwell filter supports to stabilize the cell phenotype 12].Validation of Transwell System using FITC-Insulin and Sulforhodamine B TransportUse of transwell system in determining transport of active molecules across Caco-2 monolayer was validated by using two fluorescent molecules, FITC-insulin and sulforhodamine-B, which would delineate the efficacy of system for both macromolecular and small molecular weight pharmaceutical moieties. Briefly, cells were pre-conditioned with basal seeding medium for 30 minutes before starting the experiment at 37uC. FITC-insulin andProtein Permeation across Caco-2 Monolayerssulforhodamine-B were loaded onto the individual Caco-2 monolayer filter supports at various concentrations (0.05, 0.15, 0.3, and 0.6 mg/well) dissolved in 500 ml of basal seeding medium. The basolateral chamber consisted 1400 ml of the same growth medium as per manufacturer’s protocol. The plates were incubated for 23727046 5 hrs at room temperature with gentle shaking (5 rocks/minute on a rocker, so as to mimic intestinal peristaltic movement). TEER measurements were performed at predetermined time intervals (0. 0.25, 0.5, 1, 2, 3, and 5 hrs). At the same time-points, 100 ml samples were withdrawn from the basolateral chamber to quantify the total amount of FITC-ins/sulforhodamine-B transported across the monolayer. The withdrawn sample was immediately replaced with equivalent amount of the experimental medium. Withdrawn samples were analyzed using a Tecan SaffireTM fluorescent microplate reader (Tecan Group Ltd, Mannedorf, Switzerland) at respective wavelengths for FITCinsulin (Ex 488 nm; Em 525 nm) and sulforhodamine-B (Ex 560 nm and Em 590 nm).way Analysis of Variance (ANOVA) followed by appropriate post hoc analysis. Values showing p,0.05 were considered significantly different.Results Dose-dependent Transport of FITC-insulin and Sulforhodamine-B across Caco-2 MonolayersBefore testing the transport of therapeutic peptides, the 3-day Caco-2 monolayers were validated by studying the permeation of fluorescein isothiocynate conjugated bovine insulin (FITC-insulin) and sulforhodamine-B. Only small quantities of the FITC-insulin permeated from the apical chamber to the basolateral chamber (Fig. 1). Transport of FITC-insulin was dose-dependent (r2 = 0.99) in flux as well as cumulative transport. The transported amounts were: 0.00260.0004 mg (0.05 mg loading), 0.00660.001 mg (0.15 mg loading), 0.0260.002 mg (0.3 mg loading), and 0.0460.006 mg (0.6 mg loading) after 5 hours (Fig. 1a). The apparent permeability coefficients (Papp) calculated from cumulative permeability 18204824 data ranged from 8.261.861026 cm/s to 10.561.861026 cm/s for the loading studied here (Table 1). Cumulative transport of FITC-insulin at the end of 5 hours for FITC-insulin ranged from 4.161.1 (0.15 mg loading) to 5.961.0 (0.6 mg loading) (Fig. 1b; Table 1). Transport of sulforhodamine-B also exhibited similar trends as FITC-insulin. Once again, a low percentage of applied sulforhodamine-B permeated through Caco-2 monolayer. The cumulative apical-to-basolateral transport of 0.00260.0008 mg, 0.00460.0007 mg, 0.00960.001 mg, and 0.0160.002 mg was observed at apical loadings of 0.05, 0.15, 0.3, and 0.6 mg/well at the end of 5 hours (r2 = 0.977; Fig. 2a). The cumulative transport ranged between 2.260.4 (0.6 mg loading) to 2.960.4 (0.3 mg loading) (Fig. 2b). At the same time, a consistent Papp was also ob.