Fitc dextran 4

For Fluorescein Isothiocyanate (FITC)-Dextran permeability experiments, Caco-2 monolayers differentiated for 21 days were transferred to 12-well plates containing 1.5 ml phenol red free DMEM media (pH 7.4) in the basal compartments. For the treatment, medium in the apical compartment was replaced by 0.5 ml phenol red free DMEM medium containing different concentrations of CPPs, and the plates were incubated at 37 °C for 1 h. Post treatment, inserts were transferred to a new 12-well plate containing 1.5 ml fresh phenol red free DMEM and the apical medium was replaced by medium containing 1 mg/ml FITC-dextran 4 (Sigma-Aldrich). The incubation with the permeability marker lasted for 30 min. Samples were collected from the basolateral compartments post incubation and fluorescence was measured using multi-well fluorescent plate reader (Synergy; excitation wavelength: 490 nm, emission wavelength: 520 nm). The concentration of FITC-dextran in the samples was calculated by comparing the relative fluorescence values to the FITC-dextran standard curve. The apparent permeability coefficient (P_app, cm/s) was calculated according to Eq. 1. $P_{app}=\frac{dQ}{dt}\times \frac{1}{\left(A\times C_0\right)},$ Where dQ/dt is the drug permeation rate (μg/s); a is the surface area of the inserts (cell monolayer) (cm²); and C₀ is the initial concentration at the apical side (μg/ml).

In vivo intestinal permeability was assessed using fluorescein dextran (FITC-Dextran 4, Sigma-Aldrich) as previously described [42 (link)]. The mice were orally gavaged with 0.75 mg/g body weight of 4 kDa FITC-labeled dextran, and blood samples were obtained from the retro-orbital venous plexus 5 h after this administration. Blood samples were centrifuged for 10 min at 5000 rpm, and plasma was taken and frozen at − 20 °C and analyzed the following day. Intestinal permeability to 4 kDa FITC-labeled dextran was determined by measuring the fluorescence intensity in plasma at 485 nm/525 nm using an automatic Infinite M200 microplate reader (Tecan, Lyon, France).

Mice were fasted for 6 h and orally gavaged with FITC-dextran-4 (0.2 mg/g, Sigma-Aldrich, MO, United States). An hour later, blood was collected from tail vein and fluorescent signals in serum were detected with CLARIOSTAR spectrometer (BMG Labtech, Germany) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm to determine the level of penetrated FITC-dextran in the circulation.