hCMEC/D3, cultured as reported above, were seeded at 100,000/well in a collagen-coated polytetrafluoroethylene (PTFE) membrane Transwell (Corning Life Sciences, Chorges, France) device (6.5 mm insert diameter, 3 μm pore size) and grown for 4 days up to confluence. Permeability of 0.15 mg/mL FITC-dextran, taken as a parameter of paracellular transport across hCMEC/D3 monolayer, was measured spectrofluorimetrically (λexc = 490 nm, λem = 540 nm) with a multilabel plate reader (Victor3 1420, Perkin Elmer, Waltham, MA, USA), in order to assess the monolayer’s integrity.
At day 4, the receiving medium in the basal chamber was replaced with 3-day F98 cell culture supernatant, which should contain specific F98 chemo-attractant factors. Then, selected doxorubicin loaded formulations were dropped in the apical chamber at a concentration of 2.8 μg/mL. After 1, 3, and 6 h, the medium in basal chamber was collected and doxorubicin concentration was measured spectrofluorimetrically (λexc = 490 nm, λem = 615 nm), using a multilabel plate reader (Victor3 1420, Perkin Elmer, Waltham, MA, USA), owing to a previously set calibration curve (n = 5).
At day 4, the receiving medium in the basal chamber was replaced with 3-day F98 cell culture supernatant, which should contain specific F98 chemo-attractant factors. Then, selected doxorubicin loaded formulations were dropped in the apical chamber at a concentration of 2.8 μg/mL. After 1, 3, and 6 h, the medium in basal chamber was collected and doxorubicin concentration was measured spectrofluorimetrically (λexc = 490 nm, λem = 615 nm), using a multilabel plate reader (Victor3 1420, Perkin Elmer, Waltham, MA, USA), owing to a previously set calibration curve (n = 5).