Cellular Uptake of Leflunomide in MDA-MB-231 Cells

MDA-MB-231 cell line was used to study the cellular uptake of the provided LEF and the selected LEF loaded cubosomal formulation. On the first day, the cells were seeded in T25 flask. On the second day, the media were removed, and media supplemented with predetermined drug concentration were added to cells. A confluent T25 flask 0.7 × 106 of MDA-MB-231 cells was harvested after 6 and 24 hours of incubation. The pellets were collected at each time interval were subjected to lysis according to the method previously reported by Srisongkram et al. (Srisongkram & Weerapreeyakul, 2019 (link)). Sample preparation for HPLC analysis was carried out as follows: 0.5 mL methanol was added to each sample then sonicated for 15 min. This step was followed by centrifugation for 10 min at 4500 rpm and filtration using 0.22 μm Nylon syringe filter.
Amount of LEF taken by the cells were quantified using HPLC (Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector). The Mobile phase consisted of (50%:50%) acetonitrile: phosphate buffer (0.02 M) pH was adjusted to 2.6 with orthophosphoric acid. Flow rate was 1 mL/min and the measurements were carried out at 260 nm (Zewail et al., 2019 (link)).