Uv spectroscopy

A quantitative cell uptake assay was performed as per our previous reports [55 (link)]. Briefly, 12 well plates were seeded with MCF-7 and MDA-MB-231 cells at the density of 5 × 10⁵ cells/well and incubated overnight with complete media. After incubation for 24 h, media was aspirated, and fresh media was added comprising of PAC, 5-FU, and their formulations (equivalent concentrations of 1 µM and 2 µM of PAC and 5-FU, respectively) and again incubated for different time intervals (0.5, 1, 2, 4 h). After different time intervals, media were aspirated and cells were washed with cold PBS to extrude the extracellular drugs, followed by treatment with methanol. Following this, the cells were harvested with trypsin and centrifuged for 10 min at 10,000 rpm. The supernatant obtained was then subjected to UV-spectroscopy (Agilent, Santa Clara, CA, USA) for the quantification of PAC and 5-FU.

The piperine content in the in situ gel formulation was determined by UV spectroscopy (Agilent Technologies, Santa Clara, CA, USA). Briefly, 1 mL of the gel formulation was diluted to 10 mL with ethanol and sonicated using bath sonicator until a clear solution is obtained. Solution was filtered and piperine content in the in situ gel was determined at 343 nm using ethanol as a blank [58 (link)].