Sensitizing 4T1 Cells to Doxorubicin

We seeded 4T1 cells at a density of 5 × 10³ cells/well in 96-well plates and after 24 h we transfected the cells with two different concentrations of uPAR siRNA or nonspecific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After uPAR-siRNA treatment, we added the eEVs-(ScuPA) PLGA-antimiRNA-21-Cy5 or eEVs-(uPA) PLGA-antimiRNA-21-Cy5 (1×10⁷ EVs/well) to the cell culture and acquired fluorescence imaging using Celigo, as described previously.^{[33 (link)]} To investigate the chemosensitizing functional effect of 4T1-eEVs-PNCs, we seeded the 4T1 cells at a density of 1 × 10⁵ cells/well in 12-well plates. We then treated the cells with eEV-NPs (5×10⁷ EVs/well) for 24 h and treated them with doxorubicin 0.5 μM for a further 48 h. We investigated the synergistic cytotoxicity effect of 4T1-eEVs-PNCs independently loaded with either antisense miRNAs-21 or antisense miRNAs-10b + DOX cells using the Cell titer blue (CTB) assay, as described previously with minor modifications.^{[34 (link)]}