Tamoxifen citrate salt

The tamoxifen-sensitive human breast cancer cell line MCF7 was obtained from the American Type Culture Collection (ATCC, HTB-22). Tamoxifen-resistant cells (MCF7/TAM) were established by culturing MCF7 cells in medium with 1 μM tamoxifen citrate salt (Sigma-Aldrich, T9262-1G) over 6 months, as previously described [30 (link), 31 (link)]. All of the cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) at 37 °C in the presence of 5% CO₂.

Acetylsalicylic acid (>99% pure, Sigma-Aldrich, Steinheim, Germany) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 550 mM stock solution, which was stored in aliquots at −20°C. Tamoxifen citrate salt (≥99% pure, Sigma-Aldrich, Steinheim, Germany) was dissolved in methanol (99.8% pure, Sigma-Aldrich, Steinheim, Germany) to prepare a 1 mM stock solution, which was aliquoted, wrapped in aluminum foil (light-sensitive) and stored at 4°C. 5-Fluorouracil (>99% pure, Sigma-Aldrich, Steinheim, Germany) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 28 mM stock solution and was stored at 4 °C.
Water-insoluble tamoxifen and 5-Fluorouracil (5-Fu) were used for nanoparticle loading purposes. The powder forms of 5-Fu and tamoxifen were loaded in excess. Before the experiment, excess 5-Fu and tamoxifen were allowed to sediment, and the final concentrations of treatment loaded nanopolymers were 3 μM for both large FA-DABA-SMA (350,000 g/mol) and small FA-DABA-SMA (20,000 g/mol) made in the standard culture medium.