In vitro anti-tumor activity

The cytotoxicity of DOX • HCl, SPIO-loaded micelles and DOX/SPIO-loaded micelles against BEL-7402, HepG2 and LO2 cells was evaluated by MTT assay. Briefly, 1.0 × 10⁴ cells per well were placed in a 96-well microtiter plate (Nalge Nunc International, Naperville, IL, USA) and incubated for 24 h. Then the cells were incubated with a series of concentration of micelles for another 48 h. After that, cells were incubated with 20 μL MTT solution (5 mg mL⁻¹) for further 4 h at 37 °C. Finally, the product was dissolved by 100 μL DMSO and the absorbance was determined at 570 nm using an automatic reader (Bio-Rad, Model 680, USA). All the experiments were repeated three times. The 50% cellular growth inhibitions (IC₅₀) with 48 h were determined.

Colorimetric cell viability assay^42,43 was further used to investigate the cytotoxicity of DOX • HCl, DOX/SPIO-loaded micelles against BEL-7402 and HepG2 cells. The drug concentration of DOX • HCl, DOX/SPIO-loaded micelles were same as the IC₅₀ value of A54-Dex-PLGA/DOX/SPIO micelles. After a 24 h drug exposure, cells were stained with 100 μL calcein AM (Invitrogen, Carlsbad, CA) (0.5 μL dissolved in 1 mL PBS) for 30 min at room temperature. Cells were observed using a fluorescence microscope (Leica DM4000 B, Leica, Solms, Germany).