Evaluating Cytotoxicity of Drug Formulations

The in vitro cytotoxicity of various formulations was studied by MTT assay.30 (link) Following attachment, cells were exposed to blank or DOX-loaded compounds (β-CD, mPEG5000-CD, PELA54, PELA54-CD) at a previously determined set concentration and incubated in thermostatic incubator for 48 hours. Then, 5 mg/mL MTT reagents were added into each well. After incubating for additional 4 hours in thermostatic incubator, the medium was discarded. Thereafter, 200 μL of DMSO was added to dissolve formazan. The plate was measured at 570 nm on a microplate reader (Thermo MK3; Thermo Fisher Scientific, Waltham, MA, USA). The inhibition rate of cell growth was calculated using the formula: Inhibitory rate (%) = (A_control − A_sample)/A_control ×100. The cytotoxicity of various compounds was expressed as half maximal inhibitory concentration (IC₅₀) values, which referred to the concentration that caused 50% inhibition of cell growth compared with untreated cells. The resistance reversion index (RRI) was defined as the ratio of IC₅₀ of free DOX vs that of DOX-loaded micelles (RRI = IC_{50,free DOX}/IC_50,micelles).31 (link)