Model 500

The cytotoxicity effect of Dox/HCVPs on Hela and H460 cells was evaluated by MTT assay. Briefly, the cells (5000 cells per well) in a logarithmic growth phase were seeded in 96 well plates and incubated for 24 h. Then they were treated with different concentrations of various formulations for 48 h. The solutions were aspirated, and then 100 μL fresh medium and 20 μL of MTT solution (5 mg/mL in PBS) were added. After incubation for 4 h at 37 °C, the contents were removed and 200 μL DMSO was added to dissolve the purple formazan products. The absorbances at 570 nm were read using a BioRad microplate reader (Model 500, USA). The median inhibitory concentration (IC₅₀) was determined with the software GraphPad Prism 5 (GraphPad Software, San Diego, USA). The Dox/HCVPs were pretreated under pH 6.5 condition for 30 min at 37 °C before use.

The proliferation inhibition of various formulations was evaluated using classical MTT method. Cells were plated in 96-well cell culture plates (5000 cells/well) and incubated overnight at 37 °C. Cells were treated with varies of concentrations of Dox, Dox/HCV-7, Dox/HCV-63, Dox/HCV-102 for 48 h. After aspirating the treatment medium, 100 µl of fresh medium and 20 ul of MTT solution (5 mg/ml) were added to the cells. After 4 h incubation, plates were read at 570 nm using a BioRad microplate reader (Model 500, USA). The cells treated with medium without drugs were treated as negative controls. The log dose-response curve was plotted and the median inhibitory concentration (IC₅₀) was calculated with the software GraphPad Prism 5.

To evaluate cell proliferation, an MTT assay was performed. A549 cells were seeded into a 96-well plate at a density of 6×10³ cells/well. At 24 h after seeding, cells were treated with various concentrations of tunicamycin (1.25–10 µg/ml) for 8 h. For combined treatment, cells were treated with 1.25 µg/ml tunicamycin for 8 h followed by 24 h of cisplatin treatment (1.25–40 µg/ml). Cells without drug treatment were used as the negative control. Five wells were examined for each group.
Subsequent to the chemotherapeutic treatments, 20 µl MTT solution (5 mg/ml) was added to each well. Following 4 h of incubation at 37°C, the medium was removed and 200 µl dimethyl sulfoxide (MP Biomedicals, LCC, Santa Ana, CA, USA) was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 492 nm (A492) using a microplate spectrophotometer (Model 500, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The rate of growth inhibition was determined using the following formula: Growth inhibition rate (%) = (1 - [A492_Sample/A492_Control] × 100%. The half maximal inhibitory concentration (IC₅₀) was calculated using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).