MTT cytotoxicity assay

Different human colon cancer cell lines (HCT-116, HT-29 and Caco-2) were grown in the tissue culture lab of Egyptian Company for Vaccines and Sera (Vacsera, Giza, Egypt). At the time of the experiment, the cells were checked for viability and confluency before being grown on the appropriate growth medium, RPMI 1640, supplemented with 1% mixture of 100 mg/ml of streptomycin, 100 units/ml of penicillin and 10% of heat-inactivated fetal bovine serum in a humidified, 5% (V/V) CO₂ atmosphere at 37 °C.

Exponentially growing cells from different cancer cell lines were trypsinized, counted and seeded at the appropriate densities (2000–10,000 cells/0.33 cm² well) into 96-well microtiter plates. Cells were then incubated in a humidified atmosphere at 37 °C for 24 h. Afterwards, cells were exposed to a solution of TQ in DMSO or to dialyzed dispersions of TQ-loaded nanocapsules at different concentrations for 24 or 48 h. The cytotoxicity of each concentration was assessed in triplicate. The viability of treated cells was determined using MTT technique as follows: the media were removed; cells were incubated with 200 μl of 5% MTT solution/well and were allowed to metabolize the dye into colored-insoluble formazan crystals for 2 h. The remaining MTT solution was discarded from the wells and the formazan crystals were dissolved in 200 µl/well acidified isopropanol for 30 min, covered with aluminum foil and shaken using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc., Mississauga, Canada) at room temperature. Absorbance readings were measured at 570 nm using a Stat Fax 4,200 plate reader (Awareness Technology, Inc., Florida, USA). The cell viability was expressed as the percentage of treated cells relative to untreated control cells and the concentration that induces 50% of maximum inhibition of cell proliferation (IC₅₀) was determined using GraphPad Prism version 5 software. Blank formulations (prepared without TQ) were also assessed for their cytotoxic potential⁶⁷.