*2.13. In Vitro Cytotoxicity Study*

Cytotoxicity of the formulations was investigated as the loss of cell membrane integrity accompanied by release of a cytoplasm enzyme lactate dehydrogenase (LDH). The cytotoxicity study was conducted by the Cytotoxicity Detection Kit (LDH) (Roche, France) according to manufacturer's instructions after 120 min of the transport experiment in the Caco-2 cells. Briefly, a 40-µL sample of apical medium was diluted 5-fold by DI water. Fifty microliters of the diluted sample were pipetted into a clear 96-well multiwell plate in duplicates, and 50 µL of LDH reaction mixture was added. The 96-well plate was incubated for 30 min at 37 ◦C in a horizontal shaker in the dark. Subsequently, the absorbance was measured at 492 nm.

Results are depicted as cell viability (%). Relative cytotoxicity (%) was calculated according to the manufacturer's instructions, and the resulting values were deducted from 100% to obtain cell viability (%).
