Cytotoxicity study

A549 human epithelial alveolar cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were grown in F-12K medium supplemented with 10 % heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin (ATCC recommended). Cells were cultured at 37 °C in a 5 % CO₂ and 95 % humidity atmosphere (Fisher brand Isotemp CO₂ Incubator, Thermo Fisher Scientific Inc, Hanover Park, IL, USA). The cell viability potential of free drug, blank liposome and drug-loaded liposome were performed by the MTT assay (30). Briefly, the cells were seeded into 96-well plated at a density of 1×10⁴ cells/well and cultured for 24 hrs. Then, the free drug (ciprofloxacin/colistin, 2 mg/ 2 mg, 1 mL) which was dissolved in 10 % sucrose solution and drug-loaded liposome were diluted with culture medium to obtain a series of drug solution, i.e. 20/20, 10/10, 5/5 and 2.5/2.5 μg/mL. For the blank liposomes, the lipid concentration were kept as the same to that of corresponding drug-loaded liposome (i.e. 36, 18, 9 and 4.5 μg/mL). The culture medium and 10% sucrose were added as the positive control and the cells were incubated for 24 hrs. At the end of incubation, the culture medium containing drugs was aspirated out of the 96 well plates and serum free medium and MTT solution (5 mg/mL dissolved in PBS) were added. The incubation time was 4 hrs. The medium was removed, the formazan crystals were dissolved by the addition of DMSO and the absorbance was measured at 570 nm using a microplate absorbance reader (BioTek, Bio Tek Instruments, Inc., Winooski, VT, USA).