Penicillin g sodium

The cytotoxicity of the optimized IM-LPs was investigated using a MTT assay in the PASMCs. The PASMCs was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone, Logan, UT) supplemented of 20% fetal bovine serum (Everygreen, Zhejiang, China), 100 U/mL of penicillin G sodium, and 100 μg/mL of streptomycin sulfate (Beyotime, Shanghai, China). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. Briefly, cells were seeded in a 96 well plates at a density of 5×10⁴ cells per well and incubated in DMEM for 24 h attachment. Then, the cells were treated with (i) saline (negative control), (ii) 0.1% sodium dodecyl sulfate (0.1%SDS, positive control), (iii) plain IM (25 μM), (iv) IM-LPs (equivalent to 25 μM IM) and (v) plain liposomes. Following a 24 h period of incubation, the cells were washed with PBS and then incubated with 20 μL MTT solution (5 mg/mL in sterile PBS) for 4 h at 37°C. The formazan crystals were solubilized in 100 μL DMSO with appropriate shaking on a plate shaker for 10 min. The absorbance value was read on a microplate reader at 570 nm (Tecan Infinite 200 PRO, Austria, Switzerland). The cell viability was calculated as follow: Cell viability (%) = (A_sample – A_blank)/(A_control – A_blank) × 100%; A_sample is the absorbance for the treated cells, A_control is the absorbance for saline treatment and A_blank is the absorbance for no treatment.

All cell lines, including Ad293, Ad293CR, Ad293CreER (generated from Ad293 cells by transduction of lentiviral particles carrying a CreER expression cassette), and MIN6 were cultured in DMEM (Life Technologies, Rockville, MD) supplemented with 10% fetal calf serum (HyCLONE, Logan, UT), 2 mM glutamine, 50 U/mL penicillin G sodium, and 50 μg/mL streptomycin sulfate (Beyotime, Haimen, China). Cells were cultivated in a humidified incubator at 37°C supplied with 5% CO₂.