2.6.2. Membrane Destabilization Study

16-HBE cells were plated on a 48-well plate at a cell density of 10.000 cells/well in DMEM containing 10% FBS. After 24 h of incubation, the medium was removed and then the cells were incubated with 100 μL of DPBS (pH 7.4) or 20 mM MES (pH 5.5, 130 mM NaCl) containing PHEA*-g-*bAPAE (0.5 mg/mL) for 20 min at 37 ◦C; in the same condition, blank analysis was also performed. After this time, 100 μL of Tripan Blue 0.2% was added to each well. After 1 min of incubation, the supernatant was removed and cells were observed with miscroscope (Axio Cam MRm, Zeiss, Oberkochen, Germany). All analysis was performed in triplicate. Simultaneously, 16-HBE cells were plated on a 24-well plate at a cell density of 100,000 cells/well in DMEM containing 10% FBS. After 24 h of incubation, the medium was removed and then the cells were incubated with 100 μL of PBS (pH 7.4) or 20 mM MES (pH 5.5, 130 mM NaCl) containing PHEA*-g-*bAPAE (0.5 mg/mL) for 20 min at 37 ◦C; in the same condition, blank analysis was also performed. After this time 500 μL of Trypsin was added in each well and after 5 min the content of 3 well, treated with the same condition, was reunited in a small centrifuge tube and mixed with 200 μL of Trypan Blue 0.2%. After 1 min the supernatant was removed by centrifugation at 2000 rpm and the residual pellet was washed with 2 mL of DPBS. After washing, the supernatant was removed by centrifugation at 2000 rpm and the pellet in each tube was treated with 250 μL of TRITON X100 1% and plated on a 96-well plate. After 1 h of incubation, the absorbance at 580 nm was read using a Microplate reader (Multiskan Ex, Thermo Labsystems, Vantaa, Finland). All analysis was performed in duplicate.

#### 2.6.3. Cell Viability Assay

Cell viability was assessed by a MTS assay on 16-HBE cells, using a commercially available kit (Cell Titer 96 Aqueous One Solution Cell Proliferation assay, Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and phenazine ethosulfate. 16-HBE cells were plated on a 96-well plate at a cell density of 25,000 cells/well in DMEM containing 10% FBS. After 24 h of incubation, the medium was removed and then the cells were incubated with 200 μL per well with an aqueous dispersion (DMEM containing 10% FBS) of each copolymer at concentrations between 2 mg/mL to 0.0625 mg/mL. All polymers dispersions were sterilized by filtration using 220 nm filter. After 24 and 48 h incubation, the polymers dispersions were

removed and each plate was washed with sterile DPBS; after this, cells in each well were incubated with with 100 μL of fresh DMEM and 20 μL of a MTS solution and plates were incubated for 2 h at 37 ◦C. The absorbance at 490 nm was read using a Microplate reader (Multiskan Ex, Thermo Labsystems, Finland). Relative cell viability (percentage) was expressed as (Abs490 treated cells/Abs490control cells) × 100, on the basis of three experiments conducted in multiple of six. Cells incubated with the medium were used as negative control.
