Fetal bovine serum albumin

HeLa cells were first grown in Dulbecco’s modified Eagle medium with 10% (v/v) fetal bovine serum albumin, 100 U/mL penicillin and 100 mg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO₂ atmosphere and then collected via centrifugation (1000× g, 5 min).

The cytotoxicity of the FFS of ZnO/CNC/PC III film was evaluated using the MMT (3-(4, 5 Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric assay^{29 (link)}. Human colorectal adenocarcinoma cells (Caco-2, HTB-37) and normal lung fibroblasts (WI-38, CCL-75) obtained from the American Type Culture Collection (ATCC, Rockville, MD); were seeded independently in flat-bottom 96 well plate (Corning, USA) at a density of 1 × 10⁴ cell/well (100 µL) in high glucose DMEM culture medium (Biosera, France) supplemented with 10% fetal bovine serum albumin (Sigma-Aldrich, USA) and 1% Penicillin streptomycin (Sigma Aldrich, USA). Plates were incubated at 37 °C for 18 h in a humidified 5% CO₂ incubator (Eppendorf, Germany) to allow cells attachment. The FFS was serially two-fold diluted, added in triplicate in each well, and incubated for 48 h under the previously mentioned culture conditions. Supernatants from each well were withdrawn and replaced with fresh medium (100 µL/well) containing MTT reagent with a final concentration of 0.5 mg/mL, and plates were re-incubated at 37 °C for 4 h. Media over the cells were removed, and dimethyl sulfoxide (Merck, Germany) was added (100 µL/well), plates were shaken for 15 min, and the absorbance was measured at 570 nm using a microplate reader (AccuReader M965 + , Metertech, Taiwan).

DOPC (1,2 dioleoyl-sn-glycero-3-phosphocholine) was from Avanti polar lipids (Alabaster, AL, USA), poloxamer 407 (P407) was provided by BASF (Ludwigshafen, Germany), and branched polyethylenimine, PEI was provided by Sigma-Aldrich (St. Louis, MO, USA). The cationic lipid DMAPAP (2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methyl-acetamide or dimyristoylaminopropylaminopropyl) was synthesized as described [3 (link)]. Chloroform and ethanol were purchased from Carlo-Erba reagents (Val-de-Reuil, France). Ultrafiltration units NANOSEP 300k were from PALL filtron (Rehoboth, MA, USA), polyether ether ketone (PEEK) tubes and T-shaped connectors and ferrules were from IDEX Health and Science (Oak Harbor, WA, USA), microfluidic chips were made out of Flexdym 150 × 150 mm² sheets (Eden Microfluidics, Paris, France). Endothelial cell growth medium 2 and trypsin/EDTA were obtained from Cedarlane Labs (Burlington, ON, Canada). The medium DMEM/F12 was from Thermo Fisher Scientific (Waltham, MA, USA), recombinant murine TNF-alpha from PeproTech (Neuilly-Sur-Seine, France), fetal bovine serum albumin, Embryomax^® 0.1% Gelatin Solution, and Penicillin-Streptomycin from Sigma-Aldrich (St. Louis, MO, USA). The EA.hy926 (ATCC^® CRL2922™) cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).