Protanal lf 10 60 ft

Dexamethasone acetate was purchased from Aventis (Gentilly, France). Kolliphor-P407 was from BASF (Mississauga, Canada). Hydroxy-propyl-beta-cyclodextrin was purchased from Roquette (Paris, France). Mucoadhesive agents used in the experiments were xanthan gum (Satiaxane from Cargill, Puteaux, France), carbomer (Carbopol C971P from Lubrizol, Brussels, Belgium) and sodium alginate (Protanal LF 10/60 FT from FMC Biopolymer, Cork, Ireland). Porcine gastric mucin type II was from Sigma Aldrich (Saint-Louis, MO, USA). All the experiments were performed using sterile water from Fresenius Kabi (Sévres, France).
For the HBSS medium preparation, the chemical products used were calcium chloride × 2H₂O, potassium chloride, potassium hydrogen phosphate anhydrate, sodium chloride, disodium dihydrogen phosphate × H₂O, sodium hydrogen carbonate, magnesium chloride × 6H₂O and magnesium sulfate × 7H₂O. All these products were purchased from Sigma Aldrich (Saint-Louis, MO, USA).

Alginate beads containing bacteria were prepared as previously described (23 (link)). PAO1 was inoculated in alginate beads using seaweed alginate (Protanal LF 10/60 FT; FMC Biopolymer, Drammen, Norway). Then, 9.5 mL 2% alginate was mixed with 0.5 mL ON culture OD₄₅₀ adjusted to reach final optical densities in the beads of 0.1, 0.01, 0.001, and 0.0001. For the starting inoculum of OD₄₅₀ = 1, a 4% alginate solution was mixed 1:1 with bacterial culture adjusted to OD₄₅₀ = 2 to reach the same alginate concentration. OD₄₅₀ from each cell culture tube was measured in triplicates, and the average was used. The alginate-bacterial solution was transferred to a 20 mL syringe connected to a 21 G hypodermic needle through a sterile silicone tube (internal diameter = 2 mm). The needle was placed 4.5 cm above the surface of a stirred 0.25 M CaCl₂ solution and 12 mm from the edge of the beaker. Droplets of the alginate-bacteria solution were dispensed at 30 mL h⁻¹ via a syringe pump (Perfusor Compact, Braun, Germany) into the CaCl₂ solution and hardened for 1 h, and then washed three times in 0.9% NaCl. Twenty beads were gently transferred with sterile plastic tweezers to a 250-mL Erlenmeyer flask containing 50 mL R2A and incubated at 37°C, 100 rpm.