Deacetylated chitosan

Stock solutions of pig gastric mucin (Sigma-Aldrich, M1778, type III) and sodium alginate (Sigma) were prepared in phosphate-buffered saline (PBS) buffer, pH 6.8, adjusted to an ionic strength I = 0.1 M by the addition of NaCl, according to Green, 1993. Deacetylated chitosan was also purchased from Sigma Aldrich (Kitozyme, 740,179-5 g, Dorset, UK). The carboxymethylcellulose (CMC) and pullulan samples were purchased from Carbosynth, UK. DMAE-pullulan was obtained as described previously [30 (link)]. Briefly, 5 g of pullulan was dissolved in 25 ml of distilled water and mixed with a 25 mL 10 M sodium hydroxide solution. Then, 35 g of 2-chloro-N,N dimethylethylamine hydrochloride was added to the mixture and left stirring at 60 °C for 1 h. After the reaction was completed, the mixture was washed four times with 50 ml diethyl ether and after was diluted in water to a concentration of 10 mgmL⁻¹ and adjusted to pH 7 using HCl. The solution was further cleaned of organic solvents and concentrated in a rotary evaporator, after which was dialysed in PBS buffer on a 14,000 Da (g/mol) membrane for two days. The resulting solution was freeze-dried which resulted in the formation of white odourless powder. The powder was stored at 4 °C until needed.

Deacetylated chitosan (medium viscosity), fluorescein isothiocyanate (FITC), fluorescein (Fluo), rhodamine isothiocyanate (Rho), eosin isothiocyanate (E), 5(6)-carboxynaphthofluorescein (CNF), 5(6)-carboxy-2′,7′-dichlorofluorescein (CCF), 7-amino-4-methyl-3-coumarinylacetic acid (CNH2), 7-hydroxy-4-methylcoumarin (COH), glycerol, Tween20, and 1–4 dioxane were all purchased from Sigma-Aldrich (Buchs, SG, Switzerland). Montmorillonite Dellite^® LVF (CEC: 105 meq/100 g) was donated by Laviosa Chimica Materia (Livorno, Italy). Fluorescamine was purchased from TCI (TCI Germany GmbH, Eschborn, Germany). Phosphate-buffered saline (PBS) (10 mM) was prepared by solubilizing NaCl (0.137 M), KCl (2.7 mM), Na₂PO₄ (0.01 M) and KH₂PO₄ (1.8 mM) in DI water. The pH was adjusted by adding NaOH and HCl.