*2.2. Procedure of Preparation the DEX-Loaded Liposomes Stabilized with Chitosan*

The liposomes were designed by entrapping DEX inside L-α-phosphatidylcholine lipid vesicles, which were obtained by dissolving 25 mg of lipid in 1 mL chloroform. Through evaporation, a dry lipid film was achieved, which was hydrated by adding 50 mL of DEX stock solution in deionized water at 10 mg/L. During this process, the solution was moderately ultrasonically stirred (10% amplitude mode at 20 kHz ± 500 Hz standard frequency, Sonoplus Bandeline set-up), at room temperature, for 10 min, in order to disrupt the monolayer structure. To transform the multilamellar into unilamellar vesicles, the lipid solution was sonicated. The chitosan solution 0.5% (*w*/*w*) dissolved in 0.5% (*v*/*v*) acetic acid [28,29] was used to coat the vesicles, Thereafter, the colloidal solution was dialyzed at room temperature for 10 h (using a Sigma D6191-25EA dialysis tubing cellulose membrane with pore size of 12,000 Da MWCO, Sigma Aldrich Chemical Co, Schnelldorf, Germany), to remove its acidity.

For the preparation of lipid vesicles, 15 mg of lipid were dissolved in 1 mL chloroform; through solvent evaporation, a lipid dry film was obtained; the lipid layers were hydrated with 30 mL DEX solution (10 mg/L) in deionized water. The mixture was put to magnetically stirred for 2 h for complete dissolution. A total of 19.2 mL chitosan 0.5% (*w*/*w*) was added to the blurry vesicle suspension; in order to transform the multilamellar vesicles to unilamellar vesicles, the suspension was ultrasonicated for 20 min (amplitude modulus 10% at 20 kHz ± 500 Hz standard frequency, Sonoplus Bandeline configuration). Through ultrasonication, a suspension with a transparent aspect was obtained, with a pH value of 4 [28,29]. Given that stabilizing the vesicles with chitosan led to an acid pH, the suspension was further dialyzed for 10 h, up to a pH value of 6.7.

Due to its protonated amino groups (in acid to neutral solutions), which are incompatible with the stability of the bilayer hydrophobic core, the polymer chitosan coats the surface of the vesicles. Its physicochemical parameters, such as intra- and intermolecular hydrogen bonding and cationic transformation in acidic environment, make this polymer very attractive and useful for the development of suitable drug delivery formulations [30–33].

The anionic polyelectrolyte properties of chitosan provide interaction with anionic molecules of the vesicles' bilayer, as well as with the cells' membranes. In acid-to-neutral solutions, the amino groups of chitosan become protonated through bonding to intra- and intermolecular hydrogen of the vesicle bilayer. Formation of a stable positively charged layer around the unilamellar vesicles is related to the saturation absorption of chitosan molecules. These phenomena lead to obtaining confined, small-sized vesicles, with a morphology close to spherical. From this perspective, this polymer is very attractive and useful for designing modified release dosage forms [30–33].
