*2.3. Preparation of Liposomes, Transferosomes and Ethosomes*

Several formulations of liposomes (L), transferosomes (T), and ethosomes (E) were prepared by the classic film-hydration method [42,43]. Their quantitative composition, reconstitution conditions, and the methods used to purify them are shown in Table 1. Three batches of each lipid vesicle type were prepared, empty and loaded with a variable amount of B12. Cyanocobalamin was added to the organic phase dispersion or included in the liposomal reconstitution solution (80% *w/v* of the solubility limit to achieve the maximum loading possible). Briefly, P90G, cholesterol (P90G:Chol molar ratio 17:1) or surfactants (15% *w/w* of lipid), and cyanocobalamin (except in the blank formulations and formulations reconstituted with B12 solution) were dissolved in methanol. The solvent was evaporated using a rotary evaporator (BUCHI R-210) under stirring (Heidolph RZR-2021) at 50 ◦C and 100 mbar. The resulting thin film was hydrated by addition of PBS 7.4 pH or B12 solution and then stirred for 1 h at 50 ◦C to obtain a multi-lamellar vesicle (MLV) dispersion. Transfersome batches were prepared by the same process with the exception that cholesterol was replaced by Tween 80. The ethosomal batches were prepared by the Touitou method [44], dissolving P90G and B12 in ethanol. Then, an appropriate amount of water was added at 12 ± 0.5 mL/h in a sealed beaker under stirring (710 ± 5 rpm), in a water bath tempered at 30 ◦C. Concurrently, the option of adding B12 dissolved in the water flow was also explored. The system was kept under stirring for 5 min after the addition of the water.

**Table 1.** Quantitative composition of the different lipid vesicle systems, reconstitution conditions, and purification method.


<sup>1</sup> B12 added in reconstitution solution. <sup>2</sup> B12 added in the organic-lipid phase. <sup>3</sup> B12 added dropwise in solution. <sup>4</sup> C: centrifugation; D: dialysis (24 h). PBS: Phosphate Buffer Saline.

Once MLV were obtained, their size was reduced by sonication and membrane extrusion. In the case of L and T, they were firstly sonicated at 50 ◦C for 2 h (Elmasonic S60H), then cooled down to 4 ◦C and extruded through a 200 µm membrane at 30 ◦C, using a LiposoFast-Basic Extruder, Avestin (20 times) [45]. Ethosomes were sonicated for 1 h at room temperature and reduced by the same extrusion process. After size reduction, the samples were purified by centrifugation (12,800× *g*, 30 min) or washed in 2 L PBS at 4 ◦C for 24 h to compare both methods [46,47]. The batches were then stored at 4 ◦C protected from the light.
