2.2 Liposome formulation
A lipid film was prepared by rotary evaporation from a mixture of phosphatidylcholine (PC), cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Brucella ovisHS extract (LPS), and the α1,2-Mannobiose-PEG2kDa-DOPE derivative (Manα) (Pappalardo et al., 2013) in chloroform. The B. ovis HS extract was kindly provided by Mgtr. Carlos Robles (Robles, 2009). The lipid film was rehydrated for 30 minutes in sterile normal saline (0.9% NaCl), pH 7.4, at a PC concentration of 1.2 mg/mL (mice vaccines) or 4.8 mg/mL (bovine vaccine) in the final suspension and then vortexed for 5 minutes. The liposomes were prepared by extrusion of the suspension through a 200 nm pore Whatman® Nucleopore® Polycarbonate membrane using an Avanti Polar Lipids® Mini Extruder™.
For mice vaccines: formulation of Man-L had PC:Chol:DOTAP:LPS:Manα with a 60:30:10:4.3:2 molar ratio [2.13 mg/mL total lipids]. These were incubated ON with plasmids resulting in a final dose of 15 µg DNA/300 µL liposomes.
For bovine vaccines: formulation of Man-L had PC:Chol:DOTAP:LPS:Manα with a 60:30:30:4.3:2 molar ratio [9.96 mg/mL total lipids]. These were incubated ON with plasmids, resulting in a final dose of 600 µg DNA/1500 µL liposomes. “Bovine vaccine” was formulated with a higher ratio of DOTAP with respect to “mice vaccine” to increase liposome stability due to the higher concentration of lipids in medium.