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.