100 nm nuclepore membrane

The nontargeted gadolinium-loaded liposomes (NTLs) containing Gd⋅DTPA⋅BSA, DSPC, cholesterol, and DSPE-mPEG₂₀₀₀ at a molar ratio of 0.75:1.10:1:0.15 were produced by lipid film hydration and extrusion method.23 (link),36 (link),37 (link) In short, the lipids were dissolved in a mixture of methanol and chloroform at a ratio of 1:1 (v/v). In some experiments, 0.1 mole percent of coumarin-6 was added to lipids as a fluorescent marker. The organic solvents were evaporated, and a lipid film was formed. Then, the resulting thin film was hydrated by vortexing in a HEPES-buffered saline solution (pH 7.4, containing 25 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂) and sonicated for 5 minutes with a sonifier cell disrupter. The liposome suspensions were obtained and extruded 20 times through a 100 nm nuclepore membrane (Whatman, Kent, UK) using a mini-extruder set (Avanti Polar Lipids Inc.). The above procedures were all handled at 55°C.