Liposome Preparation

Liposomes were prepared using standard techniques. We produced a mixture of DOPC, PEG2K-PE, and rhodamine-PE (molar ratio 94.2, 5.0, 0.8), or DOPC, DOPS, PEG2K-PE, and rhodamine-PE (79.2:15:5:0.8), or DOPC, cholesterol, PEG2K-PE, and rhodamine-PE (79.2, 15.0, 5.0, 0.8) in chloroform in a glass tube. Chloroform was evaporated using a dry N₂ line, and the film was placed under vacuum overnight. We added a volume of encapsulation buffer (5 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl₂, 10 mM NaCl) to the film. Typically, we would add 300 μL of buffer to a total of 4.5 μmol of lipid. The film was resuspended by shaking on a Thermomixer (Eppendorf) at 850 rpm, room temperature for 1 h. The solution was then put through seven rounds of freezing and thawing by passing between liquid N₂ and a room temperature water bath, then extruded with 21 passes through a Mini-Extruder (Avanti Polar Lipids) and a 0.2 μm polycarbonate membrane. Liposomes were stored at 4 °C, light-protected for up to 4 weeks.

The 50 nm empty vesicles prepared for in vitro and in vivo experiments were prepared in the same manner, except with a 50 nm extrusion membrane after extrusion through the 200 nm membrane.