Liposome preparation

Lipids (67 mol % POPC, 20% DOPE, 10% chol, 1% biotin-DPPE, and 2% GD1a) were mixed in chloroform, which was evaporated under nitrogen to a thin film. Residual chloroform was removed by keeping the samples in vacuum for at least 3 h. The lipid films were hydrated in 10 μM DiYO or 16 mM calcein in 10 mM NaH₂PO₄, 90 mM sodium citrate, 150 mM NaCl (pH 7.45). The lipid suspension at a final lipid concentration of 0.56 mM was subjected to 15 freeze-thaw cycles, and large unilamellar vesicles were obtained by extruding the suspension through polycarbonate membrane filters (Avestin, Mannheim, Germany) with a nominal diameter of 100 or 200 nm. Excess unincorporated dye was removed using a Sephadex G-25 desalting column (Cytiva, Marlborough, MA, USA). Labeled liposomes used for individually correlating liposome diameter and content mixing times were prepared using including 0.5% 655 Atto DOPE in the initial lipid mixture and following the same procedure.