The liposomes were prepared based on the conventional dehydration–rehydration method as previously reported.9 (link)–11 Ten micromoles of lipids in chloroform (a lipid composition of DOPC:DOPG:MPB = 4:1:5 molar ratio was typically used) were dispensed into small round-bottom flasks, and the organic solvents were evaporated under nitrogen overnight to prepare dried thin lipid films. The lipid films were rehydrated at room temperature in 1 mL 0.2 M bis-tris buffer at pH 7.0 for 1 h with rigorous vortexing for 30 s every 5 min, and then sonicated in alternating power cycles of 8% amplitude (~30W) in 30 s intervals for 5 min on ice. The resulting liposomes were extruded 21 times through a 0.2 µm polycarbonate membrane (Whatman, Piscataway, NJ, USA) using an Avanti Mini-Extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA). The liposome solutions were freshly made and used shortly after preparation to avoid the ring-opening (hydrolysis) reactions of the maleimide moieties, which might prevent them from further reactions.66 (link) The average size of the monodisperse liposomes was analyzed via DLS at 25 °C on a Malvern Zetasizer Nano ZS apparatus with Malvern Instruments DTS software (v.6.01) using the cumulants fit (Malvern Instruments, Malvern, Worcestershire, UK).