Preparation and Characterization of PEGylated Liposomes

In the preparation using the bulk mixing post-insertion method, KK-(EK)₄-lipid micelles (6 mol%) were incubated with PEGylated liposomes for 1 h at 60 °C. In the preparation using the microfluidic post-insertion method, the heat block of NanoAssemblr Benchtop (Precision NanoSystems Inc., Vancouver, BC, Canada) was set at 60 °C, and PEGylated liposomes and 6 mol% KK-(EK)₄-lipid micelles were mixed at a total flow rate (TFR) of 1 mL and a flow rate ratio (FRR) of 1:1 onto the microfluidic chip as previously described (Sugimoto et al., 2022 (link)). After reaching room temperature, the isotonic properties of the liposomes were adjusted by the addition of 10 × phosphate buffered saline (PBS, pH 7.4), and the samples were filtered through a 0.45-μm filter. The physicochemical properties of the samples were measured using a Zetasizer Nano ZS and a Zetasizer Pro (Malvern Instruments Ltd., Worcestershire, UK).