Syringe extruder

Lipids at the following molar ratios were mixed and dried under a nitrogen stream: HSPC/Chol/DSPE-PEG2000 (57/38/5) or EPC/DSPE-PEG2000 (95:5) with addition of 0.2% ICL and/or 0.2% FPL. The dry lipid cake was resuspended in PBS for a total lipid concentration of 20 mM and then incubated at 60 °C for 30 min. The solution was then vortexed for 2 min and bath sonicated. Liposomes were extruded by a syringe extruder (Avestin, Ottawa, Canada) through Whatman Nucleopore Track-Etch Membranes (200 nm pore size, 15 times). HSPC-based liposomes were extruded at 60 °C, EPC-based liposomes were extruded at room temperature. Size and ζ potential were measured at room temperature in 5% phosphate buffered saline using Zetasizer Nano (Malvern, UK). Liposomes were stored at 4 °C at a final concentration of 10 mM (total lipid) for a maximum period of 8 weeks before use.

Lipids were mixed at the following molar ratios: EPC/DSPE-PEG2000/DiD (mole %: 95/5/0.4) in chloroform and dried under a nitrogen stream. The dry lipid cake was resuspended in PBS for a total DiD concentration of 35μM, then incubated at 37°C for 30 minutes. The solution was extruded using a syringe extruder (Avestin, Ottawa, Canada) through Whatman Nucleopore Track-Etch Membranes (100 nm pore size 15 times). To prepare PLNs, DiI or DiD was mixed with DSPE-PEG2000 at 1:2 mole ratio in ethanol, dried, and resuspended in PBS at 35μM DiD. NanoSight 300 (Malvern, UK) was used to measure the size of the particles. Liposomes and PLNs were stored at 4°C for a maximum period of 4 weeks.