Anionic T Helper Liposome Generation

The generation of anionic T helper liposomes by thin-film hydration with a lipid composition of 45 mol% DSPC, 40 mol% cholesterol and 15 mol% DSPG was previously described in detail [24 (link)]. OT2 peptide solution was used for hydration of the lipid film. Subsequently, extrusion was performed at 55 °C to minimize peptide loss using a LIPEX™ Extruder (Northern Lipids Inc., Burnaby, BC, Canada) with Whatman^® Nucleopore track-etched membranes (5 cycles with 400 nm pore size, then 5–10 cycles with 200 nm or 100 nm pore sizes). Finally, the nanoparticle suspension was sterile-filtered (0.2 µm) and stored at 4 °C. Non-encapsulated peptides were removed by tangential flow filtration using a 100 kDa mPES MicroKros^® module (Repligen, Waltham, MA, USA) with a surface area of 20 cm² [24 (link)].