Preparation of fluorescent liposomes and size determination by dynamic light scattering.

A fluorescent lipid mixture composed of 62 mol% 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 16 mol% 1,2-dimyristoyl-sn-glycero-3-phospho(1’-rac-glycerol) (sodium salt) (DMPG), 16 mol% cholesterol, 5 mol% 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-1000] (ammonium salt) (DMPE PEG 1000), and 1 mol% (DiI) (ThermoFisher, D282) was dried under nitrogen gas and resuspended in 10 mM HEPES in Hank’s Balanced Salt Solution (HBSS, components purchased from Sigma-Aldrich). The resulting lipid mixture was vortexed for 5 minutes and shaken at 500 rpm overnight before being hand-extruded through a Nucleopore track-etched membrane with pore sizes of 0.03 µm, 0.1 µm, 0.2 µm, or 0.4 µm (Whatman). Liposome sizes after extrusion were determined by dynamic light scattering (DLS) using a Wyatt DynaPro Plate Reader II and Dynamics V7 software (Supplemental Table S1 and Fig. S1). Ten repeats were measured for each condition and the average liposome size was reported along with the polydispersity.