200 nm polycarbonate filter

Both niosomes and liposomes were formed from the corresponding amphiphiles by thin film hydration. The amphiphiles, lipids or non-ionic surfactants, dissolved in chloroform:methanol (9:1) were mixed and dried by rotary evaporation and subsequently hydrated in the appropriate buffer. Liposomes were flash-frozen in liquid nitrogen and thawed (5x) and stored in liquid N₂, after which the multilamellar vesicles were extruded 15x through a 200 nm polycarbonate filter (Avestin). Unless indicated otherwise, niosomes were not frozen in liquid nitrogen prior to extrusion, since this reduces the size of the vesicles (see Results section). Niosomes formed by thin film hydration were stored at room temperature and were extruded 15x through a 200 nm polycarbonate filter (Avestin). The liposome and niosome compositions are presented in Table 1.

For cryo-EM, niosomes composed of unsaturated surfactants or liposomes composed of unsaturated lipids were subjected to five freezing and thawing cycles as indicated above and extruded 15x through a 200 nm polycarbonate filter (Avestin). To examine the effect of the freezing and thawing, similar vesicles were formed with the freezing and thawing step omitted. Vesicles (10 mM of lipids or surfactants) were placed on a grid and vitrified using a vitrobot (FEI). Images were recorded on a Tecnai T20 microscope (FEI) with a Gatan cryo-stage (model 626) operating at 200 keV. Images were recorded under low-dose conditions on a slow scan CCD Camera. DLS was performed using the Dynapro Nanostar apparatus, and the results were analyzed with dynamics software, version 7.