Analysis auto version 3

The diameter and size distribution of the aqueous NP dispersions were measured by cryo-TEM. Briefly, a small droplet was placed on a perforated lacey carbon film supported on a TEM copper grid. The drop was blotted with a piece of filter paper, resulting in formation of a thin film of 100–300 nm. The specimen was subsequently plunged into a reservoir of liquid ethane cooled by liquid nitrogen to ensure vitrification (rapid freezing) and prevent ice crystal formation. The vitrified specimen was transferred under liquid nitrogen and mounted on a cryogenic sample holder cooled to −170 °C. All samples were observed under low-dose conditions. Vitrified samples were examined using a FEI T12 G2 Cryo-TEM (Hillsboro, Oregon, USA) operating at 120 kV and equipped with a Gatan 626 cryo-holder system. The mean diameter was determined by measuring at least 200 particles using the image analysis software AnalySIS Auto version 3.2 (Soft Imaging System GmbH, Münster, Germany). In addition, the hydrodynamic diameter and size distribution of the aqueous NP dispersions were measured at room temperature with a particle DLS analyzer (Nanophox, Sympatec GmbH, Clausthal-Zellerfeld, Germany).

The hydrodynamic diameter and size distribution of the NCs were measured with a cryogenic transmission electron microscope (cryo-TEM). Aqueous NC dispersions (3 μL) were loaded on a glow discharged (EmiTech K100 machine, Quorum Technologies, East Sussex, UK) Quantifoil or lacey grid, which was blotted and plunged into liquid ethane using a Gatan CP3 automated plunger (Gatan, Pleasanton, CA, USA) and stored in liquid nitrogen until use. Frozen specimens (samples with vesicles embedded in vitreous ice) were transferred to a Gatan 914 cryo-holder and maintained at temperatures below −176 °C inside the microscope. Samples were inspected with a FEI Tecnai G2 microscope (now Thermo-Fisher Scientific, Waltham, MA, USA) with an acceleration voltage of 120 kV equipped with a cryobox decontaminator. Images were taken using a digital micrograph (Gatan, ver. 1.83.842) with a mulitiscan camera (Gatan, model 794) at varying resolution. The mean diameter was determined by measuring at least 200 particles using the image analysis software AnalySIS Auto version 3.2 (Soft Imaging System GmbH, Münster, Germany). Each experiment was repeated at least three times.