Nanoparticle Characterization by Multitechnique

The hydrodynamic diameter of the nanoparticles was assessed using nanoparticle tracking analysis (NS500 Nanosight, Malvern-Panalytical, Malvern, UK) according to protocols published previously [18 (link),19 (link)], and now an established protocol for the EUNCL (EU Nanomedicine Characterisation Laboratory) [20 ]. These results were confirmed with dynamic light scattering (DLS) measurements (Malvern Nano- ZS, Malvern-Panalytical, Malvern, UK), following the EUNCL protocol for DLS size analysis [21 ]. Zeta potential data was provided by the nanoparticle supplier (at pH 7), Chemicell. For determining the dry diameter of the nanoparticles, transmission electron microscopy was used. Here, nanoparticles were diluted 1 in 1000 from the stock (100 mg/mL) in double distilled water (ddH₂0) and adhered to Lacey carbon grids (AGAR Scientific, Stansted, UK). Images were taken using the JOEL 2100 (JOEL, Tokyo, Japan) at an acceleration of 200 kV and a beam current of 100–110 µA. The longest diameter of 200 individual nanoparticles was measured using ImageJ software to generate a size distribution (for a description of hydrodynamic size, zeta potential and dry size analysis, see Figure S1).