Cells were seeded into 6-well plates (in duplicate) at the density of 20,000 cells/cm2. Once cells were attached to the plate, different doses of NBTXR3 were added overnight, according to the cell line considered (ranging from 100µM to 800µM), as indicated on Figure 1. Cells were then pre-fixed with 2.5% glutaraldehyde in cacodylate 0.1M at pH 7.4 (Sigma Aldrich) for 2 hours. Cells were subsequently post-fixed with 1% osmium tetroxide (Sigma Aldrich) for 45 minutes, washed and dehydrated in graded concentrations of ethanol (50%, 70%, 95% and 100%) (Sigma Aldrich). Cell samples were embedded with EPON (Oxford Instruments). The resin sample block was trimmed, 90µm thin sectioned to thickness of 70nm, (diatome diamond knives, Euromedex). Thin sections (70nm) were collected onto 200 mesh cooper palladium grids, and counterstained with lead citrate before examination with a Zeiss EM 902 transmission electron microscope (TEM) at 80KV (UR 1196 GPL: Génomique et Physiologie de la Lactation - MIMA2 – plateau de Microscopie Electronique – Jouy-en-Josas). Microphotographs were acquired using MegaView III CCD camera and analyzed with ITEM software (Eloïse). Ten (10) cells were observed to estimate NBTXR3 nanoparticle cell uptake.

NBTXR3 nanoparticles are endocytosed by cancer cells and form clusters in the cytoplasm. Transmission electronic microscopy (TEM) representative images of NBTXR3 nanoparticles uptake, forming clusters in CAL-33, FaDu, NCI-H460-Luc2, MDA-MB-231-luc-D3-H2LN (upper panel) and DU-145, LNCaP, PC-3, Detroit 562 cells (lower panel). Cells were treated overnight with NBTXR3. 100µM, 400µM and 800µM indicate concentrations of NBTXR3 added to cells; arrows indicate NBTXR3 clusters.

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