Figure 1.

Inhibition of lipid raft/caveolae-mediated endocytosis blocks internalization of NP1, and NP1 rapidly enters cells by endocytosis. (A) MC3T3-E1 cells were pretreated for 1 h with inhibitors of lipid rafts/caveolae (MβCD), macropinocytosis (Chloropromazine), and clathrin-mediated (Amiloride), followed by addition of NP1 (60 μg/mL) for 20 h. Fluorescent microscopy (top panels) was used to determine internalization of the particles and light microscopy for cell viability (bottom panels) (20× magnification). (B) The concentrations of multiple inhibitors used in the assay and whether fluorescent NP1 was detected in the cytoplasm. (C) MC3T3-E1 cells were treated with NP1 (60 μg/mL) (red) for 1 h, and the lysomal tracker (blue) (lysotracker, Invitrogen Molecular Bioprobes) and endosome tracker (green) (Transferrin-GFP, Invitrogen MB) were added according to the manufacturer’s protocol. Images were captured after 1 h by Zeiss LSM 510 META point scanning laser confocal microscope. Merged images identify overlap (yellow) of the nanoparticles and endosome tracker (white arrow) and a lack of overlap with lysosome tracker.