Figure 3.

Colocalization of nanoparticles with rabankyrin-5, EEA-1, caveolin-1, clathrin, rab11, and Lamp-1. hCMEC/D3 endothelial cells were incubated with nanoparticles for 30 minutes at 4 °C. Subsequently, cells were incubated for (a–c) 30 minutes, (d,e) 60 minutes, (f) 90 minutes, and (g) 18 hours at 37 °C to allow particle internalization. (a) NBs and PrPBs do not colocalize with rabankyrin-5. A significant fraction of PEIBs is located in rabankyrin-5-positive vesicles. (b) NBs do not show significant colocalization with the early endosomal marker EEA-1, while 33% of the PrPBs show colocalization with EEA-1. Fifty-five percent of the PEIBs colocalize with EEA-1. (c) NBs and PrPBs show a similar extent of colocalization with caveolin-1, around 20%, and clathrin, <5%. In contrast, PEIBs colocalize extensively with clathrin. (d) The profile of colocalization of nanoparticles with the markers caveolin and clathrin at 60 minutes is comparable to that at earlier time points. (e) NBs and PrPBs lack rab11-colocalization. Around 30% of PEIBs reach a rab11-positive compartment after 60 minutes of internalization. (f) NBs are not identified within caveolin- or clathrin-positive compartments. PrPBs show an increase in colocalization with caveolin and clathrin in time. PEIBs continually colocalize with clathrin. (g) After 18 hours of incubation of hCMEC/D3 cells with nanoparticles, NBs and PEIBs sporadically colocalize with Lamp-1, whereas PrPBs are not detected in Lamp-1-positives vesicles. Cav, caveolin; clath, clathrin; EEA-1, early endosomal antigen-1; NB, noncoated bead; PEIB, polyethyleneimine-coated bead; PrPB, prion-coated bead.