Tracking the endocytic pathway of recombinant protein toxin delivered by multiwalled carbon nanotubes

The endocytic pathway of a recombinant protein toxin, ricin A-chain (RTA), delivered by multiwalled carbon nanotubes (MWCNTs) was tracked in HeLa cells by tagging RTA with enhanced green fluorescent protein (EGFP). EGFP-RTA was found to accumulate in the endosome and to be retrogradely transported to the endoplasmic reticulum, from which it translocated into the cytosol. Nuclear staining, Z-axis scanning with a laser scanning confocal microscope (LSCM), and transmission electron microscopy (TEM) indicated that the RTA exerted its toxic effects. Endocytosis-inhibiting tests with LSCM and flow cytometry showed that MWCNT-EGFP-RTA conjugates penetrated cells principally via clathrin-mediated endocytosis. These studies are beneficial to understanding the MWCNT-based intracellular drug delivery mechanism and provide guidelines for designing promising MWCNT-based vectors for targeting diagnostic or therapeutic compounds, not only to specific cells, but even to specific cellular compartments.