Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism.

ABSTRACT

BACKGROUND: There is a huge effort in developing ligand-mediated targeting of nanoparticles to diseased cells and tissue. The plant toxin ricin has been shown to enter cells by utilizing both dynamin-dependent and -independent endocytic pathways. Thus, it is a representative ligand for addressing the important issue of whether even a relatively small ligand-nanoparticle conjugate can gain access to the same endocytic pathways as the free ligand. RESULTS: Here we present a systematic study concerning the internalization mechanism of ricinBQuantum dot (QD) nanoparticle conjugates in HeLa cells. Contrary to uptake of ricin itself, we found that internalization of ricinBQDs was inhibited in HeLa cells expressing dominant-negative dynamin. Both clathrin-, Rho-dependent uptake as well as a specific form of macropinocytosis involve dynamin. However, the ricinBQD uptake was not affected by siRNA-mediated knockdown of clathrin or inhibition of Rho-dependent uptake caused by treating cells with the Clostridium C3 transferase. RicinBQD uptake was significantly reduced by cholesterol depletion with methyl-ß-cyclodextrin and by inhibitors of actin polymerization such as cytochalasin D. Finally, we found that uptake of ricinBQDs was blocked by the amiloride analog EIPA, an inhibitor of macropinocytosis. Upon entry, the ricinBQDs co-localized with dextran, a marker for fluid-phase uptake. Thus, internalization of ricinBQDs in HeLa cells critically relies on a dynamin-dependent macropinocytosis-like mechanism. CONCLUSIONS: Our results demonstrate that internalization of a ligand-nanoparticle conjugate can be dependent on other endocytic mechanisms than those used by the free ligand, highlighting the challenges of using ligand-mediated targeting of nanoparticles-based drug delivery vehicles to cells of diseased tissues.