Quantum dots (QDs) have high photostability and brightness that allow single-particle tracking in cells and tissues by fluorescence microscopy. But over a decade after they were first introduced in biology, it remains challenging to use QDs to label specific biomolecules because of their tendency to aggregate and technical difficulties in functionalizing them. To overcome this limitation, Bhatia et al.1 encapsulated QDs in DNA icosahedra, artificial 20-faced DNA 3D structures that can be functionalized in a uniform, stable fashion. The combination of QDs with targetable DNA icosahedra allows detailed live imaging of endocytosis and in-depth characterization of endosome dynamics along different endocytic routes.

DNA polyhedra have notable advantages as small-molecule carriers. They can be readily loaded with nanoscale particles, such as QDs, without compromising the properties of the cargo. Previous work from the same laboratory showed that DNA polyhedra can be loaded with fluorescent molecules for imaging studies2. In their new paper, the authors encapsulated one CdSe/CdS/ZnS QD per DNA icosahedron by incubating QDs with a mix of two half-icosahedra in solution. The QDs were 5 nm in diameter, but the internal space of the icosahedra can potentially hold QDs up to 11 nm in diameter.

To identify the best residues in the DNA shell for displaying targeting molecules, the authors used AMBER (a software package for molecular dynamics simulations) to assess the stability of DNA icosahedra following monofunctionalization with folic acid at different positions of the scaffold. The simulations revealed icosahedra residues that exposed folate to the outside, and one of the seven conformations tested in vitro showed efficient uptake by cells in a folate-dependent fashion.

The ability to functionalize QDs with small molecules enabled real-time tracking of subcellular endocytic pathways. Using different endocytic ligands, such as folic acid, galectin-3 (Gal3) and Shiga toxin B-subunit (STxB), the authors could follow QDs along endocytic pathways specific for each ligand. As a first example, icosahedra functionalized with folate were seen to colocalize with the folic-acid endocytosis pathway, and their uptake was inhibited by adding excess folate, suggesting that uptake was folate-specific. The authors also showed that Gal3-functionalized icosahedra revealed the morphology of endosomal compartments at different stages along the Gal-3 endocytic pathway, including early and late endosome-like structures.

Finally, QDs tagged with STxB were used to image the dynamics of endosomal trafficking. Particle diffusion behavior on the extracellular face of the plasma membrane of HeLa cells was quantified by internal reflection fluorescence microscopy—an observation made possible thanks to the photostability of QDs. The results suggested a picket-fence-type compartmentalization of the underlying actin meshwork. The STxB QDs also enabled detailed study of the dynamics of endosome trafficking along the microtubule network. Internalized particles actively moved and fused with early endosomes, and individual endosomes carrying the nanoparticles alternated between active movement and diffusive behavior.

This innovative approach to target QDs could in principle be adapted to image dynamic biological processes other than endocytosis. “The cellular imaging and tracking results are very impressive,” says Shuming Nie, a researcher at Emory University and the Georgia Institute of Technology, who was not involved in the study. Nevertheless, he says, “one major limitation is that this class of QD probes is still fairly bulky (about 10 nm in diameter), which could restrict diffusion and binding inside the highly crowded cytoplasm.” In addition to reducing particle size, functionalization with multiple ligands may offer another approach to optimize the technology.