Non-covalent delivery of proteins into mammalian cells

Substances that mediate the import of proteins into cells, “carriers”, have many potential applications. The most potentially useful carriers do not have to be covalently linked to their protein cargoes. However, a common problem with all carrier molecules is that they tend to deposit the cargo proteins into endosomes; diffuse distribution in the cytosol is the desired outcome. This paper describes the import of four different labeled (Alexa Fluor® 488) proteins (avidin, recombinant streptavidin, bovine serum albumin, and β-galactosidase), with the well-known non-covalent carrier called pep-1 (also known as Chariot™), with R8 (a molecule that is not widely appreciated to import protein cargoes via a non-covalent mode of action), and with a new molecule called azo-R8. The data collected from fluorescence microscopy and flow cytometry indicate that all three non-covalent carriers can facilitate transport. At 37 °C, import into endocytic compartments dominates, but at 4 °C weak, diffuse fluorescence is observed in the cytosol, indicative of a favorable mode of action.