Trehalose-Functionalized Magnetic Affinity Probe Provides Biochemical Evidence of Nanoparticle Internalization in Mycobacteria

We developed a magnetic affinity probe (MAP), consisting of iron oxide magnetic nanoparticles (MNP) functionalized with a photoaffinity labeling agent perfluorophenyl azide (PFPA), to characterize the internalization of nanoparticles by <i>Mycobacterium smegmatis</i>. Two MAPs were synthesized: a trehalose-functionalized MAP, PFPA-MNP-Tre, and an ethanol-functionalized MAP, PFPA-MNP-OH. Following incubation of MAP with bacteria, the samples were irradiated to trigger covalent bond formation between PFPA and bacterial proteins. The captured proteins were isolated by cleaving the disulfide bond in the linkers and removing the magnetic nanoparticles by using a magnet. For PFPA-MNP-Tre incubated with <i>M. smegmatis</i> for 24 h, proteomic analysis revealed that the captured proteins are cytoplasmic mycobacterial proteins, which provided biochemical evidence for the internalization of nanoparticles in bacteria. Additionally, PFPA-MNP-Tre accumulated at the poles of the mycobacteria, and the amount of captured proteins decreased with increasing concentration of added free trehalose. These results underscore the role the surface ligand plays in modulating the uptake of nanoparticles. The modular MAP platform may find broad applications in studying mechanisms and processes involving nanoparticle-cell interactions.