Trehalose-FunctionalizedMagnetic Affinity Probe ProvidesBiochemical Evidence of Nanoparticle Internalization in Mycobacteria

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We developed a magneticaffinity probe (MAP), consistingof ironoxide magnetic nanoparticles (MNP) functionalized with a photoaffinitylabeling agent perfluorophenyl azide (PFPA), to characterize the internalizationof nanoparticles by Mycobacterium smegmatis. Two MAPs were synthesized: a trehalose-functionalized MAP, PFPA-MNP-Tre,and an ethanol-functionalized MAP, PFPA-MNP-OH. Following incubationof MAP with bacteria, the samples were irradiated to trigger covalentbond formation between PFPA and bacterial proteins. The captured proteinswere isolated by cleaving the disulfide bond in the linkers and removingthe magnetic nanoparticles by using a magnet. For PFPA-MNP-Tre incubatedwith M. smegmatis for 24 h, proteomicanalysis revealed that the captured proteins are cytoplasmic mycobacterialproteins, which provided biochemical evidence for the internalizationof nanoparticles in bacteria. Additionally, PFPA-MNP-Tre accumulatedat the poles of the mycobacteria, and the amount of captured proteinsdecreased with increasing concentration of added free trehalose. Theseresults underscore the role the surface ligand plays in modulatingthe uptake of nanoparticles. The modular MAP platform may find broadapplications in studying mechanisms and processes involving nanoparticle–cellinteractions.

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