Neural Tracing Protein‐Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons

Abstract Neural tracing proteins like horseradish peroxidase‐conjugated wheat germ agglutinin (WGA‐HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood–brain barrier (BBB). Conjugating WGA‐HRP to nanoparticles may enable the creation of BBB‐bypassing nanomedicine. Microfluidics and two‐photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run‐and‐pause pattern along the axon with endosomes carrying WGA‐HRP‐conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell‐based functional assay of neuron‐targeted nanoparticles with the goal of developing BBB‐bypassing nanomedicine for the treatment of nervous system disorders.