Aim: Propose a nanoparticle for neuron-targeted retrograde gene delivery and describe a microfluidic-based culture system to provide insight into vector performance and safety. Methods: Using compartmentalized neuron cultures we dissected nanoparticle bioactivity upon delivery taking advantage of (quantitative) bioimaging tools. Results: Targeted and nontargeted nanoparticles were internalized at axon terminals and retrogradely transported to cell bodies at similar average velocities but the former have shown an axonal flux 2.7-times superior to nontargeted nanoparticles, suggesting an improved cargo-transportation efficiency. The peripheral administration of nanoparticles to axon terminals is nontoxic as compared with their direct administration to the cell body or whole neuron. Conclusion: A neuron-targeted nanoparticle system was put forward. Microfluidic-based neuron cultures are proposed as a powerful tool to investigate nanoparticle bio-performance.