Copper-Free Click Chemistry Enables High-Fidelity Engineering of Mitochondria-Targeted Brain-Derived Exosomes

Area of Science:

• Biochemistry
• Neuroscience
• Cell Biology

Background:

• Mitochondrial dysfunction is implicated in neurodegenerative and neuroinflammatory diseases.
• Current methods for targeted mitochondrial delivery are limited in safety and precision.

Purpose of the Study:

• To establish strain-promoted azide-alkyne cycloaddition (SPAAC) as a platform for engineering mitochondria-targeted brain-derived exosomes (BR-EVs).
• To assess the biocompatibility, fidelity, and in vivo performance of SPAAC-modified BR-EVs.

Main Methods:

• Utilized copper-free click conjugation to attach mitochondrial-targeting ligands to BR-EVs under mild aqueous conditions.
• Evaluated vesicle morphology, proteomic composition, and uptake dynamics using imaging and FRAP.
• Performed in vivo neuroinflammation and biodistribution studies after intracerebroventricular administration.
• Conducted proteomic profiling of BR-EVs from Sprague-Dawley and hypertensive Dahl salt-sensitive rats.

Main Results:

• >75% mitochondrial colocalization achieved with preserved exosome morphology, proteomic integrity, and uptake kinetics.
• In vivo studies showed immunological neutrality, strong CNS retention, and minimal peripheral distribution.
• Hypertension-associated BR-EVs exhibited enriched oxidative and complement pathways, correlating with mitochondrial fragmentation and ROS generation.

Conclusions:

• SPAAC-mediated ligand conjugation is a biocompatible and precise method for creating mitochondria-targeted exosomes.
• This approach preserves exosome identity, biodistribution, and signaling fidelity.
• Establishes a foundational platform for organelle-specific delivery and mechanistic imaging in the CNS.