Macromolecule‐Loaded Hybrid Extracellular Vesicles via Ionic Lipid–Based Post‐Loading for Intracellular Delivery: Functional Evaluation for Neurodegenerative Therapy

Extracellular vesicles (EVs) are natural carriers that show promise as drug delivery systems (DDS). We developed a non‐invasive post‐loading method to encapsulate macromolecules in EVs using our proprietary ionic lipid base (ILB), which enables electrostatically driver hybridisation without disrupting EV structure. Using EVs isolated from bovine milk and adipocyte‐derived mesenchymal stem cell culture medium, hybrid‐EVs (H‐EV) with different ILB contents encapsulating protein or nucleic acid molecules for delivery were produced in a reproducible manner. The H‐EV retained EV surface markers comparable to those of native EVs after preparation. After cellular uptake, the encapsulated molecules escaped from endosomes into the cytoplasm and exhibited intended functions. In an experiment using SH‐SY5Y neuroblastoma cells in which α‐synuclein (αSyn) aggregation was induced, the introduction of H‐EV encapsulating anti‐αSyn antibodies (Abs) significantly suppressed αSyn aggregation. Furthermore, delivery of anti‐phospho‐AKT Abs using H‐EV promoted caspase 3/7 activity and cell apoptosis. Intravenous administration of H‐EV encapsulating a model Ab into mice resulted in detectable Ab signals in the cerebral cortex, cerebellum, hippocampus and other cells associated with neurodegenerative diseases within 24 h, as assessed by ex vivo imaging. H‐EV enabled loading of various molecules and their targeted transport to specific organs and the cytoplasm, highlighting their potential as a versatile platform for Ab therapies targeting intracellular proteins.