Loading of Extracellular Vesicles with Nucleic Acids via Hybridization with Non‐Lamellar Liquid Crystalline Lipid Nanoparticles

The translation of cell‐derived extracellular vesicles (EVs) into biogenic gene delivery systems is limited by relatively inefficient loading strategies. In this work, the loading of various nucleic acids into small EVs via their spontaneous hybridization with preloaded non‐lamellar liquid crystalline lipid nanoparticles (LCNPs), forming hybrid EVs (HEVs) is described. It is demonstrated that LCNPs undergo pH‐dependent structural transitions from inverse hexagonal ( H _II ) phases at pH 5 to more disordered non‐lamellar phases, possibly inverse micellar ( L ₂ ) or sponge ( L ₃ ) phases, at pH 7.4, which are particularly suitable for inducing a controlled hybridization process with EVs. State‐of‐the‐art single‐particle analysis techniques reveal that LCNPs interact with various EV subpopulations at physiological conditions and that ≈40% of HEVs are loaded with the genetic cargo. Importantly, this study demonstrates that EV membrane proteins remain accessible on HEV surfaces, with their intrinsic enzymatic activity unaffected after the hybridization process. Finally, HEVs show in vitro improved transfection efficiencies compared to unhybridized LCNPs. In summary, this versatile platform holds potential for loading various nucleic acid molecules into native EVs and may help developing EV‐based therapeutics.