Loading of extracellular vesicles with nucleic acids via hybridization with sponge-like 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, we describe the loading of various nucleic acids into small EVs via their spontaneous hybridization with preloaded non-lamellar liquid crystalline lipid nanoparticles (LCNPs) under physiological conditions, forming hybrid EVs (HEVs). We correlate LCNPs’ topological characteristics with their propensity to fuse/aggregate with EVs and found that sponge (L ₃ ) phases at pH 7.4 were particularly suitable to induce a controlled hybridization process. State-of-the-art single-particle analysis techniques revealed that L ₃ -based LCNPs interact with various EV subpopulations and that around 40% of HEVs were 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 showed 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.