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Extracellular vesicles (EVs) are cell-derived lipid membrane capsules that can deliver functional molecules, such as nucleic acids, to target cells. Currently, the application of EVs is limited because of the difficulty of loading cargo into EVs. We constructed hybrid EVs by the fusion of liposomes and insect cell-derived EVs expressing recombinant programmed cell death 1 (PD-1) protein and baculoviral fusogenic glycoprotein gp64, and evaluated delivery of the model cargo molecule, Texas Red-labeled dextran (TR-Dex), into the cytosol. When PD-1 hybrid EVs were added to HeLa cells, the intracellular uptake of the hybrid EVs was increased compared with hybrid EVs without PD-1. After cellular uptake, the PD-1 hybrid EVs were shown to be localized to late endosomes or lysosomes. The results of fluorescence resonance energy transfer (FRET) indicated that membrane fusion between the hybrid EVs and organelles had occurred in the acidic environment of the organelles. When TR-Dex-loaded liposomes were fused with the PD-1 EVs, confocal laser scanning microscopy indicated that TR-Dex was distributed throughout the cells, which suggested that endosomal escape of TR-Dex, through membrane fusion between the hybrid EVs and acidic organelles, had occurred. These engineered PD-1 hybrid EVs have potential as delivery carriers for biopharmaceuticals.

Extracellular vesicles (EVs) have potential biomedical applications, particularly as a means of transport for therapeutic agents. There is a need for rapid and efficient EV‐liposome membrane fusion that maintains the integrity of hybrid EVs. We recently described Sf9 insect cell‐derived EVs on which functional membrane proteins were presented using a baculovirus‐expression system. Here, we developed hybrid EVs by membrane fusion of small liposomes and EVs equipped with baculoviral fusogenic proteins. Single‐particle analysis of EV‐liposome complexes revealed controlled introduction of liposome components into EVs. Our findings and methodology will support further applications of EV engineering in biomedicine.

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