Fusogenic Hybrid Extracellular Vesicles with PD-1 Membrane Proteins for the Cytosolic Delivery of Cargos

SIMPLE SUMMARY: We developed a method using cell-derived lipid membrane capsules—called extracellular vesicles (EVs)—to deliver a model cargo into cytosol. These EVs were fused with liposomes (to form hybrid EVs) because cargo molecules can be more easily encapsulated within liposomes than EVs. EVs were engineered that expressed programmed cell death 1 (PD-1) and baculoviral envelope glycoprotein (gp64), which enabled the hybrid EVs to be internalized in cells and fuse with acidic organelles. The model cargo, Texas Red-labeled dextran, was shown to be released to the cytosol from the hybrid EVs by fusion with acidic organelles, such as late endosomes and lysosomes. Thus, these hybrid EVs are potential drug delivery carriers. ABSTRACT: 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.