Abstract
Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.
Highlights
Extracellular vesicles (EVs) including exosomes (30–200 nm in diameter) are cell-secreted vesicles with a lipid bilayer
We demonstrated that the modification of EVs with octaarginine peptide, which is a representative arginine-rich cell-penetrating peptide (CPP), results in the effective induction of macropinocytosis and uptake of cellular EVs11
Before conducting the cellular EV uptake assay, we tested the cytotoxicity of Rn-EMCS-modified EVs (20 μg/ ml, 1.1 × 108 EV particles/ml) on Chinese hamster ovary (CHO)-K1 cells for 24 h at 37 °C in 10% fetal bovine serum (FBS)-containing medium prior to the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-t etrazolio]-1,3-benzene disulfonate) analysis and microscope observation (Supplementary Fig. 2)
Summary
Extracellular vesicles (EVs) including exosomes (30–200 nm in diameter) are cell-secreted vesicles with a lipid bilayer. We demonstrated that the modification of EVs with octaarginine peptide, which is a representative arginine-rich cell-penetrating peptide (CPP), results in the effective induction of macropinocytosis and uptake of cellular EVs11. Octaarginine peptide, which is a representative arginine-rich CPP, has been shown to induce clustering of syndecan-4 proteoglycan on plasma membranes, which results in the binding of PKCα to the V domain of the proteoglycan in the cytosol[19]. The number of arginine residues in the sequence of oligoarginine peptides has been shown to influence their cellular uptake and cytosolic release efficiency[20]. In this research, we studied how modifying the EV membranes using oligoarginine peptides with a different number of arginine residues in the peptide sequence impacts macropinocytosis induction, cellular EV uptake, and cytosolic release of EV contents.
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