Abstract
Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.
Highlights
Given the importance of the artificial induction of macropinocytosis for the development of EV-based intracellular delivery systems, in this study, we demonstrate that modification of arginine-rich cell-penetrating peptides (CPPs) on EV membranes results in the effective induction of macropinocytosis and cellular EV uptake (Fig. 1)
Our research group found that the octaarginine peptide, which is a representative arginine-rich CPP, induces the clustering of the syndecan-4 proteoglycan on plasma membranes, resulting in the binding of PKCαto the V domain of the proteoglycan in the cytosol and the activation of PKCα25
EV membranes were modified with r8, which is a representative arginine-rich CPP18,19, by simple mixing with stearyl-r8, where the stearyl moiety served as an anchoring unit to membranes (Fig. 1)
Summary
Given the importance of the artificial induction of macropinocytosis for the development of EV-based intracellular delivery systems, in this study, we demonstrate that modification of arginine-rich cell-penetrating peptides (CPPs) on EV membranes results in the effective induction of macropinocytosis and cellular EV uptake (Fig. 1). Our research group found that the octaarginine peptide, which is a representative arginine-rich CPP, induces the clustering of the syndecan-4 proteoglycan on plasma membranes, resulting in the binding of PKCαto the V domain of the proteoglycan in the cytosol and the activation of PKCα25. We achieved the efficient cytosolic delivery of a ribosome-inactivating protein, saporin, using arginine-rich CPP-modified EVs, leading to the efficient induction of cytotoxicity in targeted cells
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