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.

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.

We demonstrate a novel system for inducing clustering of cell surface receptors via recognition peptide segments displayed on exosomes, leading to receptor activation. With this system, targeting of receptor-expressing cells and facilitation of the endocytic uptake of exosomes, which contained the anti-cancer protein saporin, were successfully achieved, leading to cell death.

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Background/Aim: Extracellular vesicles (exosomes, EVs) (30-200 nm in diameter) are secreted by various cells in the body. Owing to the pharmaceutical advantages of EVs, an EV-based drug delivery system (DDS) for cancer therapy is expected to be the next-generation therapeutic system. However, preservation methods for functional and therapeutic EVs should be developed. Here, we developed the method of lyophilization of arginine-rich cell penetrating peptide (CPP)modified EVs and investigated the effects of lyophilization on the characteristics of EVs. Materials and Methods: Particle size, structure, zeta-potential, and cellular uptake efficacy of the arginine-rich CPP-modified EVs were analyzed. The model protein saporin (SAP), having anti-cancer effects, was encapsulated inside the EVs to assess the cytosolic release of EV content after cellular uptake. Results: Lyophilization of the EVs did not affect their particle size, structure, zeta-potential, and cellular uptake efficacy; however, the biological activity of the encapsulated SAP was inhibited by lyophilization. Conclusion: Lyophilization of EVs may affect SAP structures and/or reduce the cytosolic release efficacy of EV's content after cellular uptake and needs attention in EV-based DDSs.Extracellular vesicles (exosomes, EVs) are cellular membrane vesicles (ca. 30-200 nm diameter) that are secreted from almost all the cells of the body (1-3). EVs are generated in multivesicular endosomes (MVEs) by inward budding of cellular membranes. After fusion of plasma and MVE membranes, EVs are secreted and taken up by other cells via membrane fusion and endocytosis (1-3). Because of encapsulation of biofunctional molecules including microRNAs and enzymes in the MVEs, EVs participate in cell-to-cell communication and regulate cellular/body homeostasis and the progression of diseases such as cancer (1-4). In cancer cells, cell-to-cell communication based on EVs induces angiogenesis, tumor cell migration, metastasis, and immune response modulation (2, 3). EV-based diagnostics have gained increased attention owing to ability EVs to encapsulate disease-related proteins and microRNAs (3, 4). In addition, the development of EV-based drug delivery systems (DDSs) is highly expected to become the next-generation therapeutic system because of the several advantages of EVs, such as immunological control, usage of EV-based cell-to-cell communication routes, low toxicity, controlled and designed expression of protein in/on EVs, infinite secretion, and artificial/natural encapsulation of therapeutic molecular cocktails (3,4). A receptor target system has been developed by expressing on EV membranes, through genetic engineering, a sequence that interacts with a receptor target. Examples include the central nervous system-specific rabies viral glycoprotein (RVG) peptide for targeting acetylcholine receptor (5), anti-epidermal growth factor receptor (EGFR) nanobodies fused with glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF) (6),...

Exosomes (extracellular vesicles/EVs) participate in cell–cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP‐fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV‐based cell–cell communication.

The antimicrobial protein CAP18 (approximate molecular weight: 18 000), which was first isolated from rabbit granulocytes, comprises a C-terminal fragment that has negatively charged lipopolysaccharide binding activity. In this study, we found that CAP18 (106–121)-derived (sC18)2 peptides have macropinocytosis-inducible biological functions. In addition, we found that these peptides are highly applicable for use as extracellular vesicle (exosomes, EV)-based intracellular delivery, which is expected to be a next-generation drug delivery carrier. Here, we demonstrate that dimerized (sC18)2 peptides can be easily introduced on EV membranes when modified with a hydrophobic moiety, and that they show high potential for enhanced cellular uptake of EVs. By glycosaminoglycan-dependent induction of macropinocytosis, cellular EV uptake in targeted cells was strongly increased by the peptide modification made to EVs, and intriguingly, our herein presented technique is efficiently applicable for the cytosolic delivery of the biologically cell-killing functional toxin protein, saporin, which was artificially encapsulated in the EVs by electroporation, suggesting a useful technique for EV-based intracellular delivery of biofunctional molecules.

Boron neutron capture therapy (BNCT) is a radiation therapy for cancer. In BNCT, the internalization of boron-10 atoms by cancer cells induces cell death through the generation of α particles and recoiling lithium-7 nuclei when irradiated with lowenergy thermal neutrons. In this study, we aimed to construct exosomes [extracellular vesicles (EVs)]-based drug delivery technology in BNCT. Because of their pharmaceutical advantages, such as controlled immune responses and effective usage of cell-to-cell communication, EVs are potential next-generation drug delivery carriers. In this study, we successfully developed polyhedral borane anion-encapsulated EVs with modification of hexadeca oligoarginine, which is a cell-penetrating peptide, on the EV membrane to induce the actin-dependent endocytosis pathway, macropinocytosis, which leads to efficient cellular uptake and remarkable cancer cell-killing BNCT activity. The simple and innovative technology of the EV-based delivery system with "cassette" modification of functional peptides will be applicable not only for BNCT but also for a wide variety of therapeutic methodologies.