Abstract
Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.
Highlights
Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells
In our novel findings related to exosome cellular uptake mechanisms, we here demonstrate the active induction of macropinocytosis by stimulation of cancer-related receptors, including the epidermal growth factor receptor (EGFR), which significantly enhances the cellular uptake of exosomes (Fig. 1)
The results showed that the addition of Stromal cell-derived factor 1α (SDF-1α) increased the internalisation of exosomes by the cells (approximately 2.3-fold by co-treatment with SDF-1α (200 nM) with the exosomes) even in 10% foetal bovine serum (FBS)-containing cell culture medium, suggesting that CXCR4-dependent macropinocytosis increases the cellular uptake of exosomes
Summary
Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. We demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. Exosomes are highly anticipated to be next-generation therapeutic carriers with certain pharmaceutical advantages, including non-immunogenicity, constitutive secretion from cells, very low cytotoxicity, original and artificial encapsulation of bioactive molecules (especially microRNAs), and the protein engineering of the exosomal membrane[19]. In our novel findings related to exosome cellular uptake mechanisms, we here demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of the plasma membrane, and engulfment of large volumes of extracellular fluid24,25) by stimulation of cancer-related receptors, including the epidermal growth factor receptor (EGFR), which significantly enhances the cellular uptake of exosomes (Fig. 1). We here demonstrate that the electroporation of exosomes for the artificial encapsulation of EGF significantly enhanced their cellular uptake, and the encapsulation of saporin with EGF in exosomes using our simple electroporation method resulted in superior induction of cytotoxicity compared with that of saporin-encapsulated exosomes without co-encapsulation of EGF
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