Abstract
Exosomes, which are approximately 100 nm vesicles secreted by cells, have been studied with respect to cell-to-cell communication, disease diagnosis, and intracellular delivery. The cellular uptake of exosomes occurs by endocytosis; however, the cytosolic release efficiency of encapsulated molecules inside cells is low. To address this issue, here we demonstrate a simple technique for enhancing the cellular uptake and cytosolic release of exosomes by combining a pH-sensitive fusogenic peptide for the fusion of endosomal and exosomal membranes inside cells. This method stimulates the efficient cytosolic release of the exosomal contents with cationic lipids that act as a “glue” to support cellular uptake. Using this simple combined technique, the effective cellular uptake and cytosolic release of an artificially encapsulated dextran macromolecule (70 kDa) in exosomes are achieved, and a marked improvement in bioactivity is attained with the artificially encapsulated ribosome-inactivating protein saporin. Our method will contribute to many biological research fields, including the assessment of the activities of exosomal contents and the development of candidate tools enabling intracellular visualisation and cellular regulation for future therapeutic applications.
Highlights
Most cells constitutively secrete exosomes, which are vesicles ~100 nm in diameter with a lipid bilayer morphology found in abundance in body fluids including blood, saliva, urine, and breast milk[1,2]
We previously reported that a combination of cationic lipids and a pH-sensitive fusogenic peptide, GALA, significantly enhanced endosomal release of proteins that were conjugated with the GALA peptide[20,21] into the cytosol, and we have applied this methodology for enhanced cytosolic delivery of exosomal contents
We examined the cellular uptake of exosomes using a commercially available transfection reagent, Lipofectamine LTX, which contains cationic lipids that can act as a “glue” for pasting exosomes to targeted cells (Fig. 1)
Summary
Most cells constitutively secrete exosomes, which are vesicles ~100 nm in diameter with a lipid bilayer morphology found in abundance in body fluids including blood, saliva, urine, and breast milk[1,2]. New technology to enhance the cytosolic release of exosome contents is strongly needed to achieve effective biological and therapeutic activities of molecules contained in exosomes inside targeted cells. Cellular targeting of exosomes by fusion of cell receptor recognition proteins such as rabies viral glycoprotein (RVG)[16] and integrin-specific iRGD peptide for α v integrin targeting[19] with exosomal membrane proteins was reported; new techniques for enhanced cellular uptake and the cytosolic release of exosomal contents need to be developed to achieve sophisticated delivery vehicles. We achieved the efficient cytosolic release of exosomal contents in an endocytotic pathway using the GALA peptide by the combination of exosomes and cationic lipids that work as “glue” to accumulate targeted cellular membranes and enhance cellular uptake and cytosolic release (Fig. 1). Intracellular delivery of a ribosome-inactivating protein, saporin, using exosomes was achieved using cationic lipids and GALA peptides, leading to the efficient induction of cytotoxicity in targeted cells
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