Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles

• Published in: Scientific reports Vol. 10; no. 1; p. 18718
• Main Authors: Ishida, Takenori, Hashimoto, Takuma, Masaki, Kanako, Funabashi, Hisakage, Hirota, Ryuichi, Ikeda, Takeshi, Tajima, Hideji, Kuroda, Akio
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 30.10.2020; Nature Publishing Group
• Subjects: 631/1647; 631/61; 692/53; Automation; Biomarkers - chemistry; Breast cancer; Cell culture; Drug development; Exosomes; Exosomes - chemistry; Extracellular vesicles; Extracellular Vesicles - chemistry; Humanities and Social Sciences; Humans; Immunomagnetic Separation; Lipids - chemistry; Lysine; Lysine - chemistry; MCF-7 Cells; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; multidisciplinary; Particle Size; Peptides; Peptides - chemistry; Phosphatidylserine; Phosphatidylserines - chemistry; Phospholipids; Phospholipids - chemistry; Protein purification; Reproducibility of Results; Science; Science (multidisciplinary); Sodium chloride; Ultracentrifugation
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-75561-0

Extracellular vesicles (EVs), such as exosomes, have garnered increasing interest because of their potential clinical applications that range from diagnostics to therapeutics. The development of an automated and reproducible EV purification platform would therefore aid the introduction of EV biomarkers and therapies into the clinic. Here, we demonstrate that K8- as well as K-16 peptides (containing 8 and 16 lysine residues with dissociation constants of 102 nM and 11.6 nM for phosphatidylserine, respectively) immobilized on magnetic beads can capture small EVs (< 0.2 µm) from culture supernatants of MCF7 human breast cancer cells. Importantly, the bound EVs could be dissociated from the beads under mild conditions (e.g. 0.5 M NaCl), and the isolated EVs had the typical shapes of EVs under SEM and TEM with a mean particle size of 99 nm. Using the peptide-immobilized beads, we adapted a pre-existing bench top instrument for magnetic separation to perform automated EV purification with higher purity and yield than that obtained using the standard ultracentrifugation method.