Abstract
Purpose
Phosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS(−) sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS(−) sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed.
Methods
Bulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS(−) sEVs was estimated using magnetic beads that bind to PS(+) sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated.
Results
Enzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS(−) sEVs. PSD treatment hardly changed the physicochemical properties of PS(−) sEVs. Moreover, the serum concentration profile and PK parameters of the PS(−) sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life.
Conclusions
Treatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS(−) sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS(−) sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.
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References
Yáñez-Mó M, Siljander PRM, Andreu Z, Zavec AB, Borràs FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. Co-Action Publishing; 2015. p. 1–60.
Raposo G, Stoorvogel W. Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol. 2013. p. 373–83.
Takahashi Y, Nishikawa M, Shinotsuka H, Matsui Y, Ohara S, Imai T, et al. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. J Biotechnol. 2013;165:77–84.
Imai T, Takahashi Y, Nishikawa M, Kato K, Morishita M, Yamashita T, et al. Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice. J Extracell Vesicles Co-Action Publishing. 2015;4:1–8.
Morishita M, Takahashi Y, Nishikawa M, Sano K, Kato K, Yamashita T, et al. Quantitative analysis of tissue distribution of the B16BL6-derived exosomes using a streptavidin-lactadherin fusion protein and Iodine-125-Labeled biotin derivative after intravenous injection in mice. J Pharm Sci. John Wiley and Sons Inc.; 2015;104:705–13.
Takakura Y, Matsumoto A, Takahashi Y. Therapeutic Application of Small Extracellular Vesicles (sEVs): Pharmaceutical and Pharmacokinetic Challenges. Biol. Pharm. Bull. 2020.
Matsumoto A, Takahashi Y, Nishikawa M, Sano K, Morishita M, Charoenviriyakul C, et al. Role of Phosphatidylserine-Derived Negative Surface Charges in the Recognition and Uptake of Intravenously Injected B16BL6-Derived Exosomes by Macrophages. J Pharm Sci. Elsevier B.V.; 2017;106:168–75.
Charoenviriyakul C, Takahashi Y, Morishita M, Nishikawa M, Takakura Y. Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles. Mol Pharm American Chemical Society. 2018;15:1073–80.
Yamamoto A, Yasue Y, Takahashi Y, Takakura Y. Determining The Role of Surface Glycans in The Pharmacokinetics of Small Extracellular Vesicles. J Pharm Sci. Elsevier B.V.; 2021;110:3261–7.
Matsumoto A, Takahashi Y, Ogata K, Kitamura S, Nakagawa N, Yamamoto A, et al. Phosphatidylserine-deficient small extracellular vesicle is a major somatic cell-derived sEV subpopulation in blood. iScience. Elsevier Inc.; 2021;24.
Subra C, Laulagnier K, Perret B, Record M. Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies. Biochimie. 2007. p. 205–12.
Llorente A, Skotland T, Sylvänne T, Kauhanen D, Róg T, Orłowski A, et al. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2013;1831:1302–9.
Matsumoto A, Takahashi Y, Chang HY, Wu YW, Yamamoto A, Ishihama Y, et al. Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance. J Extracell Vesicles. Taylor and Francis Ltd.; 2020;9.
Yamashita T, Takahashi Y, Nishikawa M, Takakura Y. Effect of exosome isolation methods on physicochemical properties of exosomes and clearance of exosomes from the blood circulation. Eur J Pharm Biopharm. Elsevier B.V.; 2016;98:1–8.
Charoenviriyakul C, Takahashi Y, Morishita M, Matsumoto A, Nishikawa M, Takakura Y. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics. Eur J Pharm Sci. Elsevier B.V.; 2017;96:316–22.
Liu W, Takahashi Y, Morishita M, Nishikawa M, Takakura Y. Development of CD40L-modified tumor small extracellular vesicles for effective induction of antitumor immune response. Nanomedicine Future Medicine Ltd. 2020;15:1641–52.
Rozhdestvensky TS, Tang TH, Tchirkova I v, Brosius J, Bachellerie J-P, Hüttenhofer A. Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 2003;31:869–77.
Patel GK, Khan MA, Zubair H, Srivastava SK, Khushman M, Singh S, et al. Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications. Sci Rep. Nature Publishing Group; 2019;9.
Vance JE, Tasseva G. Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells. Biochim Biophys Acta Mol Cell Biol Lipids. 2013. p. 543–54.
Drechsler C, Markones M, Choi JY, Frieling N, Fiedler S, Voelker DR, et al. Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase. Biophys J Biophysical Society. 2018;115:1509–17.
Funding
This research was supported in part by JSPS KAKENHI grants (number JP20H04533 and JP21H02616) from the Japan Society for the Promotion of Science (JSPS).
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Kobayashi, Y., Kitamura, S., Takahashi, Y. et al. Development of Enzymatic Depletion Methods for Preparation of Small Extracellular Vesicles with Long Blood-Circulation Half-Life. Pharm Res 40, 855–861 (2023). https://doi.org/10.1007/s11095-022-03405-9
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DOI: https://doi.org/10.1007/s11095-022-03405-9