Exosomes separation with aqueous two-phase systems from bovine milk

doi:10.1016/j.cjche.2024.12.016

Abstract

Abstract: The exosomes hold significant potential in disease diagnosis and therapeutic interventions. The objective of this study was to investigate the potential of aqueous two-phase systems (ATPSs) for the separation of bovine milk exosomes. The milk exosome partition behaviors and bovine milk separation were investigated, and the ATPSs and bovine milk whey addition was optimized. The optimal separation conditions were identified as 16% (mass) polyethylene glycol 4000, 10% (mass) dipotassium phosphate, and 1% (mass) enzymatic hydrolysis bovine milk whey. During the separation process, bovine milk exosomes were predominantly enriched in the interphase, while protein impurities were primarily found in the bottom phase. The process yielded bovine milk exosomes of 2.0 × 10¹¹ particles per ml whey with high purity (staining rate>90%, 7.01 × 10¹⁰ particles per mg protein) and high uniformity (polydispersity index <0.03). The isolated exosomes were characterized and identified by transmission electron microscopy, zeta potential and size distribution. The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey, presenting a novel approach for the large-scale isolation of exosomes.

Key words: Aqueous two-phase extraction, Bovine milk exosomes, Separation, Purification

摘要： The exosomes hold significant potential in disease diagnosis and therapeutic interventions. The objective of this study was to investigate the potential of aqueous two-phase systems (ATPSs) for the separation of bovine milk exosomes. The milk exosome partition behaviors and bovine milk separation were investigated, and the ATPSs and bovine milk whey addition was optimized. The optimal separation conditions were identified as 16% (mass) polyethylene glycol 4000, 10% (mass) dipotassium phosphate, and 1% (mass) enzymatic hydrolysis bovine milk whey. During the separation process, bovine milk exosomes were predominantly enriched in the interphase, while protein impurities were primarily found in the bottom phase. The process yielded bovine milk exosomes of 2.0 × 10¹¹ particles per ml whey with high purity (staining rate>90%, 7.01 × 10¹⁰ particles per mg protein) and high uniformity (polydispersity index <0.03). The isolated exosomes were characterized and identified by transmission electron microscopy, zeta potential and size distribution. The results demonstrated aqueous two-phase extraction possesses a robust capability for the enrichment and separation of exosomes directly from bovine milk whey, presenting a novel approach for the large-scale isolation of exosomes.

关键词: Aqueous two-phase extraction, Bovine milk exosomes, Separation, Purification

Jingjing Sun, Ruoque Mao, Xiaoqian Fu, Shanjing Yao, Dongqiang Lin. Exosomes separation with aqueous two-phase systems from bovine milk[J]. Chinese Journal of Chemical Engineering, 2025, 81(5): 1-10.

Jingjing Sun, Ruoque Mao, Xiaoqian Fu, Shanjing Yao, Dongqiang Lin. Exosomes separation with aqueous two-phase systems from bovine milk[J]. 中国化学工程学报, 2025, 81(5): 1-10.

References

[1] R.J. Lobb, M. Becker, S.W. Wen, C.S.F. Wong, A.P. Wiegmans, A. Leimgruber, A. Moller, Optimized exosome isolation protocol for cell culture supernatant and human plasma, J. Extracell. Vesicles 4 (2015) 27031.
[2] R. Kalluri, V.S. LeBleu, The biology, function, and biomedical applications of exosomes, Science 367 (6478) (2020) eaau6977.
[3] D. Yu, Y.X. Li, M.Y. Wang, J.M. Gu, W.R. Xu, H. Cai, X.J. Fang, X. Zhang, Exosomes as a new frontier of cancer liquid biopsy, Mol. Cancer 21 (1) (2022) 56.
[4] M.X. Fu, Q.B. Li, D.H. Sun, Y.H. Lu, N. He, X. Deng, H.X. Wang, J.L. Huang, Rapid preparation process of silver nanoparticles by bioreduction and their characterizations, Chin. J. Chem. Eng. 14 (1) (2006) 114-117.
[5] J. Zhao, Z.G. Duan, X.X. Ma, Y.N. Liu, D.D. Fan, Recent advances in systemic and local delivery of ginsenosides using nanoparticles and nanofibers, Chin. J. Chem. Eng. 30 (2021) 291-300.
[6] Y. Wang, Y.F. Wu, S. Shen, Y.Y. Liu, Y. Xia, H.M. Xia, Z.L. Xie, Y.X. Xu, Engineered plant extracellular vesicles for natural delivery across physiological barriers, Food Funct. 15 (4) (2024) 1737-1757.
[7] M.Y. Tian, D.X. Hao, Y. Liu, J. He, Z.H. Zhao, T.Y. Guo, X. Li, Y. Zhang, Milk exosomes: an oral drug delivery system with great application potential, Food Funct. 14 (3) (2023) 1320-1337.
[8] C. Thery, S. Amigorena, G. Raposo, A. Clayton, Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Curr. Protoc. Cell Biol. Chapter 3 (2006) Unit3.22.
[9] S. Cho, H.C. Yang, W.J. Rhee, Development and comparative analysis of human urine exosome isolation strategies, Process. Biochem. 88 (2020) 197-203.
[10] S.M. Langevin, D. Kuhnell, M.A. Orr-Asman, J. Biesiada, X. Zhang, M. Medvedovic, H.E. Thomas, Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum, RNA Biol. 16 (1) (2019) 5-12.
[11] G.W. Hu, Q. Li, X. Niu, B. Hu, J. Liu, X.L. Shen, Y. Wang, Z.F. Deng, Stirring ultrafiltration: a new method to isolate exosome, Acad. J. Second. Mil. Med. Univ. 35 (6) (2014) 598.
[12] M.A. Rider, S.N. Hurwitz, D.G. Meckes Jr, ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles, Sci. Rep. 6 (2016) 23978.
[13] Z. Andreu, M. Yanez-Mo, Tetraspanins in extracellular vesicle formation and function, Front. Immunol. 5 (2014) 442.
[14] C.Y. Liu, C.Q. Su, Design strategies and application progress of therapeutic exosomes, Theranostics 9 (4) (2019) 1015-1028.
[15] N. Zarovni, A. Corrado, P. Guazzi, D. Zocco, E. Lari, G. Radano, J. Muhhina, C. Fondelli, J. Gavrilova, A. Chiesi, Integrated isolation and quantitative analysis of exosome shuttled proteins and nucleic acids using immunocapture approaches, Methods 87 (2015) 46-58.
[16] S. Guan, H.L. Yu, G.Q. Yan, M.X. Gao, W.B. Sun, X.M. Zhang, Characterization of urinary exosomes purified with size exclusion chromatography and ultracentrifugation, J. Proteome Res. 19 (6) (2020) 2217-2225.
[17] P.A. Albertsson, Partition of cell particles and macromolecules in polymer two-phase systems, Adv. Protein Chem. 24 (1970) 309-341.
[18] E. Atefi, R. Joshi, J.A. Mann Jr, H. Tavana, Interfacial tension effect on cell partition in aqueous two-phase systems, ACS Appl. Mater. Interfaces 7 (38) (2015) 21305-21314.
[19] Y.K. Yau, C.W. Ooi, E.P. Ng, J.C. Lan, T.C. Ling, P.L. Show, Current applications of different type of aqueous two-phase systems, Bioresour. Bioprocess. 2 (1) (2015) 49.
[20] M. Iqbal, Y. Tao, S. Xie, Y. Zhu, D. Chen, X. Wang, L. Huang, D. Peng, A. Sattar, M.A. Shabbir, H.I. Hussain, S. Ahmed, Z. Yuan, Aqueous two-phase system (ATPS): an overview and advances in its applications, Biol. Proced. Online 18 (2016) 18.
[21] D. Rajagopalu, P.L. Show, Y.S. Tan, S. Muniandy, V. Sabaratnam, T.C. Ling, Recovery of laccase from processed hericium erinaceus (bull.: Fr) pers. fruiting bodies in aqueous two-phase system, J. Biosci. Bioeng. 122 (3) (2016) 301-306.
[22] P.J. Rosa, A.M. Azevedo, I.F. Ferreira, J. de Vries, R. Korporaal, H.J. Verhoef, T.J. Visser, M.R. Aires-Barros, Affinity partitioning of human antibodies in aqueous two-phase systems, J. Chromatogr. A 1162 (1) (2007) 103-113.
[23] H.Y. Leong, X.Q. Fu, X.Y. Liu, S.J. Yao, D.Q. Lin, Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems, Chin. J. Chem. Eng. 57 (2023) 72-78.
[24] Y. Savci, O.K. Kirbas, B.T. Bozkurt, E.A. Abdik, P.N. Tasli, F. Sahin, H. Abdik, Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing, Food Funct. 12 (11) (2021) 5144-5156.
[25] O.K. Kirbas, B.T. Bozkurt, A.B. Asutay, B. Mat, B. Ozdemir, D. Ozturkoglu, H. Olmez, Z. Islek, F. Sahin, P.N. Tasli, Optimized isolation of extracellular vesicles from various organic sources using aqueous two-phase system, Sci. Rep. 9 (1) (2019) 19159.
[26] S. Williams, M. Fernandez-Rhodes, A. Law, B. Peacock, M.P. Lewis, O.G. Davies, Comparison of extracellular vesicle isolation processes for therapeutic applications, J. Tissue Eng. 14 (2023) 20417314231174609.
[27] H. Shin, C. Han, J.M. Labuz, J. Kim, J. Kim, S. Cho, Y.S. Gho, S. Takayama, J. Park, High-yield isolation of extracellular vesicles using aqueous two-phase system, Sci. Rep. 5 (2015) 13103.
[28] J. Kim, H. Shin, J. Kim, J. Kim, J. Park, Isolation of high-purity extracellular vesicles by extracting proteins using aqueous two-phase system, PLoS One 10 (6) (2015) e0129760.
[29] H. Shin, Y.H. Park, Y.G. Kim, J.Y. Lee, J. Park, Aqueous two-phase system to isolate extracellular vesicles from urine for prostate cancer diagnosis, PLoS One 13 (3) (2018) e0194818.
[30] A. Torres-Bautista, M.A. Torres-Acosta, J. Gonzalez-Valdez, Characterization and optimization of polymer-polymer aqueous two-phase systems for the isolation and purification of CaCo₂ cell-derived exosomes, PLoS One 17 (9) (2022) e0273243.
[31] M.M. Bahr, M.S. Amer, K. Abo-El-Sooud, A.N. Abdallah, O.S. El-Tookhy, Preservation techniques of stem cells extracellular vesicles: a gate for manufacturing of clinical grade therapeutic extracellular vesicles and long-term clinical trials, Int. J. Vet. Sci. Med. 8 (1) (2020) 1-8.
[32] L. Simon, V. Lapinte, M. Morille, Exploring the role of polymers to overcome ongoing challenges in the field of extracellular vesicles, J. Extracell. Vesicles 12 (12) (2023) e12386.
[33] Q. Wu, D.Q. Lin, Q.L. Zhang, D. Gao, S.J. Yao, Evaluation of a PEG/hydroxypropyl starch aqueous two-phase system for the separation of monoclonal antibodies from cell culture supernatant, J. Sep. Sci. 37 (4) (2014) 447-453.
[34] M. Sancho-Albero, N. Navascues, G. Mendoza, V. Sebastian, M. Arruebo, P. Martin-Duque, J. Santamaria, Exosome origin determines cell targeting and the transfer of therapeutic nanoparticles towards target cells, J. Nanobiotechnology 17 (1) (2019) 16.
[35] C. Subra, K. Laulagnier, B. Perret, M. Record, Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies, Biochimie 89 (2) (2007) 205-212.
[36] J.R. Soohoo, G.M. Walker, Microfluidic aqueous two phase system for leukocyte concentration from whole blood, Biomed. Microdevices 11 (2) (2009) 323-329.
[37] M. Singla, P. Nandan Sit, Theoretical aspects and applications of aqueous two-phase systems, ChemBioEng Rev. 10 (1) (2023) 65-80.
[38] H.Y. Leong, X.Q. Fu, P.L. Show, S.J. Yao, D.Q. Lin, Downstream processing of virus-like particles with aqueous two-phase systems: Applications and challenges, J. Sep. Sci. 45 (12) (2022) 2064-2076.
[39] X.Q. Fu, H.Y. Leong, G.A. Wang, X.H. Zhou, Y.L. Li, S.J. Yao, D.Q. Lin, Enrichment and separation of recombinant adeno-associated virus particles by aqueous two-phase extraction, Biochem. Eng. J. 200 (2023) 109115.
[40] C.E. de Araujo Padilha, S.D. de Oliveira Jr, D.F. de Santana Souza, J.A. de Oliveira, G.R. de Macedo, E.S. dos Santos, Partition coefficient prediction of Baker’s yeast invertase in aqueous two phase systems using hybrid group method data handling neural network, Chin. J. Chem. Eng. 25 (5) (2017) 652-657.
[41] J.W. Zhang, Y. Wang, Q.J. Peng, Extraction of theanine from waste liquid of tea polyphenol production in aqueous two-phase systems with cationic and anionic surfactants, Chin. J. Chem. Eng. 21 (1) (2013) 31-36.
[42] H. Breisig, M. Wessling, Droplet formation and shrinking in aqueous two-phase systems using a membrane emulsification method, Biomicrofluidics 9 (4) (2015) 044122.
[43] P.J. Shiu, Y.H. Ju, H.M. Chen, C.K. Lee, Facile isolation of purple membrane from Halobacterium salinarum via aqueous-two-phase system, Protein Expr. Purif. 89 (2) (2013) 219-224.
[44] K. Vaswani, M.D. Mitchell, O.J. Holland, Y. Qin Koh, R.J. Hill, T. Harb, P.S.W. Davies, H. Peiris, A method for the isolation of exosomes from human and bovine milk, J. Nutr. Metab. 2019 (2019) 5764740.
[45] R. Weiskirchen, S.K. Schroder, S. Weiskirchen, E.M. Buhl, B. Melnik, Isolation of bovine and human milk extracellular vesicles, Biomedicines 11 (10) (2023) 2715.
[46] D. Bachurski, M. Schuldner, P.H. Nguyen, A. Malz, K.S. Reiners, P.C. Grenzi, F. Babatz, A.C. Schauss, H.P. Hansen, M. Hallek, E. Pogge von Strandmann, Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView, J. Extracell. Vesicles 8 (1) (2019) 1596016.
[47] J.K. Lee,B. Liu,K.L. Rubow, J.G. Zahka, Surface charge effects on particulate retention by microporous membrane filters in liquid filtration, Environ. Eng. Res. 3 (2) (1998) 97-104.
[48] T. Soares Martins, J. Catita, I. Martins Rosa, O.A.B. da Cruz E Silva, A.G. Henriques, Exosome isolation from distinct biofluids using precipitation and column-based approaches, PLoS One 13 (6) (2018) e0198820.
[49] J. Kim, S.Y. Li, S.Y. Zhang, J.X. Wang, Plant-derived exosome-like nanoparticles and their therapeutic activities, Asian J. Pharm. Sci. 17 (1) (2022) 53-69.
[50] V.S. Chernyshev, R. Rachamadugu, Y.H. Tseng, D.M. Belnap, Y.L. Jia, K.J. Branch, A.E. Butterfield, L.F. Pease 3rd, P.S. Bernard, M. Skliar, Size and shape characterization of hydrated and desiccated exosomes, Anal. Bioanal. Chem. 407 (12) (2015) 3285-3301.
[51] M. Skliar, V.S. Chernyshev, Imaging of extracellular vesicles by atomic force microscopy, J. Vis. Exp. (151) (2019) (151).
[52] S. Wijenayake, S. Eisha, Z. Tawhidi, M.A. Pitino, M.A. Steele, A.S. Fleming, P.O. McGowan, Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk, PLoS One 16 (9) (2021) e0257633.