Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes


Extracellular vesicles (EVs) can mediate intercellular communication by transferring cargo proteins and nucleic acids between cells. The pathophysiological roles and clinical value of EVs are under intense investigation, yet most studies are limited by technical challenges in the isolation of nanoscale EVs (nEVs). Here, we report a lipid-nanoprobe system that enables spontaneous labelling of nEVs for subsequent magnetic enrichment in 15 minutes, with isolation efficiency and cargo composition similar to what can be achieved by the much slower and bulkier method of ultracentrifugation. We also show that this approach allows for downstream analyses of nucleic acids and proteins, enabling the identification of EGFR and KRAS mutations following nEV isolation from the blood plasma of non-small-cell lung-cancer patients. The efficiency and versatility of the lipid-nanoprobe approach opens up opportunities in point-of-care cancer diagnostics.

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Figure 1: Schematic of the LNP system for nEV enrichment and downstream analyses.
Figure 2: Morphological characterization of the materials and optimization of the LNP system for isolation efficiency.
Figure 3: Isolated nEVs provide flexibility in downstream molecular analyses.
Figure 4: Detection of DNA mutations in nEVs isolated from plasma samples from NSCLC patients.


  1. 1

    Colombo, M., Raposo, G. & Thery, C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell. Dev. Biol. 30, 255–289 (2014).

    CAS  Article  Google Scholar 

  2. 2

    Raposo, G. & Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200, 373–383 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Cocucci, E. & Meldolesi, J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol. 25, 364–372 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Yáñez-Mó, M. et al. Biological properties of extracellular vesicles and their physiological functions. J. Extracell. Vesicles 4, 27066 (2015).

    Article  Google Scholar 

  5. 5

    Thery, C., Zitvogel, L. & Amigorena, S. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2, 569–579 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Li, Y., Shen, Z. & Yu, X.-Y. Transport of microRNAs via exosomes. Nat. Rev. Cardiol. 12, 198–198 (2015).

    Article  Google Scholar 

  7. 7

    Alderton, G. K. Diagnosis: fishing for exosomes. Nat. Rev. Cancer 15, 453–453 (2015).

    CAS  Article  Google Scholar 

  8. 8

    Melo, S. A. et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 523, 177–182 (2015).

    CAS  Article  Google Scholar 

  9. 9

    Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766–769 (2014).

    CAS  Article  Google Scholar 

  10. 10

    Kahlert, C. et al. Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer. J. Biol. Chem. 289, 3869–3875 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Costa-Silva, B. et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat. Cell Biol. 17, 816–826 (2015).

    CAS  Article  Google Scholar 

  12. 12

    Melo, S. A. et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell 26, 707–721 (2014).

    CAS  Article  Google Scholar 

  13. 13

    Yeo, R. W. Y. et al. Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv. Drug Deliv. Rev. 65, 336–341 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Lai, R. C., Yeo, R. W. Y., Tan, K. H. & Lim, S. K. Exosomes for drug delivery—a novel application for the mesenchymal stem cell. Biotechnol. Adv. 31, 543–551 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Azmi, A. S., Bao, B. & Sarkar, F. H. Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review. Cancer Metast. Rev. 32, 623–642 (2013).

    CAS  Article  Google Scholar 

  16. 16

    Ohno, S.-i. et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21, 185–191 (2013).

    CAS  Article  Google Scholar 

  17. 17

    Liga, A., Vliegenthart, A. D. B., Oosthuyzen, W., Dear, J. W. & Kersaudy-Kerhoas, M. Exosome isolation: a microfluidic road-map. Lab Chip 15, 2388–2394 (2015).

    CAS  Article  Google Scholar 

  18. 18

    Christianson, H. C., Svensson, K. J., van Kuppevelt, T. H., Li, J.-P. & Belting, M. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity. Proc. Natl Acad. Sci. USA 110, 17380–17385 (2013).

    CAS  Article  Google Scholar 

  19. 19

    Bechstein, D. J. B. et al. High performance wash-free magnetic bioassays through microfluidically enhanced particle specificity. Sci. Rep. 5, 11693 (2015).

    Article  Google Scholar 

  20. 20

    Caballero, J. N., Frenette, G., Belleannée, C. & Sullivan, R. CD9-positive microvesicles mediate the transfer of molecules to bovine spermatozoa during epididymal maturation. PLoS ONE 8, e65364 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Tauro, B. J. et al. Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids. Mol. Cell. Proteomics 12, 587–598 (2013).

    CAS  Article  Google Scholar 

  22. 22

    Thery, C. et al. Indirect activation of naive CD4+ T cells by dendritic cell-derived exosomes. Nat. Immunol. 3, 1156–1162 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Lambertz, U. et al. Small RNAs derived from tRNAs and rRNAs are highly enriched in exosomes from both old and new world Leishmania providing evidence for conserved exosomal RNA packaging. BMC Genomics 16, 1–26 (2015).

    CAS  Article  Google Scholar 

  24. 24

    Gormally, E., Caboux, E., Vineis, P. & Hainaut, P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat. Res. 635, 105–117 (2007).

    CAS  Article  Google Scholar 

  25. 25

    Wei, Z., Batagov, A. O., Carter, D. R. & Krichevsky, A. M. Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA. Sci. Rep. 6, 31175 (2016).

    CAS  Article  Google Scholar 

  26. 26

    Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad. Sci. USA 113, E968–E977 (2016).

    CAS  Article  Google Scholar 

  27. 27

    Asano, H. et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin. Cancer Res. 12, 43–48 (2006).

    CAS  Article  Google Scholar 

  28. 28

    Vlassov, A. V., Magdaleno, S., Setterquist, R. & Conrad, R. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta 1820, 940–948 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Klymchenko, A. S. & Kreder, R. Fluorescent probes for lipid rafts: from model membranes to living cells. Chem. Biol. 21, 97–113 (2014).

    CAS  Article  Google Scholar 

  30. 30

    Wijesinghe, D., Arachchige, M. C. M., Lu, A., Reshetnyak, Y. K. & Andreev, O. A. pH dependent transfer of nano-pores into membrane of cancer cells to induce apoptosis. Sci. Rep. 3, 3560 (2013).

    Article  Google Scholar 

  31. 31

    Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).

    Article  Google Scholar 

  32. 32

    Jeong, S. et al. Integrated magneto–electrochemical sensor for exosome analysis. ACS Nano 10, 1802–1809 (2016).

    CAS  Article  Google Scholar 

  33. 33

    Im, H. et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat. Biotechnol. 32, 490–495 (2014).

    CAS  Article  Google Scholar 

  34. 34

    Zhao, Z., Yang, Y., Zeng, Y. & He, M. A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. Lab Chip 16, 489–496 (2016).

    CAS  Article  Google Scholar 

  35. 35

    Weber, R. J., Liang, S. I., Selden, N. S., Desai, T. A. & Gartner, Z. J. Efficient targeting of fatty-acid modified oligonucleotides to live cell membranes through stepwise assembly. Biomacromolecules 15, 4621–4626 (2014).

    CAS  Article  Google Scholar 

  36. 36

    Charbonneau, D. M. & Tajmir-Riahi, H.-A. Study on the interaction of cationic lipids with bovine serum albumin. J. Phys. Chem. B 114, 1148–1155 (2010).

    CAS  Article  Google Scholar 

  37. 37

    Bobrie, A., Colombo, M., Krumeich, S., Raposo, G. & Théry, C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J. Extracell. Vesicles 1, 18397 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Van Niel, G. et al. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 121, 337–349 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Colombo, M. et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J. Cell Sci. 126, 5553–5565 (2013).

    CAS  Article  Google Scholar 

  40. 40

    Rupp, A. K. et al. Loss of EpCAM expression in breast cancer derived serum exosomes: role of proteolytic cleavage. Gynecol. Oncol. 122, 437–446 (2011).

    CAS  Article  Google Scholar 

  41. 41

    Boch, C. et al. The frequency of EGFR and KRAS mutations in non-small cell lung cancer (NSCLC): routine screening data for central Europe from a cohort study. BMJ Open 3, e002560 (2013).

    Article  Google Scholar 

  42. 42

    Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies . Sci. Transl. Med. 6, 224ra224 (2014).

    Article  Google Scholar 

  43. 43

    Newman, A. M. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat. Med. 20, 548–554 (2014).

    CAS  Article  Google Scholar 

  44. 44

    Cheng, G. et al. The GO/rGO-Fe3O4 composites with good water-dispersibility and fast magnetic response for effective immobilization and enrichment of biomolecules. J. Mater. Chem. 22, 21998–22004 (2012).

    CAS  Article  Google Scholar 

  45. 45

    Cheng, G., Zhang, J.-L., Liu, Y.-L., Sun, D.-H. & Ni, J.-Z. Synthesis of novel Fe3O4@SiO2@CeO2 microspheres with mesoporous shell for phosphopeptide capturing and labeling. Chem. Comm. 47, 5732–5734 (2011).

    CAS  Article  Google Scholar 

  46. 46

    Deng, Y., Qi, D., Deng, C., Zhang, X. & Zhao, D. Superparamagnetic high-magnetization microspheres with an Fe3O4@ SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins. J. Am. Chem. Soc. 130, 28–29 (2008).

    CAS  Article  Google Scholar 

  47. 47

    Morel, A.-L. et al. Sonochemical approach to the synthesis of Fe3O4@SiO2 core−shell nanoparticles with tunable properties. ACS Nano 2, 847–856 (2008).

    CAS  Article  Google Scholar 

  48. 48

    Ding, H. et al. Fe3O4@SiO2 core/shell nanoparticles: the silica coating regulations with a single core for different core sizes and shell thicknesses. Chem. Mater. 24, 4572–4580 (2012).

    CAS  Article  Google Scholar 

  49. 49

    Wan, Y. et al. Surface-immobilized aptamers for cancer cell isolation and microscopic cytology. Cancer Res. 70, 9371–9380 (2010).

    CAS  Article  Google Scholar 

  50. 50

    Wang, S., Wan, Y. & Liu, Y. Effects of nanopillar array diameter and spacing on cancer cell capture and cell behaviors. Nanoscale 6, 12482–12489 (2014).

    CAS  Article  Google Scholar 

  51. 51

    Xue, P. et al. Isolation and elution of Hep3B circulating tumor cells using a dual-functional herringbone chip. Microfluid. Nanofluid. 16, 605–612 (2014).

    CAS  Article  Google Scholar 

  52. 52

    Lu, N.-N . et al. Biotin-triggered decomposable immunomagnetic beads for capture and release of circulating tumor cells. ACS Appl. Mater. Interfaces 7, 8817–8826 (2015).

    CAS  Article  Google Scholar 

  53. 53

    Wan, Y. et al. Nanotextured substrates with immobilized aptamers for cancer cell isolation and cytology. Cancer 118, 1145–1154 (2012).

    CAS  Article  Google Scholar 

  54. 54

    Lynch, T. J. et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non–small-cell lung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139 (2004).

    CAS  Article  Google Scholar 

  55. 55

    Li, H & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 25, 1754–17320 (2009).

    CAS  Article  Google Scholar 

  56. 56

    Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    CAS  Article  Google Scholar 

  57. 57

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

    Article  Google Scholar 

  58. 58

    Dobin, A . et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  Article  Google Scholar 

  59. 59

    Karolchik, D., Hinrichs, A. S. & Kent, W. J. The UCSC Genome Browser. Curr. Protoc. Bioinform. Ch. 1, Unit 1 4 (2007).

  60. 60

    Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).

  61. 61

    Alam, S. et al. The eleventh and twelfth data releases of the Sloan Digital Sky Survey: final data from SDSS-III. Astrophys. J. Suppl. Ser. 219, 12 (2015).

    Article  Google Scholar 

  62. 62

    Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R016 (2010).

    Article  Google Scholar 

  63. 63

    Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2009).

  64. 64

    Wan, Y. et al. Dataset for rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes. figsharehttp://dx.doi.org/10.6084/m9.figshare.4728856 (2017).

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S.-Y.Z. thanks the Penn State Materials Research Institute, the Huck Institute of Life Sciences, the Penn State Hershey Cancer Institute, the Penn State proteomic and mass spectrometry facilities at Hershey and University Park, the Penn State Microscopy and Cytometry Facility, and the Penn State Genomics Facility for their support. This work was partially supported by the Pennsylvania State University start-up fund and the National Cancer Institute of the National Institutes of Health under Award Number DP2CA174508. We thank the Applied Bioinformatics Center (BFX) at the New York University (NYU) School of Medicine for providing bioinformatics support and for helping with the analysis and interpretation of the data. This work used computing resources at the High Performance Computing Facility (HPCF) of the Center for Health Informatics and Bioinformatics at the NYU Langone Medical Center. We also thank the Genome Technology Center (GTC) for library preparation and sequencing. This shared resource is partially supported by the Cancer Center Support Grant, P30CA016087, at the Laura and Isaac Perlmutter Cancer Center. We would like to thank S. Hafenstein at Penn State Hershey for discussions on cryo-SEM and C. Zhang at the Dana-Farber Cancer Institute for his advice on genomic analysis.

Author information




Y.W. and S.-Y.Z. designed the research. Y.W. conducted experiments and analysed data. G.C. prepared the MMPs, assisted with peptide-sample preparation and performed proteomic analyses. S.-J.H. assisted with the preparation of NGS samples, the analysis of RNA NGS data, and the fluorescence imaging. M.N. prepared blood plasma. C.-D.Z. and W.-Q.L. assisted with the cell culture, nEV collection and gel electrophoresis. Y.-Q.X. performed the wound-healing assay. Z.-G.W. performed the electron microscopy. W.-L.Z. assisted with the image processing. A.S and I.A analysed NGS DNA data. X.L., S.J.R. and C.P.B. recruited patients and provided blood samples, tissue NGS data and clinical support. Y.W. and S.-Y.Z. wrote the manuscript.

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Correspondence to Si-Yang Zheng.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary tables, figures and references (PDF 7817 kb)

Supplementary Dataset 1

Top-1,000 expressed mRNAs (XLSX 47 kb)

Supplementary Dataset 2

Top-1,000 expressed miRNAs (XLSX 43 kb)

Supplementary Dataset 3

Cargo proteins in the nanoscale extracellular vesicles (XLSX 384 kb)

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Wan, Y., Cheng, G., Liu, X. et al. Rapid magnetic isolation of extracellular vesicles via lipid-based nanoprobes. Nat Biomed Eng 1, 0058 (2017). https://doi.org/10.1038/s41551-017-0058

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