Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis

Nature Protocols volume 16, pages 131–163 (2021)Cite this article

Subjects

Abstract

The use of exosomes as selective delivery vehicles of therapeutic agents, such as drugs or hyperthermia-capable nanoparticles, is being intensely investigated on account of their preferential tropism toward their parental cells. However, the methods used to introduce a therapeutic load inside exosomes often involve disruption of their membrane, which may jeopardize their targeting capabilities, attributed to their surface integrins. On the other hand, in recent years bio-orthogonal catalysis has emerged as a new tool with a myriad of potential applications in medicine. These bio-orthogonal processes, often based on Pd-catalyzed chemistry, would benefit from systems capable of delivering the catalyst to target cells. It is therefore highly attractive to combine the targeting capabilities of exosomes and the bio-orthogonal potential of Pd nanoparticles to create new therapeutic vectors. In this protocol, we provide detailed information on an efficient procedure to achieve a high load of catalytically active Pd nanosheets inside exosomes, without disrupting their membranes. The protocol involves a multistage process in which exosomes are first harvested, subjected to impregnation with a Pd salt precursor followed by a mild reduction process using gas-phase CO, which acts as both a reducing and growth-directing agent to produce the desired nanosheets. The technology is scalable, and the protocol can be conducted by any researcher having basic biology and chemistry skills in ~3 d.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2: A schematic of the exosome isolation and Pd2+ loading.
Fig. 3
Fig. 4: Preparation and fixation of cells on coverslips for inmunolabeling of cells.
Fig. 5
Fig. 6: Electron microscopy analysis of Pd-loaded exosomes.
Fig. 7: Representative TEM images of Pd nanoengineered exosomes.
Fig. 8: Cell viability assays (A549 cells) at different concentrations of Pd-loaded exosomes.
Fig. 9: Confocal images of A549 cells incubated with Pd-loaded exosomes for 24 h.
Fig. 10: Confocal microscopy images of cells incubated with Pd-loaded exosomes.
Fig. 11: Exosome characterization.
Fig. 12: Pd-mediated conversion of nonfluorescent Pro-Resorufin to fluorescent resorufin.
Fig. 13: Time-lapse frame sequence during 24 h of Pro-Resorufin (negative control), Resorufin (positive control) and Pro-Resorufin with the catalytic Pd-exosomes.

Data availability

The main data supporting the examples of this protocol are available within the article and its Supplementary Information. Extra data are available from the corresponding author upon reasonable request. The source data underlying Figs. 5, 8 and 12 are provided as Source Data files with this protocol.

References

  1. Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. Engl. 48, 6974–6998 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Devaraj, N. K. The future of bioorthogonal chemistry. ACS Cent. Sci. 4, 952–959 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Luan, X. et al. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol. Sin. 38, 754–763 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Cooper, J. R. et al. Long term culture of the A549 cancer cell line promotes multilamellar body formation and differentiation towards an alveolar type II pneumocyte phenotype. PloS ONE 11, e0164438 (2016).

    PubMed  PubMed Central  Google Scholar 

  5. Lieber, M., Todaro, G., Smith, B., Szakal, A. & Nelson-Rees, W. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17, 62–70 (1976).

    CAS  PubMed  Google Scholar 

  6. Giard, D. J. et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl Cancer Inst. 51, 1417–1423 (1973).

    CAS  PubMed  Google Scholar 

  7. Yusop, R. M., Unciti-Broceta, A., Johansson, E. M. V., Sanchez-Martin, R. M. & Bradley, M. Palladium-mediated intracellular chemistry. Nat. Chem. 3, 239–243 (2011).

    CAS  PubMed  Google Scholar 

  8. Weiss, J. T. et al. Extracellular palladium-catalysed dealkylation of 5-fluoro-1-propargyl-uracil as a bioorthogonally activated prodrug approach. Nat. Commun. 5, 3277 (2014).

    PubMed  PubMed Central  Google Scholar 

  9. Li, J. et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nat. Chem. 6, 352–361 (2014).

    CAS  PubMed  Google Scholar 

  10. Weiss, J. T. et al. Development and bioorthogonal activation of palladium-labile prodrugs of gemcitabine. J. Med. Chem. 57, 5395–5404 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Rubio-Ruiz, B., Weiss, J. T. & Unciti-Broceta, A. Efficient palladium-triggered release of vorinostat from a bioorthogonal precursor. J. Med. Chem. 59, 9974–9980 (2016).

    CAS  PubMed  Google Scholar 

  12. Bray, T. L. et al. Bright insights into palladium-triggered local chemotherapy. Chem. Sci. 9, 7354–7361 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Adam, C. et al. Bioorthogonal uncaging of the active metabolite of irinotecan by palladium-functionalized microdevices. Chemistry 24, 16783–16790 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Stenton, B. J., Oliveira, B. L., Matos, M. J., Sinatra, L. & Bernardes, G. J. L. A thioether-directed palladium-cleavable linker for targeted bioorthogonal drug decaging. Chem. Sci. 9, 4185–4189 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Li, N., Lim, R. K. V., Edwardraja, S. & Lin, Q. Copper-free sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells. J. Am. Chem. Soc. 133, 15316–15319 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Spicer, C. D., Triemer, T. & Davis, B. G. Palladium-mediated cell-surface labeling. J. Am. Chem. Soc. 134, 800–803 (2012).

    CAS  PubMed  Google Scholar 

  17. Destito, P. et al. Hollow nanoreactors for Pd-catalyzed Suzuki-Miyaura coupling and O-propargyl cleavage reactions in bio-relevant aqueous media. Chem. Sci. 10, 2598–2603 (2019).

    CAS  PubMed  Google Scholar 

  18. Michel, B. W., Lippert, A. R. & Chang, C. J. A reaction-based fluorescent probe for selective imaging of carbon monoxide in living cells using a palladium-mediated carbonylation. J. Am. Chem. Soc. 134, 15668–15671 (2012).

    CAS  PubMed  Google Scholar 

  19. Mann, G., Satish, G., Meledin, R., Vamisetti, G. B. & Brik, A. Palladium-mediated cleavage of proteins with thiazolidine-modified backbone in live cells. Angew. Chem. Int. Ed. Engl. 58, 13540–13549 (2019).

    CAS  PubMed  Google Scholar 

  20. Wang, F. M., Zhang, Y., Du, Z., Ren, J. S. & Qu, X. G. Designed heterogeneous palladium catalysts for reversible light-controlled bioorthogonal catalysis in living cells. Nat. Commun. 9, 1209 (2018).

    PubMed  PubMed Central  Google Scholar 

  21. Miller, M. A. et al. Nano-palladium is a cellular catalyst for in vivo chemistry. Nat. Commun. 8, 15906 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hoop, M. et al. Mobile magnetic nanocatalysts for bioorthogonal targeted cancer therapy. Adv. Funct. Mater. 28, 1705920 (2018).

    Google Scholar 

  23. Li, X. et al. Superior antitumor efficiency of cisplatin-loaded nanoparticles by intratumoral delivery with decreased tumor metabolism rate. Eur. J. Pharm. Biopharm. 70, 726–734 (2008).

    CAS  PubMed  Google Scholar 

  24. Sancho-Albero, M. et al. Efficient encapsulation of theranostic nanoparticles in cell-derived exosomes: leveraging the exosomal biogenesis pathway to obtain hollow gold nanoparticle-hybrids. Nanoscale 11, 18825–18836 (2019).

    CAS  PubMed  Google Scholar 

  25. Balivada, S. et al. A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study. BMC Cancer 10, 119 (2010).

    PubMed  PubMed Central  Google Scholar 

  26. Shukla, R. et al. Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer. Proc. Natl Acad. Sci. USA 109, 12426–12431 (2012).

    CAS  PubMed  Google Scholar 

  27. Podesta, J. E. et al. Antitumor activity and prolonged survival by carbon-nanotube-mediated therapeutic siRNA silencing in a human lung xenograft model. Small 5, 1176–1185 (2009).

    CAS  PubMed  Google Scholar 

  28. Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).

    CAS  Google Scholar 

  29. Sancho-Albero, M. et al. Cancer-derived exosomes loaded with ultrathin palladium nanosheets for targeted bioorthogonal catalysis. Nat. Catal. 2, 864–872 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Yong, T. et al. Tumor exosome-based nanoparticles are efficient drug carriers for chemotherapy. Nat. Commun. 10, 3838 (2019).

    PubMed  PubMed Central  Google Scholar 

  31. Darband, S. G. et al. Exosomes: natural nanoparticles as bio shuttles for RNAi delivery. J. Control. Release 289, 158–170 (2018).

    CAS  PubMed  Google Scholar 

  32. Sancho-Albero, M. et al. Exosome origin determines cell targeting and the transfer of therapeutic nanoparticles towards target cells. J. Nanobiotechnology 17, 16 (2019).

    PubMed  PubMed Central  Google Scholar 

  33. Trams, E. G., Lauter, C. J., Salem, N. Jr. & Heine, U. Exfoliation of membrane ecto-enzymes in the form of micro-vesicles. Biochim. Biophys. Acta– Biomembr. 645, 63–70 (1981).

    CAS  Google Scholar 

  34. Tkach, M. & Théry, C. Communication by extracellular vesicles: where we are and where we need to go. Cell 164, 1226–1232 (2016).

    CAS  PubMed  Google Scholar 

  35. Harding, C. V., Heuser, J. E. & Stahl, P. D. Exosomes: looking back three decades and into the future. J. Cell Biol. 200, 367–371 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Gould, S. J., Booth, A. M. & Hildreth, J. E. K. The Trojan exosome hypothesis. Proc. Natl Acad. Sci. USA 100, 10592–10597 (2003).

    CAS  PubMed  Google Scholar 

  37. Gourlay, J. et al. The emergent role of exosomes in glioma. J. Clin. Neurosci. 35, 13–23 (2017).

    CAS  PubMed  Google Scholar 

  38. Alhasan, A. H., Patel, P. C., Choi, C. H. J. & Mirkin, C. A. Exosome encased spherical nucleic acid gold nanoparticle conjugates as potent microRNA regulation agents. Small 10, 186–192 (2014).

    CAS  PubMed  Google Scholar 

  39. Hessvik, N. P. & Llorente, A. Current knowledge on exosome biogenesis and release. Cell. Mol. Life Sci. 75, 193–208 (2018).

    CAS  PubMed  Google Scholar 

  40. Doyle, L. M. & Wang, M. Z. Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells 8, 727 (2019).

    CAS  PubMed Central  Google Scholar 

  41. International Agency for Research on Cancer. World Cancer Report: Cancer Research for Cancer Prevention (2020; accessed 5 April 2020). https://publications.iarc.fr/586

  42. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    PubMed  PubMed Central  Google Scholar 

  43. Ramirez, M. I. et al. Technical challenges of working with extracellular vesicles. Nanoscale 10, 881–906 (2018).

    CAS  PubMed  Google Scholar 

  44. Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V. & Laktionov, P. P. Isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res. Int. 2018, 8545347 (2018).

    PubMed  PubMed Central  Google Scholar 

  45. Sebastian, V., Smith, C. D. & Jensen, K. F. Shape-controlled continuous synthesis of metal nanostructures. Nanoscale 8, 7534–7543 (2016).

    CAS  PubMed  Google Scholar 

  46. Herrer, L. et al. High surface coverage of a self-assembled monolayer by in situ synthesis of palladium nanodeposits. Nanoscale 9, 13281–13290 (2017).

    CAS  PubMed  Google Scholar 

  47. Kuntsche, J., Horst, J. C. & Bunjes, H. Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems. Int. J. Pharm. 417, 120–137 (2011).

    CAS  PubMed  Google Scholar 

  48. Ilett, M., Brydson, R., Brown, A. & Hondow, N. Cryo-analytical STEM of frozen, aqueous dispersions of nanoparticles. Micron 120, 35–42 (2019).

    CAS  PubMed  Google Scholar 

  49. Elad, N., Bellapadrona, G., Houben, L., Sagi, I. & Elbaum, M. Detection of isolated protein-bound metal ions by single-particle cryo-STEM. Proc. Natl Acad. Sci. USA 114, 11139–11144 (2017).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge financial support from the ERC Advanced Grant CADENCE (grant no. ERC-2016-ADG-742684) and the EPSRC (Healthcare Technology Challenge award no. EP/N021134/1). M.S.-A. thanks the Spanish Government for an FPU PhD research fellowship. B.R.-R. thanks the EC (grant no. H2020-MSCA-IF-2014–658833). V.S. acknowledges the financial support of Ministerio de Ciencia, Innovación y Universidades, Programa Retos Investigación, Proyecto REF: RTI2018-099019-A-I00. M.A. acknowledges the financial support of the ERC Consolidator Grant programme (grant no. ERC-2013-CoG-614715). P.M.-D. also thanks Instituto de Salud Carlos III (PI19/01007). We also thank CIBER-BBN, an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III and by Fondo Europeo de Desarrollo Regional (Feder) ‘Una manera de hacer Europa’, with the assistance of the European Regional Development Fund. This study is also partially funded by the Aragon Government (T57_17R p) cofounded by Feder 2014–2020 ‘Building Europe from Aragon’.

Author information

Author notes

  1. Ana M. Pérez‐López

    Present address: Institut für Biotechnologie, Technische Universität Berlin, Berlin, Germany

  2. Belén Rubio‐Ruiz

    Present address: Pfizer–University of Granada–Andalusian Regional Government Centre for Genomics and Oncological Research (GENYO) and Department of Medicinal and Organic Chemistry, Faculty of Pharmacy, University of Granada, Granada, Spain

  3. These authors contributed equally: Victor Sebastian, María Sancho‐Albero, Manuel Arruebo.

Authors and Affiliations

  1. Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC–Universidad de Zaragoza, Zaragoza, Spain

    Victor Sebastian, María Sancho‐Albero, Manuel Arruebo & Jesús Santamaría

  2. Department of Chemical Engineering and Environmental Technologies, University of Zaragoza, Zaragoza, Spain

    Victor Sebastian, María Sancho‐Albero, Manuel Arruebo & Jesús Santamaría

  3. Networking Research Center on Bioengineering Biomaterials and Nanomedicine (CIBER‐BBN), Madrid, Spain

    Victor Sebastian, María Sancho‐Albero, Manuel Arruebo, Pilar Martin‐Duque & Jesús Santamaría

  4. Cancer Research UK Edinburgh Centre, MRC Institute of Genetics & Molecular Medicine, University of Edinburgh, Edinburgh, UK

    Ana M. Pérez‐López, Belén Rubio‐Ruiz & Asier Unciti‐Broceta

  5. Instituto Aragonés de Ciencias de la Salud‐Fundación Araid/IIS Aragón, Centro de Investigaciones Biomédicas de Aragón, Universidad San Jorge, Zaragoza, Spain

    Pilar Martin‐Duque

Authors
  1. Victor Sebastian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Sancho‐Albero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manuel Arruebo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana M. Pérez‐López
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Belén Rubio‐Ruiz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pilar Martin‐Duque
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Asier Unciti‐Broceta
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jesús Santamaría
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S.-A., B.R.-R., A.M.P.-L., P.M.-D. and V.S. prepared and characterized the materials, planned and performed the experiments and analyzed the data. V.S., P.M.-D., M.A., A.U.-B. and J.S. planned and supervised the research, analyzed the data and contributed to the manuscript writing. V.S. and M.A. conceived the research. V.S. designed and coordinated the research. All the authors checked the manuscript.

Corresponding author

Correspondence to Victor Sebastian.

Ethics declarations

Competing interests

The authors declare no competing interests as defined by Nature Research or other interests that might be perceived to influence the interpretation of the article.

Additional information

Peer review information Nature Protocols thanks Gonçalo Bernardes and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Sancho‐Albero, M. et al. Nat. Catal. 2, 864–872 (2019): https://www.nature.com/articles/s41929-019-0333-4

Sebastian, V., Smith, C. D. & Jensen, K. F. Nanoscale 8, 7534 (2016): https://pubs.rsc.org/en/content/articlelanding/2016/nr/c5nr08531d

Supplementary information

Reporting Summary

Supplementary Video 1

Confocal Z-stack sequence of A549 cells incubated with Pd-exosomesA549. Actin is labeled with phalloidin (green), nuclei are stained with Draq5 (blue) and the Pd-exosomes were directly visualized by reflection (red).

Source data

Source Data Fig. 5

Total protein exosome quantification by BCA assay.

Source Data Fig. 8

Cell viability assays.

Source Data Fig. 12

Fluorescent analysis and catalytic data.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sebastian, V., Sancho‐Albero, M., Arruebo, M. et al. Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis. Nat Protoc 16, 131–163 (2021). https://doi.org/10.1038/s41596-020-00406-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00406-z

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research