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


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.

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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.


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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




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.

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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.

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

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Key references using this protocol

Sancho‐Albero, M. et al. Nat. Catal. 2, 864–872 (2019):

Sebastian, V., Smith, C. D. & Jensen, K. F. Nanoscale 8, 7534 (2016):

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.

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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).

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