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Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives

Nature Chemistry volume 13, pages 530–539 (2021)Cite this article

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Abstract

Enabling the cellular delivery and cytosolic bioavailability of functional proteins constitutes a major challenge for the life sciences. Here we demonstrate that thiol-reactive arginine-rich peptide additives can enhance the cellular uptake of protein–CPP conjugates in a non-endocytic mode, even at low micromolar concentration. We show that such thiol- or HaloTag-reactive additives can result in covalently anchored CPPs on the cell surface, which are highly effective at co-delivering protein cargoes. Taking advantage of the thiol reactivity of our most effective CPP additive, we show that Cys-containing proteins can be readily delivered into the cytosol by simple co-addition of a slight excess of this CPP. Furthermore, we demonstrate the application of our ‘CPP-additive technique’ in the delivery of functional enzymes, nanobodies and full-length immunoglobulin-G antibodies. This new cellular uptake protocol greatly simplifies both the accessibility and efficiency of protein and antibody delivery, with minimal chemical or genetic engineering.

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Fig. 1: Concentration-dependent delivery of CPP-bearing red fluorescent cargoes into HeLa Kyoto cells at 37 °C and 4 °C.
Fig. 2: TNB-R10 and its performance in delivering CPP-bearing cargoes into cells.
Fig. 3: Protein transduction into cells through CPP-labelled cell membranes.
Fig. 4: Co-delivery with cysteine-reactive R10 peptides in different cell lines and with different cargoes.
Fig. 5: Delivery of cysteine-containing mCherry and recombinant R8-containing Cre recombinase.
Fig. 6: The application of TNB-R10 in IgG antibody delivery.

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Data availability

The UniProt Homo Sapiens database used is available at https://www.uniprot.org/proteomes/UP000005640. All other relevant data are included in the article and its Supplementary Information. The plasmids generated for this study are available from the authors upon request.

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Acknowledgements

We thank members of the Hackenberger laboratory for comments and discussion and K. Kemnitz-Hassanin and I. Kretzschmar for technical support. We thank D. Schumacher and H. Leonhardt for providing the brentuximab antibody. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SPP 1623, SFB 1449 and RTG 2473) to C.P.R.H. (HA 4468/9-1, 9-2) and M.C.C. (CA 198/8-1, 8-2), the GIF, the German–Israeli Foundation for Scientific Research and Development, the Einstein Foundation Berlin (Leibniz-Humboldt Professorship), the Boehringer-Ingelheim Foundation (Plus 3 award) to C.P.R.H. and the Fonds der Chemischen Industrie (FCI) to C.P.R.H. and A.F.L.S. (Chemiefonds fellowship).

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Authors and Affiliations

  1. Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

    Anselm F. L. Schneider, Martin Lehmann & Christian P. R. Hackenberger

  2. Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

    Anselm F. L. Schneider

  3. Technical University of Darmstadt, Darmstadt, Germany

    Marina Kithil & M. Cristina Cardoso

  4. Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany

    Christian P. R. Hackenberger

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Contributions

A.F.L.S., M.L., M.C.C. and C.P.R.H. conceived the experiments and wrote the manuscript. A.F.L.S. cloned, expressed, purified and characterized proteins, synthesized and characterized peptides and protein–peptide conjugates, and performed uptake, cell viability, microscopy and flow cytometry experiments. M.L. performed microscopy experiments and wrote the quantification script together with A.F.L.S. M.K. performed the calcein AM staining.

Corresponding author

Correspondence to Christian P. R. Hackenberger.

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Competing interests

The technology described in this Article for the delivery of cargoes into cells using cell-surface-reactive peptides is part of a patent application (EP 21159630.9) by A.F.L.S., M.L. and C.P.R.H.

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Peer review information Nature Chemistry thanks Roland Brock, Shiroh Futaki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–37, Methods, Tables 1 and 2 and references.

Reporting Summary

Supplementary Data

Raw proteomics data. Output from MaxQuant software.

Supplementary Table

Output from Perseus-based analysis of proteinGroups data.

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Schneider, A.F.L., Kithil, M., Cardoso, M.C. et al. Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives. Nat. Chem. 13, 530–539 (2021). https://doi.org/10.1038/s41557-021-00661-x

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