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.

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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

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.

References

  1. 1.

    Fu, A., Tang, R., Hardie, J., Farkas, M. E. & Rotello, V. M. Promises and pitfalls of intracellular delivery of proteins. Bioconjug. Chem. 25, 1602–1608 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Du, S., Liew, S. S., Li, L. & Yao, S. Q. Bypassing endocytosis: direct cytosolic delivery of proteins. J. Am. Chem. Soc. 140, 15986–15996 (2018).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Wang, F. et al. Recent progress of cell-penetrating peptides as new carriers for intracellular cargo delivery. J. Control. Release 174, 126–136 (2014).

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. Inhibition of antigen-induced lymphocyte proliferation by Tat protein from HIV-1. Science 246, 1606–1608 (1989).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Joliot, A. H., Triller, A., Volovitch, M., Pernelle, C. & Prochiantz, A. α-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol. 3, 1121–1134 (1991).

    CAS  PubMed  Google Scholar 

  6. 6.

    Derossi, D., Joliot, A. H., Chassaing, G. & Prochiantz, A. The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450 (1994).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Borrelli, A., Tornesello, A. L., Tornesello, M. L. & Buonaguro, F. M. Cell penetrating peptides as molecular carriers for anti-cancer agents. Molecules 23, 295 (2018).

    PubMed Central  Article  CAS  PubMed  Google Scholar 

  8. 8.

    Guidotti, G., Brambilla, L. & Rossi, D. Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol. Sci. 38, 406–424 (2017).

    CAS  PubMed  Article  Google Scholar 

  9. 9.

    Richard, J. P. et al. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280, 15300–15306 (2005).

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Fittipaldi, A. et al. Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J. Biol. Chem. 278, 34141–34149 (2003).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Ben-Dov, N. & Korenstein, R. The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis. Biochim. Biophys. Acta 1848, 869–877 (2015).

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Herce, H. D., Garcia, A. E. & Cardoso, M. C. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J. Am. Chem. Soc. 136, 17459–17467 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R. & Brock, R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8, 848–866 (2007).

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Futaki, S. & Nakase, I. Cell-surface interactions on arginine-rich cell-penetrating peptides allow for multiplex modes of internalization. Acc. Chem. Res. 50, 2449–2456 (2017).

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    He, L., Sayers, E. J., Watson, P. & Jones, A. T. Contrasting roles for actin in the cellular uptake of cell penetrating peptide conjugates. Sci. Rep. 8, 7318 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Patel, S. G. et al. Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Sci. Rep. 9, 6298 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. 17.

    Tunnemann, G. et al. Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J. 20, 1775–1784 (2006).

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Verdurmen, W. P., Thanos, M., Ruttekolk, I. R., Gulbins, E. & Brock, R. Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake. J. Control. Release 147, 171–179 (2010).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Takeuchi, T. et al. Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate. ACS Chem. Biol. 1, 299–303 (2006).

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Erazo-Oliveras, A. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat. Methods 11, 861–867 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Allen, J. et al. Cytosolic delivery of macromolecules in live human cells using the combined endosomal escape activities of a small molecule and cell penetrating peptides. ACS Chem. Biol. 14, 2641–2651 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Akishiba, M. et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. Nat. Chem 9, 751–761 (2017).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Morris, M. C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173–1176 (2001).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Reichart, F., Horn, M. & Neundorf, I. Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery. J. Pept. Sci. 22, 421–426 (2016).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Dougherty, P. G., Sahni, A. & Pei, D. Understanding cell penetration of cyclic peptides. Chem. Rev. 119, 10241–10287 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. 27.

    Lattig-Tunnemann, G. et al. Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides. Nat. Commun. 2, 453 (2011).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  28. 28.

    Nischan, N. et al. Covalent attachment of cyclic TAT peptides to GFP results in protein delivery into live cells with immediate bioavailability. Angew. Chem. Int. Ed. 54, 1950–1953 (2015).

    CAS  Article  Google Scholar 

  29. 29.

    Schneider, A. F. L., Wallabregue, A. L. D., Franz, L. & Hackenberger, C. P. R. Targeted subcellular protein delivery using cleavable cyclic cell-penetrating peptides. Bioconjug. Chem. 30, 400–404 (2019).

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    Herce, H. D. et al. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells. Nat. Chem. 9, 762–771 (2017).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Medina, S. H. et al. An intrinsically disordered peptide facilitates non-endosomal cell entry. Angew. Chem. Int. Ed. 55, 3369–3372 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Jones, A. T. & Sayers, E. J. Cell entry of cell penetrating peptides: tales of tails wagging dogs. J. Control. Release 161, 582–591 (2012).

    CAS  PubMed  Article  Google Scholar 

  33. 33.

    Robison, A. D. et al. Polyarginine interacts more strongly and cooperatively than polylysine with phospholipid bilayers. J. Phys. Chem. B 120, 9287–9296 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Shi, J. & Schneider, J. P. De novo design of selective membrane-active peptides by enzymatic control of their conformational bias on the cell surface. Angew. Chem. Int. Ed. 58, 13706–13710 (2019).

    CAS  Article  Google Scholar 

  35. 35.

    Aubry, S. et al. Cell-surface thiols affect cell entry of disulfide-conjugated peptides. FASEB J. 23, 2956–2967 (2009).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Gasparini, G., Sargsyan, G., Bang, E. K., Sakai, N. & Matile, S. Ring tension applied to thiol-mediated cellular uptake. Angew. Chem. Int. Ed. 54, 7328–7331 (2015).

    CAS  Article  Google Scholar 

  37. 37.

    Martin, R. M., Herce, H. D., Ludwig, A. K. & Cardoso, M. C. Visualization of the nucleolus in living cells with cell-penetrating fluorescent peptides. Methods Mol. Biol. 1455, 71–82 (2016).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Martin, R. M., Tunnemann, G., Leonhardt, H. & Cardoso, M. C. Nucleolar marker for living cells. Histochem. Cell Biol. 127, 243–251 (2007).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Chagin, V. O. et al. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons. Nat. Commun. 7, 11231 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Hunt, L. et al. Low-temperature pausing of cultivated mammalian cells. Biotechnol. Bioeng. 89, 157–163 (2005).

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Goldenthal, K. L., Pastan, I. & Willingham, M. C. Initial steps in receptor-mediated endocytosis. The influence of temperature on the shape and distribution of plasma membrane clathrin-coated pits in cultured mammalian cells. Exp. Cell. Res. 152, 558–564 (1984).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Melchior, F., Guan, T., Yokoyama, N., Nishimoto, T. & Gerace, L. GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import. J. Cell Biol. 131, 571–581 (1995).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Rittner, K. et al. New basic membrane-destabilizing peptides for plasmid-based gene delivery in vitro and in vivo. Mol. Ther. 5, 104–114 (2002).

    CAS  PubMed  Article  Google Scholar 

  45. 45.

    Rouet, R. et al. Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing. J. Am. Chem. Soc. 140, 6596–6603 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Mayle, K. M., Le, A. M. & Kamei, D. T. The intracellular trafficking pathway of transferrin. Biochim. Biophys. Acta 1820, 264–281 (2012).

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Ter-Avetisyan, G. et al. Cell entry of arginine-rich peptides is independent of endocytosis. J. Biol. Chem. 284, 3370–3378 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Gasparini, G. et al. Cellular uptake of substrate-initiated cell-penetrating poly(disulfide)s. J. Am. Chem. Soc. 136, 6069–6074 (2014).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Fu, J., Yu, C., Li, L. & Yao, S. Q. Intracellular delivery of functional proteins and native drugs by cell-penetrating poly(disulfide)s. J. Am. Chem. Soc. 137, 12153–12160 (2015).

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Wallbrecher, R. et al. Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity. J. Control. Release 256, 68–78 (2017).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Wei, Y., Tang, T. & Pang, H. B. Cellular internalization of bystander nanomaterial induced by TAT-nanoparticles and regulated by extracellular cysteine. Nat. Commun. 10, 3646 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. 52.

    Hirose, H. et al. Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells. Mol. Ther. 20, 984–993 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Colom, A. et al. A fluorescent membrane tension probe. Nat. Chem. 10, 1118–1125 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. 54.

    England, C. G., Luo, H. & Cai, W. HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Tesei, G. et al. Self-association of a highly charged arginine-rich cell-penetrating peptide. Proc. Natl Acad. Sci. USA 114, 11428–11433 (2017).

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Peitz, M., Pfannkuche, K., Rajewsky, K. & Edenhofer, F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc. Natl Acad. Sci. USA 99, 4489–4494 (2002).

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Yang, Y. S. & Hughes, T. E. Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells. Biotechniques 31, 1040–1031 (2001).

    Google Scholar 

  58. 58.

    Jue, R., Lambert, J. M., Pierce, L. R. & Traut, R. R. Addition of sulfhydryl groups to Escherichia coli ribosomes by protein modification with 2-iminothiolane (methyl 4-mercaptobutyrimidate). Biochemistry 17, 5399–5406 (1978).

    CAS  PubMed  Article  Google Scholar 

Download references

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).

Author information

Affiliations

Authors

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.

Ethics declarations

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.

Additional information

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.

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

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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. (2021). https://doi.org/10.1038/s41557-021-00661-x

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing