Skip to main content
SpringerLink
Log in

Comparison of CPP Uptake Methods

  • Protocol
  • First Online:
Cell-Penetrating Peptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 683))

Abstract

In the last 15 years, an ever expanding pool of cell-penetrating peptides (CPPs) has been discovered and recently focus has shifted towards improving already existing CPPs by different modifications. Since the number of published peptide sequences with cell-penetrating ability is now reaching several hundreds, the consensus methods to compare the efficacy of these is clearly needed. Many research groups are evaluating the applicability of CPPs as drug delivery vectors, all having their preferred methods of assessing uptake and intracellular distribution. Even when applying the same method, the use of different cell lines, peptide concentrations, exposure conditions, etc. are complicating comparison of data between different groups. This book is a welcome contribution to the CPP research field, hopefully paving the way for standardized protocols to be used in the future. Some of the most common methods used to this date are presented and compared in this chapter.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ryser, H. J. and Hancock, R. (1965) Histones and Basic Polyamino Acids Stimulate the Uptake of Albumin by Tumor Cells in Culture. Science. 150, 501–503.

    Article  CAS  PubMed  Google Scholar 

  2. Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The Third Helix of the Antennapedia Homeodomain Translocates through Biological Membranes. J. Biol. Chem. 269, 10444–10450.

    CAS  PubMed  Google Scholar 

  3. Lundberg, M. and Johansson, M. (2001) Is VP22 Nuclear Homing an Artifact? Nat. Biotechnol. 19, 713–714.

    Article  CAS  PubMed  Google Scholar 

  4. Lundberg, M. and Johansson, M. (2002) Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation. Biochem. Biophys. Res. Commun. 291, 367–371.

    Article  CAS  PubMed  Google Scholar 

  5. Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-Penetrating Peptides. A Reevaluation of the Mechanism of Cellular Uptake. J. Biol. Chem. 278, 585–590.

    Article  CAS  PubMed  Google Scholar 

  6. Säälik, P., Elmquist, A., Hansen, M., Padari, K., Saar, K., Viht, K., Langel, Ü., and Pooga, M. (2004) Protein Cargo Delivery Properties of Cell-Penetrating Peptides. A Comparative Study. Bioconjug. Chem. 15, 1246–1253.

    Article  PubMed  Google Scholar 

  7. Console, S., Marty, C., Garcia-Echeverria, C., Schwendener, R., and Ballmer-Hofer, K. (2003) Antennapedia and HIV Transactivator of Transcription (TAT) “Protein Transduction Domains” Promote Endocytosis of High Molecular Weight Cargo upon Binding to Cell Surface Glycosaminoglycans. J. Biol. Chem. 278, 35109–35114.

    Article  CAS  PubMed  Google Scholar 

  8. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. (2007) A Compre­hensive Model for the Cellular Uptake of Cationic Cell-Penetrating Peptides. Traffic. 8, 848–866.

    Article  CAS  PubMed  Google Scholar 

  9. Deshayes, S., Morris, M., Heitz, F., and Divita, G. (2008) Delivery of Proteins and Nucleic Acids using a Non-Covalent Peptide-Based Strategy. Adv. Drug Deliv. Rev. 60, 537–547.

    Article  CAS  PubMed  Google Scholar 

  10. Ter-Avetisyan, G., Tünnemann, G., Nowak, D., Nitschke, M., Herrmann, A., Drab, M., and Cardoso, M. C. (2009) Cell Entry of Arginine-Rich Peptides is Independent of Endocytosis. J. Biol. Chem. 284, 3370–3378.

    Article  CAS  PubMed  Google Scholar 

  11. El-Andaloussi, S., Järver, P., Johansson, H. J., and Langel, Ü. (2007) Cargo-Dependent Cytotoxicity and Delivery Efficacy of Cell-Penetrating Peptides: A Comparative Study. Biochem. J. 407, 285–292.

    Article  CAS  PubMed  Google Scholar 

  12. Tünnemann, G., Martin, R. M., Haupt, S., Patsch, C., Edenhofer, F., and Cardoso, M. C. (2006) Cargo-Dependent Mode of Uptake and Bioavailability of TAT-Containing Proteins and Peptides in Living Cells. FASEB J. 20, 1775–1784.

    Article  PubMed  Google Scholar 

  13. Silhol, M., Tyagi, M., Giacca, M., Lebleu, B., and Vives, E. (2002) Different Mechanisms for Cellular Internalization of the HIV-1 Tat-Derived Cell Penetrating Peptide and Recombinant Proteins Fused to Tat. Eur. J. Biochem. 269, 494–501.

    Article  CAS  PubMed  Google Scholar 

  14. Fischer, R., Waizenegger, T., Kohler, K., and Brock, R. (2002) A Quantitative Validation of Fluorophore-Labelled Cell-Permeable Peptide Conjugates: Fluorophore and Cargo Dependence of Import. Biochim. Biophys. Acta. 1564, 365–374.

    Article  CAS  PubMed  Google Scholar 

  15. Jones, S. W., Christison, R., Bundell, K., Voyce, C. J., Brockbank, S. M., Newham, P., and Lindsay, M. A. (2005) Characterisation of Cell-Penetrating Peptide-Mediated Peptide Delivery. Br. J. Pharmacol. 145, 1093–1102.

    Article  CAS  PubMed  Google Scholar 

  16. Dupont, E., Prochiantz, A., and Joliot, A. (2007) Identification of a Signal Peptide for Unconventional Secretion. J. Biol. Chem. 282, 8994–9000.

    Article  CAS  PubMed  Google Scholar 

  17. Adams, S. R. and Tsien, R. Y. (2006) Imaging the Influx of Cell-Penetrating Peptides into the Cytosol of Individual Cells, in Handbook of Cell-Penetrating Peptides (Ü. Langel, Ed.) 2nd ed., pp 505–512, CRC, Boca Raton.

    Google Scholar 

  18. Adams, S. R. and Tsien, R. Y. (2008) Preparation of the Membrane-Permeant Biarsenicals FlAsH-EDT2 and ReAsH-EDT2 for Fluorescent Labeling of Tetracysteine-Tagged Proteins. Nat. Protoc. 3, 1527–1534.

    Article  CAS  PubMed  Google Scholar 

  19. Holm, T., Johansson, H., Lundberg, P., Pooga, M., Lindgren, M., and Langel, Ü. (2006) Studying the Uptake of Cell-Penetrating Peptides. Nat. Protoc. 1, 1001–1005.

    Article  CAS  PubMed  Google Scholar 

  20. Oehlke, J., Scheller, A., Wiesner, B., Krause, E., Beyermann, M., Klauschenz, E., Melzig, M., and Bienert, M. (1998) Cellular Uptake of an Alpha-Helical Amphipathic Model Peptide with the Potential to Deliver Polar Compounds into the Cell Interior Non-Endocytically. Biochim. Biophys. Acta. 1414, 127–139.

    Article  CAS  PubMed  Google Scholar 

  21. Palm, C., Jayamanne, M., Kjellander, M., and Hällbrink, M. (2007) Peptide Degradation is a Critical Determinant for Cell-Penetrating Peptide Uptake. Biochim. Biophys. Acta. 1768, 1769–1776.

    Article  CAS  PubMed  Google Scholar 

  22. Aubry, S., Burlina, F., Dupont, E., Delaroche, D., Joliot, A., Lavielle, S., Chassaing, G., and Sagan, S. (2009) Cell-Surface Thiols Affect Cell Entry of Disulfide-Conjugated Peptides. FASEB J. 23, 2956–2967.

    Article  CAS  PubMed  Google Scholar 

  23. Lundin, P., Johansson, H., Guterstam, P., Holm, T., Hansen, M., Langel, Ü., and EL Andaloussi, S. (2008) Distinct Uptake Routes of Cell-Penetrating Peptide Conjugates. Bioconjug. Chem. 19, 2535-2542.

    Article  CAS  PubMed  Google Scholar 

  24. Säälik, P., Padari, K., Niinep, A., Lorents, A., Hansen, M., Jokitalo, E., Langel, Ü., and Pooga, M. (2009) Protein Delivery with Transportans Is Mediated by Caveolae rather than Flotillin-Dependent Pathways. Bioconjug. Chem. 20(5), 877–887.

    Article  PubMed  Google Scholar 

  25. Padari, K., Säälik, P., Hansen, M., Koppel, K., Raid, R., Langel, Ü., and Pooga, M. (2005) Cell Transduction Pathways of Transportans. Bioconjug. Chem. 16, 1399–1410.

    Article  CAS  PubMed  Google Scholar 

  26. Elmquist, A. and Langel, Ü. (2003) In Vitro Uptake and Stability Study of pVEC and its all-D Analog. Biol. Chem. 384, 387–393.

    Article  CAS  PubMed  Google Scholar 

  27. Burlina, F., Sagan, S., Bolbach, G., and Chassaing, G. (2005) Quantification of the Cellular Uptake of Cell-Penetrating Peptides by MALDI-TOF Mass Spectrometry. Angew. Chem. Int. Ed Engl. 44, 4244–4247.

    Article  CAS  PubMed  Google Scholar 

  28. Burlina, F., Sagan, S., Bolbach, G., and Chassaing, G. (2006) A Direct Approach to Quantification of the Cellular Uptake of Cell-Penetrating Peptides using MALDI-TOF Mass Spectrometry. Nat. Protoc. 1, 200–205.

    Article  CAS  PubMed  Google Scholar 

  29. Mäe, M., El Andaloussi, S., Lundin, P., Oskolkov, N., Johansson, H. J., Guterstam, P., and Langel, Ü. (2009) A Stearylated CPP for Delivery of Splice Correcting Oligonucleotides using a Non-Covalent Co-Incubation Strategy. J. Control. Release. 134, 221–227.

    Article  PubMed  Google Scholar 

  30. Abes, S., Moulton, H. M., Clair, P., Prevot, P., Youngblood, D. S., Wu, R. P., Iversen, P. L., and Lebleu, B. (2006) Vectorization of Morpholino Oligomers by the (R-Ahx-R)4 Peptide Allows Efficient Splicing Correction in the Absence of Endosomolytic Agents. J. Control. Release. 116, 304–313.

    Article  CAS  PubMed  Google Scholar 

  31. Abes, R., Moulton, H. M., Clair, P., Yang, S. T., Abes, S., Melikov, K., Prevot, P., Youngblood, D. S., Iversen, P. L., Chernomordik, L. V., and Lebleu, B. (2008) Delivery of Steric Block Morpholino Oligomers by (R-X-R)4 Peptides: Structure-Activity Studies. Nucleic Acids Res. 36, 6343–6354.

    Article  CAS  PubMed  Google Scholar 

  32. Lehto, T., Abes, R., Oskolkov, N., Suhorutsenko, J., Copolovici, D. M., Mäger, I., Viola, J. R., Simonsson, O., Guterstam, P., Eriste, E., Smith, C. I., Lebleu, B., El Andaloussi, S., and Langel, Ü. (2010) Delivery of Nucleic Acids with a Stearylated (RxR)(4) Peptide using a Non-Covalent Co-Incubation Strategy. J. Control. Release. 141(1), 42–51.

    Article  CAS  PubMed  Google Scholar 

  33. Kang, S. H., Cho, M. J., and Kole, R. (1998) Up-Regulation of Luciferase Gene Expression with Antisense Oligonucleotides: Implications and Applications in Functional Assay Development. Biochemistry. 37, 6235–6239.

    Article  CAS  PubMed  Google Scholar 

  34. El Andaloussi, S., Guterstam, P., and Langel, Ü. (2007) Assessing the Delivery Efficacy and Internalization Route of Cell-Penetrating Peptides. Nat. Protoc. 2, 2043–2047.

    Article  CAS  PubMed  Google Scholar 

  35. Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA Fusogenic Peptide Enhances Escape of TAT-Fusion Proteins After Lipid Raft Macropinocytosis. Nat. Med. 10, 310–315.

    Article  CAS  PubMed  Google Scholar 

  36. Aroui, S., Brahim, S., De Waard, M., Breard, J., and Kenani, A. (2009) Efficient Induction of Apoptosis by Doxorubicin Coupled to Cell-Penetrating Peptides Compared to Unconjugated Doxorubicin in the Human Breast Cancer Cell Line MDA-MB 231. Cancer Lett. 285(1), 28–38.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The work was supported by grants from Swedish Research Council (VR-NT); Center for Biomembrane Research, Stockholm; and Knut and Alice Wallenberg’s Foundation.

Author information

Authors and Affiliations

  1. Department of Neurochemistry, Stockholm University, Stockholm, Sweden

    Tina Holm, Samir EL Andaloussi & Ülo Langel

  2. Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia

    Ülo Langel

Authors
  1. Tina Holm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Samir EL Andaloussi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ülo Langel
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. , Department of Neurochemistry, Stockholm University, Svante Arrheniusv 21A21A, Stockholm, 106 91, Sweden

    Ülo Langel

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Holm, T., Andaloussi, S.E., Langel, Ü. (2011). Comparison of CPP Uptake Methods. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 683. Humana Press. https://doi.org/10.1007/978-1-60761-919-2_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-919-2_15

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-918-5

  • Online ISBN: 978-1-60761-919-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation