Comparison of CPP Uptake Methods

  • Tina Holm
  • Samir EL Andaloussi
  • Ülo Langel
Part of the Methods in Molecular Biology book series (MIMB, volume 683)


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.

Key words

CPP Fluorescence Microscopy HPLC FACS Spectrofluorometry Mass spectrometry Splice correction Biological response 



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


  1. 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.CrossRefPubMedGoogle Scholar
  2. 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.PubMedGoogle Scholar
  3. 3.
    Lundberg, M. and Johansson, M. (2001) Is VP22 Nuclear Homing an Artifact? Nat. Biotechnol. 19, 713–714.CrossRefPubMedGoogle Scholar
  4. 4.
    Lundberg, M. and Johansson, M. (2002) Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation. Biochem. Biophys. Res. Commun. 291, 367–371.CrossRefPubMedGoogle Scholar
  5. 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.CrossRefPubMedGoogle Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. 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.CrossRefPubMedGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 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.CrossRefPubMedGoogle Scholar
  10. 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.CrossRefPubMedGoogle Scholar
  11. 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.CrossRefPubMedGoogle Scholar
  12. 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.CrossRefPubMedGoogle Scholar
  13. 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.CrossRefPubMedGoogle Scholar
  14. 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.CrossRefPubMedGoogle Scholar
  15. 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.CrossRefPubMedGoogle Scholar
  16. 16.
    Dupont, E., Prochiantz, A., and Joliot, A. (2007) Identification of a Signal Peptide for Unconventional Secretion. J. Biol. Chem. 282, 8994–9000.CrossRefPubMedGoogle Scholar
  17. 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. 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.CrossRefPubMedGoogle Scholar
  19. 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.CrossRefPubMedGoogle Scholar
  20. 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.CrossRefPubMedGoogle Scholar
  21. 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.CrossRefPubMedGoogle Scholar
  22. 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.CrossRefPubMedGoogle Scholar
  23. 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.CrossRefPubMedGoogle Scholar
  24. 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.CrossRefPubMedGoogle Scholar
  25. 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.CrossRefPubMedGoogle Scholar
  26. 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.CrossRefPubMedGoogle Scholar
  27. 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.CrossRefPubMedGoogle Scholar
  28. 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.CrossRefPubMedGoogle Scholar
  29. 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.CrossRefPubMedGoogle Scholar
  30. 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.CrossRefPubMedGoogle Scholar
  31. 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.CrossRefPubMedGoogle Scholar
  32. 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.CrossRefPubMedGoogle Scholar
  33. 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.CrossRefPubMedGoogle Scholar
  34. 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.CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Tina Holm
    • 1
  • Samir EL Andaloussi
    • 1
  • Ülo Langel
    • 1
    • 2
  1. 1.Department of NeurochemistryStockholm UniversityStockholmSweden
  2. 2.Laboratory of Molecular Biotechnology, Institute of TechnologyTartu UniversityTartuEstonia

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