Abstract
CPPs have for numerous years been utilized as delivery vectors of various pharmaceutically interesting cargoes, both in vitro and in vivo. As CPPs are gradually approaching the bedsides, investigating toxicity associated with these highly interesting peptides becomes increasingly important and thorough initial assessment of cytotoxicity in vitro is a first step towards advancing these delivery vehicles in to the clinics. The present chapter describes protocols for four cytotoxicity assays in order to provide a toolbox for toxicity assessment of CPPs. The foci lie on membrane integrity (deoxyglucose leakage and propidium iodide assays) and cell viability (the MTT assay), but the chapter also provides a protocol for assessing an important parameter for future clinical applications, namely the hemolytic properties of CPPs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Massodi, I., Bidwell, G. L., Davis, A., Tausend, A., Credit, K., Flessner, M., Raucher, D. (2009) Inhibition of ovarian cancer cell metastasis by a fusion polypeptide Tat-ELP. Clin Exp Metastasis 26, 251–260.
Wu, B., Moulton, H. M., Iversen, P. L., Jiang, J., Li, J., Li, J., Spurney, C. F., Sali, A., Guerron, A. D., Nagaraju, K., Doran, T., Lu, P., Xiao, X., Lu, Q. L. (2008) Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A 105, 14814–14819.
Saar, K., Lindgren, M., Hansen, M., Eiriksdottir, E., Jiang, Y., Rosenthal-Aizman, K., Sassian, M., Langel, Ü. (2005) Cell-penetrating peptides: A comparative membrane toxicity study. Anal Biochem 345, 55–65.
Mueller, J., Kretzschmar, I., Volkmer, R., Boisguerin, P. (2008) Comparison of cellular uptake using 22 CPPs in 4 different cell lines. Bioconjug Chem 19, 2363–2374.
Bárány-Wallje, E., Gaur, J., Lundberg, P., Langel, Ü., Gräslund, A. (2007) Differential membrane perturbation caused by the cell-penetrating peptide Tp10 depending on attached cargo. FEBS Lett 581, 2389–2393.
Magzoub, M., Oglecka, K., Pramanik, A., Eriksson, L. E., Gräslund, A. (2005) Membrane perturbation effects of peptides of peptides derived from the N-termini unprocessed prion protein. Biochim Biophys Acta 15, 126–136.
Tréhin, R., Krauss, U., Muff, R., Meinecke, M., Beck-Sickinger, A. G., Merkle, H. P. (2004) Cellular internalization of human calcitonin derived peptides in MDCK monolayers: A comparative study with Tat(47-57) and Penetratin(43-58). Pharm Res 21, 33–42.
Jones, S. W., Christison, R., Bundell, K., Voyce, C. J., Brockbank, S. M. V., Newham, P., Lindsay, M. A. (2005) Characterisation of cell-penetrating peptide-mediated peptide delivery. Br J Pharmacol 145, 1093–1020.
Deshayes, S., Plenat, T., Charnet, P., Divita, G., Molle, F., Heitz, F., (2006) Formation of transmembrane ionic channels of primary amphipathic cell-penetrating peptides. Consequences on the mechanism of cell penetration. Biochim Biophys Acta 1758, 1846–1851.
Ziegler, A. (2008) Thermodynamic studies and binding mechanism of cell-penetrating peptides with lipids and glycosaminoglycans. Adv Drug Deliv Rev 60, 580–597.
Wu, R. P., Youngblood, D. S., Hassinger, J. N., Lovejoy, C. E., Nelson, M. H., Iversen, P. L., Moulton, H. M. (2007) Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid substitution on intracellular delivery and cytotoxicity. Nucleic Acids Res 35, 5182–5191.
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55–63.
Walum, E., Peterson, A. (1982) Tritiated 2-deoxy-D-glucose as a probe for cell membrane permeability studies. Anal Biochem 120, 8–11.
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
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lundin, P., EL Andaloussi, S., Langel, Ü. (2011). Toxicity Methods for CPPs. 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_14
Download citation
DOI: https://doi.org/10.1007/978-1-60761-919-2_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-918-5
Online ISBN: 978-1-60761-919-2
eBook Packages: Springer Protocols