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274 Cell-Penetrating Peptides: Processes and Applications
doxorubicin, in vivo, both when tested in brain perfusion and when i.v. injected.
Doxorubicin passage into the brain was quantified in in situ hybridization by radiolabeling.
In a further study of peptide-vectorized doxorubicin, a dose-dependent
inhibition of cell growth was detected with IC 50 concentrations of vectorized doxorubicin
at 3 µM and the drug alone at 65 µM. 27
12.3 SUMMARY
Methods to quantify the internalization of cell-penetrating peptides are still in development.
Those reviewed here have several drawbacks and are not ideal for all
applications. A variety of new fluorophores may solve problems of sensitivity,
photobleaching, and pH dependence, but will also be applicable for FRET or RET-
Q studies. Measuring the change in fluorescence usually has a sensitivity higher
than for direct measurement.
Today the development of mass spectrometry enables quantification of peptides
and proteins by, for example, MALDI-TOF and may yield a future tool for studying
CPPs. The main advantage would be that identification of the species present in the
experiments could be carried out simultaneously with the actual quantification.
For a higher sample throughput a cell-ELISA with a sensitive luciferase or RET-
Q system of detection would be highly convenient. Therefore, further development
of assays for CPP quantification is needed. An ideal would be an assay in which all
the methodological considerations in this chapter and the rest of this book are taken
into assessment.
REFERENCES
1. Lindgren, M. et al., Cell-penetrating peptides, Trends Pharmacol. Sci., 21, 99–103, 2000.
2. Hällbrink, M. et al., Cargo delivery kinetics of cell-penetrating peptides, Biochim.
Biophys. Acta, 1515, 101–109, 2001.
3. Fawell, S. et al., Tat-mediated delivery of heterologous proteins into cells, Proc. Natl.
Acad. Sci. U.S.A., 91, 664–668, 1994.
4. Oehlke, J. et al., Cellular uptake of an alpha-helical amphipathic model peptide with
the potential to deliver polar compounds into the cell interior nonendocytically,
Biochim. Biophys. Acta, 1414, 127–139, 1998.
5. Lindgren, M. et al., Translocation properties of novel cell-penetrating transportan and
penetratin analogues, Bioconjug. Chem., 11, 619–626, 2000.
6. Pooga, M. et al., Cell penetration by transportan, FASEB J., 12, 67–77, 1998.
7. Drin, G. et al., Physico-chemical requirements for cellular uptake of pAntp peptide.
Role of lipid-binding affinity, Eur. J. Biochem., 268, 1304–1314, 2001.
8. Scheller, A. et al., Evidence for an amphipathicity independent cellular uptake of
amphipathic cell-penetrating peptides, Eur. J. Biochem., 267, 6043–6050, 2000.
9. Bolton, A.E. and Hunter, W.M., The labelling of proteins to high specific radioactivities
by ionization to a 125 I-containing acylating agent, Biochem. J., 133, 529–538, 1973.
10. Kask, K. et al., Binding and agonist/antagonist actions of M35, galanin(1-13)-bradykinin(2-9)amide
chimeric peptide, in Rin m 5F insulinoma cells, Regul. Pept., 59,
341–348, 1995.