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Quantification of Cell-Penetrating Peptides and Their Cargoes 273 TABLE 12.1 Examples of Quantification Methods of Cargos Delivered by CPPs by Their Intracellular Activity Active Cargo Cargo Details CPP Quantification Method Peptide Autophosphorylation site of EGFR, 12 residue peptide εIF4E-binding motifs analogues, 12 residue peptides Antisense βAPP antisense, 25-mer oligonucleotide Signal sequence derived CPP pathways. 22 Furthermore, penetratin has been used for delivery of εIF4E-binding motifs analogues. 23 The effect of the peptides was dose-dependent measurements of cell survival by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. 12.2.2.2 Detection of DNA/PNA Antisense Effects The antisense effect in general is quantified either by analysis of protein content or quantification of mRNA levels. This detection of the extent of the effect is usually accompanied by a functional or biological assay (Table 12.1). The membrane translocation signal (MTS) from Ig(v) light chain has been coupled via peptidase-sensitive linker to the nuclear localization signal (NLS), residues 127–132, or SV40T-ag. 24 The MTS–NLS chimera coupled to antisense (AS) β-COM oligodeoxynucleotide (ODN), directed against L-type Ca 2+ channel, was shown to block mRNA translation. Furthermore, transportan has previously been used for the intracellular delivery of 21-mer antisense PNA complementary to the galanin receptor type 1 mRNA. 25 For further details, see Chapter 16 on antisense application of CPPs. 12.2.2.3 Quantification of Drug Activity Inhibition of Ras/MAP kinase activation measured by Ras nucleotide exchange in SAA cells 21 Penetratin Dose-dependent measurements of cell survival by the MTT assay 23 Penetratin Down-regulation of amyloid precursor protein to study effects on neurite outgrowth; quantified by immunoprecipitation and pulse-chase experiments 28 GalR1 antisense, Transportan, Estimation of pain transmission by the 21-mer PNA Penetratin flexor–reflex assay and western blot detection 25 Drug Doxorubicin Penetratin Quantification of radio-labeled doxorubicin in brain tissue 26,29 and cytotoxcicity measurements by the MTT assay 27 Rousselle et al. 26 describe the delivery of the antineoplastic agent doxorubicin across the blood–brain barrier using the peptides penetratin and SynB1. The peptides deliver
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.
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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116 Cell-Penetrating Peptides: Proc
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Cell-Penetrating Peptide Conjugatio
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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