Summary
Cell-penetrating peptides (CPPs) offer potential as delivery agents for the cellular administration of drugs. However, the pharmacological utility of CPPs that are derived from natural amino acids is limited by their rapid metabolic degradation, low membrane permeability, and toxicity. Various peptidomimetics able to overcome these problems have been described, including peptides formed by D-amino acids and β -peptides. This chapter summarizes the synthesis of γ-proline-derived peptides and polyproline dendrimers for drug delivery applications, and includes descriptions of several modifications in the γ-peptides (mimicking the side chains of the α -amino acids) or modulating the dendrimer surface. 5(6)-Carboxyfluorescein labeling of the aforementioned peptidomimetics for use in cell translocation studies is also described. Furthermore, different protocols for the study of the drug delivery capabilities of these compounds are reviewed, including enzymatic stability studies, cellular uptake measurements by plate fluorimetry and flow cytometry, confocal laser scanning microscopy, and cytotoxicity assays.
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References
Tréhin, R., and Merkle, H. P. (2004) Chances and pitfalls of cell penetrating peptides for cellular drug delivery. Eur. J. Phar. Biophar. 58, 209–223.
Zorko, M., and Langel, Ü. (2005) Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv. Drug Delivery Rev. 57, 529–545.
Davidson, B. L., and Breakefield, X. O. (2004) Neurological diseases: viral vecors for gene delivery to the nervous system. Nat. Rev. Neurosci. 4, 353–364.
Connor, J., and Huang, L. (1985) Efficient cytoplasmatic delivery of a fluorescent dye by pH-sensitive immunoliposomes. J. Cell. Biol. 101, 582–589.
Foldvari, M., Mezei, C., and Mezei, M. (1991) Intracellular delivery of drugs by liposomes containing P0 glycoprotein from peripheral nerve myelin into human M21 melanoma cells. J. Pharm. Sci. 80, 1020–1028.
Gentile, F. T., Doherty, E. J., Rein, D. H., Shoichet, M. S., Winn, S. R. (1995) Polymer science for macroencapsulation of cells for central nervous system transplantation. Reactive Polymers 25, 207–227.
Chakrabarti, R., Wylie, D. E., and Schuster S. M. (1989) Transfer of monoclonal antibodies into mammalian cells by electroporation. J. Biol. Chem. 264, 15, 494–15,500.
Leamon, C. P., and Low, P. S. (1991) Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis. Proc. Natl. Acad. Sci. USA 88, 5572–5576.
Sadler, K., Eom, K. D., Yang, J-L., Dimitrova, Y., and Tam, J. P. (2002) Translocating proline-rich peptides from the antimicrobial peptide Bactenecin 7. Biochemistry 41, 14,150–14,157.
Singh, D., Kiarash, R., Kawamura, K., LaCasse, E. C., and Gariépy, J. (1998) Penetration and intracellular routing of nucleus-directed peptide-based shuttles (loligomers) in eukaryotic cells. Biochemistry 37, 5798–5809.
Brokx, R. D., Bisland, S. K., and Gariépy, J. (2002) Designing peptide-based scaffolds as drug delivery vehicles. J. Controlled Release 78, 115–123.
Elmquist, E., Lindgren, M., Bartfai, T., and Langel, Ü. (2001) VE-cadherin-derived cell penetrating peptide, pVEC, with carrier functions. Exp. Cell Res. 269, 237–244.
Umezawa, N., Gelman, M. A., Haigis, M. C., Raines, R. T., and Gellman, S. H. (2002) Translocation of a beta-peptide across cell membranes. J. Am. Chem. Soc. 124, 368–369.
Rueping, M., Mahajan, Y., Sauer, M., and Seebach, D. (2002) Cellular uptake studies with beta-peptides. ChemBioChem 3, 257–259.
Potocky, T. B., Menon, A. K., and Gellman, S. H. (2003) Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells. J. Biol. Chem. 278, 50,188–50,194.
Garcia-Echeverria, C., and Ruetz, S. (2003) Beta-Homolysine oligomers: a new class of Trojan carriers. Bioorg. Med. Chem. Lett. 13, 247–251.
Farrera-Sinfreu, J., Zaccaro, L., Vidal, D., et al. (2004) A new class of foldamers based on cis-γ-amino-L-proline. J. Am. Chem. Soc. 126, 6048–6057.
Farrera-Sinfreu, J., Giralt, E., Castel, S., Albericio, F., and Royo, M. (2005) Cell-penetrating cis-γ-amino-L-proline-derived peptides. J. Am. Chem. Soc. 127, 9459–9468.
Crespo, L., Sanclimens, G., Royo, M., Giralt, E., and Albericio, F. (2002) Branched poly(proline) peptides: an efficient new approach to the synthesis of repetitive branched peptides. Eur. J. Org. Chem. 11, 1756–1762.
Crespo, L., Sanclimens, G., Montaner, B., et al. (2002) Peptide dendrimers based on polyproline helices. J. Am. Chem. Soc. 124, 8876–8883.
Sanclimens, G., Crespo, L., Giralt, E., Royo, M., and Albericio, F. (2004) Solid-phase synthesis of second-generation polyproline dendrimers. Biopolymers (Pept. Sci.) 76, 283–297.
Sanclimens, G., Crespo, L., Giralt, E., Albericio, F., and Royo, M., (2005) Preparation of de novo globular proteins based on proline dendrimers. J. Org. Chem. 70, 6274–6281..
Sanclimens, G., Shen, H., Giralt, E., Albericio, F., Saltzman, M. W., and Royo, M. (2005) Synthesis and screening of a small library of proline based biodendrimers for use as delivery agents. Biopolymers 80, 800–814.
Fernàndez-Carneado, J., Kogan, M. J., Castel, S., Pujals, S., and Giralt, E. (2004) Potential peptide carriers: amphipathic proline-rich peptides derived from the N-terminal domain of γ -zein. Angew. Chem. Int. Ed. 43, 1811–1814.
Fernàndez-Carneado, J., Kogan, M. J., Pujals, S., and Giralt, E. (2004) Amphipathic peptides and drug delivery. Biopolymers (Pept. Sci.) 76, 196–203.
Foerg, C., Ziegler, V., Fernàndez-Carneado, J., et al. (2005) Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. Biochemistry 44, 72–81.
Fernàndez-Carneado, J., Kogan, M. J., Van Mau, N., et al. (2005) Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells. J. Pept. Res. 65, 580–590.
Lloyd-Williams, P., Albericio, F., and Giralt, E. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton, FL.
Feichtinger, K., Zapf, C., Sings, H. L., and Goodman, M. (1998) Diprotected triflylguanidines: a new class of guanidinylation reagents. J. Org. Chem. 63, 3804–3805.
Rose, K., and Vizzavona, J. (1999) Stepwise solid-phase synthesis of polyamides as linkers. J. Am. Chem. Soc. 121, 7034–7038.
Liu, Y., Peterson, D. A., Kimura, H., and Schubert, D. (1997) Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J. Neurochem. 69, 581–593.
Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in solid-phase synthesis of peptides. Anal. Biochem. 34, 594–598.
Madder, A., Farcy, N., Hosten, N. G. C., et al. (1999) A novel sensitive colorimetric assay for visual detection of solid-phase bound amines. Eur. J. Org. Chem. 2787–2791.
Christensen, T. (1979) A qualitative test for monitoring coupling completeness in solid-phase peptide synthesis using chloranil. Acta Chem. Scan. 33, 760–766.
Kuisle, O., Lolo, M., Quiñoà, E., and Riguera, R. (1999) Monitoring the solid-phase synthesis of depsides and depsipeptides. A color test for hydroxyl groups linked to a resin. Tetrahedron 55, 14,807–14,812.
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Farrera-Sinfreu, J., Giralt, E., Royo, M., Albericio, F. (2007). Cell-Penetrating Proline-Rich Peptidomimetics. In: Fields, G.B. (eds) Peptide Characterization and Application Protocols. Methods in Molecular Biology™, vol 386. Humana Press. https://doi.org/10.1007/978-1-59745-430-8_9
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DOI: https://doi.org/10.1007/978-1-59745-430-8_9
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