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2.B.9 The Cell Penetrating Peptide (CPP) Functional Family

The major limitation in utilizing information rich macromolecules for basic science and therapeutic applications is the inability of these large molecules to readily diffuse across the cellular membrane. While this restriction represents an efficient defense system against cellular penetration of unwanted foreign molecules and thus a crucial component of cell survival, overcoming this cellular characteristic for the intracellular delivery of macromolecules has been the focus of a large number of research groups worldwide. With the discovery of RNA interference, many of these groups have applied cell delivery methodologies to synthetic short interfering RNA duplexes (siRNA) (Lebleu et al., 2008). Protein transduction domains and cell penetrating peptides have been shown to enhance the delivery of multiple types of macromolecular cargo including peptides, proteins and antisense oligonucleotides and are now being utilized to enhance the cellular uptake of siRNA molecules (Meade and Dowdy, 2008). The dense cationic charge of these peptides that is critical for interaction with cell membrane components prior to internalization has been shown to readily package siRNA molecules into stable nanoparticles that are capable of traversing the cell membrane.  Many different CPPs and their uses have been reviewed (see Table 1 in Durzyńska et al. 2015).  This table presents their characteristics, names, amino acyl sequences, organismal sources and references.  Other earlier reviews of CPPs include:  Deshayes et al. 2005; Deshayes et al. 2008; Henriques and Castanho 2008; De la Vieja et al. 2007.

CPPs can penetrate membranes by themselves and also with various cargos, provide promising tools for the cellular delivery of molecular cargos ranging in size from small molecules and peptides to proteins and quantum dots. CPPs are typically non-homologous cationic and/or amphipathic sequences that are unstructured or alpha-helical. Because they are non-homologous, this family should be considered a 'functional' family, not a phylogenetic family.  CPPs may also possess type-II polyproline (PPII) helical structures (Daniels and Schepartz, 2007). These motifs surpass the uptake efficiency of other CPPs and are not cytotoxic at concentrations 100 times greater than that necessary for delivery. By replacing the PPII helix of a miniature protein, the motif can endow intrinsic cell permeability without increasing molecular size. Some amphipathic CPPs traverse pure lipid model membranes at low micromolar concentrations although translocation under these conditions is not observed for non-amphipathic CPPs (Ziegler, 2008). Grdisa (2011) reviewed the use of arginine-rich and amphipathic carrier-type peptides.

CPPs derived from the native peptide hormone human calcitonin (hCT) have proven to translocate bioactive molecules across cellular membranes. N-terminally truncated hCT fragments have been optimised to extend their field of application (Rennert et al., 2008). hCT-derived carrier peptides are highly effective, branched peptides. The structural requirements, mechanistic assumptions and metabolic features of these peptides have been discussed (Rennert et al., 2008). At least some CPPs such as PEP-1 do not appear to form pores (Henriques et al. 2007).

CPPs and myristoylated peptides (Nelson et al., 2007) can overcome delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability (Hansen et al., 2008). Foged and Nielsen (2008) describe the application of cell-penetrating peptides as transmembrane drug delivery agents and fundamental principles of this membrane translocation (Jones, 2007). CPPs can be stable in vivo, noncytotoxic and protease resistant (Pujals et al., 2007). Peptides including the HIV-1 Tat peptide (TC# 2.B.11; Herce and Garcia, 2007) and oligoarginines represent arginine-rich membrane-permeable vectors that attain efficient intracellular delivery of bioactive molecules. The importance of the arginine residues or their guanidino functions is required for efficient internalization of the Tat peptide, and various novel arginine/guanidino-rich vectors have been developed. Endocytic and non-endocytic mechanisms of internalization have been demonstrated (Nakase et al., 2008; Torchilin, 2008).

CPPs are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. The effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content was investigated by Guterstam et al. (2009). Pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotides mediated by an oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. The pathway for cellular uptake of oligoarginine seems to be dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate (Guterstam et al., 2009).

CPPs have been described which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization, the second involving formation of stable, non-covalent complexes with cargos. They function in delivery of small chemical molecules, nucleic acids, proteins, peptides, liposomes to particles (Brasseur and Divita, 2010).

The ability of three primary amphipathic Cell-Penetrating Peptides (CPPs), CH3-CO-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH, CH3-CO-GALFLAFLAAALSLMGLWSQPKKKRKV-NH-CH2-CH2-SH, and CH3-CO-KETWWETWWTEWSQPKKKRKV-NH-CH2-CH2-SH called Pbeta, Palpha and Pep-1, respectively, to promote pore formation, has examined both in Xenopus oocytes and artificial planar lipid bilayers (Deshayes et al. 2006). A good correlation between pore formation and their structural properties, especially their conformational versatility, was established. The cell-penetrating peptides Pbeta and Pep-1 induce formation of transmembrane pores in artificial bilayers and these pores most likely provide the basis of their ability to facilitate intracellular delivery of therapeutics. Their behaviour provides some information concerning the positioning of the peptides with respect to the membrane and confirms the role of the membrane potential in the translocation process (Deshayes et al. 2006).

Members of the PEP family of CPPs such as Pep1, are amphipathic peptides which deliver peptides and proteins into cells through formation of non-covalent complexes. CADY and CADY2 are amphipathic peptides which deliver short nucleic acids, (e.g., siRNAs) as well as peptides and proteins with high efficiency (Kurzawa et al., 2010). CPPs allow uptake of proteins including antibodies up to the micromolar range. Antibodies allow knock-down in signalling pathways (Mussbach et al., 2011).

Unique characteristics, such as nontoxicity and rapid cellular internalization, allow the cell-penetrating peptides (CPPs) to transport hydrophilic macromolecules into cells, thus, enabling them to execute biological functions. However, some CPPs have limitations due to nonspecificity and easy proteolysis. To overcome such defects, the CPP amino acid sequence can be modified, replaced, and reconstructed for optimization. CPPs can also be used in combination with other drug vectors, fused with their preponderances to create novel multifunctional drug-delivery systems that increase the stability during blood circulation, and also develop novel preparations capable of targeted delivery, along with sustainable and controllable release (Zhang et al. 2016). Further improvements in CPP structure can facilitate the penetration of macromolecules into diverse biomembrane structures, such as the blood brain barrier, gastroenteric mucosa, and skin dermis. The ability of CPP to act as transmembrane vectors improves the clinical application of some biomolecules to treat central nervous system diseases, increase oral bioavailability, and develop percutaneous-delivery dosage form.

The generalized reaction catalyzes by CPPs is:

cargo/CPP (out) ⇌ cargo/CPP (in)

References associated with 2.B.9 family:

Brasseur, R. and G. Divita. (2010). Happy birthday cell penetrating peptides: already 20 years. Biochim. Biophys. Acta. 1798: 2177-2181. 20826125
Daniels, D.S., and A. Schepartz (2007). Intrinsically cell-permeable miniature proteins based on a minimal cationic PPII motif. J Am Chem Soc 129: 14578-9. 17983240
De la Vieja, A., M.D. Reed, C.S. Ginter, and N. Carrasco. (2007). Amino acid residues in transmembrane segment IX of the Na+/I- symporter play a role in its Na+ dependence and are critical for transport activity. J. Biol. Chem. 282: 25290-25298. 17606623
Deshayes, S., M. Morris, F. Heitz, and G. Divita. (2008). Delivery of proteins and nucleic acids using a non-covalent peptide-based strategy. Adv Drug Deliv Rev 60: 537-547. 18037526
Deshayes, S., M.C. Morris, G. Divita, and F. Heitz. (2005). Interactions of primary amphipathic cell penetrating peptides with model membranes: consequences on the mechanisms of intracellular delivery of therapeutics. Curr Pharm Des 11: 3629-3638. 16305499
Deshayes, S., T. Plénat, P. Charnet, G. Divita, G. Molle, and F. Heitz. (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. 17011511
Durzyńska, J., &.#.3.2.1.;. Przysiecka, R. Nawrot, J. Barylski, G. Nowicki, A. Warowicka, O. Musidlak, and A. Goździcka-Józefiak. (2015). Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine. J Pharmacol Exp Ther 354: 32-42. 25922342
Foged, C., and H.M. Nielsen (2008). Cell-penetrating peptides for drug delivery across membrane barriers. Expert Opin Drug Deliv 5: 105-17. 18095931
Grdisa, M. (2011). The delivery of biologically active (therapeutic) peptides and proteins into cells. Curr. Med. Chem. 18: 1373-1379. 21366527
Guterstam P., Madani F., Hirose H., Takeuchi T., Futaki S., El Andaloussi S., Graslund A. and Langel U. (2009). Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate. Biochim Biophys Acta. 1788(12):2509-17. 19796627
Hansen, M., K. Kilk, and U. Langel (2008). Predicting cell-penetrating peptides. Adv Drug Deliv Rev 60: 572-9. 18045726
Henriques, S.T. and M.A. Castanho. (2008). Translocation or membrane disintegration? Implication of peptide-membrane interactions in pep-1 activity. J Pept Sci 14: 482-487. 18181239
Henriques, S.T., A. Quintas, L.A. Bagatolli, F. Homblé, and M.A. Castanho. (2007). Energy-independent translocation of cell-penetrating peptides occurs without formation of pores. A biophysical study with pep-1. Mol. Membr. Biol. 24: 282-293. 17520484
Herce, H.D., and A.E. Garcia. (2007). Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes. Proc. Natl. Acad. Sci. USA 104: 20805-20810. 18093956
Jones, A.T. (2008). Gateways and tools for drug delivery: Endocytic pathways and the cellular dynamics of cell penetrating peptides. Int J Pharm 354: 34-8. 18068916
Kurzawa L., Pellerano M. and Morris MC. (2010). PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells. Biochim Biophys Acta. 1798(12):2274-85. 20188697
Lebleu, B., H.M. Moulton, R. Abes, G.D. Ivanova, S. Abes, D.A. Stein, P.L. Iversen, A.A. Arzumanov, and M.J. Gait (2008). Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev 60: 517-29. 18037527
Meade, B.R., and S.F. Dowdy (2008). Enhancing the cellular uptake of siRNA duplexes following noncovalent packaging with protein transduction domain peptides. Adv Drug Deliv Rev 60: 530-6. 18155315
Mussbach, F., M. Franke, A. Zoch, B. Schaefer, and S. Reissmann. (2011). Transduction of peptides and proteins into live cells by cell penetrating peptides. J. Cell. Biochem. 112: 3824-3833. 21826709
Nakase, I., T. Takeuchi, G. Tanaka, and S. Futaki (2008). Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides. Adv Drug Deliv Rev 60: 598-607. 18045727
Nelson, A.R., L. Borland, N.L. Allbritton, and C.E. Sims (2007). Myristoyl-based transport of peptides into living cells. Biochemistry 46: 14771-81. 18044965
Pujals, S., E. Sabidó, T. Tarragó, and E. Giralt. (2007). all-D proline-rich cell-penetrating peptides: a preliminary in vivo internalization study. Biochem Soc Trans 35: 794-796. 17635150
Pujals, S., J. Fernandez-Carneado, MD Ludevid, and E. Giralt (2008). D-SAP: A New, Noncytotoxic, and Fully Protease Resistant Cell-Penetrating Peptide. ChemMedChem 3: 296-301. 18058782
Rennert, R., I. Neundorf, and A.G. Beck-Sickinger (2008). Calcitonin-derived peptide carriers: mechanisms and application. Adv Drug Deliv Rev 60: 485-98. 18160173
Torchilin, V.P. (2008). Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers. Adv Drug Deliv Rev 60: 548-58. 18053612
Zhang, D., J. Wang, and D. Xu. (2016). Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems. J Control Release 229: 130-139. 26993425
Ziegler, A. (2008). Thermodynamic studies and binding mechanisms of cell-penetrating peptides with lipids and glycosaminoglycans. Adv Drug Deliv Rev 60: 580-97. 18045730