Cell Permeable Peptide Synthesis

Cell-penetrating peptides (CPPs) constitute a promising tool for the cellular import of drug cargos. They have been successfully applied for in vitro and in vivo delivery of a variety of therapeutic molecules including plasmids, DNA, oligonucleotides, siRNA, PNA, proteins, peptides, low molecular weight drugs, liposomes, and nanoparticles.


Delivery of therapeutic agents into cells through their membrane (cellular uptake) is a topic of profound importance to medicinal chemists and for the pharmaceutical industry and has always proved to be a challenging task especially in the delivery of large molecules. The plasma membrane prevents direct translocation of hydrophilic macromolecules by acting as a barrier to efficient and controlled intracellular delivery. A drug must be either highly lipophilic or very small to stand a chance of cellular internalization and it is difficult to ascertain a generic mechanism for drug uptake. These restrictions mean that the repertoire of possible drug molecules is limited. Similarly, novel therapeutic approaches such as gene and protein therapy also have limited potential due to the cell-impermeable nature of peptides and oligonucleotides. The existing methods for delivery of macromolecules, such as viral vectors and membrane perturbation techniques, can result in high toxicity, immunogenicity and low delivery yield.

Cell-Permeable Peptides (CPPs) are short peptides that are able to pass the cell membrane. CPPs have found use for transporting cargo molecules as fluorophores, drugs, nucleic acids or proteins into cells. Typically, CPPs contain a large proportion of positively charged amino acids, especially arginine, or an alternating sequence of charged and non-polar residues.


In 1988, Frankel and Pabo observed the remarkable ability of HIV-Tat protein to enter cells and translocate into the nucleus. Mutagenesis studies of the protein showed that the region between residues 47-57 (sequence: YGRKKRRQRRR) is important for cellular uptake. Soon after, in 1991, the group of Prochiantz and Derossi demonstrated that the antennapedia homeodomain protein of Drosophila melanogaster could be internalized by neuronal cells. This discovery subsequently led to the identification of a hexadecapeptide, penetratin, derived from the third helix of the homeodomain of antennapedia. Since then, the number of known natural and synthetic peptides with cell-penetrating capabilities has continued to grow (Table 1).

These peptides which are able to penetrate the cell membrane and enter the cell are known as cell-penetrating peptides. They are also known as protein transduction domains (PTDs), membrane translocating sequences (MTSs) or Trojan peptides. PTDs were identified in transcription factors, bacterial or viral surface proteins, toxins, amphipathic helix-forming peptides and in ligands of membrane-bound receptors or adhesion proteins. CPPs can be broadly classified as protein derived, chimeric (synthetic peptides combining partial sequences of natural peptides), or synthetic, as shown in Table 1.

These short peptides with less than 40 amino acids share common features such as positively charged amino acids, hydrophobicity and amphipathicity. The discovery and the ability of these peptides to traverse the cell membrane opened up a new avenue for drug delivery. Transport of therapeutically significant biomolecules  across the membrane into the cell can be facilitated by attaching them to CPPs. A major breakthrough in the CPP field came from the first proofs-of-concept of their in vivo application, by the groups of Dowdy, for the delivery of small peptides and large proteins and of Lange!, for delivery of peptide nucleic acids (PNAs) using the chimeric peptide transportan, derived from the N-terminal fragment of the neuropeptide galanin, linked to mastoparan, a wasp venom peptide. Since then, several CPPs that are able to trigger the movement of a cargo across the cell membrane into the cytoplasm have been designed.

Numerous cargo molecules have been attached to CPPs for cellular delivery. These include plasmid, DNA, oligonucleotides, siRNA, PNAs, proteins, peptides, liposomes, low molecular weight drugs, antibodies, nanoparticles, antibiotics, enzymes and enzyme substrates.



CPPs are usually connected via a covalent linkage to the cargo molecule. Proteins and peptides can be attached to CPPs through a disulfide bond (by modifying CPP and peptide/protein with cysteine) or through cross-linkers. Most CPP-nucleic acid complexes that have been proposed so far are formed through covalent bonding. Different strategies include cleavable disulfide, amide, thiazolidine, oxime and hydrazine linkages. Short interfering RNA (siRNA) can be covalently linked to transportan and penetratin by disulfide linkage at the 5′ -end of the sense strands of siRNA to target luciferase or eGFP mRNA reporters.

A stable covalent linkage between the cargo and CPP is not always necessary for translocation as simple mixing of two entities was shown to be efficient. The synthetic covalent bond between CPP and nucleic acid may alter the biological activity of the latter. In 1997, the first non-covalent CPP for delivery of nucleic acids, called MPG (see Table 1) was designed by the group of Heitz and Divita closely followed by development of Pep-1 for non-covalent cellular delivery of proteins and peptides by Morris et al. in 2001. The groups of Wender and of Futaki  demonstrated that oligoarginine sequences (Arg8) were sufficient to drive molecules into cells and proposed that their uptake mechanism involves a bidentate hydrogen-bonding interaction between the guanidinium moieties of the arginine residues and phosphate groups in the membrane.

Thus, a new non-covalent strategy requiring no chemical modification with short amphipathic CPPs, like MPG and Pep-1 as carriers has been successfully applied for delivery of cargoes. These non-covalent conjugates are formed through either electrostatic or hydrophobic interactions. With this method, cargoes such as nucleic acids and proteins could be efficiently delivered while maintaining full biological activity. MPG forms highly stable complexes with siRNA with a low degradation rate and can be easily functionalized for specific targeting, which are major advantages compared with the covalent CPP technology.

The natural cell-permeable peptides mastoparan and mastoparan X have been isolated from the venoms of the wasps Vespula lewisii and Vespa xanthoptera, respectively. Both show the typical alternation of positively charged and lipophilic amino acids. 

Crotamine, another natural CPP has been isolated from the venom of the South American rattlesnake Crotalus durissus terrificus.


The exact molecular pathways underlying cellular uptake of a cargo attached to a CPP are not clear.
Different CPPs have varying hydrophobicity, charge and amphiphilicity. The size and chemical properties
of cargos are also different. Hence, generalizing the interaction of these complex molecules and cell
membrane is not easy. For docking and cellular uptake, two major mechanisms have been considered: the
endosomal pathways composed of endocytotic entry followed by endosomal escape, and direct cell
membrane penetration. Peptides that have a high affinity for membranes have a higher propensity to be
intemalized by a non-endocytic mechanism than peptides with a lower affinity. CPPs with low molecular
weight cargos may also enter without vesicle formation and facilitate access to all intracellular
compartments. Different stages of cell penetration via endocytosis are depicted in Figure 1. According to
this mechanism, CPPs are first simply adsorbed at the cell, followed by endocytosis of membrane, vesicle
formation, formation of endosomes in which the conjugate is trapped, and endosomal release.



Research and clinical studies on the transport and delivery of therapeutics into cellular targets using cell-
penetrating peptides has been progressing well in recent years. Several companies started working on
clinical development of CPPs, for topical and systemic administration of different therapeutic molecules.
The first CPP clinical trial was initiated by Cellgate Inc. for topical delivery of cyclosporine linked to
polyarginine (CGC1072) and entered phase II trials in 2003 for the treatment of psoriasis. This is an
example of local application of a CPP-drug conjugate (local CPP-mediated delivery). However, despite an
efficient uptake of the chimera, the release of the free drug was not rapid enough to compete with
clearance. A list of different CPP-based drugs which entered clinical trial is shown in Table 2.

The therapeutic 28-amino acid cell-penetrating peptide p28 is derived from azurin, a redox protein
secreted from the pathogen Pseudomonas aeruginosa, produces a posttranslational increase in p53 by
inhibiting its ubiquitination in cancerous cells. In few of these cases therapeutic agents are covalently
linked either directly or through a linker to the CPP carrier. In KAI-9803, KAI-1678 and KAI-1455, the cargo
peptide is attached to Tat peptide via a disulfide bond between additional cysteines at the N-termini of
both entities. The cargo peptides SFNSYELGSL and EAVSLKPTC are 6 protein kinase C (6PKC) and E protein
kinase C (EPKC) specific inhibitors, respectively and HDAPIGYD is a EPKC activator peptide. DTS-108 is a
Vectocell® peptide-SN38 prodrug generated by esterification of the 10-hydroxyl group of 5N38 to a
heterobifunctional cross-linker (BCH) linked to Vectocell® peptide DPV1047 (CVKRGLKLRHVRPRVTRMDV).

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