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Cell penetration via endocytosis: A: membrane interaction; B: endocytosis; C: vesicle formation; D: endosome; E: endosomal release; F: CPP conjugate in cytoplasm (Source: American Peptide)  

The difficulty of delivering large molecules—proteins, peptides, and nucleic acids—into cells through the cell membrane (cellular uptake) has proven a significant impediment to medicinal chemists and the pharmaceutical industry as a whole.

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. 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. A number of non-viral strategies have been introduced such as lipid, polycationic, nanoparticle, and peptide-based formulations, but only a few methods are practically utilized in vivo at either preclinical or clinical levels. Thus, novel efficient carrier delivery methods have to be developed to impart good bioavailability of drug molecules.

Discovery and potential of cell-penetrating peptides
In 1991, researchers demonstrated that Drosophila Antennapedia homeodomain could be internalized by neuronal cells. A 16-amino acid peptide, penetratin, was subsequently derived from this protein. Since then, the number of known natural and synthetic peptides with cell-penetrating capabilities has continued to grow. Peptides which are able to penetrate the cell membrane are known as cell-penetrating peptides (CPPs). CPPs can be broadly classified as protein-derived, chimeric (derived from two or more genes which are coded for separate proteins), or synthetic. CPPs share common features such as positively charged amino acids, hydrophobicity, and amphipathicity.

The discovery of CPPs’ ability to traverse the cell membrane opens up a new avenue for drug delivery. Attaching therapeutically significant biomolecules to CPPs provides a means to transport them across the cell membrane. A major breakthrough in the field was the delivery of peptide-nucleic acids (PNAs) using the chimeric peptide transportan. A variety of 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.

Methods of attaching cargo
CPPs are usually connected to a cargo molecule via a covalent linkage. 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 to this point 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 has been shown to be sufficient. The synthetic covalent bond between CPP and nucleic acid may actually alter the biological activity of the latter. The first non-covalent CPP for delivery of nucleic acids—MPG —was designed shortly after the development of Pep-1 for non-covalent cellular delivery of proteins and peptides. These non-covalent conjugates are formed through either electrostatic or hydrophobic interactions. MPG forms highly stable complexes with siRNA, has a low degradation rate, and can be easily functionalized for specific targeting, all of which are major advantages compared with covalent CPP technology.
Mechanism of CPP translocation across cell membrane
The exact molecular pathways underlying cellular uptake of a cargo attached to CPPs are not clear. Different CPPs have varying hydrophobicity, charge, and amphiphilicity. The size and chemical properties of cargos are also different, making generalizing about the interaction of complex molecules and cell membrane difficult. Generally, 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 high affinity for membranes have a higher propensity to be internalized 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. CPPs are adsorbed at the cell surface due to the presence of anionic moieties, such as heparan sulfate, sialic, or phospholipidic acid. This is followed by endocytosis of membrane, vesicle formation, formation of endosome in which the conjugate is trapped, and endosomal release. Since endocytosis is a normal way for a cell to communicate with and acquire information from the surrounding environment, this process is neither harmful nor invasive to the cell or the organism as a whole. Consequently, CPPs are unlikely to cause an immune response, and are one of the least toxic transporters currently available.

Drug delivery: CPP-drug conjugates in clinical trials
Several companies have started working on clinical development of CPPs for topical and systemic administration of different therapeutic molecules. The first CPP clinical trial was initiated for topical delivery of cyclosporine linked to polyarginine and entered Phase 2 trials in 2003 for the treatment of psoriasis (PsorBan). However, despite an efficient uptake of the chimera, the release of the free drug was not rapid enough to compete with clearance. A number of different CPP-based drugs have entered clinical trials; in every case, 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 conjugation by modification with an additional cysteine at the N-terminus of both entities. The peptides SFNSYELGSL and EAVSLKPTC are δ protein kinase C (δPKC) and ε protein kinase C (εPKC) specific inhibitors respectively and HDAPIGYD is a εPKC activator peptide. DTS-108 is a vectocell peptide-SN38 prodrug generated by chemical coupling of the 10-hydroxyl group of SN38 via a heterobifunctional cross-linker (BCH) to the peptide DPV1047. This linker results in the generation of an ester bond between the peptide and SN38.

Conclusions and future outlook
With the aid of cell-penetrating peptides, delivery of therapeutic biomolecules such as oligonucleotides and proteins can be better managed in terms of efficiency, cytotoxicity, and biocompatibility. CPPs can associate with an extensive range of cargo types either via covalent linkage or non-covalent interaction. Unlike viral vectors, CPPs do not have the capacity to integrate the genetic material they deliver. One of the limitations of CPPs is the non-specific cellular uptake; this limits the drug delivery to specific cellular targets such as tumor cells. Fortunately, in recent years, some CPPs have shown high affinity for specific cell types or intracellular destinations. A recently discovered CPP known as crotamine has shown unusually high affinity for actively proliferating cells. Originally designed for nuclear delivery of siRNA, the MPG peptide has recently been altered to target the cytoplasm. Recently, activatable CPPs were introduced to address the problem of tissue non-specificity of CPPs. Activatable CPPs are polycationic CPPs whose adsorption and cellular uptake are minimized by a covalently linked polyanionic inhibitory domain. Cleavage of the linker by specific proteases enables the ACPP to enter cells. Cellular-specific penetration can also be achieved by combining targeting and internalization properties of cell targeting peptides and CPPs respectively.

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