Based on the interaction between cell penetrating peptides and the therapeutic agent two main sub-classes of CPPs can be distinguished. The first sub-class includes CPPs that are chemically linked to the therapeutic agents while the second sub-class encompasses those that form stable non-covalent complexes with the therapeutic agents [4].

Apart from these two main sub classes in the recent past, another method for non-covalent coupling of CPPs for a nucleic acid cargo (i.e., antisense oligomer, siRNA) has been evidenced. This entails sequence complementary hybridization, for instance the binding of the oligonucleotide NFkappaB decoy to the nonamer PNA sequence that resulted in a stable complex that was efficiently translocated across the plasma membrane [7].

This classification of CPPs is based on their ionic properties. In regard to the ionic charge induced by the CPP, this class could be subdivided into cationic or amphipathic CPPs.

This involves delivering therapeutic genes into the nucleus of target cells to achieve expression of a deficient or incorrectly expressed gene product. Difficulties in developing safe and efficient gene delivery vectors that could sustain gene expression for long periods, poor permeability of the plasma membrane of eukaryotic cells to DNA results in low concentration of DNA and other oligonucleotides at their targets [22]. To overcome this, peptide carriers such as polylysine and polyarginine that have membrane-destabilizing properties and could bind with DNA mainly through electrostatic interaction have been developed to facilitate gene transfer into cultured cells and cells in living organisms [23]. For example amphipathic peptides with pH-dependent fusogenic and endosomolytic activities such as the fusion peptide of HA₂ subunit of influenza hemaglutinin, or synthetic analogs GALA, KALA, JTS1, and histidine-rich peptides have been shown to increase transfection efficiency when associated with poly-L-lysine/DNA, condensing peptide/DNA, cationic lipids, poly-ethyleneimine or polyamidoamine cascade polymers [15].

The ability to condense DNA and to favor endosomal escape by CPPs like PPTG1, and the prevention of endosomal uptake by MPG has led to their use for gene delivery in cultured cells. PPTG1 has been reported as an important component for in in-vivo gene expression following intravenous injection [24].

Cationic liposomes, nanoparticles, cationic polymers, and CPPs have also been used as non-viral vectors in preference to the viral vectors. This is because viral vectors, despite their sustained levels of transduction and in some cases efficient and stable integration of exogenous DNA into a wide range of host genomes [25], have been noted to have some disadvantages such as immunogenicity, toxicity, difficulty of large-scale production, hence the preference of non-viral vectors for plasmid DNA delivery [22].

CPPs easily conjugate covalently or non-covalently with siRNAs. The siRNAs covalently linked to Transportan and Penetratin have been associated with a silencing response. Non-covalent complexes or aggregates formed with siRNA usually have a net positive charge when there is a surplus of positive charges over negative charges. The covalent linkage of CPPs to siRNAs results in the formation of small, monomeric CPP/siRNA conjugates of known stoichiometry with high reproducibility [26]. CPPs could therefore be used for delivery of siRNAs either by covalent or non-covalent approaches [27]. However non-covalent strategies are more efficient for siRNA delivery, for example MPG peptide has been extensively reported to improve siRNA delivery into a large panel of cell lines including adherent cell lines, cells in suspension, cancer and challenging primary cell lines biological response [28,29]. It has also been applied for in vivo delivery of siRNA targeting OCT-4 into mouse blastocytes. It has equally been used for the delivery of siRNA targeting an essential cell cycle protein, cyclin B1. Intravenous injection of MPG/cyclin B1 siRNA particles has been shown to efficiently block tumor growth [30].

TAT peptide associated with an RNA-binding motif has been reported to block in vivo epidermal growth factor (EGF) factor. The CPP complex, cholesterol-Arg₉ was also shown to enhance siRNA delivery in vivo against vascular endothelial growth factors. A small peptide derived from rabies virus glycoprotein linked to polyarginine R₉ has been reported recently to deliver siRNA in the CNS [31].

A new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers splice-correcting oligonucleotides (SCOs) to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD) was illustrated in a recent study as a new chemically modified CPP, PF14. Starting from stearyl-TP10, ornithines were utilized as the main source of positive charges instead of lysines. The superior efficiency of poly-L-ornithines was related to the higher affinity for DNA and the ability to make more stable complexes at lower charge ratios [32].

These studies show that CPPs are certainly among the most promising candidates in the development of siRNA-based therapeutics.

Antisense technology is based on the use of sequence specific oligonucleotides (ONs) that can hybridize with complementary mRNA strands to cause translational arrest or mRNA degradation by activation of the cellular enzymes of the RNaseH family and consequently block gene expression [22]. The ONs with therapeutic potential include aptamers, transcription factor-binding decoy ONs, ribozymes, triplex-forming ONs, immunostimulatory CpG motifs, antisense ONs, and antagomirs.

CPPs have been used for the delivery of ONs by using either a covalent linkage or a non-covalent linkage with the therapeutic agent. They have been used to mediate the delivery of PNAs and PMOs through covalent linkage [33]. The formation of efficient non-covalent complexes comprising CPPs and both charged and uncharged steric block oligonucleotides like 2'-O-methyl, LNA, PNA and charged PNA derivatives, has also been described [34].

PNA–CPP conjugates were first demonstrated to block the expression of the galanin receptor mRNA in human Bowes cells by a 21-mer PNA coupled to Penetratin or Transportan by Langel and group in 1998 [22]. In a different study, a model amphipathic peptide (MAP) conjugated to a PNA complementary to the nociceptin/orphanin FQ receptor mRNA was shown to improve cellular uptake and steric block effect in both CHO cells and neonatal rat cardiomyocytes [35].

The efficacy of PNA and PMO coupled to CPPs was only significant in the presence of endosomolytic agents such as chloroquine and calcium ions [36,37,38]. Since most of the existing endosomolytic agents are too toxic to be considered for in vivo applications, CPP-based strategies that are efficient in the absence of PNA and PMO such as co-treatment with endosome-disrupting peptides are currently being explored [39,40].

Certain proteins are currently being used as therapeutic agents for the treatment of various diseases. CPPs are usually coupled to proteins through covalent bonds or through fusion constructs except Pep-1, a CPP that forms non-covalent complexes [16]. There is evidence that CPPs are able to facilitate the delivery of proteins into a wide variety of cells both in vitro and in vivo; Dowdy and group revealed that the delivery of these therapeutic proteins into tissues and across the blood-brain barrier was severely limited by the size and biochemical properties of the proteins. An intraperitoneal injection of the 120-kilodalton beta-galactosidase protein, fused to the protein transduction domain from the human immunodeficiency virus TAT protein, resulted in delivery of the biologically active fusion protein to all tissues in mice, including the brain [41]. CPPs therefore constitute a powerful tool that could be used to facilitate the delivery of protein-based therapeutics in pathological conditions, such as cancer, inflammatory diseases, oxidative stress-related disorders, diabetes and brain injury [36,42]. However, protein stability relies on weak non-covalent interactions between secondary, tertiary and quaternary structures, which must be preserved throughout the delivery process [43]. This makes proteins vulnerable therapeutic agents with short in vivo half-lives and poor bioavailability. To improve the efficiency of delivery of proteins into cells, different types of lipid- and polymer-based vectors including liposomes, microparticles and nanoparticles have been used for protein delivery, but with relatively poor efficiency [44].

Liposomes, nanoparticles and other different types of pharmaceutical nanocarriers have been used to increase the stability of drugs, modulate their pharmacokinetics and biodistribution, improve their efficacy while decreasing side-effects [21]. However, the intracellular delivery of these large molecules remains a challenge because of their three-dimensional structure, spatial occupation and hydrophilic/hydrophobic nature.

CPPs have been used to functionalize these vectors with a view to increasing the cellular uptake of the encapsulated therapeutic agents [45]. CPPs have been shown to offer the opportunity to deliver therapeutic molecules that are even 200 times larger than the current bioavailability size restriction. In one such study Torchilin prepared targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles possessing several functionalities [21]. These liposomes and micelles were modified with TATp moieties attached to the surface of the nanocarriers by using TATp-short PEG-PE derivatives. This made them degradable by inserting the pH-sensitive hydrazine bond between PEG and PE (PEG-Hz-PE) whose cleavability by acidic hydrolysis makes them acquire the ability to be effectively internalized and released. These can be considered as an important step in the development of tumor-specific stimuli-sensitive delivery systems.

Dextran-coated superparamagnetic iron oxide particles (CLIO) coupled with TATp, were demonstrated to provide efficient labeling of cells, and could serve as a tool for magnetic resonance imaging (MRI). The uptake of the TATp-CLIO nanoparticles by cells was shown to be about 100-fold higher than that of the non-modified iron oxide particle [21]. Other studies reveal that modifying the surface of nanoparticles with CPPs, enhances the cell permeability of nanoparticulate-based therapeutics [46]. This is further proved by the CPP-mediated delivery of bioactive compounds into model organisms for cancer, cardiomyopathy, stroke, muscular dystrophy and viral infections [47,48]. For example PsorBanR, a cyclosporin–poly-arginine conjugate formulated as a topical treatment for psoriasis, and KAI-9803, a PKC (protein kinase C) δ peptide inhibitor–TAT conjugate for the treatment of acute MI (myocardial infarction) [49].

A family of cell-penetrating peptides named Vectocell^® peptides [also termed DPVs (Diatos peptide vectors)] originating from human heparin binding proteins and/or anti-DNA antibodies with both enhanced and safe cell penetration characteristics have been identified. These new peptidic sequences are reported to deliver small and large active molecules inside cells that would otherwise have limited or no bioavailability [45].

CPPs have been reported to act as active pharmaceutical ingredients when used alone. A TAT peptide containing a cysteine residue at its C-terminal (TAT-C) was shown to be able to inhibit infection by irreversibly inactivating virions exposed to the tat-C prior to cell infection, blocking entry of cell-adsorbed viruses, or inducing a state of resistance to infection in cells pretreated with TAT-C [50]. The mechanism of the antiviral activity is yet to be elaborated. This has set the stage for exploring the therapeutic activity of CPPs as active pharmaceutical ingredients

Apart from functional group modification of amino acids in the peptide chain, modification of cell penetrating peptides have also been shown to involve substitution of amino acids to achieve variability of the peptide properties such as hydrophobicity or cationic nature. This strategy has been noted to result in increased intracellular internalization by certain CPPs. Kaeko et al. conducting a comprehensive search for novel CPPs using an in vitro virus library of peptides consisting of 15 amino acids [19] and reported improved intracellular translocation efficiency at low concentrations due to the influence of cationic amino acids. Since the amino acid Arginine has a stronger affinity to the cell surface, a desirable modification in this case involved substituting another amino acid such as Lysine in the peptide chain with Arginine. Such substitutions were reported [66] to have remarkably improved intracellular translocation even at low concentration. Substitution (of amino acids) with Histidine residue is also a promising option as this could provide endosomal disruption by the “proton sponge effect” of Histidine residues in the acidic endosomal compartment as previously reported for LAH4; an amphiphatic peptide rich in Alanine and Leucine. It bears Lysines at its ends to condense DNA and Histidines inside the sequence which favours endosomal escape. LAH4/DNA polyplexes were reported to be able to transfect cells in the absence of serum. From a series of LAH4 derivatives, high transfection efficiencies were obtained only with peptides containing four to five Histidine residues in the central region of the peptide sequence [67].

Increasing amino acids like Tryptophan which has the tendency to be buried in the cell membrane [68] improves CPP hydrophobic interaction. It has been confirmed by Kaeko et al. that in low peptide concentrations, two Trp residues were not enough to initiate effective translocation, but three Trp enhanced cellular uptake. However, when the number of Tryptophan residues increased to four the intracellular translocation activity decreased [19]. This may be due to a decrease in solubility of the peptide as a result of the increased number of Tryptophan residues. Therefore, the increase of the number of Trp residues may be ideal but to a certain limit and such modifications should be carried out with strict consideration of the impact of amino acid substitution to the physicochemical parameters, especially changes in dissolution properties.

Functional group modification of the amino acids in the peptide usually involves the formation of masking groups or linkages to highly reactive sites. In either case, the peptide bonds formed should be labile and easily broken for regeneration of the initial cell penetrating peptide by simple variation of physiological conditions.

Helicity of the peptide chain can be exploited for modification. For example through hydrocarbon stapling the overall cell penetrating peptide’s alpha helicity could further be stabilized. This is derived from the fact that the α-helix, as a major structural motif of proteins, frequently mediates intracellular protein-protein interactions that govern many biological pathways [69].

Fei and co workers reported a modification strategy to decrease the non-specific binding and uptake of a CPP, model amphipathic peptide (MAP, for use as a potential targeted drug carrier [68]. This was illustrated using (S)-a-(2’-pentenyl)alanine containing olefin-bearing tethers to generate an all hydrocarbon “staple” by ruthenium-catalyzed olefin metathesis. The (S)-a-(2’-pentenyl)alanine peptides were made to flank three (substitution positions l and l + 4) or six (l and l + 7) amino acids within the peptide, so that reactive olefinic residues would reside on the same face of the a-helix. The modified hydrocarbon-stapled peptides were helical, relatively protease-resistant, and cell-permeable peptides that bound with increased affinity for the target [70]. Such hydrocarbon stapling could provide a useful strategy for therapeutic modulation of protein-protein interactions.

In another study the modification of MAP with citraconic anhydride (CA) blocks, the epsilon-amino groups of the Lysine residues formed acid-labile amide linkages. This was confirmed by analysis for internalization in HeLa cells which revealed that 80.8 ± 2.2% of the amino groups were modified in the CA-MAP conjugate. In the study a MAP conjugate was designed to contain acid-labile modifications to mask the cationic charge, and therefore decreasing the non-specific binding and uptake [71,72]. After binding and internalization, the acidic pH in the endosomes was realized to facilitate the cleavage of the acid-labile attachments and hence release of MAP. The combination of CA-MAP with a targeting molecule (e.g., folic acid) offers significant improvement in the use of CPPs in targeted drug delivery.