In the last decade, almost one-third of the newly discovered drugs approved by the US FDA were biomolecules and biologics. Effective delivery of therapeutic biomolecules to their target is a challenging issue. Innovations in drug delivery systems have improved the efficiency of many of new biopharmaceuticals. Designing of novel transdermal delivery systems has been one of the most important pharmaceutical innovations, which offers a number of advantages. The cell-penetrating peptides have been increasingly used to mediate delivery of bimolecular cargoes such as small molecules, small interfering RNA nucleotides, drug-loaded nanoparticles, proteins, and peptides, both in vitro and in vivo, without using any receptors and without causing any significant membrane damage. Among several different drug delivery routes, application of cell-penetrating peptides in the topical and transdermal delivery systems has recently garnered tremendous attention in both cosmeceutical and pharmaceutical research and industries. In this review, we discuss history of cell-penetrating peptides, cell-penetrating peptide/cargo complex formation, and their mechanisms of cell and skin transduction.
In recent years, tremendous efforts have been made to develop novel transdermal drug formulations. Ideally, a non-invasive drug delivery system would be painless and easy to use, which provides better patient compliance. Moreover, when an active ingredient is unstable in the gastrointestinal tract or patients require long-term or repetitive doses of a medication, transdermal delivery route is considered as an effective alternative choice.
The topical drug delivery heavily depends on the ability of active ingredient to permeate the skin in sufficient quantities to achieve its desired therapeutic effects. The stratum corneum is the exterior skin layer and the major barrier of transdermal absorption. In this layer, extracellular lipids such as ceramides, cholesterol, and free fatty acids form complexes with cellular structures within the cornified layer of intrafollicular epidermis and provide a tight barrier to microbial invasion and desiccation (1–4).
Dermal and transdermal delivery of large molecules such as peptides, proteins, and DNA has remained a significant challenge. Several attempts have been made to develop topical formulations for macromolecules using a wide variety of tools such as using delivery enhancers, delivery vehicles, and different penetration methods. For instance, the chemical enhancers such as alcohols (5), fatty acids (6), and surfactants (7), and physical enhancers such as microneedles (8), ultrasonic waves (9), and low electrical current methods (10) have been examined to improve topical delivery of macromolecules. The delivery vehicles were mainly based on dispersion systems containing nanoparticles (11), microdroplets as liposomes (12) or nanostructured liquids (13) and microemulsions (14,15), and lyotropic liquid crystals (16). Hydrogels (17) and polymer-based systems (18,19) have also been used.
Chemical and physical enhancers for modulating the penetration of macromolecules across the skin have their limitations including skin toxicity of chemical enhancers at high concentrations, inconvenience of using electrical apparatuses at home, and high production costs of sophisticated drug delivery systems (20).
To overcome the cell membrane impermeability, a novel approach for delivery of large particles and macromolecules across biological membranes and tissue barriers has been recently emerged, which is called cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs). The CPPs are relatively short (up to 30 amino acids in length), water soluble, cationic, and/or amphipathic peptides that are capable of carrying large macromolecules across cellular membranes (21–25).
The main feature of CPPs is that they are able to penetrate the cell membrane at low micromolar concentrations in vivo and in vitro without using any receptors and without causing any significant membrane damage. These peptides are capable of internalizing electrostatically or covalently bound biologically active cargoes with high efficiency and minimal toxicity (26–28).
History of CPPs
This new class of peptides initially discovered in 1988 by the discovery of the human immunodeficiency virus type 1 (HIV-1) encoded transactivator of transcription (Tat) peptide (29,30) that was the first sequence found to be capable of translocating cell membranes and gaining intracellular access. A few years later, this discovery was followed when penetratin was employed for the delivery of a small exogenous peptide (31–34). It was later shown that small domains within these peptides are often responsible for cellular uptake (35).
So these translocation sequences could be shortened to a few amino acids as compared with the first Tat peptide, without losing cell permeation capacity (30). Since then, the list of synthetic CPPs has grown dramatically and the number continues to increase (Table 1).
Table 1. Examples of synthetic CPPs with their sequences
CPP, cell-penetrating peptides.
While the choice of CPP often depends on the type of application, some of the most commonly used peptides include Tat, polyarginine, penetratin, transportan, and Pep-1. These CPPs have successfully delivered proteins (36,37), nucleic acids (38), and small molecule therapeutics (39) into cell lines.
Binding to cargoes
There are two major ways to bind CPP and cargo together. In the first approach, the CPP/cargo sequences are expressed in E. coli and then the recombinant fusion protein purified (33,34). The second way is to make a linkage between CPP and cargo and form a complex. Most of the proposed CPPs such as Tat (36), Poly-Arg (40), transportan (41), and penetratin (33) are covalently linked to their cargoes and internalized into cells. Although these covalent connections have been successfully used for the delivery of a wide range of cargoes, they have some limitations. For example, as chemical binding is based on a synthetic disulfide linkage between carrier and cargo, there is always a risk of altering the biological activity of the cargo. This issue has been recently discussed for the delivery of siRNA (42). As an alternative to covalent linkage, a new potent strategy for the delivery of biomolecules into mammalian cells, based on short amphipathic peptide carriers such as MPG and Pep-1, has been proposed (43–48). These peptide carriers form stable nanoparticles with cargoes without requiring any cross-linking or chemical modifications. MPG efficiently delivers nucleic acids such as plasmid DNA, oligonucleotides, and siRNA, while Pep-1 enhances the delivery of proteins, peptides, and protein mimics in a fully biologically active form into a variety of cell lines as well as in animal models (44–46). We have recently used Pep-1 to improve topical delivery of elastin in both in vitro and in vivo models (49). As shown in Figure 1, Pep-1 efficiently passes elastin through the plasma membranes of cultured fibroblasts.
Although many studies have been conducted on CPPs, the mechanism of their entry into cells still remains unknown. Most likely, a combination of different model systems and techniques is required to reveal their mechanisms of cellular entry.
It is suggested that the entry of CPP/cargo complex into the cell could be influenced by variety of factors, including the nature of the cargo (such as size and charge), the properties of CPP (such as molecule length, charge delocalization, hydrophobicity, other physicochemical parameters), the cell line, and the CPP concentration (50).
Historically, two major cellular uptake mechanisms, non-endocytotic or energy-independent pathways and the endocytotic pathways, have been proposed to explain how these peptides could possibly deliver various kinds of macromolecules into the cell. Both mechanisms contain (i) membrane interaction, (ii) membrane permeation, and (iii) release of CPP into the cytosol after the entry procedure. The main difference between energy-independent pathway and endocytosis is in the membrane permeation and release steps. In the non-endocytotic pathway, CPPs directly localize in cytoplasm after traversing the plasma membrane, whereas in endocytosis, CPPs may or may not be released into the cytosol and even they may end up in intracellular vesicular compartments (51). It was also proposed that CPPs, especially Tat, Antennapedia, Poly-Arg, transportan, MPG, and Pep-1, may pass through the plasma membrane via an energy-independent pathway (52).
Moreover, some elaborate models have been also proposed to explain the translocation of these peptides, such as inverted micelle formation (34), pore-like structure formation (53), the carpet-like model (54), and the membrane thinning model (55).
The first step in all these mechanisms is the adsorption at the cell surface owing to the negatively charged membrane components such as heparin sulfate, phospholipidic or sialic acid (56–58), which serve as a ‘capture or tethering platform’, prior to the ‘onset’ of internalization (59). They cause membrane alterations such as stable or transient destabilization accompanied by folding of the peptide on the lipid membrane (60–62).
In general, direct penetration is most likely occurring at high CPP concentrations (>10 μm) and for primary amphipathic CPPs such as transportan analogues and MPG (63–65).
The ‘inverted micelle’ is one model describing an early stage for the direct penetration of penetratin (66). Besides forming primarily electrostatic interactions with the extracellular matrix, hydrophobic interactions between hydrophobic residues of the CPP/cargo complex such as tryptophan and the hydrophobic part of the membrane are also shown to contribute to this mechanism. Similar mechanism is less likely applicable for the highly cationic CPPs such as Tat and poly-Arg (50).
For Tat and Poly-Arg CPPs (the two peptides with the highly positive delocalized charge), both endocytosis and direct entry mechanisms are involved in cellular membrane permeation process. At low peptide concentrations, cellular permeation is achieved by endocytosis, and above a certain concentration threshold, peptide internalization for Tat and R9 (a short chain of 9 Arg) occurs by direct uptake (50).
Pore-like structure formation has also been proposed as mechanism for MPG and Pep-1 cellular entry. Both MPG and Pep-1 peptides form nanoscale particles with cargo and, through their hydrophobic tryptophan-rich domains, strongly interact with membrane lipids, thereby facilitating insertion into the membrane and beginning of the translocation procedure (67). NMR, circular dichroism, and FTIR analysis have revealed that the interaction of MPG and Pep-1 complexes with phospholipids mediates their folding into a β-structure and α-helix, respectively (47,48). For MPG, the pore is mainly composed of a β-barrel structure, while for Pep-1, the pore is constituted of an association of α-helices (67).
When the CPP concentration is higher than a certain threshold, it follows the carpet-like model and membrane thinning model of cellular entry, in which interactions between cationic CPPs and negatively charged extracellular matrix result in a carpeting and thinning of the membrane, respectively (68). Endocytosis is an energy-dependent cellular process for macromolecule internalization, which is distinguished by vesicle formation (69). It consists of phagocytosis for uptake of large particles and pinocytosis for solute uptake.
Although a variety of entry mechanisms has been implicated as pathways for internalization of different CPPs with different types of cargo (71–76), there is a consensus on the involvement of endocytosis as the translocation mechanism for most CPPs (51,68).
Transdermal delivery with CPPs
The delivery of macromolecules to and across the skin is a difficult task owing to the barrier function of the skin. The basis for this barrier has been studied extensively (76). As mentioned earlier, the skin barrier function exists because of the highly organized structure of the stratum corneum (77). In addition to protecting the body from hostile outside environment, the epidermal permeability barrier also poses a substantial obstacle to the delivery of therapeutic agents for the treatment for systemic or primary skin diseases. Poor skin drug absorption and very low topical bioavailability of some active ingredients for the treatment for primary cutaneous disease have left no choices but systemic administration of those drugs (39).
Topical delivery of peptides has been increasingly studied owing to the importance of these compounds for the treatment for skin diseases and for the improvement in skin properties (in case of cosmeceuticals). Topical administration of several peptides would be attractive, including IGF-1, TGF-β, leptin, 14-3-3 proteins (for wound healing), interferon α (antiviral), cyclosporine (for treatment for autoimmune diseases), bacitracin (for skin infections), and palmitoyl-glycyl-histidyl-lysine tripeptide (for stimulation of collagen synthesis), among many others (39,78–83). In addition, several peptides have been applied to the skin and studied as antigens for the development of topical vaccines (84). Topical administration of conjugates of PTD peptides may have therapeutic potential for skin disorders. The use of PTDs not only successfully increases peptides’ delivery to the skin and avoids systemic side-effects but also enhances patient’s compliance and satisfaction (20).
Several studies demonstrated the efficiency of applying CPP and cargo complexes on laboratory animal skin. Rothbard et al. reported efficient topical delivery and inflammation inhibition of cyclosporin A when conjugated to arginine oligomers. They have shown that short oligomers of arginine facilitated transport across the cutaneous barrier upon topical application to either mouse or human skin (39). Hou et al. also studied transdermal delivery of proteins mediated by non-covalently associated arginine-rich peptides in the presence of chemical enhancers. In this study, they demonstrated that arginine-rich intracellular delivery peptide was able to rapidly penetrate through skin tissue and its efficiency was further increased in the presence of chemical enhancer. They proposed macropinocytosis and actin rearrangement as mechanisms involved in improving topical drug delivery (78). More recently, Cohen-Avrahami et al. (86) used penetratin embedded within a reversed hexagonal mesophase to enhance transdermal delivery of sodium diclofenac. They studied transdermal delivery rate of sodium diclofenac through porcine skin and found that the reversed hexagonal mesophase served as the solubilization reservoir and gel matrix, whereas penetratin enhanced the transdermal penetration of the drug. Instrumental analysis such as ATR-FTIR revealed that penetratin accelerated the structural transition of skin lipids from hexagonal to liquid and therefore induced efficient drug diffusion through the stratum corneum, toward the different skin layers (6,86).
Cancer immunotherapy with topical vaccination is another example of transdermal delivery of proteins. Schutze-Redelmeier et al. (87) reported that the topical application of OVA257–264 linked to Antennapedia onto mice skin resulted in enhanced delivery through the skin and increased anti-tumor immunity of the antigen, which provides a technical and theoretical basis for the further development of novel peptide-based cancer vaccines.
Transdermal penetration and stability improvement of salmon calcitonin by forming a complex with Tat peptide is another example of improving the topical delivery of bioactives (88). It has been suggested that an electrostatic interaction between the positively charged arginine groups of Tat and negatively charged skin cell surfaces was responsible for the skin binding of calcitonin-Tat complexes and also the translocation of these complexes through the excised skin.
The Pep-1 ability to pass across skin has been studied using in vivo setups. Kim et al. (89) transduced botulinum neurotoxin (BOTOX) as an effective anti-aging compound into mouse skin. Eum et al. (90), using animal model, achieved effective protection against ischemic insult by fusing superoxide dismutase to Pep-1. Pep-1 can also form nanoparticle complex with elastin to improve its delivery across stratum corneum to improve skin elasticity and prevent skin aging symptoms (49). The protective effects of Pep-1-FK506BP complex on atopic dermatitis have been recently demonstrated by Kim et al. (91). They reported that Pep-1-FK506BP complex significantly reduced the expression levels of cytokines and chemokines and, as a consequence, could be a potential therapeutic agent for the treatment for atopic dermatitis (91). Furthermore, the CPPs have been also used for transdermal systemic peptide delivery purposes. Chen et al. (92) studied transdermal protein delivery of insulin and human growth hormone when co-administered with the CPP.
Nanoparticles including solid lipid nanoparticles (SLNs), nanostructure lipid carriers, polymeric nanoparticles, and magnetic nanoparticles have been used for efficient cutaneous drug delivery. For the better and more efficient transdermal delivery of drug-loaded nanoparticles, the CPPs can also be used to cargo the nanolipid carriers, SLNs, liposomes, and polymeric nanoparticles into the skin. This strategy can be applicable for the treatment for various skin disorders such as psoriasis, fungal, bacterial, viral infections, and skin cancers (93–95). For example, Tat peptide enhanced the skin permeation of celecoxib nanoparticles by translocating the nanoparticles across the skin layers to a depth of 120 μm (97). Tat peptides are useful intracellular delivery vectors for a broad series of nanoparticles, thanks to their good-natured ability to enter cells and smallest cytotoxicity (97).
In another study (Singh et al.), peptide containing 11 arginine amino acids (R11) was used to investigate whether CPPs can improve transdermal delivery of nanoparticles. Spantide II and ketoprofen were used as model drugs for combined delivery. Their results strongly suggested that the surface modification of nanolipid carriers with R11 improved the transport of Spantide II and ketoprofen across the deeper skin layers and thus resulted in reduction in inflammation associated with allergic contact dermatitis (98).
The effective delivery of nucleic acids across a cell membrane has been associated with obstacles, mostly because of size and highly negative charge of oligonucleotides. Until recently and before using CPPs to deliver nucleic acids, viral vectors have been the only tool to overcome cell membrane hurdle for oligonucleotide delivery. Although viral vector delivery of oligonucleotides such as siRNA first appeared very promising in gene therapy of various diseases, its serious carcinogenic and immunogenic consequences casted doubt upon its therapeutic values. The positively charged or amphipathic CPPs are effective and less-toxic alternatives to the viral vectors for systemic and topical delivery of oligonucleotides such as siRNA (99–101). Recently, a Peptide Transduction Domain-ds (double-stranded) RNA Binding Domain (PTD-DRBD) fusion protein has been proposed for more efficient and non-cytotoxic siRNA delivery in variety of cellular models. DRBDs bind to siRNAs with high affinity, masking the siRNA negative charge and allow PTD-mediated cargo uptake (102). In dermatology, topical application of siRNA as a therapeutic nucleic acid tool has been studied for the treatment for skin diseases like atopic dermatitis. For instance, to overcome transdermal delivery limitations, Uchida et al. used Tat to achieve effective transdermal siRNA delivery. As the size of paracellular path in the epidermis and hair follicles was reported to be about 70 and 200 nm, respectively, they thus formed Tat/siRNA complexes in approximately 70 nm and +4 mV. Consequently, they assumed that the nanocomplex could simply go paracellularly through the tight junctions of the epidermis from either the skin surface or hair follicle routes (103). Later, this group used Tat to accelerate the delivery of siRNA to silence RelA, a member of the nuclear factor-κB family in mice skin (104).
It appears that the mechanism for the skin penetration of PTDs is likely different from those discussed above for the cell membrane crossing. A model for topical delivery of CPP/cargo complexes has been schematically represented in Figure 2. The outermost layer of the skin is composed of non-viable cells and endocytosis is not expected, although it is believed that these dead cells form a metabolically active environment. Moreover, there are some distinct differences between stratum corneum and common cell membrane in terms of intercellular lipid domain, lipid composition, water content, and lipid/protein ratio (105). It is believed that metabolically active, non-viable cells of SC directly participate in the transport of PTDs (39). In addition, interactions between lipids and PTDs may also play important role in their transport across the SC. CPPs enable to transport their cargoes across the skin by destabilizing SC and increasing PM permeability after interaction of CPPs with SC lipids (94). For example, poly-l-arginine was reported to increase the permeability of tight junctions of the nasal epithelium (106) and facilitate the transport of a dextran. This effect was initiated by interaction of positively charged polyarginine with negatively charged cellular lipids (107). Macropinocytosis is another possible transport mechanism, by which the CPPs pass through the mammalian cells. It has been shown that the macropinocytosis and actin reorganization are both involved in the cellular entry and transdermal delivery of CPP/cargo complexes (78). However, this mechanism of transport is less likely to be involved in the penetration of non-covalently attached CPP/Cargo complexes (108). Solid evidence confirms that the existence of the tight junctions is in the skin (109); therefore, the effects of PTDs on disassembly of these structures might be important for understanding their penetration into the viable layers of the skin. Finally, the gradient factor might be involved as a driving force in PTDs’ penetration into different layers of the skin (39). The exact mechanism of CPP transport through skin layers is not fully understood, and further studies are required to identify mechanism(s) responsible for the CPP-mediated transport from non-viable to viable layers of the skin.
Transdermal delivery of drugs is an attractive route of administration because it provides higher patient acceptance and compliance and it also avoids the first-pass hepatic metabolism. The stratum corneum is a physiological barrier responsible for protecting body from the outside environment and at the same time limits the skin penetration of hydrophilic macromolecules. CPPs have been shown to facilitate delivering a wide variety of biomolecules across the skin. The enormous potential of this technology resides in the high efficiency and relatively low toxicity of CPPs conjugated to bioactive cargoes. Since the discovery of Tat and penetratin, the number of CPPs has been rapidly increasing and their properties have been subjects of intensive research. The main remaining challenge is now to elucidate their exact uptake mechanisms and particularly their transdermal delivery. Different CPPs can be successfully used for the delivery of high molecular weight drugs into cells as well as for vaccine development. The application of CPPs in pharmaceutical formulations is becoming increasingly popular with a great potential in transdermal drug delivery systems.
The authors would like to thank Dr. Siavash Khalaj for his contribution in drawing Figure 2.