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 (Seisel et al. 2019). 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). The transduction domain of the HIV-1 Tat protein makes a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). Basolateral sorting in epithelial cells involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif (Soza et al. 2004).
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 include penetratin (TC# 8.A.39.1.1), Tat of HIV, (1.C.101.1.1) and octaarginine.
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). Cell-penetrating peptides behave differently from pore-Forming peptides because of the structure and stability of induced transmembrane pores (Alimohamadi et al. 2023).
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). Lower transmembrane potentials boost CPP cellular internalization, and reduction in the Δψ (Vm) can occur as a result of the activities of KCNQ5 (TC# 1.A.1.15.5), KCNN4 (TC# 1.A.1.16.2) and KCNK5 (1.A.1.8.2) (Trofimenko et al. 2021). Membrane binding strength vs pore formation determines what drives the membrane permeation of nanoparticles coated with cell-penetrating peptides (Klug et al. 2024).
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 internalization of CPPs and the design of cell penetrating peptides and drug conjugates with high efficiency and low toxicity has been studied with Arg8 peptides with and without cargos (Hu et al. 2019). Peptide-mediated membrane transport via Pep-1 drives macromolecular as a result of membrane asymmetry (Li et al. 2017). Adsorption and insertion of polyarginine peptides into membranes has been studied by Ramírez et al. 2019 revealing the trade-off between electrostatics, acid-base chemistry and pore formation energy. A common strategy has been devised to improve transmembrane transport in polarized epithelial cells based on sorting signals by guiding nanocarriers to the trans-Golgi network (TGN) rather than to the basolateral plasma membrane directly (Zhang et al. 2021).
The ability of the Pep-1 peptide to induce (1) vesicle aggregation, (2) lipidic fusion, (3) anionic lipid segregation, (4) pore or other lytic structure formation, (5) asymmetric lipidic flip-flop, and (6) peptide translocation across the bilayers in large unilamellar vesicles has been studied (Henriques and Castanho 2004). Clustering of vesicles occurs in the presence of the peptide in a concentration- and anionic lipid content-dependent manner. Lipidic fusion and anionic lipid segregation without pore or other lytic structure formation was observed, and asymmetric lipid flip-flop was not detected. A specific method related to the quenching of rhodamine-labeled lipids by Pep-1 was developed to study the eventual translocation of the peptide. Translocation does not occur in symmetrical neutral and negatively charged vesicles, except when a valinomycin-induced transmembrane potential exists. Thus, the main driving force for peptide translocation is charge asymmetry between the outer and inner leaflets of biological membranes. Pep-1 is able to perturb membranes without being cytotoxic. This nonlytic perturbation is probably mandatory for translocation to occur (Henriques and Castanho 2004).
Wang et al. 2022 reported a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. These study showed that positive charge is a dispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications (Wang et al. 2022).
Differently from cationic CPPs, which are commonly developed from naturally occurring proteins, hydrophobic CPPs are usually artificial in origin. The translocating peptide TP1 (Translocating peptide 1, sequence PLILLRLLRGQFC) has proven not only of being able to deliver polar cargo through synthetic lipid bilayers and in vitro cell membranes alike, but they are also able to cross cell barriers in living animals and deliver cargo (Muñoz-Gacitúa et al. 2022). An equilibrium structure simulates the entire translocation mechanism. After the peptide reaches its equilibrium structure, it must undergo a two-step mechanism in order to completely translocate the membrane, each step involving the flip-flop of each arginine residue in the peptide. This leads to the conclusion that the RLLR motif is essential for the translocating activity of the peptide (Muñoz-Gacitúa et al. 2022).
The generalized reaction catalyzes by CPPs is: