Cell-penetrating peptides (CPPs) are a class of polycationic transporters which have the ability to bypass the cell membrane predominantly through direct translocation (Figure 2). They were first introduced in 2000 by Rothbard and coworkers[14] taking inspiration from the fact that certain protein subunits, which were highly basic, could enter cells in an energy-independent manner.
Indeed, in 1988 it was shown that the Tat protein of the HIV-1 virus, unlike other proteins, was able to readily pass through the membrane of different cell lines.[15-16] Ten years later,[17] it was shown that the α-helical region of the protein, bearing both hydrophobic and hydrophilic residues, was not necessary for the cell entry, but that, counter-intuitively, the cation-rich sequence (Tat49-57: RKKRRQRRR) was responsible for the direct translocation and it was proven that endocytosis was not the main mechanism involved. At this point, Tat started to be used to achieve cellular entry of various cargos, as depicted in Figure 2, such as the delivery of a 120 kDa β-galactosidase protein across the blood brain barrier.[18] It was, finally, in 2000 that the guanidinium groups present in the arginine-rich sequence of the protein were determined to be responsible for its diffusion.[14] Since then, the optimal structural requirements were studied in detail such as the presence of spacers, the stereochemistry and the number of the arginine residues required for cell entry or the introduction of other cationic amino acids (histidines and lysines).[14]
Figure 2. The evolution of cell-penetrating peptides. Figure adapted from reference.[12]
As for the mechanism of entry of these arginine-rich transporters, it is hypothesized that it involves ion-pairing with anionic species. In particular, Wender and coworkers proposed a model which relies on the ability of the guanidinium cations to form strong bidentate hydrogen bonds with the anionic phosphates, carboxylates and sulfates that are located on the cell surface.[19] This bifurcated motif would explain the difference with less active ammonium cations coming from lysine groups which on the other hand can only react with anions through random electrostatic interactions. On the other hand, Matile and coworkers have also proposed that the strong ion-pairing with arginine residues stems from the very low acidity of the guanidinium group.[20] Indeed, the strong binding of anions to arginine residues compared to lysines originates from a proximity effect, as shown in Figure 3.[21] In nature, the proximity of ammonium groups of lysine residues (pKa ≈ 10.5) causes charge repulsion that can be eliminated by the deprotonation of the vicinal cation (with pKa ≈ 7). In the case of proximal arginines, due to the weak acidity of the guanidinium group (pKa ≈ 12.5) the charge repulsion cannot be eliminated by deprotonation and therefore ion-pairing with anions always occurs. In the case of the arginine-rich transporters, the ion-pairing between the guanidinium groups to the exofacial anions binds them to the cell surface increasing their hydrophobicity and enabling direct translocation. It is noteworthy to mention that depending on the size of the transporter, endocytosis is in competition or dominates with direct
Tat49-57 (RKKRRQRRR)
: Cargo
Tat Rn
NH NH2 H2N
NH NH2 H2N NH
H2N H2N
NH NH2 NH2
translocation and many CPP-like systems can be found to enter either using both pathways or only one of the two.
Figure 3. Charge repulsion elimination through ion-pairing for arginine residues and deprotonation for lysine residues. Image from reference.[21]
Once the ion-pairing occurs, direct translocation through the membrane enables the cell entry. However, the mechanism of this type of transport is still under debate. Many models have been proposed to explain how the entry occurs,[22] as shown in Figure 4. Among the various theories we can find the formation of transient micellar pores (carpet model),[23] the loosening of the cell membrane packing,[24] pore formation (barrel-stave model)[25] or an influence of the different protonation and deprotonation state of fatty acids.[26] Effort is also oriented towards activating these cationic transporters rendering them more efficient through the use of more hydrophobic counter-anions such as pyrenebutyrate[27] or ion-pair-π interactions and fluorophiles[28] to gain
N
Figure 4. Models postulating how CPPs may perturb membranes and enter into the cell. Image from reference.[22]
As mentioned earlier, oligoarginines 1 were first used as CPPs, however further investigations led also to the replacement of the peptidic backbone with a great variety of scaffolds, depicted in Figure 5, such as PNAs,[29] glycosides introduced by Tor et al.,[30] polymers such as oligophosphoesters 2[31] and oligocarbonates 3[32] developed by Wender et al. or polyprolines 4.[33] Variations of the positive charge have also been studied, replacing the guanidinium group with ammoniums 5,[34] phosphoniums 6,[35] or sulfoniums 7 introduced by Deming et al.[36] leading to less toxicity in most cases, but also less proficient ion-pairing with the exofacial anions. On the other hand, Schmuck and coworkers have shown that guanidinocarbonylpyrroles 8 can lead to better transfection efficiencies compared to the guanidinium analogues thanks to the better anion binding motif.[37]
Figure 5. Original CPP oligoarginine 1 and CPP mimics 2-8.
Even though many CPPs have been developed throughout the years leading to the efficient delivery of a wide range of cargos not only in cultured cells but also in animal models,[38] the two major drawbacks that can be encountered are endosomal entrapment and cytotoxicity. The introduction of pH-responsive residues like histidines with a weakly basic imidazole ring, such as in 9 (Figure 6),[39] can help to escape the endosomes due to the slightly acidic pH found in these vesicles (so-called proton-sponge effect). Indeed, the presence of the guanidinium groups enables 9 to enter the cell through a counterion-mediated
HN
escape. On the other hand, a common strategy to address CPP cytotoxicity is to enable their degradation as in the self-immolative oligo-α-aminoesters 10.[40]
Indeed, Waymouth et al. recently reported the dynamic CPP 10 which can undergo a rearrangement once it enters the cell, leading to the formation of the neutral piperazine 11. Moreover, it was shown that it allows the progressive reduction of the charge aiding to escape possible endosomes, releasing the cargo it carries and thus eliminating toxicity. Another common strategy involves the introduction of cleavable linkers such as disulfides like the one contained in transporter 9, which can be reduced in the cytosol of the cell by the high concentration of glutathione (GSH).
Figure 6. pH-responsive CPP 9 and biodegradable CPP mimic 10.