CPPs and cell entry

Read Also

As mentioned in the previous section, the entry mechanism of CPPs into cells is still a matter of some debate. Historically, two hypotheses were put forward to explain how these peptides could possibly deliver various kinds of molecules, and also much larger macromolecular structures, into the cell (for a review [16]). It was first proposed that CPPs, especially Tat and Antennapedia, but also others such as poly-Arg [10, 26], Transportan [27], MPG [28] or Pep-1 [20], could pass through the plasma membrane via an energy-independent pathway. Some suggestions have been put forward to explain the translocation of these peptides, such as the formation of micromicelles at the membrane [5], or direct translocation through the lipid bilayer [29, 30]. If conceivable for small CPPs, these models can not explain the passage through the plasma membrane of CPPs-cargoes of very important size [31, 32]. The hypothesis of a direct translocation through the plasma membrane became less popular when the entry mechanism for the Tat and the poly-arginine CPPs had to be re-evaluated following evidences of fixation artifacts during the preparation for samples for microscopic observation [33]. Indeed, fixation has been described to interfere with the sub-cellular localization of constructs with a high content in cationic residues, such as histones and the VP22 protein [34]. This redistribution upon fixation has been clearly demonstrated using fusion proteins made of Antennapedia, poly-Arg, or Tat peptides [35]. As a consequence, the majority of the new microscopic studies on CPP-cargoes localization have been conducted on living cells. As a result, during these last few years, numerous new works about the mechanism of entry of CPPs appeared in the literature, but the conclusions we can draw from these very elegant works could be summarized by: “the more we learn, the less we know”. As a matter of fact, there has been a profusion of publications highlighting one or another entry route, sometimes with some obvious discrepancies. CPP-mediated transport has been shown, so far, to mainly follow a cellular endocytosis-mediated uptake [36-38].

According to this mechanism, CPPs, particularly those with a high content in cationic residues, are first simply adsorbed at the cell surface thanks to the numerous anionic moieties, such as heparan sulfate, sialic or phospholipidic acid [39-41]. Then CPP-mediated transport has been reported to happen through different endocytosis routes [33]: via caveolae [42], macropinocytosis [43, 44], through a clathrin-dependent pathway [45], via a cholesterol-dependent clathrin-mediated pathway [46] or in the trans-Golgi network [47]. Some publications have provided convincing arguments against one or the other of these cellular pathways despite the use of rather similar experimental models. It has been suggested that these controversies might be due to the use of different peptide concentrations as they can trigger different endocytotic pathways [38, 48]. Higher CPP concentration (>10mM) could also lead to an energy-independent internalization [38, 49]. A molecular mechanism for a direct translocation of the Tat peptide through the plasma membrane has been also recently described [50]. In conclusion, more work is needed to highlight unambiguously the precise mechanism(s) of entry of these peptides.

In addition, since no cellular pathway appears absolutely predominant or more convincing than another one, most of these pathways could be involved depending on yet unknown events such as the concentration, the net charge, the hydrophobicity or other physico-chemical parameters of the CPPs. In several studies, initial ionic interactions at the cell membrane surface have been however shown to be key determinants for the uptake of all the cationic CPPs since the peptide entry could be strongly reduced by competition with polyanionic compounds [51-54] or by stringent cell washes with solutions at acidic pH [55].

Moreover, the way of entry into the cell could also be influenced by the nature of the cargo, the type of CPP, the cell line and the conditions of incubation (for example, the CPP concentration as shown above). There is still a major need to compare different CPP-mediated delivery systems in the same cell model. Attempts have been made recently to compare the effects of the cargo as well, but none of these studies could define precise rules that might explain all the observed discrepancies [49, 56].

Despite all the controversies about the route of entry, some consensual features are now privileged, at least for the Tat peptide and other multi-cationic CPPs. First, the entry mechanism implies the use of pathways that are sensitive to lysosomotrophic agents, such as chloroquine and sucrose [57, 58]. Moreover, the efficacy of the cell uptake is improved by co-incubation of Tat with another peptide derived from the hemagglutinin protein which has membrane fusogenic properties [59], or by the use of photochemical internalization mediated by a membrane soluble photosensitizer [60].

Since the endosomial pathway is likely to be involved in the cellular delivery of Tat-conjugated molecules whatever the initial route, a strong enzymatic degradation within this compartment and a poor cytoplasmic release of intact molecules from this compartment are expected, thus leading to a global weak transfer into the cytoplasm. Therefore, increasing the escape rate from the endosome could be a strategy to improve the intracellular delivery of CPP-attached molecules. How could this be done? One possibility could be to increase the hydrophobicity of CPPs to favor the destabilization of the endosomal membrane. To this aim, lipid moieties have been coupled to molecules to be delivered inside the cells. For example, a cholesterol unit has been attached to a free terminus of an oligonucleotide hairpin, thus enhancing its cellular delivery in comparison to conventional transfection methods [61]. In another study, cholesterol-derivatized oligonucleotides also showed a rapid binding and cytosolic partition in cells [62]. Closer to the CPP context, cell delivery improvement has been also observed upon stearylation of an octa-arginine peptide [63] or following the introduction of a proline amino acid derivative with a higher hydrophobicity into a proline-rich CPP [64]. In most of these studies, except for the last one, the derivatization of peptides with lipids was performed at the N-terminal end as it allowed easy coupling of the lipid by conventional amide bond formation directly on the peptidyl-resin after completion of the peptide sequence. We are currently developing an approach to insert lipophilic groups anywhere within the CPP sequence to increase the destabilization of cellular or endosomial membranes.