Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s).

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.

In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein-ligand interactions and conformations. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.

Basic peptides derived from the HIV-1 1 Tat protein (Tat-(48 -60)) and Drosophila Antennapedia protein (Antp-(43-58)) have been reported to have the ability to translocate through the cell membranes and to carry exogenous molecules into the cytoplasm and nucleus (1-13). A 119-kDa protein, ␤-galactosidase, genetically fused with the former peptide segment, was successfully carried into various tissues in mice including the brain via intraperitoneal injection (6). The 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside (X-gal) staining of the tissues indicated that the fusion protein was delivered in its active form. Oligo-DNAs and metal chelates were also brought into cells using the Tat-derived peptide (4, 7). Such a method to deliver bioactive molecules into cells using membrane-permeable peptides has a great potential for therapeutic fields.We have recently demonstrated that not only Tat-(48 -60) and Antp-(43-58) but also various arginine-rich RNA-or DNAbinding peptides such as HIV-1 Rev-(34 -50) and flock house virus (FHV) coat-(35-49) were membrane-permeable and have the ability to bring exogenous protein into cells (14). Even octaarginine (Arg 8 ) gave similar results based on the fluorescence microscopic observation of the fluorescein-labeled peptides (14, 15). These peptides seem to have other similarities in translocation, namely facile internalization within 5 min, little uptake inhibition at 4°C, and localization in the nucleus and cytosol. The above results suggested the possible existence of a ubiquitous mechanism for the internalization of the argininerich peptides. Since there were no sequence similarities among these peptides except that they had several arginine residues, arginine seemed to be the key amino acid for membrane permeability.Despite the great potential of the arginine-rich peptides as carriers of proteins, nucleic acids, and other bioactive compounds, little is known about the mechanism of their internalization. Involvement of the cell surface heparan sulfate (HS) and low density lipoprotein receptor-related protein (LRP) was suggested in the translocation of the full-length Tat protein (16,17). The addition of HS and the inhibitor of LRP to the culture medium produced a significant decrease in the cellular uptake of the protein. However, the uptake of the full-length Tat protein suffered a certain decrease at 4°C (16), and some energydependent endocytosis pathway seemed to play a significant role in the internalization of the Tat protein. These results suggested that the mechanisms of internalization of the Tat-(48 -60) peptide and the full-length Tat protein may not be completely parallel. Actually, the importance of the "core" domain (Tat-(37-48)) of the Tat protein has been claimed for the LRP-dependent internalization pathway (16).We have pointed out that many arginine-rich peptides showed very similar characteristics in translocation with HIV-1 Tat-(48 -60) (14). In addition to the translocation mechanisms of these arginine-rich peptides, it is unclear whether these peptides share...

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.

Arginine-rich cell-penetrating peptides (AR-CPPs) are very promising tools for the delivery of therapeutic macromolecules such as peptides, proteins, and nucleic acids. These peptides allow efficient internalization of the linked cargos intracellularly through the endocytic pathway. However, when linked to bulky cargos, entrapment in the endocytic vesicles is a major limitation to the application of these peptides in cytosolic delivery. Attachment of a compatible endosomal escape device is, therefore, necessary to allow cytosolic delivery of the peptide-attached cargo. This review presents different endosomal escape devices currently in application in combination with AR-CPPs. Applications of fusogenic lipids, membrane-disruptive peptides, membrane-disruptive polymers, lysosomotropic agents, and photochemical internalization to enhance the cytosolic delivery of AR-CPPs-attached cargos are presented. The properties of each system and its mechanism of action for the enhancement of endosomal escape are discussed, together with its applications for the delivery of different macromolecules in vitro and, if applicable, in vivo.

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

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