Elsevier

Advanced Drug Delivery Reviews

Volume 57, Issue 4, 28 February 2005, Pages 559-577
Advanced Drug Delivery Reviews

Tat peptide-mediated cellular delivery: back to basics

https://doi.org/10.1016/j.addr.2004.12.001Get rights and content

Abstract

Peptides are emerging as attractive drug delivery tools. The HIV Tat-derived peptide is a small basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The ‘transduction domain’ or region conveying the cell penetrating properties appears to be confined to a small (9 amino acids) stretch of basic amino acids, with the sequence RKKRRQRRR [S. Ruben, A. Perkins, R. Purcell, K. Joung, R. Sia, R. Burghoff, W.A. Haseltine, C.A. Rosen, Structural and functional characterization of human immunodeficiency virus tat protein, J. Virol. 63 (1989) 1–8; S. Fawell, J. Seery, Y. Daikh, C. Moore, L.L. Chen, B. Pepinsky, J. Barsoum, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 664–668; E. Vives, P. Brodin, B. Lebleu, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem. 272 (1997) 16010–16017; S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836–5840.]. The mechanism by which the Tat peptide adheres to, and crosses, the plasma membrane of cells is currently a topic of heated discussion in the literature, with varied findings being reported. This review aims to bring together some of those findings. Peptide interactions at the cell surface, and possible mechanisms of entry, will be discussed together with the effects of modifying the basic sequence and attaching a cargo.

Introduction

Over the last decade, several publications revealed a massive improvement in the cellular delivery of various biologically active molecules upon their attachment to a peptide derived from the HIV-1 Tat protein. This peptide can be reduced to a cluster of basic amino acids containing 6 arginine and 2 lysine residues within a linear sequence of 9 amino acids. Because of the high content of arginine residues within the Tat sequence, various homopolymers of arginine have also been investigated to study the mechanism of entry of various cargoes. Very similar results were obtained with these simple polymers of arginine in terms of transduction efficiency and apparent mechanism of entry compared to the Tat peptide [4].

Among others, we recently reevaluated the mechanism of entry of the Tat peptide [5] and highlighted various problems related with the FACS quantification and the fixation procedure prior to microscopy observations. Despite the possibility that the uptake of various entities previously described in the literature could have been artifactual or overestimated, it is unlikely that the efficiency of the Tat-mediated uptake could be disputed, due to the high number of examples of biological activity which have been provided upon Tat peptide-mediated cellular delivery of peptides, proteins or nucleic acids (to name but a few) [6], [7], [8]. In the large majority of the experiments, the chimera concentration used for obtaining the expected biological responses was not outrageously different to those used to assess the cell uptake of the Tat peptide itself. For instance, most of the fused compounds are active at concentrations in the 100-nM range ([9] for oligonucleotides, [10] for peptides, and [11] for protein delivery), whereas fluorescence microscopy or FACS quantification of the uptake were usually performed with 1–10 μM of the peptide [3], [4], [5], [12]. Despite a possible dose effect causing variations in the efficiency of the uptake, and potentially the cellular pathway induced in uptake, it has been assumed that the entry mechanism of the Tat peptide and of Tat-carrying chimera is similar. However, little has been done to unambiguously answer this question by comparing the uptake efficiency of two different entities under the same cellular and experimental conditions. It might be very important to consider the influence of the physicochemical character of the cargo.

Despite the high number of biological applications using these peptides, and principally the Tat peptide, the precise mechanism of entry still appears controversial and certainly requires further investigations. Contradictory results are still often obtained. They could result from experimental variations in, for example, the diversity of the Tat peptide sequence used to promote the translocating activity, the wide variety of cell lines studied, the differing protocols applied to investigate the entry mechanism or the high diversity of cargoes, all of which might well influence the behavior of the Tat peptide during the cellular entry process. This review is aimed at giving an up-to-date statement of various parameters possibly influencing the observed results during the investigations about the translocating properties of the Tat peptide and its attached cargoes.

Section snippets

Implication of the basic cluster

The initial work showing the ability of a Tat protein-derived peptide to deliver heterologous molecules into cells was provided 10 years ago by Fawell et al. [2]. In this study, the covalent coupling of a 36 amino acid peptide (Tat37–72) to large proteins, such as β-galactosidase, RNAse or peroxidase, was performed through a heterolinker. Four to five peptide molecules per protein unit were sufficient to mediate the cellular delivery of the covalently bound protein [2]. The delivery was

Influence of other components surrounding the basic domain

Since different versions of the Tat peptide have been used to promote cellular delivery of many types of cargo, it appears necessary to consider a putative effect of their molecular nature. Various results, sometimes contradictory, about the efficacy of the uptake or about the internalisation pathway have been obtained during the last years. It appears now widely accepted that the cationic nature of the Tat peptide alone promotes the cellular delivery of very different entities in terms of

Influence of the cargo

The more remarkable fact regarding the Tat peptide's ability as a vector system is the molecular diversity of the “transduced” entities, ranging from small molecules of some hundreds of daltons to massive structures with a diameter up to 200 nm such as liposomes [36], [37]. Until very recently, it was believed that the translocating activity of the Tat peptide could occur directly through the plasma membrane following an inverted micelle formation as earlier proposed for the

Tat and cell surface interactions

Because of the highly cationic nature of the Tat peptide, several anionic cellular candidates are available to influence the initial ionic cell surface interactions. These interactions, or this binding to the cell surface can, in part, be competitively inhibited with heparin [11], [24], [31], [71], along with heparin analogues, such as PPS (pentosan polysulfate) [72], the heparin-binding protein TSP (platelet thrombospondin-1) [73] and other soluble polyanions, such as suramin, suramin

Possible mechanisms of internalisation

Membrane association or binding occurs at any temperature, including the metabolically inhibiting 4°C. In traversing the extracellular membrane, however, the Tat peptide behaves in an energy-dependent manner requiring temperatures above 4 °C and ATP. Only one group [18] continues to observe uptake at 4 °C with a modified Tat peptide [Tat-(48–60:P59W)].

The initial association is followed by a rapid translocation to the cytosolic side most probably within vesicle-like structures, of which some at

Conclusions

The Tat peptide delivery strategy is now widely used to improve the cellular delivery of a very large panel of cargo molecules. The increase of the biological response of peptides, proteins or oligonucleotides upon their coupling to the Tat peptide has been assessed in several recent studies, although the precise mechanism of entry is far from being firmly identified. Major controversies still exist in the literature, probably as the direct result of previously reported misleading results, due

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