Tat peptide-mediated cellular delivery: back to basics

Advanced Drug Delivery Reviews 57 (2005) 559 – 577 www.elsevier.com/locate/addr

Tat peptide-mediated cellular delivery: back to basics Hilary Brooks, Bernard Lebleu, Eric Vive`s* De´fenses Antivirales et Antitumorales, CNRS-UMR5124-Universite´ de Montpellier II, CC086; 5, Place Euge`ne Bataillon, 34095 Montpellier Cedex 05, France Received 10 October 2004; accepted 27 October 2004 Available online 6 January 2005

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 dtransduction domainT 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. D 2004 Elsevier B.V. All rights reserved. Keywords: TAT; Uptake; Cell delivery; CPP

Contents 1. 2. 3.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Implication of the basic cluster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Influence of other components surrounding the basic domain . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +33 467 16 33 06; fax: +33 467 16 33 01. E-mail address: [email protected] (E. Vive`s). 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.12.001

560 560 561

560

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

4.

Influence of the cargo . . . . . . . . . . . . . . . . . . . 4.1. Influence of Tat peptide exposure . . . . . . . . . 4.2. Influence of the hydrophobicity . . . . . . . . . . 4.3. Influence of the chemical linkage between Tat and 4.4. Influence of the Tat peptide density . . . . . . . . 4.5. Influence of the serum . . . . . . . . . . . . . . . 5. Tat and cell surface interactions. . . . . . . . . . . . . . 6. Possible mechanisms of internalisation . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 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 peptidemediated cellular delivery of peptides, proteins or nucleic acids (to name but a few) [6–8]. 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 100nM range ([9] for oligonucleotides, [10] for peptides, and [11] for protein delivery), whereas fluorescence

. . . . . . the . . . . . . . . . . . .

. . . . . . . . . . . . cargo . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

562 563 564 565 567 568 568 571 572 573

microscopy or FACS quantification of the uptake were usually performed with 1–10 AM of the peptide [3– 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 Tatcarrying 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.

2. 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

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

Fawell et al. [2]. In this study, the covalent coupling of a 36 amino acid peptide (Tat37–72) to large proteins, such as h-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 assessed by monitoring the corresponding activity of the delivered protein, therefore, demonstrating the effectiveness of the uptake process and also, more importantly, the cell viability. This cell-penetrating peptide (CPP) encompassed a highly cationic cluster composed of 6 arginine and 2 lysine residues in the very middle of the peptide sequence and an a-helical structure on the N-terminal part [13]. Around the same time, another peptide able to translocate through the plasma membrane had been discovered [14]. This peptide was derived from the third domain of the Antennapedia homeodomain from Drosophila melanogaster known to bind DNA sequences and to activate various genes. Interestingly, this peptide also contains a high number of cationic amino acids and structural data showed an overall ahelical structure. Since the N-terminal portion of the Tat sequence used for introducing large proteins into cells was shown to adopt an a-helical conformation, and the cationic cluster was shown to adopt an extended structure [13], a structure–activity relationship study was performed to delineate which feature within the Tat peptide was responsible for the cell membrane translocating property [3]. Several peptides carrying either partial or total deletion within the ahelical structure and a peptide harboring a deletion within the cationic domain of the original peptide were synthesized [3]. It was shown, primarily by fluorescence microscopy, that the main determinant responsible for the translocating activity was the cationic cluster of amino acids and that deletion of arginine led to an apparent non-translocating peptide. The a-helical structure was shown to induce significant toxicity in HeLa cells, as assessed by MTT toxicity testing [3]. The toxicity of this region has been recently confirmed in an independent study [15]. These studies, however, found no toxicity for the cationic cluster [3,4,15], in contrast to a previous study showing a neurotoxic activity induced by the Tat basic peptide after intracerebroventricular (ICV) injection in mice [16]. From these observations, the

561

translocating property of the initial peptide could be reduced down to the sequence encompassing the cationic cluster containing 8 basic charges within a 9 amino acid linear sequence. Despite the risk of misleading results, due to fixation artifacts (see below), this short Tat cationic peptide has been used in several examples to mediate the cellular uptake of biologically active molecules. At the same time, evidence has been provided to show that the a-helical structure within the Antennapedia peptide was dispensable, since the insertion within the primary sequence of two proline residues, an amino acid known to disrupt a-helical structures, did not impair the translocating activity of this peptide [17]. The cationic content of these peptides, thus, appeared to be responsible for their ability to be taken up by cells. In the same study [17], tryptophan residues were highlighted to play a crucial role in the translocating property of the Antennapedia peptide [17]. No tryptophan residue, or other aromatic amino acids, is present within the minimal Tat peptide primary sequence shown to be able to enter cells. Solely the work of Thoren et al., using Tat peptide with a terminal tryptophan, continues to describe uptake at 4 8C [18]. Despite this latter difference and some controversial data regarding the actual mechanism of entry (see below), both peptides were initially shown to be taken up by cells very rapidly (within minutes) and at temperatures known to inhibit active transport. These findings stimulated a very high interest in the possibility of using such peptides to carry various drugs into cells.

3. 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 molecular size, structure and overall physicochemical properties. The common

562

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

sequence determinant in most of the studies performed with the Tat peptide is the GRKKRRQRRR sequence. The C-terminal part of this peptide has been extended by various sequences or coupled to different moieties (see Table 1). These include amino acids present in the native Tat protein sequence, such as the PPQ sequence [3,19–23], but also longer cargoes in order to induce their own cellular uptake ([9,11,24] and Table 1). The N-terminal part of this minimal Tat peptide has also been extended by various amino acid sequences from either the native Tat protein sequence or again attached to different entities [15,21,22,24– 27]. All of these Tat-cargo derivatives, except the one used in the work from Koppelhus and collaborators [23], showed the ability to be taken up, since fluorochromes or covalently bound molecules were recovered in cells (see Table 1 for references). From these different applications, no clear influence of the additional moiety attached either to the N-terminal or to the C-terminal end of the bcoreQ Tat peptide could be related to a variable ability of the basic Tat peptide to mediate cellular uptake. It should, however, be pointed out that comparison is rather difficult since most of these studies were performed on different cell lines following different experimental protocols, as is discussed below.

Table 1 Tat-derived peptides used for the cellular delivery of various cargoes N-terminal end

Core peptide

C-terminal end

Reference

MYGGST proteinSGYG MLGISYCYCGISYCFITKALGISYCBiotin-Y MLGISYHis6YAcetyl-

GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQ GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR GRKKRRQRRR

-GYK(FITC)C -PPQ -G -PPQC -GYK(FITC) -Cre protein -G -GFP protein -C -PPQT

[28] [19,22] [25] [9,20,29] [30] [11] [27] [24] [31] [21] [22,25] [15] [15] [23] [26] [21] [32] [33,34] [35]

4. Influence of the cargo The more remarkable fact regarding the Tat peptide’s ability as a vector system is the molecular diversity of the btransducedQ 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 Antennapedia-derived peptide [17]. Such a mechanism was originally proposed to explain the Antennapedia peptide translocation occurring even at a temperature (4 8C) known to inhibit all cellular energy-dependant pathways. Basically, ionic interactions between the cationic charges of the peptide and anionic charges of the membrane components (principally the phosphate groups of the phospholipid heads) initiated the membrane adsorption of the peptide. Then, phospholipid reorganization led to the scavenging of the peptide inside a fully hydrophilic pocket, the inverted micelle [17]. No direct evidence, however, except in one single report [38], has been provided so far to clearly demonstrate such a mechanism within homogeneous artificial membrane systems for either the Antennapedia or the Tat peptide. Nevertheless, the presence of numerous proteins anchored in cell membrane or exposed at the cell membrane surface, along with the variation in lipid composition within and between different cell types (e.g. lipid rafts), could be required for such a mechanism to function properly. It is difficult to imagine, however, that an inverted micelle mechanism could be applied to large molecules, such as proteins or even much bigger structures. As an example, ferromagnetic particles of about 45 nm in diameter [22] were shown to be taken up by cells, as demonstrated by the magnetic recovery of particlecontaining cells from a cell mixture. The particle diameter was about 15-fold larger than the entire thickness of membrane (30 angstroms or 3 nm). Although each particle carried 4 to 5 peptide molecules at its surface, it seems very unlikely that the membrane could reorganize itself completely around the ferromagnetic particle as proposed for the peptide itself.

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

The cationic charges of the Tat peptide certainly play a key role in the uptake process, since a single deletion or substitution of basic charges induced a reduction of the cell association of the peptide, therefore, probably reducing the overall intracellular uptake [3,39,40]. The guanidinium group of the arginine side chain was shown to be more potent in mediating cellular uptake than other cationic groups, such as lysine, histidine or ornithine [41]. Along these lines, very similar cell-penetrating properties were obtained with simple homopolymers containing only residues with guanidinium side chains ([4,40,42], see below). Since ionic interactions appear to initiate the uptake process, it has to be recalled that the improvement of the delivery of highly anionic moieties, namely, nucleic acids, can be mediated by the arginine-rich peptide derived from the Tat protein, as has been recently documented [25]. It is assumed that the complexation of nucleic acids to cationic polymers allows the condensation of the nucleic acid moiety and the subsequent release from endosomes, as assessed for cationic lipids [43]. In this study, the Tat peptide, with its high guanidinium group content, has been attached to polyethylene imine (PEI), an amine containing polymer, and compared to PEI alone for the transfection efficiency of a reporter gene. A marked increase, up to 100-fold, of the cellular delivery of plasmids already complexed with polycationic compounds was recorded. Thus, the cationic charge of the Tat peptide alone could not be considered responsible for the improved translocating properties of these nucleic acids, as it also markedly improved the delivery of nucleic acids complexed with PEI, which already carry a highly cationic charge due to the high amount of amino groups. In conclusion, the Tat guanidinium groups induced a massive increase in the transfection efficiency of nucleic acids delivered by complexation with the simple amine-functionalized polymer, PEI. This latter example also illustrates that particular features are required to fulfill this translocating process. These are, for instance, an appropriate charge ratio or the number of Tat peptides present on each plasmid particle. Therefore, possible factors expected to play a role in the cell entry mechanism will be discussed further.

563

4.1. Influence of Tat peptide exposure In some cases, it cannot be excluded that the direct environment of the Tat peptide once inserted into a chimera and its overall exposure within this structure could influence the behavior of the translocating process. As an example, a shorter version than the 36 amino acid Tat peptide used originally to mediate the cellular delivery of proteins was also evaluated in the same study [2]. A weaker translocating activity was reported for such covalently bound chimeras. Thus, the level of exposure of the Tat basic peptide at the molecular surface of the transported protein could be closely related with the level of the observed cellular uptake. Along these lines, it is possible that a steric hindrance of the short cationic peptide prevented interactions with cellular components promoting the uptake, since the Tat peptide coupling was mediated randomly on large proteins, such as hgalactosidase (120 kDa) [2]. In longer versions of the bound peptide, the cationic cluster is potentially better exposed and, therefore, more accessible to cell surface structures. The influence of the exposure of the short Tat basic peptide has been elegantly demonstrated for the cellular delivery of liposomes [37]. In this study, liposomes were functionalized by lipids coupled to the Tat peptide following various degrees of exposure depending on the spacer length. Only liposomes with an appropriate exposure of the peptide were taken up by cells [37]. Other studies, including one showing in vivo delivery of proteins fused to an 11 amino acid Tat peptide containing the cationic cluster, provided evidence of the importance of the exposure of the Tat peptide to fulfill the cellular delivery of the chimera [44]. A fully unfolded fusion protein, believed to expose the highly hydrophilic Tat sequence to the fluidic environment, was recovered in various tissues, including lungs, heart, spleen, kidneys, and also the brain, after intraperitoneal injection [44]. It was later discussed that the protein had to be unfolded to be efficiently taken up by cells [45,46]. Although the reasons of this requirement are still not fully understood, this work was certainly influential in stimulating the widespread use of the Tat peptide to mediate the cellular delivery of various entities into several cell types, both in vitro and in vivo (for a review, see Ref. [6]).

564

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

In another example, two very different mechanisms of Tat peptide-mediated cell entry (caveolae versus macropinocytosis) were recently proposed following strongly convincing studies [11,24]. The cargoes used in these studies were both proteins: One was made up of the Tat peptide fused to the GST tag protein at the N-terminal end and to the GFP reporter protein at the C-terminal end [24], while the second was composed of the Tat peptide fused to the Cre-recombinase at its C-terminal end [11]. Although the studies were again performed on different cell lines (HeLa and CHO cells for the GST-Tat-GFP and 3T3 cells for the Tat-Cre construct), it is conceivable that the exposure, and thus the accessibility of the peptide, could be different in both constructs, because of differences in their folding, due to the physicochemical properties of the cargo itself. This could lead subsequently to a different ability to follow one or the other cell entry pathway. Along these lines, some toxins have been shown to enter cells through caveolae, whereas other pathogens exclusively used alternative routes without the involvement of identified specific cellular receptors able to trigger one or the other entry route [47]. Biochemical evidence also highlighted that each construct followed its own individual entry pathway. Real-time microscopy experiments were performed to follow the entry route of the GFP-cargo, the kinetics of which implicated the caveolae pathway [24]. In addition, it was assumed that the GFP protein had to be trapped in a neutral environment to maintain its fluorescent property. No reduction of the fluorescence activity was recorded, therefore, confirming indirectly that the GFP-cargo was taken up by cells through the caveolae pathway, which is not acidified during the course of intracellular trafficking [48]. On the other hand, the Tat-Cre biological response was strongly increased upon co-incubation with a Tat peptide fused to the fusogenic sequence derived from the Influenza hemagluttinin protein, known to promote membrane fusion once exposed to an acidic environment [49]. This observation, therefore provided an additional argument that the Tat-Cre protein was taken up by a pathway undergoing acidification [11], while the GST-Tat-GFP construct appeared to be taken up though a mechanism of entry with a stable neutral pH environment [24]. Again, the different cell entry behaviors of these

similar constructs, both are fusion proteins, are likely due to unknown parameters that require further investigation. In addition, the orientation of the coupling of peptide to cargo has been investigated in a number of studies. For instance, morpholino oligonucleotides (PMO) were coupled to the Tat peptide either at their 5V or 3V end [50]. A higher antisense activity was recorded when the Tat peptide was attached to the 5V end. It is believed that the addition of bulky moieties to the 3V end of an oligonucleotide could decrease the expected biological activity, because of probable steric interferences affecting PMO/mRNA binding [50]. Any possible direct effect of this orientation on the uptake itself was, however, not investigated in this study. 4.2. Influence of the hydrophobicity The influence of the structure attached to the Tat peptide should also be further considered. For instance, in early studies of the Tat peptide translocating properties, it was shown that the attachment of a biotin group at the N-terminal end of the longer version of the Tat peptide could lead to a 6-fold increase of the cellular uptake [51]. In this study, the improvement of the cellular delivery of the biotinylated peptide was assessed by the increase of the biological activity mediated by the cargo-bound RNAse [51]. This associated biological effect allowed the avoidance of any risk of artifactual results, such as increased extracellular association or relocalisation upon fixation, as recently described [5]. Therefore, the attachment of such a small hydrophobic group to the Tat peptide could significantly modify the translocating efficiency of the cationic Tat peptide. The improvement of the cellular uptake upon increase of the hydrophobicity has also been indirectly shown after variation of the methylene content of the side chain of h-amino acids of arginine [40]. In this study, although performed on an arginine homopolymer, a stronger cell associated signal was observed by FACS analysis when 4 to 6 methylene groups were present between the a-carbon and the distal guanidinium group. Conversely, a reduction of the side chain length (down to 2 methylene groups) showed a weaker signal compared to the native side chain length containing 3 methylene groups [40]. Addition-

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

ally, the insertion of aminocaproic acid (aca) groups within the peptide backbone showed a stronger cell associated signal than the corresponding heptahomopolymers of arginine [42]. Aca groups allow spacing along the peptidic backbone between the a-carbon, to which the arginine side chain is attached, but also confer a highly hydrophobic character because of their five methylene groups. Although not directly related to the Tat peptide, the improvement of the cell association of a homopolymer of arginine upon attachment of a stearyl moiety has also been documented [52]. Very recently, two phenylalanine amino acids have been inserted at the C-terminal end of arginine homopolymers [50]. These homopolymers were compared directly to the Tat peptide with regard to their expression of luciferase mediated by a positive readout of the antisense action provided by a phosphorothioate oligonucleotide [53]. Despite the absence of a direct comparison of the positive effect of these two phenylalanine residues on the transfection efficiency of either Tat or arginine polymers in this study, it is noteworthy to compare the biological response induced by the native Tat sequence and by the hexapolymer of arginine to which the two phenylalanine were attached. They were found to be very similar [50], whereas without the addition of these two extra phenylalanine residues, Tat was shown to be more efficiently taken up when compared to a simple hexapolymer of arginine [54]. It thus appears that the attachment of hydrophobic residues, or the inclusion of hydrophobic patches in a polycationic cell-penetrating peptide, such as Tat or arginine polymers, could improve their overall uptake. Whether this apparent increase of the measured signal resulted from an effective improvement of the translocating process itself, an increase of the initial cell binding or the use of an additional entry pathway upon peptide modification could not be fully ascertained and, again, could be the subject of further investigations. The hydrophobicity of the peptide does not appear to be the only single characteristic promoting peptide uptake. In a closely related example, the substitution of two tryptophan residues by two phenylalanine residues within the Antennapedia peptide [17] led to the complete loss of translocating properties, although phenylalanine residues show a relatively higher hydrophobicity than tryptophan residues [55].

565

On the other hand, the Tat peptide does not contain any hydrophobic amino acids and is conversely very hydrophilic because of the presence of 8 ionic charges on the side chains and the two N- and C-terminal ionic groups. The translocating process has also been assessed for hydrophilic cargoes. For instance, the covalent coupling of the Tat peptide to a phosphorothioate oligonucleotide with a high number of anionic charges, still resulted in the uptake of the nucleic acid cargo [9]. These results appear quite convincing since they were obtained after trypsin treatment of the cells prior to analysis either by FACS or by fluorescent microscopy, therefore, allowing the removal of the nonspecific membrane-bound peptides as detailed in another section of this chapter [5]. 4.3. Influence of the chemical linkage between Tat and the cargo Surprisingly, the nature of the linkage between the Tat peptide and its cargo has not been deeply investigated. Indeed, this could be highly important if we consider first the requirement of an efficient exposure of the Tat peptide when bound to the cargo to any cell component involved in the translocating process (see above), and, secondly, the intracellular activity of the cargo itself which has to be unaffected by the nature of its chemical coupling to the Tat peptide. For instance, the Tat peptide could impair the biological response by reducing the affinity of the cargo to the targeted material. Along these lines, little in vitro evaluation of the biological activity of the cargo moiety prior to and after Tat attachment has been provided, nor has detection of the subcellular localisation of the cargo following Tat-mediated internalisation been investigated thoroughly, despite a very important number of Tat-mediated deliveries of various cargoes (see Ref. [6] for a review). Most of the studies were devoted to gaining substantial biological activity triggered by the cargo once coupled to the CPP without considering these biochemical features. Moreover, most of these biological responses have been recorded with substantially high doses of extracellular chimera (100 nM or more). Considering that an intracellular concentration of the cargo is expected [56], this could reflect either a very weak activity of the cargo moiety, or an inappropriate location within the cell upon Tat peptide attachment,

566

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

thus scavenging the active drug. Therefore, a labile bond between the Tat carrier and the desired cargo is expected to perform better. The most convenient bond formation for this purpose is that of a disulfide bridge between the CPP and the cargo. Considering the preparation of these chimeras, this strategy first allows the separate synthesis, purification and characterisation of both entities. Secondly, several strategies are available for promoting the oriented formation of the heterodimer entity upon activation of one of the sulfhydryl functions only, such as the incorporation of a thionitropyridine either on the peptides or an oligonucleotide cargo [57–59]. Thirdly, the reduction of the disulfide bridge once the cargo reaches the cytoplasmic compartment is expected to induce its release, therefore, preventing putative negative effects of the cell penetrating peptide. The efficiency of the intracellular reduction of the disulfide bridge between a CPP and its cargo has been demonstrated by using a quenched fluorescent construct whose fluorescence was activated upon cytoplasmic disulfide bond reduction [56]. In some particular examples, however, an increase in the biological response is expected upon attachment of a cationic moiety in general, and particularly upon attachment of the Tat peptide. This is the case for the Tat-mediated delivery of antisense oligonucleotides. The expected activity of an oligonucleotide delivered in this manner results from a complementary binding to its RNA/DNA target sequence. This leads to either steric hindrance of transcription or splicing, or the digestion of mRNA by RNAse H, and, thus, a modulation of the corresponding target protein expression (for a review, see Ref. [60]). The positive effect on oligonucleotide binding to a complementary oligonucleotide sequence upon their coupling to cationic sequences has been widely investigated [61,62]. Because of the highly cationic nature of the Tat peptide, the improvement of binding affinity and kinetics of a Tat-oligonucleotide chimera is also expected and should be considered an important issue for Tat-mediated oligonucleotide delivery. This increase in kinetics has been recently documented for the Tat peptide coupled to an oligonucleotide using the BioCore technology [9]. Therefore, in some cases, a stable link between the Tat peptide and its oligonucleotide cargo might be preferred in the

interest of augmenting the hybridisation of complementary sequences of nucleic acids. The placement of the peptide with respect to the cargo should also be taken into consideration [50]. A comparison of a stable versus a labile bond has been recently provided [50]. Although this study used arginine homopolymers (9 arginine residues) as a cellpenetrating peptide for delivering morpholino oligomers (PMO), similar data could be expected with the Tat peptide considering their very close behavior with regards to inducing cellular uptake of various cargoes. Interestingly, it was shown that when both constructs were labeled with fluorescein, the uptake was more efficient for the stable link construct compared to the labile bond construct, but both gave nearly identical antisense activity in a dose-dependant manner [50]. The following comment was made to explain such differences: A reduction of the disulfide bridge could occur at the membrane level of the cell by gluthatione [63], therefore, reducing the overall pool of internalised PMO. To explain the identical biological effect obtained for both constructs, it was proposed that the stable nature of the maleimide linker could induce the scavenging of the PMO to anionic cellular components rather than the targeted mRNA because of the positive charges of the CPP [50]. These results took into account the risk of a nonspecific binding at the cell surface membrane since a trypsin treatment was performed prior to FACS analysis as previously demonstrated [5]. Therefore, the nature of the chemical bond between a CPP, such as the Tat peptide, and its cargo should be probably more deeply evaluated in each application to optimize the level of the expected biological response, particularly in the case of biologically active oligonucleotides and peptides. One of the most intriguing applications observed in the field of Tat-cargo coupling was the improvement of the cellular delivery of an adenovirus simply upon the mixture of cells with a Tat peptide solution for 30 min prior to exposing the cells to the adenovirus (4 h) [19]. Interactions between Tat (and also the Antennapedia peptide) with cellular coat proteins or lipids of the cell membrane were proposed to improve the effective surface concentration of the adenovirus. It is noteworthy that high concentrations (above 100 AM) of either Tat or Ant were required to show an improvement of the Adenovirus transfection effi-

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

ciency [19]. Conversely, a simple mixture of the Tat peptide either with a peptide [39] or an oligonucleotide [64] do not lead to cargo internalisation. A simple mixture with plasmid DNA was, however, sufficient to increase the DNA transfection rate [65]. 4.4. Influence of the Tat peptide density The Tat peptide density also appears to be an important issue in explaining the cellular delivery of very large structures, such as particles, liposomes or phages [21,28,36,66]. The requirement of 4 to 5 Tat molecules to promote the uptake of native hgalactosidase [2] has been already mentioned (see above). Although the cellular uptake of chimeras with lower loading degrees was not investigated in this latter study, it was shown in another report that one single Tat peptide was sufficient to allow the cellular delivery of an unfolded fusion construct of the same protein [44]. The possible differences in the exposure of the Tat peptide within both constructs have been already discussed. Other studies, however, showed that several Tat peptides (in some cases up to several hundreds) attached at the surface of large particles were required to promote efficient cellular delivery [21,28,36,66]. Along these lines, another study was carried out, in which six to seven Tat peptides were coupled to large (45 nm diameter) ferromagnetic particles, thus promoting their efficient delivery. Again, a direct evaluation of the translocating property in accordance with the number of attached Tat peptides was not considered in this work [66] and has not been further investigated. Considering the size of such a structure, it is difficult to believe that a direct passage through the plasma membrane was possible when mediated only by six to seven Tat peptides exposed at the particle surface. They do, however, seem sufficient to induce an efficient cellular delivery, since it was possible to recover cells through magnetic collection [66]. Since no stringent washes were performed prior to cell recovery, it cannot be excluded that the ferromagnetic particles were simply strongly bound to the cell surface. Confocal microscopy pictures provided in this study could not discriminate the cellular compartimentation of the particle since it was performed after cell fixation and permeabilization prior to observation. Whether the fixation step could induce the cellular translocation of such a massive

567

structure as demonstrated for fusion proteins [67,68] remains unknown. Linear repeats from one to four Tat sequences have also been used to increase the PEI-mediated transfection of plasmids [25]. Important differences in the transfection rate between the different complexes indicated that the total content of guanidinium groups per complex appeared to be important, since dimeric or tetrameric repeats of linear Tat peptides complexed to PEI improved the overall transfection efficiency [25]. Conversely, the use of a much longer homopolymer of arginine (with a molecular weight ranging from 5000 to 15,000 Da, corresponding to a linear length of 50 to 150 residues) led to a minor increase in the transfection efficiency of the PEI-plasmid [25]. Along these lines, homopolymers containing 16 arginines were also shown to be poorly taken up by cells when compared to shorter homopolymer sequences [4]. On the other hand, cellular plasmid delivery, after complexation to branched-Tat peptide constructs, was shown to be much more effective with larger peptide repeats than the corresponding plasmid complexed with lower branched-Tat structures [65]. Basically, branched structure containing 8 Tat peptides showed a better transfection efficacy than those containing 4 Tat peptides, and even better results than those containing 2 or 1 Tat peptide [65]. Therefore, these data indicate that the Tat peptide sequence requires an appropriate number of arginines to efficiently translocate into cells and that a given cargo requires an optimal number of Tat peptides to be efficiently taken up by cells. Whether these results could be influenced by differences in the experimental protocols, the cell type used or the physicochemical properties of these cargo molecules still remains to be tested. The investigation of the influence of the Tat peptide density around these massive structures will likely highlight important features about the mechanism of their translocation. One work, which, in parallel experiments, investigated the influence of the number of attached Tat peptides to the cargo, by derivatizing Fab fragments with either 1.1 or 1.6 Tat peptide molecules [69]. The Tat peptide used in this study corresponded to a slightly different version of the Tat peptide (Tat 37–62 encompassing also the basic region), but appears relevant enough to be discussed in this chapter. The cargo substituted with more peptide was shown to be more efficiently taken

568

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

up by cells. However, when evaluated in vivo, this chimera showed a weaker cell selectivity of the Fab fragment per se. This result very likely reflects a strong nonspecific cell association of the higher loaded chimera directed by the highly cationic Tat peptide, whereas the Tat peptide loading balance for the weaker derivatized chimera still allowed the Fab fragment to preferentially reach its target cells [69]. Along these lines, tumor growth inhibition induced by Tat-Liposomes loaded with doxorubicin was also evaluated in vivo on BALB/c mice [22]. About 50% reduction of the tumor size was observed compared to the liposome control . The benefit of the Tat peptide was, however, not that important since bnudeQ doxorubicin-loaded liposomes (without Tat peptide) showed identical, or better, tumor reduction [22]. This data also indicates that the Tat peptide interferes with the delivery of the liposome, probably by adhering locally to other cells prior to reaching the targeted cells. 4.5. Influence of the serum In contrast to the cationic lipid-mediated transfection of oligonucleotides, serum has been shown to unexpectedly augment the biological response when oligonucleotides were delivered by Tat or Antennapedia CPPs [70]. However, in later data using the Kole system, where a delivered oligonucleotide corrects an aberrant splice site resulting in expression of a reporter gene [53], no serum effect was observed [9]. In a recent study, serum was even shown to decrease, but not to abolish, the biological effect of a Tat-Cre fusion construct [11]. Unpublished data from our group did not reveal a noticeable effect on the cell-associated fluorescence when Tat peptide was incubated with up to 50% serum. Although no trypsin treatment was performed at that time prior FACS analysis, a decreased signal should have been observed if serum competes with the Tat peptide for cell binding. To date, the observations of Tat peptide uptake in the presence of serum remain promising for future in vivo application.

5. Tat and cell surface interactions Because of the highly cationic nature of the Tat peptide, several anionic cellular candidates are avail-

able 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 derivatives [74] dextran sulfate [75] and CS/DS chondroitin/dermatan sulfates [29]. Many initial studies of internalisation of the Tat peptide, although now known to be false or compromised in terms of internalisation, can reveal interesting aspects of binding. Where no biological activity is used as a control for effective entry and delivery, externally bound peptide in many cases has been confused with effective delivery. FACS analysis is particularly susceptible to giving artificially high fluorescent values. Given that standard wash techniques in an isotonic buffer, such as the often used PBS, leave residually bound peptide [2,5], a more stringent treatment is required before analysis can be performed. The initial association of the Tat peptide with the cell surface membrane occurs independently of temperature, is resistant to mechanical washing with isotonic buffers, such as PBS/EDTA [2], but is sensitive to treatment with proteases, such as trypsin [5]. Trypsinisation of externally bound peptide is, therefore, an often preferred alternative to mechanical washing. Yet again, to what extent this treatment or washes with acidic buffers, high salt solutions or competing substances, such as heparin, are effective in disrupting ionic interactions has not been quantitatively and comparatively studied for different cell lines. Both the full length GST-Tat protein and fusion proteins containing the basic domain only require a high ionic strength (1.6–1.3 M NaCl) to elute them from bound heparin [74]. In contrast, the substitution of six arginine residues within the basic domain using alanine residues reduced the required ionic strength to only 0.3 M NaCl, once again highlighting the importance of these basic residues in the ionic binding profile of the Tat peptide. With respect to the use of high salt washes to eliminate excess peptide bound to the surface of cells, a 2-M wash, as used in some protocols [29], would be thought sufficient. Suzuki and co-workers, however, reported that this was not

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

the case in their study [20]. Washing the cells with a high salt buffer [20 mM HEPES containing 2 M NaCl (pH 7.4)] produced little difference in the amount of peptides bound to the cell surface compared with PBS alone [20]. In cases where the internalisation of the peptide has not been distinguished from strong extracellular attachment, the data might still be useful with respect to the analysis of binding. In one example, the uptake of Tat peptide was examined in a range of cells [31] using FACS analysis after washing the cells simply with EDTA, thereby looking at EDTA resistant bound peptide (along with any internalised). Competing anionic compounds, such as heparin and dextran sulfate, caused a significant decrease (60–70%) in cell-surface association of the peptide, whereas other glycosaminoglycans, such as chondroitin sulfate (CS) A, B, and C and hyaluronic acid, had no effect. If anything, it was noted that chondroitin sulfate A increased Tat-cell association. A similar study looking at GST-Tat-GFP in CHO cells examined only the trypsin resistant fraction of whole protein-GFP and yet came to similar conclusions with regard to the type of glycosaminoglycans (GAG) interacting with Tat. Internalisation of the Tat protein was inhibited by HS, but not by the chondroitin sulfates [76]. Suzuki et al., however, using HeLa cells washed only with PBS and then fixed with acetone:methanol, found that the basic peptide (Tat48–60) was inhibited in its binding to HeLa cells by all GAGs [20]. The uptake was significantly reduced in the presence of heparin sulfate or chondroitin sulfates A, B, and C, as well as by pretreatment of the cells with the anti-heparin sulfate antibody or heparinase III [20]. Looking at the biological activity of an effectively delivered Cre recombinase, it was shown that heparin was able to confer a total inhibition at low concentrations (2.5 Ag/ ml), followed by CS-B at only slightly higher amounts [11]. CS-C, however, was only able to inhibit recombination by 80% at 20-fold higher concentrations of the inhibitor and CS-A showed only 40% inhibition at all concentrations tested. This study effectively showed the competition for cell surface binding of the Tat-Cre construct with the free GAG and the specificity of this cell surface interaction. It was observed very early on for the full-length Tat protein that uptake and Tat-promoted transactivation of HIV-1 gene expression could be blocked by soluble

569

polyanions (heparin and dextran sulfate) [75]. Surprisingly, the same study could not show an effect for trypsinisation or heparinase treatment. The Tat protein binds to cell surface heparan sulfate (HS) and heparin [77], and consistent with its heparin-binding properties, Tat can be purified to homogeneity by heparin-affinity chromatography [78]. The group of Presta and co-workers first examined the role of the basic domain in the binding of Tat to heparin. They found that neutralization of the positive charges in the basic domain of Tat significantly reduces its interaction with the GAG. The dissociation constant of heparin to immobilised GSTTat was observed to be around 0.3 AM [74]. Work in CHO cells demonstrated that cell uptake and association of Tat constructs containing the basic peptide were effectively blocked by heparin [24,76], pre-treatment of HeLa cells with heparinase III [20] or pre-treatment of CHOs with glycosaminoglycan lyases that specifically degrade HS chains ([76] and Melikov et al., submitted for publication). Given the homology of heparin to the surface sulfated glycosaminoglycans, the observed Tat–heparin interaction could reflect the initial cell surface interactions of Tat with exposed surface HS proteoglycans, perhaps serving as the initial point of contact or even a route of entry for Tat, as observed for some other heparin-binding proteins. Sulfated glycosaminoglycans (GAGs), such as HS, increasingly implicated in cell adhesion, are distributed ubiquitously on cell surfaces as the carbohydrate component of proteoglycans [79]. Many microbial and viral particles enter cells using HS receptors via a two-step process, adhering to the cell surface by binding initially to GAGs followed by internalisation. The foot-and-mouth disease virus (FMDV) infects cells in such a way, its primary contact being with a low-affinity HS proteoglycan receptor, followed by transfer to the high-affinity integrin receptor for endocytosis [80]. HS facilitates entry of the FMDV and it was found that alteration of the HS affinity had profound consequences for the infectivity of the virus [81]. HSPG serve as cell surface receptors for a number of natural ligands, some of which include matrix proteins, such as laminin, cell adhesion molecules (N-CAM) and growth factors, such as fibroblast growth factor (FGF) [82], insulin-like growth factor-binding pro-

570

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

tein-2 (IGFBP-2) [83] and vascular endothelial cell growth factor (VEGF) [84]. The basic domain of Tat shown to be responsible for the Tat–heparin interaction [74] has homology with heparin-binding growth factors [85]. Structurally, there appears to be no conserved conformation for this domain, clusters of basic residues and a heparinbinding capacity using heparin-sepharose chromatography serve as common criteria when labelling a peptide domain as heparin binding [86]. Studies are yet to be done demonstrating direct competition of Tat with natural ligands, such as the heparin-binding proteins growth factors mentioned above, for the binding sites of these cell surface receptors. This would potentially provide useful information about the initial binding and entry of the Tat peptide. Binding to HSPGs is often followed by rapid internalisation via endocytosis. There are multiple proposals for the mechanism of internalisation via such proteoglycans, ranging from simple endocytosis via classical clathrin pathways [87] to alternative routes, such as those mediated by the syndecan HSPGs, those independent of coated pits or those utilizing a much slower pathway of internalisation, as was recently described for the perlecans [88]. In the case of syndecan HSPGs, efficient internalisation is triggered by a clustering of transmembrane and cytoplasmic domains and then proceeds via a noncoated pit pathway, possibly caveolae [88,89]. When CHO cells were pre-treated with chondroitin ABC lyase to eliminate CS/DSPGs or heparitinase/ heparinase to cleave HS chains, all proteolytic treatment resulted in a significant reduction in the uptake of Tat peptide [29]. Likewise, treatment of CHO cells with chlorate (which inhibits GAG sulfation) had a similar inhibitory effect [29]. Mutant cells defective for GAG synthesis show dramatically reduced TATmediated transmembrane transport [24,31,76]. The cell line CHO pgs D-677, which does not produce HSPGs (due to a 10-fold reduction in N-acetylglucosaminyl-transferase and glucuronosyltransferase), produces chondroitin sulfates in excess of about a 3-fold [90]. According to the work of Tyagi et al., these cells show a 50% reduction in transactivation by recombinant GST-Tat [76], equal to that observed in E-606 cells, which produce HSPGs that are undersulfated, thereby indicating the importance of the sulfation step. By contrast, the complete proteoglycan null mutant

pgs A-745 (deficient in xylosyl transferase, which catalyses the first step in PG assembly/formation [91]) lacks all surface PGs and show a much severer reduction in Tat uptake of around 80% [76]. CHO 745 cells also show no cell membrane adhesion of the basic peptide or vesicle inclusion using confocal microscopy [24], and show a reduced uptake of Tat peptide/HS complexes [29], except where the ratios of peptide to anion are particularly high (i.e., high excess of peptide). Thus, the internalisation was if anything more important in the HSPG-deficient cells. In a study examining only EDTA washed cells, the milder D-677 mutant was not observed to have any difference in its binding of Tat when compared to wild type [31]. The severe A-745 mutant, however, showed a reduction of 80–90%. Identical work in our laboratory comparing GST-Tat-GFP and fluoresceinlabelled Tat peptide in only PBS-washed cells showed that binding of the fusion protein was completely inhibited in mutants compared to wild type, whereas the peptide adhered to all three cell lines regardless of their GAG expression [92]. The initial attachment of Tat peptide to GAGs or any other molecule at the cell surface would likely be influenced by an attached or previously bound cargo (see above). The size of the cargo, the overall charge involved, the way in which it was coupled (N- versus C-terminal binding, covalent binding, fusion protein or chemical coupling as well as pure electrostatic interactions), and the degree of exposure of the basic residues would all play a role in influencing these initial cell-surface associations. Aside from the known affinity for HSPGs, other cell surface receptors have also been implicated in Tat binding. Yeast 2-hybrid screens controlled with subsequent GST pull down assays confirmed the binding of the full-length Tat protein to both HSPGs and LRP (low-density lipoprotein receptor family) [93]. They also confirmed that the domain responsible (amino acids 34–48) was just before the cluster of basic residues (49–57), meaning that any elongation of this minimal sequence to include the a-helical-like structure located just prior to the translocating domain might result in differences in affinity and the pathway of internalisation. Tat and its basic domain have been proposed to bind many cell surface receptors. One study in particular showed the Tat peptide as causing the release of acetylcholine from human and rat chol-

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

inergic terminals [94]. The release was dependent on calcium, effected through voltage sensitive calcium channels, and inhibited strongly by cadmium, as well as the mGluR and IP3R antagonists (heparin and xestosponginC). Further studies showed immunoprecipitation of the Tat peptide with various antiintegrin antibodies suggesting that the vitronectinbinding integrin (alphaVbeta5) is the cell surface protein responsible for binding to the basic domain of Tat [95]. A natural ligand of this receptor, vitronectin, also contains a related basic peptide sequence (KKQRFRHRNRKG) in its heparin-binding domain, which served to competitively inhibit binding. Another group showed in the same year that antibodies to the h-4 integrin subunit were able to inhibit cell attachment to Tat specifically [96], yet were unable to demonstrated co-precipitation. They found instead a strong relation to a 90-kDa surface membrane protein in both Molt3 and PC12cells. Although HSPGs can resolve at around this size, they vary considerably depending on the saccharide chain length from 12 kDa for the HS chain, to 61 kDa for the core protein, and 90–190 kDa for the intact PG [97]. Rusnati et al. found that a positive correlation existed between the size of heparin oligosaccharides and their capacity to inhibit the internalisation of Tat [98]. Given the size heterogeneity of HSPGs, it is most likely that the observed 90-kDa protein is an additional separate factor in Tat cell membrane adhesion. Taken together, studies on the binding of Tat would implicate more than one component involved in initial cell membrane attachment. A strong argument for the role of GAGs, in particular HS, has been assembled from the data of many independent studies; however, the lack of complete inhibition by mutant, enzymatic digestion or competition studies would tend to preclude their exclusivity.

6. Possible mechanisms of internalisation Membrane association or binding occurs at any temperature, including the metabolically inhibiting 48C. In traversing the extracellular membrane, however, the Tat peptide behaves in an energy-dependent manner requiring temperatures above 4 8C and ATP.

571

Only one group [18] continues to observe uptake at 4 8C 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 least are acidified according to colocalisation studies with pH markers [29] or inhibition of vesicle acidification [Melikov et al., submitted for publication]. The fluorescence of labelled Tat peptide when observed in live cells is often described as being punctate or vesicular [24,29,75] and more rarely as diffuse cytosolic [18,34]. It has been observed to be close to the membrane at early time points, progressing to larger aggregations with a more perinuclear type pattern at longer time points and can be observed to continue on into the nucleus [2,29,99]. Various drugs have been shown to affect the entry or distribution of the Tat peptide. For instance, Golgi destabilisation of HeLa cells (brefeldinA) converts the punctate vesicular staining to a more cytoplasmic, even distribution, while having no effect on control dextran [100]. Ammonium chloride halted the staining of the nucleus, but appeared to have no effect on the vesicular pattern, leading the authors to conclude that the basic peptide is normally released from vesicles after endosomal uptake by means of a mechanism requiring endosome acidification [99]. Chloroquine, another inhibitor of endosomal acidification appeared likewise to inhibit the release of Tat from vesicles [100], enhancing the vesicular staining for both Arg9 and Tat peptides and reducing/ eliminating any diffuse cytoplasmic staining. No effect on Tat was seen with a similar drug, bafilomycinA, nor of wortmannin, a potent PI-kinase inhibitor. In our hands, the drug monensin, which causes deacidification of cytoplasmic compartments, resulted in an increase in the fluorescent signal of FITC-labelled Tat, indicating that the internalised label had been sequestered in acidic compartments, which masked the level of fluorescence [Melikov et al., submitted for publication]. In a nice experiment using the similar Arg9 peptide, the authors co-cultured different cells and then showed that distribution of the peptide was different according to cell type (vesicular and cytosolic in MC57 cells, while only vesicular in HeLa cells) [100]. CytochalasinD is known to depolymerise the actin cytoskeleton causing clustering of caveolae at the cell

572

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

surface. A construct of Tat, namely, GST-Tat-EGFP, was found to be restricted to the plasma membrane area following exposure to cytochalasinD, whereas nocodazole treatment, which preferentially disrupts the actin microtubules, resulted in perinuclear fluorescence similar to untreated controls [24]. Transferrin, often used as a marker for early endosomes following the clathrin-coated vesicular pathway, has been observed to partially colocalise with the Tat peptide [5,31,99]. The same has been observed for the non-clathrin markers, such as cholera toxin [24,71] or the SV40 virus [21], which are internalised via caveolin-cholesterol rich domains. Inhibition of the caveolin pathway by the drug nystatin reduced the Tat peptide reporter h-gal activity by 50% in CHO and HepG2 (a rather surprising finding when it has been reported that HepG2 cells lack Cav-1 [101]), but showed some cell specificity, having no effect on buffalo green monkey (BGM) cells [34]. Nystatin also inhibited Tat-phagemediated gene transfer up to 50% of control values, whereas DEAE-dextran-mediated gene transfer remained unaffected [21]. The kinetics of internalisation of a GST-Tat-GFP conjugate and the cholera toxin were reported to be far slower than the comparatively rapid internalisation of transferrin [71]. The peptide conjugate showed no evidence of co-localisation with either transferrin, the marker EE1 (early endosome antigen-1) or lysotracker dye. The authors concluded that for their conjugate at least, Tat was internalised via a non-clathrin-dependent route, possibly caveolae. A large number of, but not all, vesicles containing Tat were positive for Caveolin-1. Tat uptake was also shown to be inhibited by the sequestration of cholesterol by methyl-hcyclodextrin [71]. It should be noted, however, that uptake of the GST-Tat-GFP conjugates were studied in the presence of 100 AM chloroquine (Tat protein (1 Ag/ml)) [76,77], which, although used as a lysosomal trophic agent to reduce degradation of the parent Tat protein [102], is nonetheless going to interfere with vesicular recycling and affect the subcellular localisation of both control markers and the Tat peptide. The pH neutral environment of caveolae would, in any case, conflict with data obtained regarding the acidic nature of at least some of the Tat-containing vesicles and the partial colocalisation observed with transferrin for non-conjugated peptide.

The data on Tat and Tat-cargoes would tend to preclude the dominance of one exclusive pathway of entry into the cell. The lack of complete inhibition by selective drugs or complete colocalisation with known markers strongly suggests a multiplicity of entry pathways for this sticky basic peptide. Aside from clathrin and caveolae, other mechanisms of crossing the plasma membrane include the non-clathrin/noncaveolin-type pathway(s) (lipid rafts or microdomains, e.g., IL-2 receptor), macropinocytosis (platelet derived growth factor), potocytosis (folate receptor) and phagocytosis (specialised cells only). Perhaps we must also entertain the idea that Tat is simply an opportunistic peptide, adhering strongly to the cell surface on the basis of its charge to any negative offerings, such as lipids or proteins, and then being internalised through natural cell membrane recycling on regions or microdomains, presumably captured by any type of endocytic vesicle. Cell plasma membrane turnover continues constitutively at an estimated rate of ~2%/min [103], in other estimates as fast as 5%/min [104], meaning 100% of the cells surface is internalised nonspecifically in less than an hour, notwithstanding the faster receptor mediated or stimulated routes of uptake. To this end, most drugs inhibiting cell membrane recycling will show an effect on Tat uptake, yet not prevent it entirely as long as other possible routes exist. Competitors for cell surface binding, however, would presumably be more effective at reducing the level of peptide internalised as is seen for heparin and the similar glycosaminoglycans.

7. 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 to the presence of fixation artifacts, even in mild conditions, affecting molecules containing a strong cationic cluster of

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

amino acids, such as the Tat peptide. Nowadays, it seems that most of the studies are performed following uptake on live (unfixed) cells. As discussed in this review, comparisons between the different reports relative to the transducing properties of the Tat peptide are very often difficult, because of the very wide diversity of the Tat sequence used as a cell-penetrating peptide, although it seems very likely that the Tat peptide corresponding to its basic domain showed the best ubiquitous positive effect. More importantly, the very large differences within the cargoes, in terms of size (from some hundreds of daltons to massive particles up to 200 nm of diameter), in terms of composition and, therefore, of physicochemical properties, make it very difficult to extract clear information allowing the definition of a universal mechanism of entry. Moreover, some reports revealed that additional parameters involving the cargo could be very important. These include the type of linkage between the peptide and the cargo, its length, the orientation of the peptide relative to the cargo, the quantity and peptide exposure at the overall surface of the cargo. In addition, most of the experiments aimed at defining the mechanism of entry of this Tat-derived cell-penetrating peptide have been performed with a large heterogeneity of cell types. Altogether, we counted about 50 different cell types used for studying the Tat delivery process, from primary cells to established cell lines, derived from various species or tissues, and studied either in vitro or in vivo. The experiments were often performed under variable conditions in terms of kinetics (from minutes to hours), concentration of the chimera (from nanomolar to hundreds of micromolars) and protocols applied to estimate the uptake efficiency or the subcellular localisation of the chimeras (from biological activity to fluorescence detection). When drugs were used to block different entry pathways proposed as candidates for Tat-mediated cellular entry of cargo, an absolute blockade of the entry or the full biological inhibition has not been always fully achieved, excluding the possibility that a discrete entry process could also be involved. Moreover, the detection of such a phenomenon is always limited in each application by detection limits of the tools available.

573

This Tat peptide (and probably some of the arginine polymers shown to be closely related to Tat) was found to induce the cellular uptake of very different molecules. It appears however, that this process will suffer from a lack of cellular specificity since it seems that it is effective in a very large number of different cell types. Anionic structures at the cell surface are probably nonspecific agents interacting with the Tat peptide to increase the local concentration of the Tat-bound cargo at the cell surface before allowing its cellular entry through general endocytosis pathways. Conditions inducing the entry through any of the possible routes (caveolae, clathrin-dependant endocytosis, macropinocytosis, fluid-phase endocytosis. . .) are far from being fully understood and will certainly require a complete study with regard to the possible influences of all the various parameters discussed here. References [1] 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. [2] 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. [3] 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. [4] 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. [5] J.P. Richard, K. Melikov, E. Vives, C. Ramos, B. Verbeure, M.J. Gait, L.V. Chernomordik, B. Lebleu, Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake, J. Biol. Chem. 278 (2003) 585 – 590. [6] M.A. Lindsay, Peptide-mediated cell delivery: application in protein target validation, Curr. Opin. Pharmacol. 2 (2002) 587 – 594. [7] M.J. Gait, Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues, Cell. Mol. Life Sci. 60 (2003) 844 – 853. [8] M. Zhao, R. Weissleder, Intracellular cargo delivery using tat peptide and derivatives, Med. Res. Rev. 24 (2004) 1 – 12. [9] A. Astriab-Fisher, D. Sergueev, M. Fisher, B.R. Shaw, R.L. Juliano, Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on

574

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577 cellular uptake, binding to target sequences, and biologic actions, Pharm. Res. 19 (2002) 744 – 754. C. Bonny, A. Oberson, S. Negri, C. Sauser, D.F. Schorderet, Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death, Diabetes 50 (2001) 77 – 82. J.S. Wadia, R.V. Stan, S.F. Dowdy, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004) 310 – 315. S. Futaki, I. Nakase, T. Suzuki, Z. Youjun, Y. Sugiura, Translocation of branched-chain arginine peptides through cell membranes: flexibility in the spatial disposition of positive charges in membrane-permeable peptides, Biochemistry 41 (2002) 7925 – 7930. E.P. Loret, E. Vives, P.S. Ho, H. Rochat, J. Van Rietschoten, W.C. Johnson Jr., Activating region of HIV-1 Tat protein: vacuum UV circular dichroism and energy minimization, Biochemistry 30 (1991) 6013 – 6023. D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 269 (1994) 10444 – 10450. S.D. Kramer, H. Wunderli-Allenspach, No entry for TAT(44– 57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides, Biochim. Biophys. Acta 1609 (2003) 161 – 169. J.M. Sabatier, E. Vives, K. Mabrouk, A. Benjouad, H. Rochat, A. Duval, B. Hue, E. Bahraoui, Evidence for neurotoxic activity of tat from human immunodeficiency virus type 1, J. Virol. 65 (1991) 961 – 967. D. Derossi, S. Calvet, A. Trembleau, A. Brunissen, G. Chassaing, A. Prochiantz, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent, J. Biol. Chem. 271 (1996) 18188 – 18193. P.E. Thoren, D. Persson, P. Isakson, M. Goksor, A. Onfelt, B. Norden, Uptake of analogs of penetratin, Tat(48–60) and oligoarginine in live cells, Biochem. Biophys. Res. Commun. 307 (2003) 100 – 107. J.P. Gratton, J. Yu, J.W. Griffith, R.W. Babbitt, R.S. Scotland, R. Hickey, F.J. Giordano, W.C. Sessa, Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo, Nat. Med. 9 (2003) 357 – 363. T. Suzuki, S. Futaki, M. Niwa, S. Tanaka, K. Ueda, Y. Sugiura, Possible existence of common internalization mechanisms among arginine-rich peptides, J. Biol. Chem. 277 (2002) 2437 – 2443. A. Eguchi, T. Akuta, H. Okuyama, T. Senda, H. Yokoi, H. Inokuchi, S. Fujita, T. Hayakawa, K. Takeda, M. Hasegawa, M. Nakanishi, Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells, J. Biol. Chem. 276 (2001) 26204 – 26210. Y.L. Tseng, J.J. Liu, R.L. Hong, Translocation of liposomes into cancer cells by cell-penetrating peptides penetratin and tat: a kinetic and efficacy study, Mol. Pharmacol. 62 (2002) 864 – 872.

[23] U. Koppelhus, S.K. Awasthi, V. Zachar, H.U. Holst, P. Ebbesen, P.E. Nielsen, Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates, Antisense Nucleic Acid Drug Dev. 12 (2002) 51 – 63. [24] A. Ferrari, V. Pellegrini, C. Arcangeli, A. Fittipaldi, M. Giacca, F. Beltram, Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time, Molec. Ther. 8 (2003) 284 – 294. [25] C. Rudolph, C. Plank, J. Lausier, U. Schillinger, R.H. Muller, J. Rosenecker, Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells, J. Biol. Chem. 8 (2003) 8. [26] J.C. Mai, H. Shen, S.C. Watkins, T. Cheng, P.D. Robbins, Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate, J. Biol. Chem. 277 (2002) 30208 – 30218. [27] P.O. Falnes, J. Wesche, S. Olsnes, Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment, Biochemistry 40 (2001) 4349 – 4358. [28] A. Nori, K.D. Jensen, M. Tijerina, P. Kopeckova, J. Kopecek, Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells, Bioconjug. Chem. 14 (2003) 44 – 50. [29] S. Sandgren, F. Cheng, M. Belting, Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans, J. Biol. Chem. 277 (2002) 38877 – 38883. [30] R. Bhorade, R. Weissleder, T. Nakakoshi, A. Moore, C.H. Tung, Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide, Bioconjug. Chem. 11 (2000) 301 – 305. [31] S. Console, C. Marty, C. Garcia-Echeverria, R. Schwendener, K. Ballmer-Hofer, Antennapedia and HIV transactivator of transcription (TAT) bprotein transduction domainsQ promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans, J. Biol. Chem. 278 (2003) 35109 – 35114. [32] E.L. Snyder, B.R. Meade, S.F. Dowdy, Anti-cancer protein transduction strategies: reconstitution of p27 tumor suppressor function, J. Control. Release 91 (2003) 45 – 51. [33] V.P. Torchilin, T.S. Levchenko, TAT-Liposomes: a novel intracellular drug carrier, Curr. Protein Pept. Sci. 4 (2003) 133 – 140. [34] I.A. Ignatovich, E.B. Dizhe, A.V. Pavlotskaya, B.N. Akifiev, S.V. Burov, S.V. Orlov, A.P. Perevozchikov, Complexes of plasmid DNA with basic domain 47–57 of the HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways, J. Biol. Chem. 278 (2003) 42625 – 42636. [35] S. Violini, V. Sharma, J.L. Prior, M. Dyszlewski, D. PiwnicaWorms, Evidence for a plasma membrane-mediated permeability barrier to Tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate, Biochemistry 41 (2002) 12652 – 12661.

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577 [36] V.P. Torchilin, T.S. Levchenko, R. Rammohan, N. Volodina, B. Papahadjopoulos-Sternberg, G.G. D’Souza, Cell transfection in vitro and in vivo with nontoxic TAT peptideliposome-DNA complexes, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 1972 – 1977. [37] V.P. Torchilin, R. Rammohan, V. Weissig, T.S. Levchenko, TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8786 – 8791. [38] P.E. Thoren, D. Persson, M. Karlsson, B. Norden, The antennapedia peptide penetratin translocates across lipid bilayers—the first direct observation, FEBS Lett. 482 (2000) 265 – 268. [39] E. Vives, C. Granier, P. Prevot, B. Lebleu, Structure activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein, Lett. Pept. Sci. 4 (1997) 429 – 436. [40] P.A. Wender, D.J. Mitchell, K. Pattabiraman, E.T. Pelkey, L. Steinman, J.B. Rothbard, The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13003 – 13008. [41] D.J. Mitchell, D.T. Kim, L. Steinman, C.G. Fathman, J.B. Rothbard, Polyarginine enters cells more efficiently than other polycationic homopolymers, J. Pept. Res. 56 (2000) 318 – 325. [42] L.R. Wright, J.B. Rothbard, P.A. Wender, Guanidinium rich peptide transporters and drug delivery, Curr. Protein Pept. Sci. 4 (2003) 105 – 124. [43] O. Zelphati, F.J. Szoka, Mechanism of oligonucleotide release from cationic liposomes, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 11493 – 11498. [44] S.R. Schwarze, A. Ho, A. Vocero-Akbani, S.F. Dowdy, In vivo protein transduction: delivery of a biologically active protein into the mouse, Science 285 (1999) 1569 – 1572. [45] S.R. Schwarze, S.F. Dowdy, In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA, Trends Pharmacol. Sci. 21 (2000) 45 – 48. [46] J.S. Wadia, S.F. Dowdy, Protein transduction technology, Curr. Opin. Biotechnol. 13 (2002) 52 – 56. [47] R. Montesano, J. Roth, A. Robert, L. Orci, Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins, Nature 296 (1982) 651 – 653. [48] L. Pelkmans, J. Kartenbeck, A. Helenius, Caveolar endocytosis of simian virus 40 reveals a new two-step vesiculartransport pathway to the ER, Nat. Cell Biol. 3 (2001) 473 – 483. [49] J. Alblas, L. Ulfman, P. Hordijk, L. Koenderman, Activation of Rhoa and ROCK are essential for detachment of migrating leukocytes, Mol. Biol. Cell 12 (2001) 2137 – 2145. [50] H.M. Moulton, M.H. Nelson, S.A. Hatlevig, M.T. Reddy, P.L. Iversen, Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides, Bioconjug. Chem. 15 (2004) 290 – 299.

575

[51] L.L. Chen, A.D. Frankel, J.L. Harder, S. Fawell, J. Barsoum, B. Pepinsky, Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin, Anal. Biochem. 227 (1995) 168 – 175. [52] S. Futaki, W. Ohashi, T. Suzuki, M. Niwa, S. Tanaka, K. Ueda, H. Harashima, Y. Sugiura, Stearylated arginine-rich peptides: a new class of transfection systems, Bioconjug. Chem. 12 (2001) 1005 – 1011. [53] P. Sazani, S.H. Kang, M.A. Maier, C. Wei, J. Dillman, J. Summerton, M. Manoharan, R. Kole, Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs, Nucleic Acids Res. 29 (2001) 3965 – 3974. [54] S. Futaki, Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms, Int. J. Pharm. 245 (2002) 1 – 7. [55] J. Kyte, R.F. Doolittle, A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157 (1982) 105 – 132. [56] M. Hallbrink, A. Floren, A. Elmquist, M. Pooga, T. Bartfai, U. Langel, Cargo delivery kinetics of cell-penetrating peptides, Biochim. Biophys. Acta 1515 (2001) 101 – 109. [57] F. Albericio, D. Andreu, E. Giralt, C. Navalpotro, E. Pedroso, B. Ponsati, M. Ruiz-Gayo, Use of the Npys thiol protection in solid phase peptide synthesis. Application to direct peptide–protein conjugation through cysteine residues, Int. J. Pept. Protein Res. 34 (1989) 124 – 128. [58] M.S. Bernatowicz, R. Matsueda, G.R. Matsueda, Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation, Int. J. Pept. Protein Res. 28 (1986) 107 – 112. [59] E. Vives, B. Lebleu, Selective coupling of a highly basic peptide to an oligonucleotide, Tetrahedron Lett. 38 (1997) 1183 – 1186. [60] B. Lebleu, I. Robbins, L. Bastide, E. Vives, J.E. Gee, Pharmacokinetics of oligonucleotides in cell culture, Ciba Found. Symp. 209 (1997) 47 – 54. discussion 54-49. [61] T. Zhu, Z. Wei, C.H. Tung, W.A. Dickerhof, K.J. Breslauer, D.E. Georgopoulos, M.J. Leibowitz, S. Stein, Oligonucleotide-poly-l-ornithine conjugates: binding to complementary DNA and RNA, Antisense Res. Dev. 3 (1993) 265 – 275. [62] Z. Wei, C.H. Tung, T. Zhu, W.A. Dickerhof, K.J. Breslauer, D.E. Georgopoulos, M.J. Leibowitz, S. Stein, Hybridization properties of oligodeoxynucleotide pairs bridged by polyarginine peptides, Nucleic Acids Res. 24 (1996) 655 – 661. [63] E.P. Feener, W.C. Shen, H.J. Ryser, Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes, J. Biol. Chem. 265 (1990) 18780 – 18785. [64] H.M. Moulton, J.D. Moulton, Peptide-assisted delivery of steric-blocking antisense oligomers, Curr. Opin. Mol. Ther. 5 (2003) 123 – 132. [65] C. Tung, S. Mueller, R. Weissleder, Novel branching membrane translocational peptide as gene delivery vector, Bioorg. Med. Chem. 10 (2002) 3609. [66] M. Lewin, N. Carlesso, C.H. Tung, X.W. Tang, D. Cory, D.T. Scadden, R. Weissleder, Tat peptide-derivatized magnetic

576

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79] [80]

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577 nanoparticles allow in vivo tracking and recovery of progenitor cells, Nat. Biotechnol. 18 (2000) 410 – 414. M. Lundberg, M. Johansson, Positively charged DNAbinding proteins cause apparent cell membrane translocation, Biochem. Biophys. Res. Commun. 291 (2002) 367 – 371. M. Lundberg, S. Wikstrom, M. Johansson, Cell surface adherence and endocytosis of protein transduction domains, Molec. Ther. 8 (2003) 143 – 150. D.C. Anderson, E. Nichols, R. Manger, D. Woodle, M. Barry, A.R. Fritzberg, Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT proteinderived peptide, Biochem. Biophys. Res. Commun. 194 (1993) 876 – 884. A. Astriab-Fisher, D.S. Sergueev, M. Fisher, B.R. Shaw, R.L. Juliano, Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates, Biochem. Pharmacol. 60 (2000) 83 – 90. A. Fittipaldi, A. Ferrari, M. Zoppe, C. Arcangeli, V. Pellegrini, F. Beltram, M. Giacca, Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 tat fusion proteins, J. Biol. Chem. 278 (2003) 34141 – 34149. M. Rusnati, C. Urbinati, A. Caputo, L. Possati, H. LortatJacob, M. Giacca, D. Ribatti, M. Presta, Pentosan polysulfate as an inhibitor of extracellular HIV-1 Tat, J. Biol. Chem. 276 (2001) 22420 – 22425. M. Rusnati, G. Taraboletti, C. Urbinati, G. Tulipano, R. Giuliani, M.P. Molinari-Tosatti, B. Sennino, M. Giacca, M. Tyagi, A. Albini, D. Noonan, R. Giavazzi, M. Presta, Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat, FASEB J. 14 (2000) 1917 – 1930. M. Rusnati, G. Tulipano, C. Urbinati, E. Tanghetti, R. Giuliani, M. Giacca, M. Ciomei, A. Corallini, M. Presta, The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists, J. Biol. Chem. 273 (1998) 16027 – 16037. D.A. Mann, A.D. Frankel, Endocytosis and targeting of exogenous HIV-1 Tat protein, EMBO J. 10 (1991) 1733 – 1739. M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J. Biol. Chem. 276 (2001) 3254 – 3261. M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d’Adda di Fagagna, M. Giacca, M. Presta, Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size, J. Biol. Chem. 272 (1997) 11313 – 11320. H.C. Chang, F. Samaniego, B.C. Nair, L. Buonaguro, B. Ensoli, HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region, Aids 11 (1997) 1421 – 1431. M. Salmivirta, K. Lidholt, U. Lindahl, Heparan sulfate: a piece of information, FASEB J. 10 (1996) 1270 – 1279. T. Jackson, F.M. Ellard, R.A. Ghazaleh, S.M. Brookes, W.E. Blakemore, A.H. Corteyn, D.I. Stuart, J.W. Newman, A.M.

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

[90] [91]

[92]

[93]

[94]

King, Efficient infection of cells in culture by type O footand-mouth disease virus requires binding to cell surface heparan sulfate, J. Virol. 70 (1996) 5282 – 5287. E.E. Fry, S.M. Lea, T. Jackson, J.W. Newman, F.M. Ellard, W.E. Blakemore, R. Abu-Ghazaleh, A. Samuel, A.M. King, D.I. Stuart, The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex, EMBO J. 18 (1999) 543 – 554. A. Yayon, M. Klagsbrun, J.D. Esko, P. Leder, D.M. Ornitz, Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor, Cell 64 (1991) 841 – 848. T. Arai, W. Busby Jr., D.R. Clemmons, Binding of insulinlike growth factor (IGF) I or II to IGF-binding protein-2 enables it to bind to heparin and extracellular matrix, Endocrinology 137 (1996) 4571 – 4575. D. Mohanraj, T. Olson, S. Ramakrishnan, A novel method to purify recombinant vascular endothelial growth factor (VEGF121) expressed in yeast, Biochem. Biophys. Res. Commun. 215 (1995) 750 – 756. A. Albini, R. Benelli, M. Presta, M. Rusnati, M. Ziche, A. Rubartelli, G. Paglialunga, F. Bussolino, D. Noonan, HIV-tat protein is a heparin-binding angiogenic growth factor, Oncogene 12 (1996) 289 – 297. Y.G. Brickman, M.D. Ford, D.H. Small, P.F. Bartlett, V. Nurcombe, Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells, J. Biol. Chem. 270 (1995) 24941 – 24948. J.S. Bartlett, R. Wilcher, R.J. Samulski, Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors, J. Virol. 74 (2000) 2777 – 2785. I.I. Fuki, R.V. Iozzo, K.J. Williams, Perlecan heparan sulfate proteoglycan. A novel receptor that mediates a distinct pathway for ligand catabolism, J. Biol. Chem. 275 (2000) 31554. K.J. Williams, I.V. Fuki, Cell-surface heparan sulfate proteoglycans: dynamic molecules mediating ligand catabolism, Curr. Opin. Lipidol. 8 (1997) 253 – 262. K.S. Rost, J.D. Esko, Microbial adherence to and invasion through proteoglycans, Infect. Immun. 65 (1997) 1 – 8. K. Lidholt, J.L. Weinke, C.S. Kiser, F.N. Lugemwa, K.J. Bame, S. Cheifetz, J. Massague, U. Lindahl, J.D. Esko, A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 2267 – 2271. M. Silhol, M. Tyagi, M. Giacca, B. Lebleu, E. Vives, Different mechanisms for cellular internalization of the HIV1 Tat-derived cell penetrating peptide and recombinant proteins fused to Tat, Eur. J. Biochem. 269 (2002) 494 – 501. Y. Liu, M. Jones, C.M. Hingtgen, G. Bu, N. Laribee, R.E. Tanzi, R.D. Moir, A. Nath, J.J. He, Uptake of HIV-1 Tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands, Nat. Med. 6 (2000) 1380 – 1387. M. Feligioni, L. Raiteri, R. Pattarini, M. Grilli, S. Bruzzone, P. Cavazzani, M. Raiteri, A. Pittaluga, The human immuno-

H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577

[95]

[96]

[97]

[98]

deficiency virus-1 protein Tat and its discrete fragments evoke selective release of acetylcholine from human and rat cerebrocortical terminals through species-specific mechanisms, J. Neurosci. 23 (2003) 6810 – 6818. B.E. Vogel, S.J. Lee, A. Hildebrand, W. Craig, M.D. Pierschbacher, F. Wong-Staal, E. Ruoslahti, A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin, J. Cell Biol. 121 (1993) 461 – 468. B.S. Weeks, K. Desai, P.M. Loewenstein, M.E. Klotman, P.E. Klotman, M. Green, H.K. Kleinman, Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90kDa cell surface binding protein, J. Biol. Chem. 268 (1993) 5279 – 5284. R. Katoh-Semba, A. Oohira, S. Kashiwamata, Nerve growth factor-induced changes in the structure of sulfated proteoglycans in PC12 pheochromocytoma cells, J. Neurochem. 59 (1992) 282 – 289. M. Rusnati, G. Tulipano, D. Spillmann, E. Tanghetti, P. Oreste, G. Zoppetti, M. Giacca, M. Presta, Multiple interactions of HIV-I Tat protein with size-defined heparin oligosaccharides, J. Biol. Chem. 274 (1999) 28198 – 28205.

577

[99] T.B. Potocky, A.K. Menon, S.H. Gellman, Cytoplasmic and nuclear delivery of a TAT-derived peptide and a {beta}peptide after endocytic uptake into HeLa cells, J. Biol. Chem. 278 (2003) 50188 – 50194. [100] R. Fischer, K. Kohler, M. Fotin-Mleczek, R. Brock, A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides, J. Biol. Chem. 279 (2004) 12625 – 12635. [101] T. Fujimoto, H. Kogo, K. Ishiguro, K. Tauchi, R. Nomura, Caveolin-2 is targeted to lipid droplets, a new bmembrane domainQ in the cell, J. Cell Biol. 152 (2001) 1079 – 1085. [102] A.D. Frankel, C.O. Pabo, Cellular uptake of the tat protein from human immunodeficiency virus, Cell 55 (1988) 1189 – 1193. [103] G. Kilic, R.B. Doctor, J.G. Fitz, Insulin stimulates membrane conductance in a liver cell line: evidence for insertion of ion channels through a phosphoinositide 3-kinase-dependent mechanism, J. Biol. Chem. 276 (2001) 26762 – 26768. [104] E.M. Neuhaus, T. Soldati, A myosin I is involved in membrane recycling from early endosomes, J. Cell Biol. 150 (2000) 1013 – 1026.