Upload
hilary-brooks
View
212
Download
0
Embed Size (px)
Citation preview
www.elsevier.com/locate/addr
Advanced Drug Delivery Rev
Tat peptide-mediated cellular delivery: back to basics
Hilary Brooks, Bernard Lebleu, Eric Vives*
Defenses Antivirales et Antitumorales, CNRS-UMR5124-Universite de Montpellier II, CC086; 5, Place Eugene 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. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
2. Implication of the basic cluster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
3. Influence of other components surrounding the basic domain . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
0169-409X/$ - s
doi:10.1016/j.ad
* Correspondi
E-mail addres
iews 57 (2005) 559–577
ee front matter D 2004 Elsevier B.V. All rights reserved.
dr.2004.12.001
ng author. Tel.: +33 467 16 33 06; fax: +33 467 16 33 01.
s: [email protected] (E. Vives).
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577560
4. Influence of the cargo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
4.1. Influence of Tat peptide exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
4.2. Influence of the hydrophobicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
4.3. Influence of the chemical linkage between Tat and the cargo . . . . . . . . . . . . . . . . . . . . . . . . 565
4.4. Influence of the Tat peptide density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
4.5. Influence of the serum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
5. Tat and cell surface interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
6. Possible mechanisms of internalisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
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 mecha-
nism of entry compared to the Tat peptide [4].
Among others, we recently reevaluated the mecha-
nism of entry of the Tat peptide [5] and highlighted
various problems related with the FACS quantification
and the fixation procedure prior to microscopy obser-
vations. 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) [6–8]. 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 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 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 controver-
sial and certainly requires further investigations. Con-
tradictory 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 mol-
ecules into cells was provided 10 years ago by
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577 561
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 perox-
idase, was performed through a heterolinker. Four to
five peptide molecules per protein unit were suffi-
cient 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 a-
helical 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 relation-
ship 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 a-
helical 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 signifi-
cant 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
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 dis-
pensable, 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 high-
lighted 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
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577562
sequence determinant in most of the studies per-
formed 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
GRKKRRQRRR -GYK(FITC)C [28]
GRKKRRQRRR -PPQ [19,22]
GRKKRRQRRR -G [25]
GRKKRRQRRR -PPQC [9,20,29]
GRKKRRQRRR -GYK(FITC) [30]
GRKKRRQRRR -Cre protein [11]
MYG- GRKKRRQRRR -G [27]
GST protein- GRKKRRQRRR -GFP protein [24]
SGYG GRKKRRQRRR -C [31]
MLGISY- GRKKRRQRRR -PPQT [21]
CY- GRKKRRQRRR [22,25]
CGISY- GRKKRRQRRR [15]
CFITKALGISY- GRKKRRQ [15]
C- GRKKRRQRRR [23]
Biotin-Y GRKKRRQRRR [26]
MLGISY- GRKKRRQRRR [21]
His6- GRKKRRQRRR [32]
Y- GRKKRRQRRR [33,34]
Acetyl- GRKKRRQRRR [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 trans-
location occurring even at a temperature (4 8C)known to inhibit all cellular energy-dependant path-
ways. 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) ini-
tiated 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. Never-
theless, the presence of numerous proteins anchored
in cell membrane or exposed at the cell membrane
surface, along with the variation in lipid composi-
tion 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 particle-
containing 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) 559–577 563
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 guanidi-
nium group content, has been attached to poly-
ethylene 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.
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 pre-
vented interactions with cellular components promot-
ing the uptake, since the Tat peptide coupling was
mediated randomly on large proteins, such as h-galactosidase (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]).
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577564
In another example, two very different mecha-
nisms 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 con-
struct), 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 recep-
tors 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, con-
firming 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, there-
fore 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 5Vend. 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 trans-
locating 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 biotiny-
lated 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 trans-
locating 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) 559–577 565
ally, the insertion of aminocaproic acid (aca) groups
within the peptide backbone showed a stronger cell
associated signal than the corresponding heptahomo-
polymers 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 trans-
fection 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 poly-
cationic 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 trans-
locating process itself, an increase of the initial cell
binding or the use of an additional entry pathway
upon peptide modification could not be fully ascer-
tained 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].
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 phosphor-
othioate 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,
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577566
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 oligo-
nucleotide cargo [57–59]. Thirdly, the reduction of the
disulfide bridge once the cargo reaches the cytoplas-
mic compartment is expected to induce its release,
therefore, preventing putative negative effects of the
cell penetrating peptide. The efficiency of the intra-
cellular 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 comple-
mentary 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 cell-
penetrating 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 oligo-
nucleotides 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 Anten-
napedia 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) 559–577 567
ciency [19]. Conversely, a simple mixture of the Tat
peptide either with a peptide [39] or an oligonucleo-
tide [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 h-galactosidase [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
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 trans-
fection 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 differ-
ences 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 mecha-
nism 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
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577568
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 doxor-ubicin-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 trans-
fection of oligonucleotides, serum has been shown to
unexpectedly augment the biological response when
oligonucleotides were delivered by Tat or Antenna-
pedia 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 ana-
logues, such as PPS (pentosan polysulfate) [72], the
heparin-binding protein TSP (platelet thrombospon-
din-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 techni-
ques 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 quanti-
tatively 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) 559–577 569
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 pre-
treatment 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 concentra-
tions 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
polyanions (heparin and dextran sulfate) [75]. Sur-
prisingly, 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 signifi-
cantly reduces its interaction with the GAG. The
dissociation constant of heparin to immobilised GST-
Tat 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 inter-
action 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 glycosaminogly-
cans (GAGs), such as HS, increasingly implicated in
cell adhesion, are distributed ubiquitously on cell
surfaces as the carbohydrate component of proteogly-
cans [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-
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577570
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 heparin-
binding capacity using heparin-sepharose chromatog-
raphy 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 non-
coated 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 treat-
ment 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 TAT-
mediated 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-acetylgluco-
saminyl-transferase and glucuronosyltransferase), pro-
duces 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 recombi-
nant 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 EDTAwashed 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 fluorescein-
labelled 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) 559–577 571
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 immunopre-
cipitation of the Tat peptide with various anti-
integrin antibodies suggesting that the vitronectin-
binding 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-bind-
ing 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, how-
ever, the Tat peptide behaves in an energy-dependent
manner requiring temperatures above 4 8C and ATP.
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 stain-
ing 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 acid-
ification 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, bafilomy-
cinA, nor of wortmannin, a potent PI-kinase inhibitor.
In our hands, the drug monensin, which causes de-
acidification 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 cyto-
solic 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
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577572
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 fluo-
rescence similar to untreated controls [24]. Trans-
ferrin, 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-galactivity 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-phage-
mediated 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-depend-
ent 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-h-cyclodextrin [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 local-
isation 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/non-
caveolin-type pathway(s) (lipid rafts or microdo-
mains, 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 con-
stitutively 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 nonspecifi-
cally 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 oligonu-
cleotides 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) 559–577 573
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 correspond-
ing 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 nano-
molar to hundreds of micromolars) and protocols
applied to estimate the uptake efficiency or the
subcellular localisation of the chimeras (from bio-
logical 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.
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 func-
tional 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
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577574
cellular uptake, binding to target sequences, and biologic
actions, Pharm. Res. 19 (2002) 744–754.
[10] 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.
[11] 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.
[12] 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, Biochem-
istry 41 (2002) 7925–7930.
[13] 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.
[14] 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.
[15] 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.
[16] 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.
[17] D. Derossi, S. Calvet, A. Trembleau, A. Brunissen, G.
Chassaing, A. Prochiantz, Cell internalization of the third
helix of the Antennapedia homeodomain is receptor-inde-
pendent, J. Biol. Chem. 271 (1996) 18188–18193.
[18] 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.
[19] 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.
[20] 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.
[21] 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.
[22] 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, Biochem-
istry 40 (2001) 4349–4358.
[28] A. Nori, K.D. Jensen, M. Tijerina, P. Kopeckova, J. Kopecek,
Tat-conjugated synthetic macromolecules facilitate cytoplas-
mic 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 suppres-
sor 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. Piwnica-
Worms, Evidence for a plasma membrane-mediated perme-
ability 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) 559–577 575
[36] V.P. Torchilin, T.S. Levchenko, R. Rammohan, N. Volodina,
B. Papahadjopoulos-Sternberg, G.G. D’Souza, Cell trans-
fection in vitro and in vivo with nontoxic TAT peptide-
liposome-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 evalua-
tion 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 internal-
ization of cholera and tetanus toxins, Nature 296 (1982)
651–653.
[48] L. Pelkmans, J. Kartenbeck, A. Helenius, Caveolar endocy-
tosis of simian virus 40 reveals a new two-step vesicular-
transport 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.
[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 intra-
cellular 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, Oligonucleo-
tide-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 poly-
arginine 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
H. Brooks et al. / Advanced Drug Delivery Reviews 57 (2005) 559–577576
nanoparticles allow in vivo tracking and recovery of
progenitor cells, Nat. Biotechnol. 18 (2000) 410–414.
[67] M. Lundberg, M. Johansson, Positively charged DNA-
binding proteins cause apparent cell membrane trans-
location, Biochem. Biophys. Res. Commun. 291 (2002)
367–371.
[68] M. Lundberg, S. Wikstrom, M. Johansson, Cell surface
adherence and endocytosis of protein transduction domains,
Molec. Ther. 8 (2003) 143–150.
[69] 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 protein-
derived peptide, Biochem. Biophys. Res. Commun. 194
(1993) 876–884.
[70] 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. Pharma-
col. 60 (2000) 83–90.
[71] 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.
[72] M. Rusnati, C. Urbinati, A. Caputo, L. Possati, H. Lortat-
Jacob, M. Giacca, D. Ribatti, M. Presta, Pentosan polysulfate
as an inhibitor of extracellular HIV-1 Tat, J. Biol. Chem. 276
(2001) 22420–22425.
[73] 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.
[74] 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 polysulfo-
nated heparin-mimicking extracellular Tat antagonists, J.
Biol. Chem. 273 (1998) 16027–16037.
[75] D.A. Mann, A.D. Frankel, Endocytosis and targeting of
exogenous HIV-1 Tat protein, EMBO J. 10 (1991)
1733–1739.
[76] M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization
of HIV-1 tat requires cell surface heparan sulfate proteogly-
cans, J. Biol. Chem. 276 (2001) 3254–3261.
[77] 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.
[78] 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-associ-
ated heparan sulfate proteoglycans through its basic region,
Aids 11 (1997) 1421–1431.
[79] M. Salmivirta, K. Lidholt, U. Lindahl, Heparan sulfate: a
piece of information, FASEB J. 10 (1996) 1270–1279.
[80] 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.
King, Efficient infection of cells in culture by type O foot-
and-mouth disease virus requires binding to cell surface
heparan sulfate, J. Virol. 70 (1996) 5282–5287.
[81] 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.
[82] 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.
[83] T. Arai, W. Busby Jr., D.R. Clemmons, Binding of insulin-
like 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.
[84] 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.
[85] 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.
[86] 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.
[87] 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.
[88] 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.
[89] K.J. Williams, I.V. Fuki, Cell-surface heparan sulfate
proteoglycans: dynamic molecules mediating ligand catabo-
lism, Curr. Opin. Lipidol. 8 (1997) 253–262.
[90] K.S. Rost, J.D. Esko, Microbial adherence to and invasion
through proteoglycans, Infect. Immun. 65 (1997) 1–8.
[91] 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.
[92] M. Silhol, M. Tyagi, M. Giacca, B. Lebleu, E. Vives,
Different mechanisms for cellular internalization of the HIV-
1 Tat-derived cell penetrating peptide and recombinant
proteins fused to Tat, Eur. J. Biochem. 269 (2002) 494–501.
[93] 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.
[94] 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) 559–577 577
deficiency virus-1 protein Tat and its discrete fragments
evoke selective release of acetylcholine from human and rat
cerebrocortical terminals through species-specific mecha-
nisms, J. Neurosci. 23 (2003) 6810–6818.
[95] 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.
[96] 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 90-
kDa cell surface binding protein, J. Biol. Chem. 268 (1993)
5279–5284.
[97] R. Katoh-Semba, A. Oohira, S. Kashiwamata, Nerve growth
factor-induced changes in the structure of sulfated proteo-
glycans in PC12 pheochromocytoma cells, J. Neurochem. 59
(1992) 282–289.
[98] 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.
[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.