Volume 2 † Number 1 † March 2009
10.1093/biohorizons/hzp001
.........................................................................................................................................................................................................................................
Review
Cell-penetrating peptides and their therapeutic
applications
Victoria Sebbage*
21 Westminster Gardens, London, SW1P 4JD, UK.
* Corresponding author: Tel: 07743426792. Email: [email protected]
Supervisor: Dr J.H. Walker, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
........................................................................................................................................................................................................................................
The process of introducing drugs into cells has always proved a major challenge for research scientists and for the pharmaceutical industry. The cell membrane is selectively permeable and supports no generic mechanism for their uptake. A drug must be either highly
lipophilic or very small to stand a chance of cellular internalization. These restrictions mean that the repertoire of possible drug molecules is limited. Similarly, novel therapeutic approaches such as gene and protein therapy also have limited potential due to the
cell-impermeable nature of peptides and oligonucleotides. The existing methods for delivery of macromolecules, such as viral
vectors and membrane perturbation techniques, can result in high toxicity, immunogenicity and low delivery yield. However, in 1988
the remarkable ability of a peptide to traverse a cell’s plasma membrane independent of a membrane receptor was revealed. Known
as Tat, the transcription activator of the human immunodeficiency virus type 1 (HIV-1) viral genome was shown to enter cells in a
non-toxic and highly efficient manner. In light of such properties Tat became known as the first ‘cell-penetrating peptide’ (CPP).
CPPs have demonstrated themselves to be capable of delivering biologically active cargo to the cell interior and the vehicular capabilities
of CPPs have already been harnessed for use as laboratory tools. However, it is believed that their true potential lies within the field of
therapeutics. Attached to a CPP, therapeutic cargo could be delivered to an intracellular target, thus overcoming the entry restrictions set
by the plasma membrane. Since the discovery of Tat, the number of known peptides with cell-penetrating capabilities has grown and in
2003, the first CPP-based drug reached phase II clinical trials. This review discusses the controversial mechanism of entry employed by
CPPs, their potential applications in vitro and in vivo, and the ways in which CPP properties have been optimized to maximize their
potential as future therapeutics.
Key words: Drug, delivery, vector, peptide, intracellular, penetrating.
........................................................................................................................................................................................................................................
Cell-penetrating peptides (CPPs) have the ability to enter
cells independent of a membrane receptor,1 and they show
no cell-type specificity.2 They are small (10 –30 residues in
length), often positively charged sequences of amino acids.3
A consensus as to what defines a CPP is yet to be agreed
upon, as there seem to be a number of diverse criteria that
can define a peptide as ‘penetrating’. Current research is
focused on two CPPs in particular; Tat, originating from
the retrovirus human immunodeficiency virus type 1
(HIV-1) and pAntp, derived from the transcription factor
Antennapedia.4 Their penetrating capabilities were revealed
unexpectedly, in 1988 and 1991, respectively.
In the late 1980s, research in the field of AIDS and its
causative agent HIV-1 was focused on potential targets for
AIDS therapy. One such target was identified as Tat, the
86 amino acid transcriptional activator coded for by the
HIV-1 genome, which is expressed upon HIV-1 infection
of a host cell.5 Studies carried out in 1988 aimed to characterize the structural and functional domains of Tat.6, 7
Unexpectedly, researchers discovered two things: that Tat
could be taken up from the surrounding medium by HeLa
cells in vitro; and that it could directly trans-activate its
target HIV LTR gene located in the nucleus7 (Fig. 1).
Using mutagenesis studies, the biological activity and
penetrating capability of Tat was found to reside in the
region between residues 48–60.7 In this report, unless
otherwise stated, ‘Tat’ refers to this commonly used
peptide region.
.........................................................................................................................................................................................................................................
#
2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any
64
medium, provided the original work is properly cited.
Bioscience Horizons
Review
† Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
Figure 1. Transactivation of the LTR gene in HeLa cells by the Tat protein.
(A) HeLa cells were transformed with a plasmid coding for the CAT gene
fused to an HIV-LTR promoter ( pHIV-LTRCAT) and incubated with Tat
before fixation and analysis. Trans-activation of pHIV-LTRCAT by (B) coinjection with a plasmid encoding Tat cDNA ( pCV-1) or (C) by incubation with
Tat protein. Source: Taken from Green and Loewenstein 1988.
In 1991 it was established that the properties conferred by
Tat were not unique. The receptor-independent penetrating
capabilities of the Antennapedia transcription factor were
discovered during investigations into the neural development
of Drosophila.8 The 1 –60 amino acid region of the
transcription factor (known as the ‘homeodomain’) was
shown to enter live, cultured neuronal cells, undergo targeting to the nucleus and cause cellular morphological
changes.8 Specifically residues 43–58, the ‘internalization
helix’ or ‘pAntp’, were responsible for cell penetration.9
Since then, the number of known natural and synthetic
peptides with cell-penetrating capabilities has continued to
grow (Table 1).10
The vehicular potential of CPPs was realized in 1995 when
studies on pAntp demonstrated that the peptide could be
attached to a bioactive compound (forming a ‘conjugate’)
and used to achieve its intracellular delivery.11 Significantly,
the attached cargo (a protein kinase C inhibitor) retained its
function upon internalization into a live neurone. The potential for CPPs to act as vectors for therapeutically active macromolecules was realized as a result of preliminary in vivo
experiments using Tat. Beta-galactosidase, a macromolecule
previously considered to be impermeable to cells, was covalently cross-linked to Tat.12 The delivery of beta-galactosidase
to the cytoplasm of several tissues was achieved in a cell-type
independent manner.
Until these studies, the peptide-mediated cytoplasmic
delivery of macromolecules had not been achieved. Tat and
pAntp had provided the mechanism with which it was possible. It was a promising prospect that potentially any molecule could be attached to a CPP in order to achieve its
successful intracellular delivery. However, there remain
gaps in our knowledge regarding the mechanism by which
CPPs enter cells and traffic once internalized. This currently
limits their clinical use.
Since the discovery of Tat in 1988, the disparity among
results from laboratories attempting to elucidate the uptake
mechanism has caused controversy. Experimental procedures
used commonly throughout the pioneering phase of CPP
investigation (namely, the period from their discovery until
2003) provided much of the evidence supporting a
non-endocytic, energy-independent direct traversal of the
cell membrane by CPPs. The validity of those experimental
procedures has since been called into question.13
One example of this is the use of flow cytometry to quantify accurately the cellular uptake of fluorescently labelled
CPPs, which was a technique put into doubt in 2003.14
The source of the ambiguity was considered to be the
inability of the procedure to discriminate between extracellular and internalized CPPs. Studies carried out after 2004
incorporated external trypsin protease washing (‘trypsinization’) after CPP incubation15 in an attempt better to represent the quantity of CPP determined within the cell.15
The splice correction assay designed in 1998 has further
improved the reliability of CPP quantification (Fig. 2).15
This recombinase system only gives a positive signal in the
event of successful (and non-toxic) nuclear delivery of bioactive cargo.16 It was successfully employed in 2004 to quantify Tat internalization.15
In vivo techniques possible since 1999 have shown that
in vitro models may not be accurately representative of
in vivo systems.15 In vivo studies carried out on the brain
have suggested that plasma proteins may bind the conjugated
CPP, prohibiting release of the bioactive cargo to the
tissues.17
An additional source of unreliability in the pioneering
phase was the fixation protocol employed for visualization
.........................................................................................................................................................................................................................................
65
Review
Bioscience Horizons † Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
Table 1. A selection of currently known CPPs, their origins and sequences
Name
Origin
Sequence
Tat (48-60)
HIV-1 protein
GRKKRRQRRRPPQQ
Oligoarginine
Tat derivative
Rn
p-Antp
Antermapedia homeodomain
RQIKIWFQNRRMKWKK
plsl
Igl-1 homeodomain
RVIRVWFQNKRCKDKK
Galanin-mastoparan
GWTLNSAGYLLGKINLKALAALAKKIL
Pb
gp41-SV40
GALFLGFLGAAGSTMGAWSQPKKKRKV
Pa
gp41-SV40
GALFLAFLAAALSLMGLWSQPKKKRRV
Trp-rich motif-SV40
KETWWETWWTEWSQPKKKRRV
........................................................................................................................................................................................................................................
Tat family
Penetralia family
Chimeric CPPs
Transportan
MPG peptides
Pep-1
Source: Taken from Foerg and Merkle.18
Figure 2. Nuclear delivery of Tat-Cre is required for the expression of
enhanced green fluorescent proteins (EGFP) in reporter T cells. (A) The
nuclear presence of Tat-Cre mediates the excision of the STOP gene
segment, permitting transcription of EGFP. The STOP gene is flanked by
loxP recombination sites, which undergoes site-specific recombination
mediated by Cre recombinase protein. Pr corresponds to the promoter.
(B) The results of flow cytometry quantification of EGFP in untreated
cells, cells incubated with 2 mM Tat-Cre and cells incubated with 2 mM
control Cre for at least 5 min. All cells were trypsinized post-incubation.
Source: Taken from Wadia et al.15
of CPPs by fluorescence microscopy.18 As early as 1992 it
was noted that fluorescently labelled cellular proteins were
localized to ‘inappropriate’ locations as a result of cell fixation,13 attributed to the permeating activity of the ethanol
and acetic acid on the nuclear membrane.19 Since then CPP
studies have avoided nuclear artefacts by using live, unfixed
cells to show CPPs colocalized with fluorescent endosomal
membrane markers.15 Studies using asolectin liposomes
(known as ‘giant unilamellar vesicles’ or ‘GUVs’) have also
avoided problems associated with cellular fixation.20
Figure 3. The three proposed routes of CPP entry. Model 1: The inverted
micelle model. Model 2: The direct penetration ( pore formation) mechanism. Model 3: An endocytic mechanism of uptake. Source: Taken from
Trehin and Merkle.13
These techniques have been used in attempt to determine,
amongst other CPP features, the mechanism of CPP internalization. The current view is that each CPP may be internalized
via an idiosyncratic mechanism (Fig. 3),13 the major route
being endocytic.21 But despite today’s clear evidence for an
endosomal route of entry there is also strong evidence for a
minor route of cell entry, specifically a ‘direct translocation’
of CPP across the membrane bilayer.21 However, evidence
for direct translocation was obtained mostly during the
.........................................................................................................................................................................................................................................
66
Bioscience Horizons
Review
† Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
pioneering phase, often via unreliable experimental techniques.
The evidence for direct translocation is considered first.
Since endocytic uptake is energy-dependent, experiments
in the pioneering phase were commonly carried out at 48C
and in the absence of ATP22 in attempt to investigate an
energy-independent (and therefore non-endocytic) internalization mechanism. Evidence for a direct translocation through
the membrane bilayer was accumulated for both Tat and
pAntp from studies using the temperature- and ATP-dependent
properties of the ATPase required for endocytosis.21, 23
Multiple experiments were carried out in conditions of
reduced ATP concentration and low temperatures, which
failed to inhibit internalization of Tat23 and pAntp,9 and thus
supported a non-endocytic uptake mechanism. In addition,
early studies using a caveolae-mediated endocytosis inhibitor
also failed to inhibit Tat internalization.23 Furthermore, in
2000, a landmark study found that pAntp was able to move
directly into and out of fluorescently labelled GUVs. GUVs do
not permit the formation of internal endosomal structures,
thus pAntp was shown to have the capacity for non-endosomal
entry.20 Investigation using pAntp analogues provided evidence
for a stereospecific receptor-independent endocytic route of
internalization.1
In 1996, researchers proposed the inverted micelle model
in attempt to explain the penetration of the bilayer in
terms of receptor-, energy-, and endocytosis-independence.13
The ‘inverted micelle’ was defined as the hydrophilic ‘cavity’
present within the two bilayer leaflets, which accommodates
the CPP within the wider hydrophobic environment of the
phospholipid tails.1 This micellular phase is transient, and
the CPP is subsequently released, directly into the cytosol
and independent of any vesicular body, as the micelle
passes to the cytosolic side of the bilayer.13 An alternative
model of direct translocation of the membrane was proposed
with respect to conjugate complexes known as ‘nanoparticles’.24 This model proposes that the nanoparticle induces
the formation of a transient b-barrel pore in the cell membrane to allow the passage of the nanoparticle into the cell.25
The evidence for endocytic routes of entry, which
mounted after 2002 with the increased use of reliable experimental techniques, is now considered. This evidence was in
direct contradiction to that supporting a direct translocation
mechanism, gathered during the pioneering phase.
Supporting an endocytic route of entry for pAntp, fluidphase endocytosis inhibitors were used successfully to
prevent pAntp internalization.3 In 2004 evidence for a
macropinocytotic mechanism of Tat uptake in live mouse
cells was published. These studies showed colocalization of
an endosome marker with Tat, suggesting that CPPs were
localized to endosomes (Fig. 4).15 Studies carried out in
2006 demonstrated that endosome lysis resulted in an
increase in splice correction efficacy,26 suggesting that Tat
was localized to vesicles, possibly macropinosomes. Indeed,
endosomal disruption agents (such as the viral haemagglutinin protein ‘HA2’) were shown to cause the artificial release
of Tat-Cre from the endosome.15
Today, it is thought that each CPP expresses its own preferred mechanism of uptake.13 Although studies using the
most recent, reliable techniques have provided evidence for
endocytic internalization, studies such as the experiment
using GUVs (the data from which supported a direct
bilayer translocation) are yet to be refuted.
One detail of CPP internalization, the interaction with
membrane components, has remained a common feature of
both major and minor routes of CPP internalization mechanisms. With the arrival of evidence that pAntp could not cross
pure phospholipid bilayers in 2001, it was proposed that
pAntp may require electrostatic interaction with components
other than membrane phospholipids for cell penetration.27
Evidence for such interaction with respect to Tat was published in 2004.15 The involvement of extracellular matrix
components, in particular heparin sulphate (HS) and chondroitin sulphate (CS), was considered in the study in order
to elucidate the ionic interaction between Tat and the cell
surface.15 Pre-incubation of Tat-Cre with soluble HS and
CS prevented the uptake of Tat-Cre when the conjugate
Figure 4. Confocal images showing the colocalization of Tat-Cre-488 with FM4-64 in live 3T3 cells. From left to right: Tat-Cre-488 alone, the fluorescent
general marker of endosomes FM4-64 alone and the colocalization of Tat-Cre-488 with FM4-64. Incubation was carried out with 2 mM Tat-Cre-488 and
4 mM of FM4-64. After 8 h cells were washed and images acquired. The fluorescent label used for Tat-Cre is referred as 2488. Source: Taken from
Wadia et al.15
.........................................................................................................................................................................................................................................
67
Review
Bioscience Horizons † Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
mixture was subsequently incubated with cells for uptake. It
was therefore suggested that the electrostatic interaction
( prior to internalization) of positively charged Tat with
negatively charged cell-surface glycoproteins was necessary
for successful translocation.15, 28
In addition to interaction with the membrane, methods of
cargo attachment and cargo type are also factors that have
been shown to affect the uptake mechanism.29 – 31 Lipid raftmediated CPP uptake has even been implicated.15, 32
Regarding the route of intracellular trafficking, evidence
exists for both nuclear and cytoplasmic delivery of
CPP-internalized cargo.7, 33 However, nuclear delivery is
considered to occur as a result of cytoplasmic delivery.14
There is much contradictory evidence with regards to endosomal accumulation and release, retrograde trafficking and
lysosomal degradation.34, 35
Evidence for cytosolic delivery after endosomal localization was published in 2005.33 Colocalization studies of
CPP conjugates with HeLa cells showed only a transient
localization to early endosomes, prior to conjugate release
to the cytoplasm. It has been proposed that the excessive
accumulation of CPPs may result in endosomal destabilization and conjugate release.31
Endosome-localized CPPs have been shown to undergo
retrograde delivery, through the Golgi apparatus and endoplasmic reticulum (ER), prior to their cytosolic release.34
This study pointed out similarities to the trafficking of the
ricin and Shigella toxins, which are known to bind to
the host cell surface, internalize by endocytosis, traffic to
the Golgi and ER via retrograde transport and then release
into the cytosol. There also exists evidence for the trafficking
of endosome-localized Tat to late endosomes and lysosomal
bodies.35 This has negative therapeutic implications.
Early studies on Tat verified its nuclear translocation using
fluorescence microscopy and a positive transcription assay.7
Later studies using the splice correction assay also provided
evidence for CPP-mediated nuclear delivery.15 No study
has yet established the detailed mechanism of nuclear entry
or localization.
In spite of the lack of a complete understanding, CPPs are
currently being manufactured for use as transduction tools in
laboratory studies.21 A selected example is that of ChariotTM ,
a peptide delivery vector manufactured by Panomics, advertised as the ‘Revolutionary new transfection agent’.36 There
are numerous advantages conferred by CPPs that define them
as a more appropriate means to intracellular delivery than
the principle methods available for intracellular cargo delivery
today (which include viruses and liposomes in addition to electroporation and microinjection techniques).37, 38
Unlike liposomes, CPPs have exhibited successful delivery to
numerous cell types, including human Jurkat and HeLa cell
lines and those notoriously impermeable to retroviral vectors
such as osteoclasts.2 Unusually, CPPs exhibit remarkably low
toxicity and have an extensive range of possible cargo
types.21, 17 Unlike viral vectors, CPPs do not have the capacity
to integrate the genetic material they deliver, and therefore
there has been no evidence for CPP use resulting in oncogene
activation.39 The immunogenicity raised in response to conjugated CPPs alone is said to be very unlikely.2, 39 There is also
evidence of (the highly desirable) sustained presence and functionality of CPP-delivered protein cargo.18, 38, 40 In contrast to
the membrane rupturing techniques of electroporation and
microinjection there is no evidence for membrane perturbation
caused by CPP cell entry.20, 38
CPPs have enormous potential for novel therapeutics.18
The potential therapeutic benefits of intracellular delivery
of macromolecules such as protein and nucleic acids
in vivo have not been realized due to the ineffective
uptake of such large, hydrophobic compounds. However,
in 1999 biologically active cargo was successfully internalized for the first time, mediated by Tat.41 Tat was used to
deliver active protein 116 KDa beta-galactosidase (b-Gal)
to all tissues in a mouse model in vivo, including the brain
(Fig. 5).
Figure 5. Active Tat-b-Gal was successfully transduced across the blood –brain barrier. The X-Gal staining of brain sections from mice 8 h after intraperitoneal injection with (A) control b-Gal or (B) Tat-b-Gal. b-Gal activity was localized to the cell nuclei indicated by the arrows. Source: Taken from Schwarze
et al.41
.........................................................................................................................................................................................................................................
68
Bioscience Horizons
Review
† Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
This set a precedent. In 2004, a CPP was used for the
delivery of an siRNA in order to down-regulate a reporter
gene representative of a therapeutically detrimental gene.42
The siRNA, complementary to reporter protein ‘firefly
luciferase’, was covalently attached to pAntp. The
CPP-mediated delivery of anti-luciferase siRNA was successfully achieved. Decreased luciferase activity was observed
after CPP-siRNA incubation with the luciferase-expressing
cells. CPP-mediated siRNA delivery achieved both increased
efficiency of delivery and of sustained expression of the
cargo, when compared with liposomal delivery of the same
cargo.
In another study, the virally derived CPP ‘VP22’ was successfully used to deliver the transcription factor Gata4 into
cardiac cells in vivo.38 Gata4 controls the stress responsiveness of the heart.43 It is a transcription factor naturally
expressed in cardiomyocytes in order to upregulate genes
of therapeutic benefit in the conditions of increased stress
or pressure.44 Ischaemic cardiomyopathy is one such condition, in which decreased blood supply to the heart results
in damage to cardiomyocytes. If these conditions are prolonged, cells undergo necrosis and the function of the heart
is compromised. Low oxygen levels induce a stress response,
one of which is the upregulation of the transcription activator Gata4 by local cells.45 Gata4 acts on multiple routes
within the cell to cause hypertrophy.43 Increased dimensions
of functional, hypertrophic myocytes enhance their performance and thus compensate for those that have reduced function due to damage.38, 46 This study demonstrated the
therapeutic benefits of Gata4 over-expression on a murine
model of ischaemic cardiomyopathy.38 Fibroblasts, transformed with expression plasmids constructed to express
Gata4-VP22 fusion proteins, were injected into the site of
induced cardiac damage.38 Improved cardiac function,
increased cardiomyocyte diameter and other favourable
effects on tissue repair could be seen after 4 weeks. A survival rate of 6 weeks beyond the time frame required for
mounting an antibody response was observed. This provides
evidence that protein-based gene therapy could permit
sustained release of a functional therapeutic protein,
without causing toxicity. In the context of today’s increased
prevalence of cardiomyopathy, this study has particular
significance.
CPPs also have potential in the treatment of strokes. In
2002 Tat was conjugated to an anti-apoptotic protein in
order to inhibit the apoptosis of an ischaemic neuronal cell
in the brain of a stroke model.47 Cerebral ischaemia is the
result of a blood vessel blockage in the brain. The resulting
loss of oxygen supply to the surrounding neurones is
known to result in the stimulation of their apoptotic pathways. This is mediated by the release of pro-apoptotic proteins. Anti-apoptotic proteins such as Bcl-xL counteract the
pro-apoptotic process. Bcl-xL is a mitochondrial integral
membrane protein, which acts to inhibit the release of
cytochrome C and other apoptotic factors from the mitochondrial intermembrane space. Bcl-xL thus has the potential to prevent neuronal apoptosis, after an ischaemic
episode. However, failure to deliver Bcl-xL proteins to cells
successfully, let alone to the brain, has always stifled such
protein therapy. Tat was selected for the cerebral delivery
of Bcl-xL because of its ability to traverse the blood–brain
barrier (BBB) upon systemic injection.41
The conjugate-treated brains exhibited anti-apoptotic
effects. They showed a 60% reduction in farction volume,
even when administered up to 45 min after the end of an
ischaemic episode. Of greatest clinical significance, administration of Tat-Bcl-xL has since been shown to reduce neuronal apoptosis and infarct volume in a murine stroke
model when administered, both before and after a focal
ischaemic episode, intravenously.48
CPPs have also been shown to have potential uses in
cancer therapy. The cell-specific in vivo delivery of Caspase
3 was achieved in 2002 using an oxygen-dependent degradation (ODD) domain as a means to artificially stimulate
the apoptotic process in cancer cells (Fig. 6).49 Such an
approach has long been identified as a potential means to
cancer therapy.39 However, bioactive agents causing apoptosis are required to be tumour-cell specific. Through the use of
a cell-specific tag, the function of which was based on the
differential oxygen concentration of cancer cells, Tat was
used to carry out tumour-specific delivery.41
Cancer cell proliferation is mediated by transcription
factors, some of whose activities are regulated by
hypoxia.49 Hypoxia-Inducible Factor-1a (HIF-1a) is one
such transcription factor. The stability of HIF-1a is dependent on its ODD domain. When too much oxygen is
present, the ODD domain is activated and HIF-1a becomes
instable. Thus, hypoxic conditions are required to maintain
its stability. Solid tumour cells are characteristically
hypoxic. Harada et al.49 proposed that linking an ODD
Figure 6. CPP-ODD mediated delivery of pro-apoptotic protein Caspase 3
in normal (20% O2) and hypoxic (1% O2) conditions. Mouse 3T3 cells were
treated with buffer, mutant Tat-ODD-Caspase 3 or wild-type (functional)
Tat-ODD-Caspase 3 for 24 h. Tat-ODD-Caspase 3 was administered as a
fusion protein by intraperitoneal injection. Apoptotic features are clearly
visible only in hypoxic cells after treatment with functional
Tat-ODD-Caspase 3. Source: Taken from Harada et al.49
.........................................................................................................................................................................................................................................
69
Review
Bioscience Horizons † Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
domain to a CPP-Caspase 3 conjugate would stabilise the
Caspase cargo in an hypoxic environment only.49 This
could achieve hypoxic-cell specific delivery of Caspase 3.
The Caspase 3-mediated stimulation of apoptosis resulted
in tumour mass reduction.49 No evidence of toxic side effects
on surrounding tissues was observed. The evidence suggested
that the Tat-ODD-Caspase 3 conjugate accessed all cells in
the tissue but, since it was degraded in normoxic cells by
the ODD domain, only retained its stability in hypoxic
cells. A reduction in tumour mass was demonstrated.
With regards to cerebral therapy, access of pharmaceutical
agents to the brain is notoriously difficult to achieve. As a
result of P-glycoprotein expression (P-gp) at the BBB and its
over-expression in tumour cells, the therapeutic potential of cerebral anti-cancer agents has been strictly limited.17 The brain is
said to exhibit ‘multidrug resistance’ and most methods that
achieve brain uptake are invasive, causing high toxicity.17
The potential of CPPs to act as peptide vectors able to
penetrate the BBB in a non-toxic fashion was demonstrated
in 1999 when a Tat fusion protein was shown to internalize
throughout the brain, 8 h after intraperitoneal administration.41 Investigation in 2000 demonstrated that pAntp
and SynB vectors were able to deliver the anticancer agent
Doxorubicin to the brain upon intravenous administration.17
A study carried out by the same laboratory in 2003 was able
to confirm that anticancer agents delivered by SynB retained
their pharmacological effects.50
As well as exploring their current applications, researchers
have been looking for ways to optimize CPPs in order to
tailor them to suit the needs of each therapeutic application.
The release from endosomes was considered to be the ratelimiting step in the CPP-mediated delivery of Tat-Cre.15
Exploiting the mechanism of endosomal escape employed
by the HIV viral protein haemagglutinin (HA2) has provided
a novel mechanism of endosomal escape for CPPs (Fig. 7).15
Cells were co-incubated with Tat-HA2 and Tat-Cre in order
to enhance the efficacy of Tat-mediated transduction and
cargo release.
Some CPPs have shown high affinity for specific cell types
or intracellular destinations. A recently discovered CPP
known as ‘Crotamine’ has shown unusually high affinity for
actively proliferating cells.51 Another example lies with
MPG, a synthetic CPP derived from the SV40 virus.
Originally designed for nuclear delivery of siRNA, it has
recently been altered to target the cytoplasm.24, 25 By tampering with the nuclear localization sequence present within its
hydrophobic region, the destination of the cargo release can
be selected. MPG has been successfully used for the targeted
delivery of siRNA to mouse blastocytes in vivo.25, 52
CPPs also have to resist degradation once internalized. It
has been documented that peptide delivery to neurones
induces the activity of endogenous neuronal proteases.53
However, D-peptide forms of pAntp confer resistance to
the neuronal proteolytic activity.53 D-isomers generally
confer the loss of biological activity; however, the synthesis
of pAntp in reverse order using D-amino acids maintains
its biological activity.
Furthermore, the form in which the cargo is presented to
the cell has implications for its stability within the cellular
environment. Peptide nucleic acids (PNA) are more effective
than oligonucleotides because only the latter are subject to
nuclease degradation in the cytosol.54 The PNA is comprised
of a polyamide backbone, which replaces the phosphodiester
backbone of nucleic acids.
The ability of CPPs to cross membranes has been attributed to their high content of positive residues such as arginine
and lysine.27 Studies carried out on the Tat sequence necessary for internalization have suggested that the feature
required for efficient uptake may not be the cationic nature
of the CPP, but rather the guanidinium head group of the
arginine residues contained within it.55 Polyarginine uptake
was comparable with that of a synthesized polymer of the
concerned guanidinium head group of arginine, known as
a ‘peptoid’. A peptoid is made up of a polyglycine backbone,
which incorporates one guanidium group at every fourth
position on the backbone. The high degree of peptoid
Figure 7. A model for the facilitated release of Tat-Cre from an endosome using Tat-conjugated haemagglutinin (Tat-HA2). Co-treatment with Tat-HA2 and
Tat-Cre results in Tat-Cre release from the endosome. Key: Blue, Cre cargo; Pink, Tat CPP; Red, HA2. Source: Adapted from Alberts et al.58
.........................................................................................................................................................................................................................................
70
Bioscience Horizons
Review
† Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
uptake was attributed to the guanidine head group of the
arginine residue.
Polyguanines are promising drug candidates. Designed for
the maximum efficiency of uptake, these peptoids are of high
stability and resistant to proteolysis.55 Their preparation is
achieved with more ease than Tat, with favourable economic
implications. Racemization is straightforward in peptoid synthesis and therefore peptoid analogues suitable for individual
applications could be simply produced.
The repertoire of possible drug structures has been greatly
expanded by the advent of peptide vectors. Macromolecules
can be transported into cells using these molecular vehicles,
which offer a highly efficient, non-toxic and nonimmunogenic route for cell entry. Currently, their only limiting feature is their lack of cell-type specificity, although this
has been overcome.49 Advantageously, no cell is known to be
excluded from the possibility of CPP internalization. The
mechanism of entry, trafficking route and degradation
pathway of CPPs remains elusive, though technological
advances promise to shed more light on what is presently a
rather mysterious field.
Current clinical trials of CPP-based drugs are, however,
transforming the conceptual ideas into rather more practical
ones. One trial concerns the drug cyclosporine A (CsA) used
in ointment for the topical treatment of psoriasis.56 Previous
formulations were ineffective due to the impermeable nature
of the skin. However, a CPP-based formulation permits skin
penetration. A polyarginine heptamer with a pH-sensitive
linker allows the release of CsA at functional pH within
the skin. Phase 1 trials were successful. An unconfirmed
Internet source has stated that the phase 2 trials of
‘Psorban’ are still underway.57 The limited information
available on this drug prevents further comment.
Despite the enormous gaps in our understanding, experiments continue to demonstrate the highly desirable features
of CPPs. Existing restrictions on the structures of therapeutic
agents could be lifted as a consequence of CPP-mediated
delivery. The range of potential therapeutic targets could
greatly expand as a result. A similar effect was seen in the
wake of commercial prodrug manufacture. Until the complete picture is revealed however, the therapeutic potential
of CPPs cannot be realized, only predicted.
References
1. Derossi D et al. (1996) Cell internalization of the third helix of the
Antennapedia homeodomain is receptor-independent. J Biol Chem 271:
18188 –18193.
2. Kerkis A, Hayashi MA, Yamane T, Kerkis I (2006) Properties of cell penetrating
peptides (CPPs). IUBMB Life 58: 7 –13.
3. Drin G, Cottin S, Blanc E et al. (2003) Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem 278: 31192 –31201.
4. Vives E (2005) Present and future of cell-penetrating peptide mediated
delivery systems: ‘is the Trojan horse too wild to go only to Troy?’
J Control Release 109: 77–85.
5. Hauber J, Perkins A, Heimer EP et al. (1987) Trans-activation of human immunodeficiency virus gene expression is mediated by nuclear events. Proc Natl
Acad Sci USA 84: 6364 –6368.
6. Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human
immunodeficiency virus. Cell 55: 1189 –1193.
7. Green M, Loewenstein PM (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.
Cell 55: 1179 –1188.
8. Joliot A, Pernelle C, Deagostini-Bazin H et al. (1991) Antennapedia homeobox peptide regulates neural morphogenesis. Proc Natl Acad Sci USA 88:
1864 –1868.
9. Derossi D, Joliot AH, Chassaing G et al. (1994) The third helix of the
Antennapedia homeodomain translocates through biological membranes.
J Biol Chem 269: 10444 –10450.
10. Joliot A, Prochiantz A (2004) Transduction peptides: from technology to
physiology. Nat cell Biol 6: 189– 196.
11. Theodore L et al. (1995) Intraneuronal delivery of protein kinase C pseudosubstrate leads to growth cone collapse. J Neurosci 15: 7158 –7167.
12. Fawell S et al. (1994) Tat-mediated delivery of heterologous proteins into
cells. Proc Natl Acad Sci USA 91: 664 –668.
13. Trehin R, Merkle HP (2004) Chances and pitfalls of cell penetrating peptides
for cellular drug delivery. Eur J Pharm Biopharm 58: 209– 223.
14. Richard JP et al. (2003) Cell-penetrating peptides. A reevaluation of the
mechanism of cellular uptake. J Biol Chem 278: 585 –590.
15. Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide
enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat
Med 10: 310 –315.
16. Kang SH, Cho MJ, Kole R (1998) Up-regulation of luciferase gene expression
with antisense oligonucleotides: implications and applications in functional
assay development. Biochemistry 37: 6235 –6239.
17. Rousselle C et al. (2000) New advances in the transport of doxorubicin
through the blood-brain barrier by a peptide vector-mediated strategy.
Mol Pharmacol 57: 679 –686.
18. Foerg C, Merkle HP (2007) On the biomedical promise of cell penetrating
peptides: limits versus prospects. J Pharm Sci 97: 144 –162.
19. Carrasco MP, Jimenez-Lopez JM, Segovia JL et al. (2007) Effects of ethanol on
the remodeling of neutral lipids and phospholipids in brain mitochondria
and microsomes. Neurochem Int 50: 858 –865.
20. Thoren PE, Persson D, Karlsson M et al. (2000) The antennapedia peptide
penetratin translocates across lipid bilayers – the first direct observation.
FEBS letters 482: 265 –268.
21. Jarver P, Langel U (2006) Cell-penetrating peptides – a brief introduction.
Biochim Biophys Acta 1758: 260 –263.
22. Kawasaki-Nishi S, Nishi T, Forgac M (2001) Yeast V-ATPase complexes
containing different isoforms of the 100-kDa a-subunit differ in
coupling efficiency and in vivo dissociation. J Biol Chem 276:
17941 –17948.
23. Vives E, Brodin P, Lebleu B (1997) A truncated HIV-1 Tat protein basic
domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272: 16010 –16017.
24. Deshayes S et al. (2004) On the mechanism of non-endosomial peptidemediated cellular delivery of nucleic acids. Biochim Biophys Acta 1667: 141–147.
25. Crombez L et al. (2007) A non-covalent peptide-based strategy for siRNA
delivery. Biochem Soc Trans 35: 44 –46.
26. El-Andaloussi S, Johansson HJ, Lundberg P et al. (2006) Induction of splice
correction by cell-penetrating peptide nucleic acids. J Gene Med 8:
1262 –1273.
27. Drin G et al. (2001) Physico-chemical requirements for cellular uptake of
pAntp peptide. Role of lipid-binding affinity. Eur J Biochem/FEBS 268:
1304 –1314.
.........................................................................................................................................................................................................................................
71
Review
Bioscience Horizons † Volume 2 † Number 1 † March 2009
.........................................................................................................................................................................................................................................
28. Belting M (2003) Heparan sulfate proteoglycan as a plasma membrane
carrier. Trends Biochem Sci 28: 145 –151.
43. Oka T et al. (2006) Cardiac-specific deletion of Gata4 reveals its requirement
for hypertrophy, compensation, and myocyte viability. Circ Res 98: 837 –845.
29. Console S, Marty C, Garcia-Echeverria C et al. (2003) Antennapedia and HIV
transactivator of transcription (TAT) ‘protein transduction domains’ promote
endocytosis of high molecular weight cargo upon binding to cell surface
glycosaminoglycans. J Biol Chem 278: 35109 –35114.
44. Bisping E et al. (2006) Gata4 is required for maintenance of postnatal cardiac
function and protection from pressure overload-induced heart failure. Proc
Natl Acad Sci USA 103: 14471 –14476.
30. Fittipaldi A et al. (2003) Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J Biol Chem 278: 34141 –34149.
31. Ross MF, Filipovska A, Smith RA et al. (2004) Cell-penetrating peptides do not
cross mitochondrial membranes even when conjugated to a lipophilic
cation: evidence against direct passage through phospholipid bilayers.
Biochem J 383: 457–468.
45. Frey N, Olson EN (2003) Cardiac hypertrophy: the good, the bad, and the
ugly. Annu Rev Physiol 65: 45 –79.
46. Wilkins BJ, Molkentin JD (2002) Calcineurin and cardiac hypertrophy: where
have we been? Where are we going? J Physiol 541: 1–8.
47. Cao G et al. (2002) In vivo delivery of a Bcl-xL fusion protein containing the
Tat protein transduction domain protects against ischemic brain injury and
neuronal apoptosis. J Neurosci 22: 5423 –5431.
32. Jones SW et al. (2005) Characterisation of cell-penetrating peptide-mediated
peptide delivery. Br J Pharmacol 145: 1093 –1102.
48. Kilic E, Dietz GP, Hermann DM et al. (2002) Intravenous TAT-Bcl-Xl is protective after middle cerebral artery occlusion in mice. Ann Neurol 52: 617 –622.
33. Foerg C et al. (2005) Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape.
Biochemistry 44: 72 –81.
49. Harada H, Hiraoka M, Kizaka-Kondoh S (2002) Antitumor effect of
TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically
stabilized and activated in hypoxic tumor cells. Cancer Res 62: 2013 –2018.
34. Fischer R, Kohler K, Fotin-Mleczek M et al. (2004) A stepwise dissection of the
intracellular fate of cationic cell-penetrating peptides. J Biol Chem 279:
12625 –12635.
50. Rousselle C et al. (2003) Improved brain uptake and pharmacological activity
of dalargin using a peptide-vector-mediated strategy. J Pharmacol Exp Ther
306: 371– 376.
35. Al-Taei S et al. (2006) Intracellular traffic and fate of protein transduction
domains HIV-1 Tat peptide and octaarginine. Implications for their utilization
as drug delivery vectors. Bioconjug Chem 17: 90 –100.
51. Kerkis A et al. (2004) Crotamine is a novel cell-penetrating protein from the
venom of rattlesnake Crotalus durissus terrificus. FASEB J 18: 1407 –1409.
36. URL1: Active Motif: Tools to analyse nuclear function. ,http://www.activemotif.com/catalog/37/chariot-protein-delivery-reagent/description.html
[Accessed September 2008].
37. Akhtar S (2006) Non-viral cancer gene therapy: beyond delivery. Gene Ther
13: 739 –740.
38. Bian J et al. (2007) Effect of cell-based intercellular delivery of
transcription factor GATA4 on ischemic cardiomyopathy. Circ Res 100:
1626 –1633.
52. Morris MC, Vidal P, Chaloin L et al. (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res 25: 2730–2736.
53. Brugidou J, Legrand C, Mery J et al. (1995) The retro-inverso form of a
homeobox-derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system. Biochem
Biophys Res Commun 214: 685 –693.
54. Jarver P, Langel U (2004) The use of cell-penetrating peptides as a tool for
gene regulation. Drug Discov Today 9: 395 –402.
39. Snyder EL, Dowdy SF (2004) Cell penetrating peptides in drug delivery.
Pharm Res 21: 389 –393.
55. Wender PA et al. (2000) The design, synthesis, and evaluation of molecules
that enable or enhance cellular uptake: peptoid molecular transporters. Proc
Natl Acad Sci USA 97: 13003 –13008.
40. Elliott G, O’Hare P (1999) Intercellular trafficking of VP22-GFP fusion proteins.
Gene Ther 6: 149 –151.
56. Price VH (2003) Therapy of alopecia areata: on the cusp and in the future.
Journal Invest Dermatol Symp Proc 8: 207–211.
41. Schwarze SR, Ho A, Vocero-Akbani A et al. (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:
1569 –1572.
57. URL2: New and emerging psoriasis treatment information from Psoriasis Cure
Now. ,http://psoriasis-cure-now.org/hope.php [Accessed September 2008].
42. Muratovska A, Eccles MR (2004) Conjugate for efficient delivery of short
interfering RNA (siRNA) into mammalian cells. FEBS Lett 558: 63–68.
58. Alberts B, Johnson A, Lewis J et al. (2002) Molecular Biology of the Cell, 4th
edn, Chapter 13. Intracellular Vesicular Traffic. Accessed online at www.ncbi.
nlm.gov. [accessed September 2008].
........................................................................................................................................................................................................................................
Submitted on 30 September 2008; accepted on 18 January 2009; advance
access publication 17 February 2009
.........................................................................................................................................................................................................................................
72