???????? ??? ???????
peptide-mediated drug smuggling: A proof of mechanism for trypanosomatid
Juan Rom´ an Luque-Ortega, Beatriz G. de la Torre, Valent´ ın Hornillos,
Jean-Mathieu Bart, Cristina Rueda, Miguel Navarro, Francisco Amat-Guerri,
A. Ulises Acu˜ na, David Andreu, Luis Rivas
To appear in:
Journal of Controlled Release
20 February 2012
11 May 2012
Please cite this article as: Juan Rom´ an Luque-Ortega, Beatriz G. de la Torre, Valent´ ın
Hornillos, Jean-Mathieu Bart, Cristina Rueda, Miguel Navarro, Francisco Amat-Guerri,
A. Ulises Acu˜ na, David Andreu, Luis Rivas, Defeating Leishmania resistance to Mil-
tefosine (hexadecylphosphocholine) by peptide-mediated drug smuggling: A proof of
mechanism for trypanosomatid chemotherapy, Journal of Controlled Release (2012), doi:
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Defeating Leishmania resistance to Miltefosine (hexadecylphosphocholine) by
peptide-mediated drug smuggling: a proof of mechanism for trypanosomatid
Luque-Ortega, Juan Román a, de la Torre, Beatriz G. b, Hornillos, Valentín c,d, Bart,
Jean-Mathieu e, Rueda, Cristina a, Navarro, Miguel e, Amat-Guerri, Francisco c†, Acuña,
A. Ulises d, Andreu, David b,*, Rivas, Luis a,*.
aCentro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid,
bUniversitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona, Dr. Aiguader 88,
08003 Barcelona, Spain.
cInstituto de Química Orgánica General, CSIC, Juan de la Cierva 3, 28006 Madrid,
dInstituto de Química Física “Rocasolano”. CSIC, Serrano, 28006, Madrid, Spain.
eInstituto de Parasitología “López Neyra”, CSIC, Conocimiento s/n, 18100 Armilla,
† This work is dedicated to the memory of Prof. Francisco Amat-Guerri, who passed
away during the preparation of this article.
Luis Rivas, Centro de Investigaciones Biológicas(CSIC). Ramiro de Maeztu 9, 28040-
Madrid Spain. E-mail: firstname.lastname@example.org. Phone: +34918373112.
David Andreu, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona,
Dr. Aiguader 88, 08003 Barcelona, Spain, E-mail: email@example.com, Phone
Conflict of interest.- The authors declare the total absence of conflict of interests in the
Miltefosine (hexadecylphosphocholine, HePC), the first orally active drug successful
against leishmaniasis, is especially active on the visceral form of the disease. Resistance
mechanisms are almost exclusively associated to dysfunction in HePC uptake systems.
In order to evade the requirements of its cognate receptor/translocator, HePC-resistant
Leishmania donovani parasites (R40 strain) were challenged with constructs consisting
of an ω-thiol-functionalized HePC analogue conjugated to the cell-penetrating peptide
(CPP) Tat(48-60), either through a disulfide or a thioether bond. The conjugates enter
and kill both promastigote and intracellular amastigote forms of the R40 strain.
Intracellular release of HePC by reduction of the disulfide-based conjugate was
confirmed by means of double tagging at both the CPP (Quasar 670) and HePC
(BODIPY) moieties. Scission of the conjugate, however, is not mandatory, as the
metabolically more stable thioether conjugate retained substantial activity. The disulfide
conjugate is highly active on the bloodstream form of Trypanosoma b. brucei, naturally
resistant to HePC. Our results provide proof-of-mechanism for the use of CPP
conjugates to avert drug resistance by faulty drug accumulation in parasites, as well as
the possibility to extend chemotherapy into other parasites intrinsically devoid of
membrane translocation systems.
Key words: Miltefosine, Leishmania, Trypanosoma, Tat, resistance reversion, cell-
substantial literature on CPPs focuses mostly on the release into mammalian [16, 17],
much less on protozoan cells [18-20]. For trypanosomatids like Leishmania in
particular, a plasma membrane structure and functionality quite unlike that of
mammalian cells may underlie the relative paucity and lack of conclusiveness of reports
on CPP applications for these parasites.
In the present work, we have used Tat(48-60) (GRKKRRQRRRPPQ-amide), a
CPP prototypical for mammalian cells, to redress the failure of HePC-resistant R40 L.
donovani strain to uptake HePC, and to expand the range of HePC-susceptible parasites
into the naturally HePC-resistant African trypanosome Trypanosoma brucei brucei. Our
approach uses conjugates with either a redox-scissile (disulfide) or stable (thioether)
linkage of HePC to the CPP, which enter and kill R40 parasites. For the disulfide-linked
conjugate, intracellular reductive release of HePC from the CPP was established, by
double tagging of both HePC and Tat moieties with fluorescent dyes. Thus, by
disregarding the non-functional LdMT transporter, CPP vector-based delivery
overcomes HePC resistance and facilitates the killing of R40 promastigotes. Our
Among human protozoan diseases, leishmaniasis ranks second only to malaria in terms
of mortality and morbidity, with an incidence of 2 million cases a year in 2010, of
which 0.5 million correspond to visceral leishmaniasis (VL), fatal if untreated . A
reliable human vaccine remains elusive, and chemotherapy has traditionally relied on
pentavalent antimonials, dating back over 50 years and imperiled by resistance
phenomena in endemic foci . More recently miltefosine (hexadecylphosphocholine,
HePC), liposomal amphotericin B and paromomycin  have been added as first-line
drugs. Clearly, novel therapeutic approaches, particularly those aimed at bypassing or
inhibiting resistance mechanisms and currently limited to combination therapies , are
required to curb this negative scene.
HePC, the only leishmanicidal clinical drug orally active on VL, is currently
administered as an alternative to antimonials in areas of India where resistance to these
drugs is rampant. Although no HePC-resistant clinical isolates have yet been reported,
they can somehow be anticipated given that (i) resistant strains are relatively easy to
generate in the laboratory ; (ii) in animal models, resistant parasites retain the
virulence of the wild type ; (iii) partial cross-resistance with antimonials has been
observed in both laboratory  and field  isolates, and (iv) irregular treatment
compliance and poor outpatient surveillance (hospitalization is only required in extreme
cases) may eventually succeed in generating resistance in the field.
HePC resistance is easily induced by growing Leishmania parasites under
increasing drug concentrations . Although substantial (low mM) intracellular HePC
concentrations are rapidly achieved in susceptible promastigotes, a resistant phenotype
can soon be observed , characterized by a severely diminished intracellular HePC
concentration, due to overexpressed efflux pumps  and, to a much larger extent, to
the failure of the HePC-dedicated transporter, the aminophospholipid translocase LdMT
(Leishmania donovani miltefosine transporter). Variations in subunit expression of this
uptake system underlie Leishmania HePC susceptibility, at both inter- and intra-species
level . This is yet another example of how the selective permeability of plasma
membrane to various bioactive molecules poses a serious obstacle for effective drug
targeting . In such a scenario, the development of molecular tools for bypassing the
resistance posed by faulty uptake mechanisms becomes a highly desirable goal.
Cell-penetrating peptides (CPPs) are ideal candidates in this regard, due to their
ability to transport various bioactive cargo molecules –complexed or covalently
conjugated– across plasma or endosomal membranes into target cells [13-15]. The
construct also penetrates and reduces the parasite load of macrophages infected with the
R40 strain, predictably by accumulation within the intracellular amastigotes followed by
reductive cleavage and HePC release.
This first account –to the best of our knowledge–of reversing the therapeutic
failure of a leishmanicidal drug by CPP-mediated delivery constitutes proof-of-
mechanism of the potential of CPP vehicles in trypanosomatid chemotherapy.
Materials and Methods
HePC-Tat conjugates. The synthesis of HePC analogues with ω-thiol-functionalization
(HePC-SH) and with an internal BODIPY tag has been previously described [21, 22].
For the present work, an analogue featuring both thiol and BODIPY groups (HePC-
BODIPY-SH) was synthesized. Both HePC-SH and HePC-BODIPY-SH building
blocks were subsequently conjugated to a Tat(48-60) sequence (hereafter named Tat)
bearing (or not) a Quasar 670 fluorescent moiety and appropriate modifications to allow
conjugation. Tat and its variants were conveniently made by Fmoc-based solid phase
peptide synthesis methods and were conjugated to HePC-BODIPY either via disulfide
(HePC-BODIPY-SS-Tat, reduction-sensitive) or thioether (HePC-BODIPY-S-Tat,
reduction-stable) linkages (see Scheme 1 for structures). In the first case, the disulfide
conjugate resulted from reaction of HePC-BODIPY-SH with the Npys (3-nitro-2-
pyridylsulfenyl)-activated thiol group of a Cys residue added to the N-terminus of Tat.
For the thioether conjugate, Tat was N-terminally elongated –while still on the solid
phase– with an ε-Mmt (4-methoxytrityl)-protected Lys residue, followed by selective
deprotection of the ε-amino group, acylation with chloroacetic acid and deprotection/
cleavage. After purification, the resulting chloroacetyl-functionalized Tat underwent
nucleophilic substitution by HePC-BODIPY-SH to give the target thioether conjugate.
A detailed description of the synthesis of these constructs exceeds the scope of this
paper and will be given elsewhere. For Tat and all conjugates, purities of 95% or higher
were established by HPLC, and identities satisfactorily confirmed by MALDI-TOF MS.
Scheme 1.- Structures of the HePC surrogates
was followed, except that Giemsa staining was substituted by Hoechst 342, as detailed
below. BALB/c peritoneal macrophages were seeded in a Lab-Tek™ 8-well
Permanox™ chamber slide system, at 105 cells/well in RPMI 1640 + 10% HIFCS, and
incubated for 6 h with R40 L. donovani promastigotes at a 1:10
macrophage:promastigote ratio. After removal of unbound parasites, infection was
allowed to progress for 72 h at 37°C, followed by treatment with HePC-SS-Tat or HePC
for 48 h in the same culture medium. Cells were finally stained with 10 µg/ml of the
vital dye Hoechst 342 for 45 min, fixed in 4% (w/v) paraformaldehyde, mounted with
Mowiol and observed in a Leitz Dialux 20 fluorescence microscope coupled to a Leica
DFC340FX CCD camera, using a standard DAPI filter. The infection index, taken as
the amastigote: macrophage ratio, averaged over 200 macrophages regardless of
whether they were infected or not, was measured in triplicate.
Assessment of plasma membrane permeabilization. Alterations in the plasma
membrane permeability were evaluated by vital dye entrance . Briefly, R40
Cell culture. L. donovani promastigotes (strain MHOM/ET/67/L82) (WT), and the
HePC resistant strain MHOM/ET/67/L82R40 (R40), kindly provided by Prof. S. L.
Croft (London School of Hygiene and Tropical Medicine), were grown at 26 °C in
RPMI medium supplemented with 10% heat-inactivated fetal calf serum, gentamycin,
penicillin, and 2 mM glutamine. For the resistant strain, 40 µM HePC was added to the
growth medium to maintain the resistant phenotype. As standard procedure, prior to
each assay, Leishmania parasites were harvested at late exponential phase and washed
twice in Hanks balanced salt solution buffer supplemented with 10 mM D-glucose (pH
Peritoneal macrophages from BALB/c mice were obtained by sodium
thioglycolate elicitation of mice three days before extraction according to the protocol
approved by the animal welfare committee of CIB-CSIC; cells were harvested by
peritoneal washing of the intraperitoneal cavity, and maintained in RPMI+HIFCS under
5% CO2 atmosphere .
The bloodstream forms (BSF) of T. b. brucei 221 were cultured at 37 °C in 5%
CO2 in HMI-9 medium supplemented with 10% fetal bovine serum and 10% Serum
Plus (SAFC Biosciences, Spain, ) . Unless otherwise stated, samples were assayed
by triplicate, and experiments were repeated at least twice regardless of the parasite
Measurement of cytotoxic effects. For inhibition of proliferation, R40 promastigotes
were seeded in full growth medium devoid of phenol red at 2 × 106 cells/ml. Tat, HePC-
BODIPY, HePC-BODIPY-SS-Tat, and HePC-BODIPY-S-Tat, were dissolved in the
same buffer, and added at their corresponding concentrations to the parasite suspension.
Parasites were allowed to proliferate for 72 h at 26 ºC. Inhibition of proliferation was
assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
reduction (0.5 mg/ml final concentration) solubilized with 5% (w/v) SDS and detected
at 600 nm with a Bio-Rad microplate reader 640 .
T. b. brucei BSF were harvested at exponential phase, resuspended in growth
medium (2 106 cells/ml) into 96-well microplates and incubated with the different
reagents. After 24 h, resazurin (440 µM final concentration) was added to each well.
Fluorescence was read in a Varioskan Flash (Thermo Scientific) microplate reader (λexc
= 528 nm; λem = 590 nm).
For intracellular amastigote killing measurements, the protocol described in 
centrifugation, resuspended in 10 µl of TDB plus 40 µl of 0.8% low melting point
agarose at 37 ºC, and freshly observed in an Olympus R IX81in vivo fluorescence
microscope. At least 100 parasites in different fields were observed; pictures are
representative of the whole population.
HePC conjugation to Tat bypasses Leishmania HePC resistance. As a first step, we
tested whether the conjugation of HePC to Tat was able to abrogate resistance in R40
promastigotes. This is characterized by a faulty HePC uptake, as previously evidenced
using radioactive  or fluorescent HePC analogues [21, 28]. As shown in Fig. 1 the
HePC-BODIPY-SS-Tat conjugate inhibited proliferation of resistant R40 promastigotes
in a dose-dependent manner with an IC50 of 2.4 ± 0.8 µM, while incubation with either
HePC-BODIPY or Tat, added separately or together (molar ratio 1:1), was innocuous
(<10% inhibition of proliferation) up to 40 µM. In comparison, the IC50 for the WT
strain was slightly higher, 3.1 ± 0.9 µM.
promastigotes in complete growth medium (2 × 107 cells/ml) were treated with the
reagents and after 4 h propidium iodide (PI) (5 µg/ml, final concentration) was added.
After 15 min incubation, fluorescence was measured in a Polarstar Galaxy microplate
reader (λexc = 544 nm; λem = 612 nm). Results are expressed as the percentage of
fluorescence relative to parasites fully permeated with 0.1% Triton X-100 (TX-100).
Intracellular accumulation of fluorescent analogues in R40 promastigotes.
Promastigotes were resuspended in complete growth medium (2 × 107 cells/ml) devoid
of phenol red and incubated with the corresponding reagent for 3 h at 26ºC. Next,
parasites were washed twice with 10 mg/ml fatty acid-free bovine serum albumin
(BSA) in HBSS-Glc at 4 ºC, resuspended in 100 µl of the same buffer, transferred into a
96-well black microplate and lysed with SDS (1% final concentration). Fluorescence
was then measured in a Varioskan microplate reader (λexc = 480nm; λem =520 nm).
Results were compared to a standard curve for each of the different reagents
Intracellular distribution of fluorescent analogues. R40 promastigotes were
resuspended at 2 107 cells/ml in HBSS-Glc plus Hoechst 342 (10 µg/ml final
concentration). After 1 h, the different HePC reagents were added at 2.5 µM final
concentration and fluorescence observed up to 3 h on a Leica TCS-SP2-AOBS-UV
ultraspectral confocal microscope (Leica Microsystems, Heidelberg, Germany) without
fixation. Excitation/emission wavelengths were respectively 350/460 nm for Hoechst
342, 488/520 nm for BODIPY-labeled compounds, and 644/670 nm for the HePC-
BODIPY-SS-Tat (Quasar 670) conjugate.
BALB/c peritoneal macrophages, seeded on 35 mm glass bottom culture dishes
(MatTek), were infected with SNARF®-1 (carboxylic acid, acetate, succinimidyl ester)-
pre-stained R40 promastigotes (10 µM, 30 min) at a 1:10 macrophage:parasite ratio for
4 h in growth medium. Infection was allowed to progress for 48 h at 37 ºC, followed by
treatment with HePC-BODIPY or HePC-BODIPY-SS-Tat(Quasar 670) at 2.5 µM for
different time periods. Cells were washed and freshly observed on a Leica TCS SP2
confocal microscope. Excitation and emission wavelengths were as above; for SNARF-
1 visualization they were set at 514 and 578 nm, respectively.
For short-term observation of T. b. brucei BSFs, parasites were resuspended in
trypanosome dilution buffer (TDB)  at 107 cells/ml, supplemented with 1 µg/ml
DAPI. 40-µl aliquots of this suspension were incubated for 2 min with the
corresponding reagents at 5 µM concentration. Parasites were then harvested by
results on Fig. 2 we conclude: (i) HePC conjugation to Tat is required for entrance in
R40 promastigotes; (ii) uptake of Tat conjugates (both disulfide and thioether) was
greater for R40 than for WT parasites, with a slightly larger accumulation for the
thioether over the disulfide; (iii) in the WT strain, none of the conjugates challenged
HePC uptake by its cognate receptor, as evidenced by the higher IC50 of the disulfide
conjugate (see previous heading).
We next calculated HePC-BODIPY concentration inside R40 promastigotes.
Using the intracellular volume reported in the literature , a 0.8 ± 0.3 mM value was
determined, in the same range than other fluorescent  and radioactive HePC
The uptake process is highly dependent on temperature; at 4ºC it is only 18.9 ±
2.5% of that at standard 26ºC conditions. This is not unusual for CPPs such as Tat, for
which temperature-sensitive and –insensitive uptake mechanism e.g., endocytosis and
membrane translocation, respectively have been shown to coexist in mammalian cells,
their respective contribution depending on multiple factors [31, 32].
To discard that the lethal effect might result from membrane permeabilization by
the different reagents, entrance of the vital dye propidium iodide (PI) in R40
promastigotes was assayed. After 4 h incubation, the percentage of PI uptake (referred
to cells fully permeabilized with 0.1% TX-100) was 6.9 ± 3.0, 7.9 ± 2.8, 3.2 ± 2.3, 3.8 ±
2.5, and 2.6 ± 1.2, for HePC-BODIPY-SS-Tat, HePC-BODIPY-S-Tat, HePC-BODIPY,
unconjugated Tat + HePC-BODIPY (1:1 ratio), and Tat, respectively, while for
untreated promastigotes it was 1.3 ± 0.9. Thus, the lethal effect was clearly due to the
action of the drug once inside the parasite.
Figure 1. HePC resistance in R40 Leishmania donovani promastigotes is averted by
conjugation to Tat. Panel A: Inhibition of R40 proliferation by () HePC-BODIPY,
() HePC-BODIPY-SS-Tat, () HePC-BODIPY-S-Tat, () Tat and () Tat +
HePC-BODIPY (molar ratio 1:1).Panels B-D: Uptake of HePC-BODIPY, HePC-
BODIPY-SS-Tat and HePC-BODIPY-S-Tat, respectively, by R40 promastigotes.
Parasites were pre-stained with Hoechst 342 (blue fluorescence), then incubated for 3 h
with 2.5 µM BODIPY-labelled reagents. Excitation/emission wavelengths were
350/460 nm and 488/520 nm for Hoechst 342 and BODIPY, respectively. Bar =10 µm.
Tat promotes HePC-BODIPY uptake on HePC-resistant promastigotes. Having
discarded membrane permeabilization by HePC analogues as the cause of R40
promastigote killing, we proceeded to quantify uptake in both WT and R40 strains, with
the accumulation endpoint set up at 3 h to prevent massive parasite death. From the
pocket. After 3h incubation, substantial changes were noted: fluorescence spread to
most intracellular space of the parasite except the nucleus and the kinetoplast and, most
importantly, areas where a given fluorophore (green or red) prevailed could be observed
(Fig. 3) and assigned to HePC-BODIPY-SH or HS-Tat(Quasar 670) enrichment,
respectively, over either unsplit conjugate or other byproducts. Reductive intracellular
HePC release, however, is not required for leishmanicidal activity, as shown by HePC-
BODIPY-S-Tat, whose HePC and Tat moieties are connected by a metabolically stable
thioether bond, yet displays significant activity (IC50 = 5.9 ± 1.4 µM), not much unlike
its disulfide counterpart (IC50 =2.4 ± 0.8 µM, Fig 1).
Figure 2. Intracellular accumulation of HePC fluorescent analogues in WT
and R40 L. donovani promastigotes. Parasites were incubated witheither HePC-
BODIPY (square), HePC-BODIPY-SS-Tat (triangle), or HePC-BODIPY-S-Tat (circle)
for 3h in complete growth medium, as described in Materials and methods. Afterwards,
cells were lysed and dye was measured by fluorometry at λEXC = 480nm and λEM = 520
nm excitation and emission wavelengths . Filled and empty symbols stand for WT and
R40 promastigotes, respectively
A scissile HePC moiety is not mandatory for leishmanicidal activity. Our rationale
for choosing a disulfide conjugate was to ensure payload (HePC) release into the
cytoplasm by the redox system of the parasite. In order to confirm this point, a doubly
tagged conjugate, HePC-BODIPY-SS-Tat(Quasar 670), was synthesized, where the
green-emitting HePC-BODIPY moiety was linked to a non-overlapping, far red-
emitting Tat derivative bearing a Quasar 670 fluorophore (Scheme 1). Parasites
incubated with 2.5 µM conjugate in HBSS-Glc showed after 5 min a rather homogenous
fluorescence pattern, mostly associated to the plasma membrane and the flagellar
privileged localization and with nil accumulation inside intracellular R40 amastigotes.
In contrast, incubation with 2.5 µM HePC-BODIPY-SS-Tat(Quasar 670) under
identical conditions produced far more intense fluorescence, with both HePC-BODIPY
and Quasar 670 fluorophores co-localizing almost exclusively inside the intracellular
amastigotes (Fig. 4). Shorter incubation times (1h) produced a similar pattern, albeit
with lower intensity (not shown). Most importantly, infected macrophages treated with
2.5 µM HePC-BODIPY-SS-Tat showed a sharp decrease in parasite:macrophage ratio;
thus, whereas for untreated and HePC-BODIPY-treated macrophages averaged ratios
were 4.2 ± 0.7 and 3.9 ± 0.4, respectively (Supplemental material, Fig S1), for
conjugate-treated macrophages the ratio was 0.3 ± 0.2. To discard artifacts due to
fluorophore inclusion, non-fluorescent HePC-SH and HePC-SS-Tat  controls were
assayed and gave ratios of 5.1 ± 0.2 and 0.6 ± 0.4, respectively.
Figure 3. Time-dependence of HePC-BODIPY-SS-Tat(Quasar 670)
intracellular distribution in R40 parasites. Cells were pre-stained with Hoechst 342
and incubated with 2.5 µM HePC-BODIPY-SS-Tat(Quasar 670) for 5 min (A) or 3 h
(B), then observed unfixed by confocal microscopy. Excitation/ emission wavelengths
were: BODIPY fluorescence (green fluorescence), 488/520 nm; QUASAR 670 (red
fluorescence), 644/670 nm. Fluorescence settings for Hoechst 342 were 350/460 nm.
Bar =10 µm.
The HePC-SS-Tat conjugate abrogates HePC resistance in intracellular R40
amastigotes. In a further step, we assessed the efficacy of HePC-Tat conjugates on
intracellular amastigotes, as the intracellular form fully retained the HePC resistance
phenotype . To this end, peritoneal macrophages were infected with R40
promastigotes and, three days later, were challenged with either HePC-BODIPY-SS-
Tat(Quasar 670) or HePC-BODIPY and analyzed by confocal microscopy in unfixed
form. As shown in Fig. 4, live infected macrophages incubated overnight with 2.5 µM
HePC-BODIPY displayed only a faint, widely distributed fluorescence, with no
Figure 4. Effect of Tat conjugation on uptake by live R40-infected murine
peritoneal macrophages. BALB/c mouse peritoneal macrophages were infected with
SNARF-1-prelabeled R40 promastigotes and treated overnight with either HePC-
BODIPY-SS-Tat(Quasar 670) (A) or HePC-BODIPY (B), both 2.5 µM.
Excitation/emission wavelengths were: 514 /578 nm for SNARF-1 (blue fluorescence,
false colored to highlight differences with QUASAR 670), 488/520 nm for BODIPY
(green fluorescence), and 644/670 for QUASAR 670. Bar = 25 µm.
Conjugation to Tat broadens HePC activity spectrum into the bloodstream forms
of African trypanosomes. Bloodstream trypomastigotes of T.b. brucei are much less
susceptible to HePC than L. donovani promastigotes . Upon 15 min incubation of
BSF with 5 µM HePC-BODIPY-SS-Tat, these parasites showed an initial fluorescence
pattern, mostly accumulated at a defined spot, most likely the lysosome (Fig. 5, column
A). After 90 min, fluorescence had spread throughout the cell body (Fig 5, column B)
and the parasite was devoid of movement and with severe structural damage leading to
death (Fig. 5)
parasites is confined to the work of Corradin et al. , who conjugated a
leishmanolysin substrate peptide to Tat to inhibit the proteolytic activity of the enzyme;
while the construct was successfully translocated, it did not decrease parasite viability.
In this work, we demonstrate the efficacy of HePC-CPP conjugates in averting
resistance in Leishmania. This model constitutes an excellent proof-of-mechanism
because, in the R40 strain we have used, HePC resistance is almost exclusively
associated to deficient drug uptake, hence recovered lethality is univocally linked to
efficient drug delivery by Tat. Identical CPP-drug conjugate strategy was used to
overcome resistance to daunomycin , methotrexate  or doxorubicin [41, 42] in
To visualize drug entrance and localization we have used HePC-BODIPY, a new
analogue that (i) preserves key structural features of HePC such as a phosphocholine
polar head group or a long (18 carbon atoms, vs. 16 in HePC) alkyl chain, (ii) has
improved photostability over previous fluorescent HePC derivatives , and (iii)
retains the leishmanicidal activity of HePC as well as the recognition by its cognate
transporter in Leishmania.
Figure 5. HePC-BODIPY-SS-Tat uptake by T. b. brucei trypomastigotes. Parasites
resuspended in trypanosome dilution buffer (107 cells/ml) were prelabeled with 1 µg/ml
DAPI (blue) (350 nm/460 nm and incubated with 5 µM HePC-BODIPY-SS-Tat (green)
for 15 (column A) or 90 min (column B). Excitation/emission wavelengths: 350/460nm
and 488/520 nm for DAPI and BODIPY respectively. Bar = 10 µm.
Translocation across trypanosomatid membranes has been documented for some
antimicrobial peptides in Leishmania [34-36], as well as for standard CPPs such as
TP10 in Trypanosoma brucei , or Tat in Leishmania . Nevertheless, to the best
of our knowledge the use of CPPs for release of chemotherapeutic reagents into
obtain efficient interaction of HePC with its targets upon intracellular reductive
cleavage of the disulfide, a process that in trypanosomatids is enhanced by the high
redox potential of trypanothione. That this was indeed the case was confirmed by the
time-dependent changes in intracellular fluorescence of parasites challenged with the
dual tagged conjugate HePC-BODIPY-SS-Tat(Quasar 670). The structure of the
conjugate ruled out fluorophore separation by proteolysis instead of disulfide reduction.
Even so, cleavage is not mandatory for HePC activity, as the non-cleavable thioether
conjugate HePC-BODIPY-S-Tat is also active. Although metabolically stable drug-CPP
linkages may severely affect the activity of the cargo molecule , a thioether is
advantageous over the disulfide in that it prevents promiscuous exchange with surface
thiol groups . It is worth noting that accumulation of the thioether conjugate is
slightly higher than the disulfide, albeit with lower toxicity for the parasite. Given the
variety of targets reported for HePC in Leishmania [60, 61] and the intracellular
concentrations in the low-mM range achieved by the conjugates (see above), interaction
with even low affinity targets must be expected. While the non-cleavable thioether bond
the resistant strain; the HePC-BODIPY-SS-Tat conjugate entered R40 promastigotes
while unconjugated drug showed only a faint fluorescence associated to parasite
membrane. Although Tat has some activity on bacteria and fungi [43-45], its IC50 values
in the present system, much above those of the conjugate, discard any determining
effect in the leishmanicidal activity.
Endocytosis accounts for most Tat uptake in Leishmania. The special features of
its plasma membrane exclude macropinocytosis, as for other trypanosomatids , and
genome mining appears to rule out also caveolin-mediated endocytosis from the feasible
uptake pathways. Clathrin-mediated endocytosis, on the other hand, is essential for T.b.
brucei [47, 48] and may be in effect in the present case. The uptake mechanism
involves a receptor, which for Tat in mammalian cells is an anionic glycosaminoglycan
such as heparan sulfate [49, 50], although a direct role for it in CPP membrane
translocation has been questioned . On the other hand, about one fifth of the total
uptake was still taking place at low temperature, suggesting a simple crossover of the
plasma membrane by the conjugate, aside from energy-demanding mechanisms. Indeed,
Tat translocation to the lumen of giant plasma membrane vesicles has been reported
, and histatin 5, a leishmanicidal peptide targeting mitochondria, is known to
translocate across the plasma membrane of the parasite . Also, in CHO mammalian
cells direct translocation has been found to coexist with endocytosis, and to be favored
for conjugates with low molecular weight cargoes . In the present case, this pathway
might be additionally facilitated by the considerable hydrophobicity of the HePC moiety
of the conjugate, which would increase partition into the membrane . Direct
interaction between the membrane and the conjugate may also explain the small
increase in uptake of R40 vs. WT promastigotes. The higher phosphatidylethanolamine
content of the plasma membrane external leaflet  would favor negative membrane
curvature, hence Tat translocation by formation of an inverted micelle 
In trypanosomatids, particularly African BSFs, endocytic membrane turnover
rates are high enough to make a specific receptor unnecessary , a situation that may
explain the results found for T. brucei in this work; hence, simple electrostatic binding
of Tat to the external anionic phospholipids of the plasma membrane will suffice for
engulfment through endocytic entrance .
The type of linkage between a CPP and its cargo molecule may affect the
intracellular fate of the conjugate, particularly the interaction of the payload with its
target . Our choice of a disulfide bond  for HePC-Tat conjugation aimed to
Conjugation to Tat is required to restore the leishmanicidal activity of HePC on
that in Leishmania is only second in importance to faulty uptake. The impact of this
pathway in the present model is currently under investigation. A second benefit might
reside in the reported preferential accumulation of CPPs in the liver and the spleen ,
both main reservoirs for visceral leishmaniasis.
To summarize, we have provided proof-of-mechanism for the fitness of Tat and other
CPPs as vectors for otherwise hard-to-deliver leishmanicidal drugs, using Tat-
conjugated miltefosine to avert resistance in Leishmania parasites. Secondly, release of
the drug by the parasite redox system can be visualized by a doubly tagged conjugate.
Finally, we have shown how this strategy can broaden the range of susceptible
pathogens. Nonetheless, several issues remain to be solved before the potential of this
approach in trypanosomatid chemotherapy is fully realized. Organelle specificity, for
one thing, must be addressed, possibly by inclusion of structural motifs whose exclusive
recognition by macrophage receptors improves selective uptake, as reported for
branched poly-lysine carriers conjugated to methotrexate [66, 67]. Particulate
presentation of conjugates, a strategy exploiting the high endocytosis rate of
may hamper some of these interactions, the substantial fraction remaining is likely to
inflict damage to the parasite.
We have also explored the activity of HePC conjugates on intracellular
amastigotes, the pathological form of the parasite in vertebrate hosts. As R40 parasites
showed close-to-nil uptake of non-conjugated drug, accumulation in intracellular
amastigotes requires that the conjugate, in full or in part, arrive intact to the
parasitophorous vacuole where amastigotes reside. Since, to the best of our knowledge,
Leishmania does not induce a preferential permeability of this parasitophorous vacuole
which might account for the privileged accumulation of both Tat and HePC, the more
plausible explanation is that, similar to other mammalian cells , the typical pathway
is followed of (i) interaction of conjugate with the macrophage membrane, presumably
via glucosaminoglycans, and (ii) subsequent inclusion into an endosomal vacuole that
fuses with the parasitophorous vacuole. The alternative, direct translocation across the
macrophage membrane and further traffic into the parasitophorous vacuole, is most
unlikely, since to reach the parasite the conjugate would have to cross both plasma and
parasitophorous vacuole membranes and, additionally, risks reductive cleavage by
glutathione in the macrophage cytoplasm. A dedicated transporter for cytoplasmic
HePC into the parasitophorous vacuole remains to be described, even less for the Tat
moiety. Nonetheless, the faint fluorescence pattern observed in the cytoplasm of the
macrophage might be likely ascribed to this secondary pathway.
The HePC-BODIPY-SS-Tat conjugate was also tested on the BSFs of T. b.
brucei, an extracellular parasite endowed with a natural higher resistance to HePC and
other alkyl lysophospholipids . The conjugate accumulated first in the flagellar
pocket and then spread on the entire parasite, with ensuing death. Bloodstream
trypomastigotes have an extremely high endocytic activity, and may accumulate HePC
non-specifically adsorbed in the membrane; toxic effects, however, are much lower than
for the conjugate. It is worth noting that, after conjugate incubation, an internal giant
vesicle, phenotypically similar to the big-eye phenotype, was formed. This is typical of
blocked endocytosis, but has also described for parasite knockouts for neutral
sphingomyelinase , an endoplasmic reticulum enzyme inhibited in vitro by HePC,
hence it is tempting to speculate that HePC-Tat conjugation may afford intracellular
access to this enzyme.
There may be additional advantages to the metabolically stable (thioether-
linked) HePC-Tat conjugates. For instance, they may attenuate the effect of transporters
promoting the efflux of intracellular drug [10, 63, 64], a mechanism of HePC resistance
favours the acquisition of multidrug resistance in Leishmania, Cell Death Dis., 2 (2011)
 D. Kumar, A. Kulshrestha, R. Singh, P. Salotra, In vitro susceptibility of field
isolates of Leishmania donovani to Miltefosine and amphotericin B: correlation with
sodium antimony gluconate susceptibility and implications for treatment in areas of
endemicity, Antimicrob. Agents Chemother., 53 (2009) 835-838.
 F.J. Pérez-Victoria, F. Gamarro, M. Ouellette, S. Castanys, Functional cloning of the
miltefosine transporter. A novel P-type phospholipid translocase from Leishmania
involved in drug resistance, J. Biol. Chem, 278 (2003) 49965-49971.
 J.M. Pérez-Victoria, F. Cortés-Selva, A. Parodi-Talice, B.I. Bavchvarov, F.J.
Pérez-Victoria, F. Muñoz-Martnez, M. Maitrejean, M.P. Costi, D. Barron, A. Di Pietro,
S. Castanys, F. Gamarro, Combination of suboptimal doses of inhibitors targeting
different domains of LtrMDR1 efficiently overcomes resistance of Leishmania spp. to
Miltefosine by inhibiting drug efflux, Antimicrob. Agents Chemother., 50 (2006) 3102-
macrophages to increase drug concentration in the parasitophorous vacuole [68-70],
also needs to be further explored. Finally, CPPs with intrinsic leishmanicidal properties
[e.g.,VIP or histatin 5 ] appear to be promising in that the synergy between
payload and carrier may reduce the amount of drug required. In conclusion, while open
questions remain about the practical application of CPPs, it is fair to say that workable
routes for circumventing such problems also exist, opening new chemotherapeutic
prospects into a field where they are sorely needed.
Research supported by funds from the European Union (HEALTH-2007-223414 to L.R.
and D.A.), Fondo de Investigaciones Sanitarias (RICET RD 06/0021/0006 and PI09-
01928 to L.R.; RD 06/0021/0010 to M.N.), MICINN (BIO2008-04487-CO3 to D.A.)
and Generalitat de Catalunya (SGR09-00492 to D.A.). and CTQ2010-16457 (AUA) and
 Control of the leishmaniases, World Health Organ Tech Rep Ser, (2010) xii-xiii, 1-
186, back cover.
 F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, S. Rijal, R.W. Peeling, J. Alvar, M.
Boelaert, Visceral leishmaniasis: what are the needs for diagnosis, treatment and
control?, Nature Rev. Microbiol., 5 (2007) 873-882.
 M.L. den Boer, J. Alvar, R.N. Davidson, K. Ritmeijer, M. Balasegaram,
Developments in the treatment of visceral leishmaniasis, Expert. Opin. Emerg. Drugs,
14 (2009) 395-410.
 J. van Griensven, M. Balasegaram, F. Meheus, J. Alvar, L. Lynen, M. Boelaert,
Combination therapy for visceral leishmaniasis, Lancet Infect. Dis., 10 (2010) 184-194.
 F.J. Pérez-Victoria, S. Castanys, F. Gamarro, Leishmania donovani resistance to
miltefosine involves a defective inward translocation of the drug, Antimicrob. Agents
Chemother., 47 (2003) 2397-2403.
 K. Seifert, F.J. Pérez-Victoria, M. Stettler, M.P. Sánchez-Cañete, S. Castanys, F.
Gamarro, S.L. Croft, Inactivation of the miltefosine transporter, LdMT, causes
miltefosine resistance that is conferred to the amastigote stage of Leishmania donovani
and persists in vivo, Int. J. Antimicrob. Agents, 30 (2007) 229-235.
 W. Moreira, P. Leprohon, M. Ouellette, Tolerance to drug-induced cell death
remodeling of expression sites, EMBO J., 18 (1999) 2265-2272.
 J.R. Luque-Ortega, S. Martínez, J.M. Saugar, L.R. Izquierdo, T. Abad, J.G. Luis, J.
Piñero, B. Valladares, L. Rivas, Fungus-elicited metabolites from plants as an enriched
source for new leishmanicidal agents: antifungal phenyl-phenalenone phytoalexins from
the banana plant (Musa acuminata) target mitochondria of Leishmania donovani
promastigotes, Antimicrob. Agents Chemother., 48 (2004) 1534-1540.
 J.R. Luque-Ortega, O.M. Rivero-Lezcano, S.L. Croft, L. Rivas, In vivo monitoring
of intracellular ATP levels in Leishmania donovani promastigotes as a rapid method to
screen drugs targeting bioenergetic metabolism, Antimicrob. Agents Chemother., 45
 C.G. Grunfelder, M. Engstler, F. Weise, H. Schwarz, Y.D. Stierhof, G.W. Morgan,
M.C. Field, P. Overath, Endocytosis of a glycosylphosphatidylinositol-anchored protein
via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via
RAB11-positive carriers, Mol. Biol. Cell, 14 (2003) 2029-2040.
 M.P. Sánchez-Cañete, L. Carvalho, F.J. Pérez-Victoria, F. Gamarro, S. Castanys,
Low plasma membrane expression of the miltefosine transport complex renders
Leishmania braziliensis refractory to the drug, Antimicrob. Agents Chemother., 53
 K. Sugano, M. Kansy, P. Artursson, A. Avdeef, S. Bendels, L. Di, G.F. Ecker, B.
Faller, H. Fischer, G. Gerebtzoff, H. Lennernaes, F. Senner, Coexistence of passive and
carrier-mediated processes in drug transport, Nat. Rev. Drug Discov., 9 (2010) 597-614.
 A. Chugh, F. Eudes, Y.S. Shim, Cell-penetrating peptides: Nanocarrier for
macromolecule delivery in living cells, IUBMB Life, 62 (2010) 183-193.
 M. Grdisa, The delivery of biologically active (therapeutic) peptides and proteins
into cells, Curr. Med. Chem., 18 (2011) 1373-1379.
 S.B. Fonseca, M.P. Pereira, S.O. Kelley, Recent advances in the use of
cell-penetrating peptides for medical and biological applications. Adv. Drug Deliv., 61
 M. Lindgren, U. Langel, Classes and prediction of cell-penetrating peptides,
Methods Mol. Biol., 683 (2011) 3-19.
 J.M. Waugh, J. Lee, M.D. Dake, D. Browne, Nonclinical and clinical experiences
with CPP-based self-assembling peptide systems in topical drug development, Methods
Mol. Biol., 683 (2011) 553-572.
 V. Nain, S. Sahi, A. Verma, CPP-ZFN: a potential DNA-targeting anti-malarial
drug, Malar. J., 9 (2010) 258.
 L. Rivas, J.R. Luque-Ortega, D. Andreu, Amphibian antimicrobial peptides and
Protozoa: lessons from parasites, Biochim. Biophys. Acta, 1788 (2009) 1570-1581.
 N.S. Santos-Magalhaes, V.C. Mosqueira, Nanotechnology applied to the treatment
of malaria, Adv. Drug Deliv. Rev., 62 (2010) 560-575.
 V. Hornillos, E. Carrillo, L. Rivas, F. Amat-Guerri, A.U. Acuña, Synthesis of
BODIPY-labeled alkylphosphocholines with leishmanicidal activity, as fluorescent
analogues of miltefosine, Bioorg. Med. Chem. Lett., 18 (2008) 6336-6339.
 V. Hornillos, J.M. Saugar, B.G. de la Torre, D. Andreu, L. Rivas, A.U. Acuña, F.
Amat-Guerri, Synthesis of 16-mercaptohexadecylphosphocholine, a miltefosine analog
with leishmanicidal activity, Bioorg. Med. Chem. Lett, 16 (2006) 5190-5193.
 J.R. Luque-Ortega, L. Rivas, Characterization of the leishmanicidal activity of
antimicrobial peptides, Methods Mol. Biol., 618 (2010) 393-420.
 M. Navarro, G.A. Cross, E. Wirtz, Trypanosoma brucei variant surface
glycoprotein regulation involves coupled activation/inactivation and chromatin
substrate)-related protein, Biochem. J., 367 (2002) 761-769.
 R. Szabo, Z. Banoczi, G. Mezo, O. Lang, L. Kohidai, F. Hudecz, Daunomycin-
polypeptide conjugates with antitumor activity, Biochim. Biophys. Acta, 1798 (2010)
 M. Lindgren, K. Rosenthal-Aizman, K. Saar, E. Eiriksdottir, Y. Jiang, M. Sassian,
P. Ostlund, M. Hallbrink, U. Langel, Overcoming methotrexate resistance in breast
cancer tumour cells by the use of a new cell-penetrating peptide, Biochem. Pharmacol.,
71 (2006) 416-425.
 S. Aroui, S. Brahim, J. Hamelin, M. De Waard, J. Breard, A. Kenani, Conjugation
of doxorubicin to cell penetrating peptides sensitizes human breast MDA-MB 231
cancer cells to endogenous TRAIL-induced apoptosis, Apoptosis, 14 (2009) 1352-1365.
 Z. Zheng, H. Aojula, D. Clarke, Reduction of doxorubicin resistance in P-
glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide,
J. Drug Target, 18 (2010) 477-487.
 J.M. Saugar, J. Delgado, V. Hornillos, J.R. Luque-Ortega, F. Amat-Guerri, A.U.
Acuña, L. Rivas, Synthesis and biological evaluation of fluorescent leishmanicidal
analogues of hexadecylphosphocholine (miltefosine) as probes of antiparasite
mechanisms, J. Med. Chem., 50 (2007) 5994-6003.
 D. Zilberstein, H. Philosoph, A. Gepstein, Maintenance of cytoplasmic pH and
proton motive force in promastigotes of Leishmania donovani, Mol. Biochem.
Parasitol., 36 (1989) 109-117.
 J.M. Pérez-Victoria, F.J. Pérez-Victoria, A. Parodi-Talice, I.A. Jiménez, A.G.
Ravelo, S. Castanys, F. Gamarro, Alkyl-lysophospholipid resistance in multidrug-
resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like
transporter modulator, Antimicrob. Agents Chemother., 45 (2001) 2468-2474.
 H. Brooks, B. Lebleu, E. Vives, Tat peptide-mediated cellular delivery: back to
basics, Adv. Drug Deliv. Rev., 57 (2005) 559-577.
 C.Y. Jiao, D. Delaroche, F. Burlina, I.D. Alves, G. Chassaing, S. Sagan,
Translocation and endocytosis for cell-penetrating peptide internalization, J. Biol.
Chem., 284 (2009) 33957-33965.
 S.L. Croft, D. Snowdon, V. Yardley, The activities of four anticancer
alkyllysophospholipids against Leishmania donovani, Trypanosoma cruzi and
Trypanosoma brucei, J. Antimicrob. Chemother., 38 (1996) 1041-1047.
 M. Delgado, P. Anderson, J.A. García-Salcedo, M. Caro, E. González-Rey,
Neuropeptides kill African trypanosomes by targeting intracellular compartments and
inducing autophagic-like cell death, Cell Death Differ., 16 (2009) 406-416.
 J.R. Luque-Ortega, L.J. Cruz, F. Albericio, L. Rivas, The Antitumoral
Depsipeptide IB-01212 Kills Leishmania through an Apoptosis-like Process Involving
Intracellular Targets, Mol. Pharm. 7 (2010). 1608–1617.
 J.R. Luque-Ortega, W. van't Hof, E.C. Veerman, J.M. Saugar, L. Rivas, Human
antimicrobial peptide histatin 5 is a cell-penetrating peptide targeting mitochondrial
ATP synthesis in Leishmania, FASEB J., 22 (2008) 1817-1828.
 R.B. Arrighi, C. Ebikeme, Y. Jiang, L. Ranford-Cartwright, M.P. Barrett, U.
Langel, I. Faye, Cell-penetrating peptide TP10 shows broad-spectrum activity against
both Plasmodium falciparum and Trypanosoma brucei brucei, Antimicrob. Agents
Chemother., 52 (2008) 3414-3417.
 S. Corradin, A. Ransijn, G. Corradin, J. Bouvier, M.B. Delgado, J. Fernández-
Carneado, J.C. Mottram, G. Vergeres, J. Mauel, Novel peptide inhibitors of Leishmania
gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase
membranes, Chemphyschem., 7 (2006) 2134-2142.
 R. Begley, T. Liron, J. Baryza, D. Mochly-Rosen, Biodistribution of intracellularly
acting peptides conjugated reversibly to Tat, Biochem. Biophys. Res. Commun., 318
 G. Saito, J.A. Swanson, K.D. Lee, Drug delivery strategy utilizing conjugation via
reversible disulfide linkages: role and site of cellular reducing activities, Adv. Drug
Deliv. Rev., 55 (2003) 199-215.
 M.P. Barnes, W.C. Shen, Disulfide and thioether linked cytochrome c-
oligoarginine conjugates in HeLa cells, Int. J. Pharm., 369 (2009) 79-84.
 S. Aubry, F. Burlina, E. Dupont, D. Delaroche, A. Joliot, S. Lavielle, G. Chassaing,
S. Sagan, Cell-surface thiols affect cell entry of disulfide-conjugated peptides, FASEB
J., 23 (2009) 2956-2967.
 S.L. Croft, K. Seifert, M. Duchene, Antiprotozoal activities of phospholipid
analogues, Mol. Biochem. Parasitol., 126 (2003) 165-172.
 H.J. Jung, K.S. Jeong, D.G. Lee, Effective antibacterial action of tat (47-58) by
increased uptake into bacterial cells in the presence of trypsin, J. Microbiol. Biotechnol.,
18 (2008) 990-996.
 H.J. Jung, Y. Park, K.S. Hahm, D.G. Lee, Biological activity of Tat (47-58) peptide
on human pathogenic fungi, Biochem Biophys Res Commun, 345 (2006) 222-228.
 W.L. Zhu, S.Y. Shin, Effects of dimerization of the cell-penetrating peptide Tat
analog on antimicrobial activity and mechanism of bactericidal action, J. Pept. Sci., 15
 M.J. Soares, Endocytic portals in Trypanosoma cruzi epimastigote forms, Parasitol.
Res., 99 (2006) 321-322.
 C.L. Allen, D. Goulding, M.C. Field, Clathrin-mediated endocytosis is essential in
Trypanosoma brucei, EMBO J., 22 (2003) 4991-5002.
 P. Overäth, M. Engstler, Endocytosis, membrane recycling and sorting of GPI-
anchored proteins: Trypanosoma brucei as a model system, Mol. Microbiol., 53 (2004)
 G.M. Poon, J. Gariepy, Cell-surface proteoglycans as molecular portals for cationic
peptide and polymer entry into cells, Biochem. Soc. Trans., 35 (2007) 788-793.
 A. Ziegler, J. Seelig, Binding and clustering of glycosaminoglycans: a common
property of mono- and multivalent cell-penetrating compounds, Biophys. J., 94 (2008)
 J.M. Gump, R.K. June, S.F. Dowdy, Revised role of glycosaminoglycans in TAT
protein transduction domain-mediated cellular transduction, J. Biol. Chem., 285 (2010)
 P. Saalik, A. Niinep, J. Pae, M. Hansen, D. Lubenets, U. Langel, M. Pooga,
Penetration without cells: Membrane translocation of cell-penetrating peptides in the
model giant plasma membrane vesicles, J. Control. Release, 153 (2011) 117-125
 G. Tunnemann, R.M. Martin, S. Haupt, C. Patsch, F. Edenhofer, M.C. Cardoso,
Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and
peptides in living cells, FASEB J., 20 (2006) 1775-1784.
 A. Weingartner, B. Drobot, A. Herrmann, M.P. Sánchez-Cañete, F. Gamarro, S.
Castanys, T. Gunther Pomorski, Disruption of the lipid-transporting LdMT-LdRos3
complex in Leishmania donovani affects membrane lipid asymmetry but not host cell
invasion, PloS one, 5 (2010) e12443.
 S. Afonin, A. Frey, S. Bayerl, D. Fischer, P. Wadhwani, S. Weinkauf, A.S. Ulrich,
The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC
 J.R. Luque-Ortega, L. Rivas, Miltefosine (hexadecylphosphocholine) inhibits
cytochrome c oxidase in Leishmania donovani promastigotes, Antimicrob. Agents
Chemother., 51 (2007) 1327-1332.
 S.A. Young, T.K. Smith, The essential neutral sphingomyelinase is involved in the
trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma
brucei, Mol. Microbiol., 76 (2010) 1461-1482.
 E. Castanys-Muñoz, N. Alder-Baerens, T. Pomorski, F. Gamarro, S. Castanys, A
novel ATP-binding cassette transporter from Leishmania is involved in transport of
phosphatidylcholine analogues and resistance to alkyl-phospholipids, Mol. Microbiol.,
64 (2007) 1141-1153.
 E. Castanys-Munoz, J.M. Pérez-Victoria, F. Gamarro, S. Castanys,
Characterization of an ABCG-like transporter from the protozoan parasite Leishmania
with a role in drug resistance and transbilayer lipid movement, Antimicrob. Agents
Chemother., 52 (2008) 3573-3579.
 P. Jarver, I. Mager, U. Langel, In vivo biodistribution and efficacy of peptide
mediated delivery, Trends. Pharmacol. Sci., 31 (2010) 528-535.
 F. Hudecz, J. Remenyi, R. Szabo, G. Koczan, G. Mezo, P. Kovacs, D. Gaal, Drug
targeting by macromolecules without recognition unit?, J. Mol Recognit. : JMR, 16
 G. Koczan, A.C. Ghose, A. Mookerjee, F. Hudecz, Methotrexate conjugate with
branched polypeptide influences Leishmania donovani infection in vitro and in
experimental animals, Bioconj. Chem., 13 (2002) 518-524.
 K. Padari, K. Koppel, A. Lorents, M. Hallbrink, M. Mano, M.C. Pedroso de Lima,
M. Pooga, S4(13)-PV cell-penetrating peptide forms nanoparticle-like structures to gain
entry into cells, Bioconjug. Chem,. 21 (2010) 774-783.
 P.E. Saw, Y.T. Ko, S. Jon, Efficient Liposomal Nanocarrier-mediated
Oligodeoxynucleotide Delivery Involving Dual Use of a Cell-Penetrating Peptide as a
Packaging and Intracellular Delivery Agent, Macromol. Rapid Commun., 31 (2010)
 V.A. Sethuraman, Y.H. Bae, TAT peptide-based micelle system for potential active
targeting of anti-cancer agents to acidic solid tumors, J. Control Release, 118 (2007)
ACCEPTED MANUSCRIPT Download full-text