Unsaturated acyl chains dramatically enhanced cellular uptake by direct translocation of a minimalist oligo-arginine lipopeptide

Article abstract of DOI:10.1039/c5cc06116d

The recurring issue with cell penetrating peptides is how to increase direct translocation vs. endocytosis, to avoid premature degradation. Acylation by a cis unsaturated chain (C22:6) of a short cationic peptide provides a new rational design to favour diffuse cytosolic and dense Golgi localisations.

Full text of DOI:10.1039/c5cc06116d

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Unsaturated acyl chains dramatically enhanced
cellular uptake by direct translocation of a
minimalist oligo-arginine lipopeptide†
Cite this:DOI: 10.1039/c5cc06116d
Received 22nd July 2015,
Accepted 12th August 2015
J.-M. Swiecicki,‡abc M. Di Pisa,‡abc F. Lippi,abc S. Chwetzoff,acdef C. Mansuy,abc
G. Trugnan,acde G. Chassaing,abc S. Lavielle*abc and F. Burlina*abc
DOI: 10.1039/c5cc06116d
www.rsc.org/chemcomm
The recurring issue with cell penetrating peptides is how to increase that purpose. Acylation by a saturated chain of linear or cyclic CPPs
direct translocation vs. endocytosis, to avoid premature degradation. has been previously shown to increase cellular internalisation,9,16–20
Acylation by a cis unsaturated chain (C22:6) of a short cationic but mostly by promoting endocytosis.9 We reasoned that acylation
peptide provides a new rational design to favour diffuse cytosolic with a cis-unsaturated fatty acid should promote direct translocation
and dense Golgi localisations.
over endocytosis. Single or multiple cis-unsaturation(s) must
decrease the aggregation propensity, (which was shown on cells
Cell penetrating peptides (CPPs) can enter cells by energy-dependent to induce endocytosis),9 and should participate to the ‘‘inter-
(endocytosis) and energy-independent pathways (direct trans- facial activity’’ of the peptide,21 favouring its insertion within
location).1–7 The relative contribution of translocation, which disordered domains and meanwhile locally perturb PL packing
depends on the CPP and on the cell line may account for 10 to (increasing direct translocation).15
70% of the total internalisation.8,9 The central but tricky question is
The scope of this study was to compare the impact of acylation
how to increase direct translocation vs. endocytosis?
with cis-unsaturated vs. saturated fatty acid chain of a minimalist
The key role of phospholipids (PLs) in the transfer of CPPs cationic peptide containing only four arginines and an acyl chain.9
via the formation of inverted micelles has been proposed at the A C-terminal lysine was added to this short cationic sequence, for
early stage of the CPP discovery,10 and later extended to the labelling with a small neutral fluorescent probe: 4-nitrobenzo-2-oxa-
‘‘mutual adaptation’’ concept.7 We recently demonstrated that 1,3-diazole (NBD).11–13 We showed by FACS and confocal micro-
cationic CPPs recruit anionic PLs on the outer leaflet of large scopy that the acylation of (Arg)4-Lys(NBD)-NH2,with an all-cis-C22:6
unilamellar vesicles (LUVs), in the fluid phase, and consequently fatty acid dramatically enhanced the internalisation at 4 1C in four
diffuse through the bilayer as a neutral lipophilic CCP/PL different cell lines, compared to C18:0, C18:1, C18:2, C12:0 and
complex.11–13 CPPs may also translocate into LUVs presenting C12:1 acyl chains. The cellular uptake by direct translocation was
fluid domains in gel phase or LUVs made of ternary mixture selectively increased for the C22:6 lipopeptide, as ascertained
including cholesterol.14 All these data led us to propose that by diffuse cytosolic and dense Golgi localisations at both 4 1C
direct translocation might be enhanced if CPPs could target and/ and 37 1C.
or induce disordered domains into cell membrane.15 Acylation
The length of the fatty acid chain and the number of unsatura-
of the CPP by an unsaturated fatty acid might be a key feature for tions were varied from C12 to C22, and from 1 to 6, respectively. The
commercially available C22:6 fatty acid (docosahexaneoic acid)
a
was chosen, this o3-fatty acid represents about 40% of the poly-
unsaturated fatty acids in the brain,22 and it should be the best
compromise between the length and the number of unsaturations
to decrease the aggregation by micellisation of long fatty acyl chain.
All the lipopeptides, C12:0-Arg4, C12:1-Arg4, C18:0-Arg4, C18:1-
Arg4, C18:2-Arg4 and C22:6-Arg4, (Scheme 1), were prepared via Boc
strategy on solid support. The NBD, on the eNH2 of the lysine, and
the saturated acyl chains C12:0 and C18:0 at the N-terminus were
introduced on solid support. The N-hydroxysuccinimic ester of the
unsaturated acyl chains, C12:1, C18:1, C18:2 and C22:6, had to be
coupled in solution to the crude precursor (Arg)4-Lys(NBD)-NH2.
The final peptides and lipopeptides were purified either by HPLC or
´
´
Sorbonne Universites, UPMC Univ Paris 06, Laboratoire des Biomolecules,
UMR 7203, 4, Place Jussieu 75005, Paris, France.
E-mail: solange.lavielle@upmc.fr, fabienne.burlina@upmc.fr
b
´
Ecole Normale Superieure – PSL Research University Institution,
´
Departement de Chimie, 24, Rue Lhomond, 75005 Paris, France
c CNRS – UMR7203, Paris, France
d INSERM – ERL 1157, Paris, France
e
ˆ
AP-HP, Hopital Saint Antoine, 75012 Paris, France
f
´
INRA, UR892, Virologie et Immunologie Moleculaires, 78350, Jouy-en-Jossas,
France
† Electronic supplementary information (ESI) available: All the experimental
procedures are described in SI with the characterization of all the lipopeptides.
See DOI: 10.1039/c5cc06116d
‡ J.-M. Swiecicki and M. Di Pisa: equal contribution.
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After 5 min incubation of the LUVs (10 mM) with the fluorescent
peptide (100 nM, peptide : phospholipid, ratio 1 : 100), the non-
permeate reducing anion dithionite (DT) was added to quantitatively
reduce and thus quench the fluorescence of the NBD-labelled
peptide, which has not been internalised inside LUVs,11 (Fig. 1A
and cf. S4 in ESI†). The lipopeptides accumulate to a similar extent
inside LUVs in fluid phase (DOPG, 20 1C). Interestingly, the
accumulation of the lipopeptides in LUVs presenting fluid
domains in gel phase (DPPG, 37 1C) increases with the number
of unsaturations; from C18:0-Arg4 to C18:2-Arg4 and C22:6-Arg4.
Altogether the validity of our hypothesis was ascertained:
unsaturations favour translocation of a short cationic lipopeptide
into LUVs presenting nanodomains with packing defects, such as
LUVs made of DPPG incubated at 37 1C.
Our hypothesis was then confronted to the complexity of
living cells, using four different cell lines: MA-104 cells (monkey
kidney epithelial cells), RAW 264.7 cells (mouse leukemic
monocyte macrophage), HepG2 cells (human hepatocellular
carcinoma) and Caco-2 cells (epithelial cells from a human
colorectal adenocarcinoma) and four lipopeptides C18:0-Arg4,
C18:1-Arg4, C18:2-Arg4 and C22:6-Arg4.
Scheme 1 Formula and abbreviations of the NBD-labelled lipopeptides.
by Fast Centrifugal Partition Chromatography, (cf.§ and S1
in ESI†). Two control peptides were also used in this study:
Ac-(Arg)4-Lys(NBD)-NH2, abbreviated as Ac-Arg4 (with Ac: acetyl)
and the N-ter modified nona-arginine: NBD-(Arg)9-NH2, already
used in previous experiments with LUVs.11
The internalisation of the lipopeptides was first analysed with two
model systems, which have been found to be the most appropriate
ones to screen the entry of CPPs.14 LUVs (100 nm) were prepared
from either 100% 1,2-dioleyl-sn-glycero-3-phosphoglycerol, (DOPG)
or 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, (DPPG).
These NBD-labelled peptides at concentrations from 1 to 10 mM
did not affect the viability of the cells, only a slight but significant
decrease in cell viability was observed at 10 mM for C18:0-Arg4 and
C18:1-Arg4, on RAW 264.7 and HepG2 cells, (cf. S6 in ESI†).
Consequently, the lipopeptides were incubated at 5 mM, con-
sidering also the aggregation propensity of such amphiphiles,
around 1 to 10 mM depending of the acyl chain, (cf. S2 in ESI†).
Fig. 1 (A) Internalisation of the indicated NBD-labelled peptides (100 nM)
into LUVs (100 nm, 10 mM, peptide : phospholipid ratio 1 : 100) made of
100% DOPG (at 20 1C, fluid phase) or 100% DPPG (at 37 1C, fluid domains
in a gel phase), data are means of 3–5 experiments Æ s.d., (cf. S4 in ESI†).11
(B) Uptake into MA-104 cells (105 cells) at 4 1C or 37 1C of the indicated
NBD-labelled peptides (5 mM) analysed by FACS. At the end of the
incubation (10 or 60 min), dithionite was added to quench the fluores-
cence of the non-internalized NBD-peptides. For untreated cells and cells
incubated with the control peptide Ac-Arg4, data are reported in the ESI,†
(cf. S7 in ESI†). Data are means of 2–4 experiments Æ s.d. Student test: with
**: p r 0.01, ***: p r 0.001 and ****: p r 0.0001, respectively.
Fig. 2 Confocal laser scanning microscopy experiments. Internalisation
of lipopeptides C18:0-Arg4, C18:1-Arg4, C18:2-Arg4 or C22:6-Arg4, with
four cells lines (105 cells per dish): (A) MA-104 cells, (B) RAW 264.7 cells,
(C) HepG2 cells and (D) Caco-2 cells. Peptides (5 mM) were incubated at
4 1C for 60 min, the non-internalised NBD-labelled peptide was quenched
by dithionite. Direct observation after washings.
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Fig. 3 Colocalisation studies of NBD-labelled C18:0-Arg4 or C22:6-Arg4 with either Golgi Tracker or Lyso Tracker using confocal laser scanning
microscopy. After incubation with Golgi Tracker (A) or Lyso Tracker (B) followed by incubation with the indicated lipopeptide, cells were immediately
treated with dithionite (3 min) to quench the staining of labelled peptides remaining at the cell surface. Upper panels of A and B correspond to the medial
section of an image stack of a field observed with an Â63 objective, while lower panels of A and B correspond to acquisitions performed at the same
depth using a numerical zoom adjusted to observe a single cell, with for the merged pictures the lipopeptide in green (C18:0-Arg4 and C22:6-Arg4) and
the tracker in red. (A) MA-104 cells (105 cells), were incubated for 30 min at 4 1C, then 30 min at 37 1C with Golgi Tracker (Bodipy TR Ceramide, 5 mM),
C18:0-Arg4 (left panels) or C22:6-Arg4 (right panels) were then added, at 5 mM, and further incubated for 60 min at 37 1C. (B) MA-104 cells (105 cells), were
incubated for 30 min at 37 1C with Lyso Tracker Deep Red (100 nM), C18:0-Arg4 (left panels) or C22:6-Arg4 (right panels) were added, at 5 mM, and further
incubated for 60 min at 37 1C.
This concentration is also the best compromise for FACS and depending on the chain length and number of unsaturation(s),
confocal microscopy experiments and the sensitivity of the NBD two trends are observed. C12:0-Arg4 and C22:6-Arg4 are rapidly
probe. In the literature, most of the experiments with CPPs, internalised, with no increase in intracellular fluorescence after
bearing different types of probe, are in the 1–20 mM range.23
10 min incubation, whereas for C18:0-Arg4 a 4.8-fold increase in
The plasma membranes of the MA-104 cells incubated with the intracellular fluorescence is observed when increasing the
lipopeptides (5 mM) are densely labelled, but this fluorescence incubation time from 10 to 60 min. The delayed internalisation
was totally quenched by reduction/quenching of the NBD for C18:0-Arg4, as also observed for NBD-Arg9, should be related
fluorophore with dithionite, to only detect the internalised to a slow onset of the endosomal machinery. These data there-
fluorescent peptides. This quenching of fluorescence on the fore suggested that the main internalisation pathway differs for
external leaflet was quantitative, as shown by flow cytometry and both groups of CPPs. This was confirmed by incubating the
confocal microscopy assays (cf. S7 and S8 in ESI†). The inter- lipopeptides at 4 1C.
nalisation into MA-104 cells was then analysed by flow cytometry
Indeed, at 4 1C, the internalisation via active mechanisms
after incubation for 10 or 60 min at 37 1C, and 3 min reduction (endocytosis) is abolished, and incubation at this temperature
with dithionite (Fig. 1B). Since Ac-Arg4 is not internalized (cf. S7 allowed to unveil the involvement of direct translocation, an
in ESI†), the acyl chain is required for the internalisation of energy independent mechanism. The fluidity of the plasma
this short cationic peptide as observed before.9 Importantly, membrane should also change at 4 1C, but translocation can
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still occur even if probably reduced. Interestingly, at 4 1C the Notes and references
amount of internalisation via direct translocation increases
§ Syntheses of the lipopeptides: Boc-Arg(Tos)4-Lys(Fmoc)-MBHA was
synthesised by SPPS on a MBHA Resin. Activation was accomplished
with DCC/HOBt in NMP. After Fmoc deprotection by piperidine, the
fluorophore was introduced on the lysine side chain by reacting, in
DMF, NBD-Cl (4 eq.) with DIEA (10 eq.). The saturated acyl chains were
introduced at the N-terminus of this peptidyl resin, after Boc deprotec-
tion by trifluoroacetic acid, and on-resin acylation of the N–ter arginine
with the corresponding fatty acid pre-activated by DCC/HOBT. The final
lipopetides were cleaved from the support by HF in the presence of
anisole and Et2S. For the preparation of the unsaturated lipopeptides,
Arg4-Lys(NBD)-NH2 was obtained after cleavage from the support and
this crude peptide was acylated in solution with the corresponding
N-hydroxysuccinimic ester of the unsaturated fatty acid. The crude
peptides were lyophilised before purification by HPLC or Fast Centri-
fugal Partition Chromatography leading to purity Z97%. The purified
peptides were characterised by MALDI-TOF mass spectrometry,
(cf. S1 in ESI†).
significantly with the number of insaturation(s) from C18:0-Arg4
to C18:1-Arg4 and C18:2-Arg4. The most efficient lipopeptide at 4 1C
was found to be as hypothetised C22:6-Arg4, when the C18:0-Arg4
lipopeptide and the control NBD-Arg9 are poorly internalised. Thus,
the direct translocation pathway is dramatically enhanced for
the polyunsaturated C22:6-Arg4. The entry at 4 1C of the four
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was further analysed with the four different cell lines by confocal
microscopy (Fig. 2). In every cases the most unsaturated lipopeptide
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an intense labelling of the cells at 4 1C after 60 min incubation,
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