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Photoresponsive Polymers DOI: 10.1002/anie.201106777
Photocontrol of the Translocation of Molecules, Peptides, and
Quantum Dots through Cell and Lipid Membranes Doped with
Azobenzene Copolymers**
Sarra C. Sebai, Dimitra Milioni, Astrid Walrant, Isabel D. Alves, Sandrine Sagan, Ccile Huin,
Loic Auvray, Dominique Massotte, Sophie Cribier, and Christophe Tribet*
Permeabilization of lipid membranes is a major challenge for
the development of biocides and for improving the delivery of
DNA, anticancer agents, and small proteins within the cell
cytosol. Effective membrane permeabilization can be ach-
ieved by chemical techniques that often involve an intra-
membrane assembly of permeabilizing agents such as pep-
tides,[1–3] amphiphilic molecules, or polymers.[4,5] Alternative
physical techniques are electroporation or microinjection. A
present challenge for chemical approaches lies in targeting
cells while maintaining cell viability.[6,7] The development of
permeabilizers for the remote control of membrane proper-
ties is accordingly in high demand to compete with micro-
injection, because the former avoids mechanical damage and
addresses large cell subsets.
The strategy consists of using so-called “smart” systems,
that is, tailoring stimuli-responsive molecules to affect lipid
membranes.[4,8–12] The variation of the pH value has been the
major focus for the design of smart bilayers doped with
polymers[11,12] or peptides[13] with the goal of delivering
substances of interest in the late endosomes or targeting
acidic tumors.[14] However, light-responsive systems would
offer a more versatile and external trigger. To photocontrol
self-assembly and/or interaction with lipid bilayers several
authors had recourse to amphiphilic azobenzene deriva-
tives.[15,16] The best achievements in this field show a
modulation of the rate of transmembrane release of small
molecules from liposomes, the membranes of which contain
either a pore-forming peptide, gramicidin, conjugated to
azobenzene[17] or an azobenzene-containing lipid.[18] How-
ever, except for highly specific photogating by engineered
membrane proteins,[19] no artificial system (likely less specific
than protein gates) enabled phototriggered permeabilization
on living cells. So far, in regard to large molecules (e.g.
peptides), their pH-controlled passage was essentially ach-
ieved using polymer carriers. Herein, we show that azoben-
zene-modified polymers (AMPs; Scheme 1)[20] can impart
plasma membranes of living cells and vesicles with this
desirable photoresponse.
The photocontrolled delivery of a small peptide was
tested in CHO mammalian cells. A well-characterized basic
peptide, RL9 (RRLLRRLRR-NH2;R=Arg =arginine, L =
Leu =leucine), that binds to cell surfaces without crossing
membranes[22] was added to cells and incubated in the
presence of AMP, which is predominantly in its cis form.
cis-AMP was prepared as described in the Supporting
Information by pre-exposure to UV light (365 nm) so that
cells were not exposed to UV light. trans-AMP was generated
in situ by exposing half of the wells containing cells to blue
light (436 nm) for two minutes. Images of cells in Figure 1a
were obtained by using a biotinylated RL9 derivative; the
cells were incubated for one hour with either cis-ortrans-
AMP and labeled with streptavidine-AlexaFluor488 (see the
Experimental Section). The diffuse green fluorescence in
samples exposed to blue light (trans-AMP) shows a homoge-
neous internalization that differs from punctuated images
Scheme 1. Structure of AMPs and cis–trans photoisomerization. Typi-
cally x=10 and y=15 mol% (see text and the Supporting Informa-
tion).
[*] Dr. S. C. Sebai, Dr. C. Tribet
Ecole Normale Suprieure, Departement de Chimie, UMR 8640
CNRS-ENS-UPMC
24, rue Lhomond, 75005 Paris (France)
E-mail: christophe.tribet@ens.fr
D. Milioni, Dr. A. Walrant, Dr. I. D. Alves, Dr. S. Sagan,
Prof. S. Cribier
UPMC Univ Paris 06, UMR 7203 CNRS-UPMC-ENS
4, Place Jussieu, 75005 Paris (France)
Dr. C. Huin, Dr. L. Auvray
LAMBE UMR8587, UEVE-CNRS-CEA
Bd F. Mitterrand, 91025 Evry (France)
D. Massotte
IGBMC
1 rue Laurent Fries, 67400 Illkirch (France)
[**] This work was supported by the ANR for the award of Grant photo-
channels N
8
ANR-07-BLAN-0278. The authors thank Dborah
Cardoso for providing Figure S4 and F. Pinaud and M. Dahan for
providing Qdots.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106777.
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commonly obtained after endocytosis. The fluorescence of
cells incubated with cis-AMP was significantly lower and is
not distinguished from the fluorescence achieved with the
parent chain poly(acrylic acid) (PAA), which is devoid of
hydrophobic side groups (see controls in the Supporting
Information). This observation suggests a markedly lower
internalization with cis-AMP (or PAA) compared to that
achieved with trans-AMP. Quantification of internalized RL9
was performed by MALDI-TOF mass spectrometry[23] (see
the Experimental Section for protocols). We can see from
Figure 1b that RL9 internalization is dependent on the AMP
isomerization state. cis-AMP does
not significantly improve RL9 inter-
nalization, compared to control
experiments in the absence of
AMPs ((1.5 1) vs. (0.3
0.2) pmol of peptide in one million
cells, respectively), whereas RL9 is
significantly internalized in cells
treated with trans-AMP. About
4.5 pmoles of RL9 (corresponding
to an intracellular concentration of
approximately 4 mmunder the
assumption of an intracellular
volume of 1 pL) are delivered to
the cytosol of cells, an amount
similar to the cell-penetrating pep-
tide penetratin.[24] In the absence of
AMPs, the amount of internalized
RL9 remained negligible as
reported.[22] We can therefore pos-
tulate that trans-AMP delivers
small peptides to cells in a light-
responsive manner. These condi-
tions ensured the absence of tox-
icity. Toxicity of AMP is not
detected up to a polymer concen-
tration of 1 mg mL1, that is, three
orders of magnitude above the
present conditions of photopermea-
bilization (for toxicity assays see the
Supporting Information).
Light-driven permeabilization
could be nonspecific and share
similarities with polymer-triggered
pore formation described with
hydrophobically modified poly-
(acrylic acid) devoid of photores-
ponsive groups.[9,25] This hypothesis
would imply that the permeabiliza-
tion controlled by AMPs should not
markedly depend on a specific pro-
tein machinery and should in addi-
tion induce photorelease of cyto-
solic molecules as well as delivery.
To confirm this hypothesis, we
investigated first the leakage of
cell-encapsulated probes. Second,
membrane permeabilization in the
absence of proteins was illustrated by experiments on
artificial lipid membranes.
Phototriggered release from living cells was demonstrated
by using a cell-encapsulated fluorescein probe. Figure 1d
shows viable mammalian COS cells that were grown on glass
coverslips and fluorescently labeled with 6-carboxyfluores-
cein diacetate (CFDA), a molecule that infuses through the
membrane in its ester form and is trapped in cells in its anionic
form after hydrolysis by cytosolic esterases (see the Exper-
imental Section). cis-AMP was added at time zero after
washing out the excess CFDA. No CFDA leakage is observed
Figure 1. Effect of a cis-ortrans-AMP (x=0.10, y=0.15 in Scheme 1) on cell permeabilization. cis-
AMP was added to cells at 2 mgmL1and switched to the trans form in half of the wells by exposure
to blue light. a) Fluorescence images of CHO cells incubated for one hour with biotinylated RL9 and
cis-ortrans-AMP as quoted. Cells were labeled with both streptavidine-AlexaFluor488 (green) and
4’,6-diamidino-2-phenylindole (DAPI; blue) after fixation. Scale bars correspond to 10 mm. Over-
exposed image on the upper right panel shows more clearly the RL9 distribution in the cell upon
incubation with trans-AMP. b) Quantification of internalized RL9 peptide in CHO cells in the presence
of cis-AMP or trans-AMP (see text for details). Significance was tested using one-way analysis of
variance (ANOVA) and subsequent Tukey’s multiple comparison test, ns: p>0.05, *** : p<0.001.
c) Histogram of CFDA intracytosolic fluorescence intensity over a group of 100 COS cells after
40 min and incubation with cis-ortrans-AMP. d) CFDA-labeled COS cells shown after addition of
AMP at t=0 min. Cells permeabilized at t=40 min are indicated by arrows. e) Release kinetics of
CFDA measured by detecting the intracytosolic fluorescence DFI (averaged on three independent
groups of 10 cells each) during incubation with cis-AMP (black squares) or trans-AMP (blue dots).
.
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in cells exposed to predominantly cis-AMP (Figure 1e). In
contrast, the leakage curve shown in Figure 1 e shows that a
gradual drop of the average intracellular fluorescence is
triggered by exposure to blue light at time zero, that is,
immediately upon formation of trans-AMP. The AMPs
polarity hence controls the level of fluorescence within the
cells. For this kinetics study, however, the reference experi-
ment with cis-AMP (no leakage) needed short (1 s) exposures
to UV light to preserve predominantly the cis state while
taking pictures (requiring blue light) of fluorescein distribu-
tion (one picture every 10 min). Exposure of cells to UV light
is not needed in practice to achieve the photocontrol of
leakage: in another set of experiments, fluorescently labeled
COS cells were supplemented with preformed cis-AMP. Cells
were then incubated in the dark for 40 min. In the dark the
predominant cis form of AMP can be preserved for hours.
trans-AMP was formed in situ by exposure to blue light at
time zero. Figure 1c presents the distribution of the fluores-
cence intensity of CFDA in cells treated with either cis- or
trans-AMP (population of N100 cells). Cells incubated with
cis-AMP are characterized by a more intense average
fluorescence (FI; DFI 1100) compared to cells treated
with trans-AMP (DFI 500). Impermeable and fully loaded
cells in the absence of AMPs (as a control) exhibited
fluorescence around the average DFI 1400. As reflected
by the broad fluorescence distribution, heterogeneities exist
in the cell population. The significant shift of the mean
fluorescence intensity noticeable between cells treated with
cis-AMP and trans-AMP confirms, however, that trans-AMP
allows a more rapid release of a small anionic soluble dye
through the plasma membrane. We then wondered if mole-
cules with larger radii, such as small endogenous soluble
proteins, are released. We expressed soluble enhanced green
fluorescent protein (eGFP) in COS cells and monitored the
fluorescence of the protein in the cell after treatment with
AMP. No effects of either trans-orcis-AMP were detected
over an hour, thus suggesting that we are dealing with a size
limitation in the permeabilization efficiency (see the Support-
ing Information). The retention of intracytosolic proteins is of
major importance and is consistent with the absence of
toxicity of AMP (see the Supporting Information) and
preservation of cell viability upon incubation with trans-
AMP that we previously showed under similar conditions.[21]
Electrophysiology experiments were then conducted on
artificial planar lipid membranes (also called black lipid
membranes, BLM). This method enables direct monitoring of
membrane permeability by probing current traces across the
bilayer. A typical signature for an impermeable membrane is
identified by a stable current trace with no associated
conductance at a fixed applied voltage of 100 mV across
the membrane (signal similar to that shown in Figure 2d in
the presence of cis-AMP). In contrast, the presence of the
same concentration (1 mgmL1)oftrans-AMP generates
variable currents (Figure 2a, b). These signals are character-
istic of passage of ions, and abrupt jumps of current from zero
to plateau values suggest the fast opening of pores in the
bilayer.[26] Typically, the current varies slightly during the first
15 min after membrane formation (e.g. Figure 2a, part 1).
Hence, the membrane likely remains mainly closed at the
onset of the interaction with trans-AMP (see the Supporting
Information for current histograms). However, after about
30 min incubation (Figure 2a, part 2) discrete current jumps
persist till the end of the run, indicating long-lasting permeant
structures. Figure 2c shows the histogram of the times lapsed
by the membrane in its “open” states. A broad distribution of
currents is revealed, with one peak corresponding to “closed”
(0 pA), a second centered at 8 pA, and a third broader peak
at about 60 pA. This broad distribution reflects a multi-
plicity, and/or fluctuation, of structures of trans-AMP-formed
pores, which we have not investigated yet. The dispersity of
opening events suggests that AMPs in their more hydro-
phobic trans form induce dynamic pore formation. Penetra-
tion of hydrophobic polymers in the hydrophobic core of a
bilayer can scramble lipid organization.[10] In contrast, pres-
ervation of the impermeable state when AMPs were added in
their more polar cis form suggests a penetration to a lesser
extent of cis-AMP in the membrane (note that cis-AMP was
shown however to tightly bind to membranes, as shown for
the preparation of giant unilamellar vesicles (GUV); see the
Supporting Information).
The structure of polymers significantly affects their
interaction with the membrane, and for instance longer
polymer chains destabilize lipid bilayers at higher pH
values.[8] We compared AMPs having the same backbone
but a slightly different degree of modification with octyl and
azobenzene side groups to vary the hydrophobicity of the
chains (x=10 or 5 mol% in Scheme 1). Giant vesicles with
AMPs inserted in their membrane were prepared by electro-
formation (see the Supporting Information) in buffer con-
taining the water-soluble fluorescein-labeled probe, dextran-
FITC. The diameters of the spherical giant vesicles, called pol-
GUVs, ranged from 10 to 80 mm (Figure 3a). Pol-GUVs were
Figure 2. Current-trace measurements through a black lipid membrane
monitored after supplementation with AMP in the anodic compart-
ment (pH 7.0). The voltage applied is 100 mV (for detailed exper-
imental conditions see the Supporting Information). a) Incubation
with trans-AMP. b) Magnification of a region of fluctuations between
permeant states. c) Histograms of current occurrence during measure-
ment over approximately one hour for five runs (five different
membranes). d) Current trace in the presence of cis-AMP at pH 7
showing no permeabilization.
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diluted in the microscope observation chamber under expo-
sure to UV light to reach the predominantly cis-AMP state.
The fluorescence profile across the vesicle diameter is
indicative of the amount of GUV-encapsulated dextran. For
simplicity, we distinguished between “empty” and “loaded”
GUVs on the basis of the absence or presence of internal
fluorescence (the fluorescence intensity threshold for empty
GUVs was fixed arbitrarily at the background fluorescence
plus twice the noise). In a first set of experiments, pol-GUVs
prepared at an AMP/lipid ratio of 0.05 or 0.01 g/g were
exposed to blue light (two minutes) just after dilution at
pH 7.0. In Figure 3 c, the fraction of “dextran-containing” pol-
GUV is plotted against increasing incubation times after
switching on the light. In the absence of AMP or in the
presence of AMP having lower hydrophobicity (here
15 mol % octyl and 5 mol % azobenzene), dextran is essen-
tially kept entrapped in GUV for one hour. In contrast, a
significant leakage of dextran is observed after exposure of
pol-GUV containing AMPs with 15% octyl and 10 %
azobenzene (i.e. the polymer also used in experiments on
cells) to blue light. This feature confirms the ability of AMPs
to help translocation of large molecules. Membrane integrity
was preserved while permeabilization occured, which was
already observed for other hydrophobically modified poly-
mers.[9,25]
Moreover, we confirmed the possibility for nanometer
particles to cross the membrane by performing leakage
experiments on isolated GUVs loaded with soluble quantum
dots (QDs) as fluorophores (see the Supporting Information,
radius 4.2 nm)[27] and AMP (10 mol% azobenzene) in the
membrane. QDs were not bleached during the experiment,
which enabled us to record leakage kinetics. Fluorescence
intensity across a diameter indicates the presence of soluble
QDs as described in reference [28]. Release of QDs upon
exposure of pol-GUVs to blue light is shown in Figure 3 b, d.
In contrast, no leakage was observed during one hour when
UV light was constantly shone on pol-GUV (leakage can then
be triggered after 60 min; Figure 3d shows a representative
example). Hence, we show that AMPs with a high enough
density of azobenzene in the chain allow controlled release of
macromolecules and nanoparticles such as dextran and QDs.
On the mechanistic side, polymer binding to lipid bilayers
is known to affect the local assemblies of lipids and induces,
for instance, lateral lipid segregation or membrane thin-
ning.[4,9] The interaction between amphiphilic copolymers and
liposomes is modulated by their apparent hydrophobicity,[25]
which in AMPs is controlled by light:[21] destabilization of the
membrane occurs upon cis to trans isomerization of the
AMPs, likely owing to deeper penetration of trans-azoben-
zene hydrophobic groups in the bilayer.[29] AMPs are
promising nonspecific tools for controlling the permeability
of plasma membranes in physiological conditions (such as in
culture medium) and more importantly phototriggered inter-
nalization of peptides in living cells. Furthermore, they bring
on a control of the opening of artificial pores and passage of
macromolecules through lipid bilayers; these latter points will
help to study the dynamics of biomimetic gates and delivery
systems.
Experimental Section
The Supporting Information describes polymer synthesis, toxicity
assays, cis–trans isomerization, controls, GFP transfection, and details
on current measurements.
RL9 (RRLLRRLRR-NH2) cellular uptake: Chinese Hamster
Ovary CHO-K1 cells were cultured in Dulbeccos modified Eagles
medium (DMEM) supplemented with 10% foetal calf serum (FCS),
penicillin (100000 IU/L), streptomycin (100000 IU/L), and ampho-
tericin B (1 mg L1) in a humidified atmosphere containing 5% CO2
at 37
8
C. AMP (2 or 16 mgmL1) was added to preseeded CHO cells
(106) in the presence of RL9 (10 mm) or added to cells 30 min prior to
RL9 addition. The order of addition made no difference to the results.
Experiments were carried out in triplicate, on different cell batches.
RL9 was incubated for one hour at 37
8
C in both conditions. RL9
cellular uptake was quantified by using the method described by
Burlina et al.[23] except that membrane-bound RL9 was efficiently
removed by sequential washing steps made of Hank’s buffered salt
solution (HBSS), 1mNaCl/HBSS, trypsin, 1mNaCl/HBSS, HBSS, so
that only internalized RL9 would be quantified. Final trypsinization
Figure 3. Leakage of dioleoylphosphatidylcholine (DOPC) pol-GUVs
revealed by epifluorescence. a) Images of one pol-GUV of approx-
imately 37 mm in diameter with encapsulated QDs (green) at pH 7 and
DPPE-rhodamine (DPPE=dipalmitoylphosphatidylethanolamine sulfo-
rhodamine; red). b) Fluorescence profile along diameter (white line in
(a)) showing the decrease of internal fluorescence from a GUV
containing trans-AMPs (0.1 g/g AMPs/DOPC ratio) at 0 min (black
line), 10 min (red line), 20 min (blue line), and 40 min (green line)
after irradiation with blue light. Scale: 10 pixels =2.2 mm. c) Fractions
of dextran-containing pol-GUV in a random sampling of approximately
150 GUVs. Two polymers were used with fractions of azobenzene (x in
Scheme 1) of 0.1 (“10azo”) or 0.05 (“5azo”). AMP/DOPC ratios are
0.01 g/g for the yellow (5azo) and light blue (10azo) bars and 0.05 g/g
for the red (5azo) and dark blue (10azo) bars. GUVs were diluted
under UV light and exposed to blue light at time zero (see text for
definition of empty GUVs). d) Delayed release of QDs from an isolated
pol-GUV in cis-AMP form (t=0 min to t =40 min, then switched
under blue light to the trans-AMP form (t =60 min to t =130 min).
Error bars indicate standard deviations calculated from the spatial
noise of the fluorescence in both the background and in the GUV.
.
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(5 min at 37
8
C) resulted in removal of external peptide and the
detachment of cells, which were then collected and lysed in Triton
X100 (0.3%) and NaCl (1m) at 100
8
C. Lysates were incubated with
streptavidin-coated magnetic beads to extract RL9, which was later
eluted with a-cyano-4-hydroxycinnamic acid (HCCA) matrix and
spotted on the MALDI plate. Deuterated RL9 (1.5 pmol) was used as
an internal standard, and RL9 in cell lysates was quantified by
MALDI-MS.[22,24] Fluorescence images of the distribution of RL9
biotinylated at the N terminus were recorded after incubation in the
same conditions as for RL9 prior to the fixation of cells (4%
paraformaldehyde pH 7.4 for 15 min at room temperature), TX100
permeabilization (0.3% for 5 min), and labeling by streptavidine-
AlexaFluor488. Nuclei were stained with DAPI. All images were
recorded under the same excitation conditions by using the same
exposure time and objective (x100 PlanFluor, inverted NikonEclipse
microscope).
Carboxyfluorescein diacetate cell encapsulation and release:
COS cells were grown in DMEM supplemented with 10 % FCS and
glutamine (2 mm)at37
8
C and 5% CO2. COS cells were preseeded
(50000 cells) on MatTek (MatTek Corporation Ashland, MA, USA)
glass bottom dishes (35 mm) in complete medium the day before the
experiment. 6-carboxyfluorescein diacetate (Sigma Aldrich) was
added at 100 mmin serum-free DMEM for 30 min at 37
8
CinaCO
2
chamber. Esterases present in the cytoplasm hydrolyze it into
encapsulated carboxyfluorescein. Excess CFDA was removed by
washing the cells in serum-free DMEM. AMP (2 mgmL1) was
irradiated with UV light for 15 min in serum-free DMEM (1 mL) and
then added to COS cells. For “cis conditions”, cells were maintained
in the dark. For “trans conditions”, cells were irradiated for 30
seconds either at 436 nm (or 476 nm through the microscope
objective). Fluorescence images were recorded by using a LEICA
DM-IRE2 inverted microscope under fixed exposure conditions
(typical exposure time 500 ms, binning 2 2). To determine fluores-
cence intensities, the average background intensity was subtracted
from the intensity measured inside the vesicle or the cell for every
single frame.
Received: September 23, 2011
Revised: November 9, 2011
Published online: && &&,&&&&
.
Keywords: azobenzenes · membranes · photochromism ·
polymers · vesicles
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Communications
Photoresponsive Polymers
S. C. Sebai, D. Milioni, A. Walrant,
I. D. Alves, S. Sagan, C. Huin, L. Auvray,
D. Massotte, S. Cribier,
C. Tribet*
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Photocontrol of the Translocation of
Molecules, Peptides, and Quantum Dots
through Cell and Lipid Membranes
Doped with Azobenzene Copolymers
Light opens: Photocontrolled transmem-
brane passage of soluble dyes and deliv-
ery of small peptides into mammalian
cells has been achieved using azoben-
zene-modified polymers (AMPs) as per-
meabilizing agents. Irradiation with UV
and visible light triggers polarity switches
upon cis–trans isomerization of the azo-
benzene moieties. Photoresponsive per-
meability and pore opening promoted by
trans-AMPs, but not cis-AMPs, in sup-
ported lipid bilayers are observed.
.
Angewandte
Communications
6www.angewandte.org 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2012,51,1–6
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