Development of a nonionic azobenzene amphiphile for remote photocontrol of a model biomembrane

Article abstract of DOI:10.1021/acs.jpcb.6b00303

We report the synthesis and characterization of a simple nonionic azoamphiphile, C₁₂OazoE₃OH, which behaves as an optically controlled molecule alone and in a biomembrane environment. First, Langmuir monolayer and Brewster angle micr

Full text of DOI:10.1021/acs.jpcb.6b00303

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Article
Development of a Non-Ionic Azobenzene Amphiphile
for Remote Photocontrol of a Model Biomembrane
Luciano A. Benedini, Maria Alejandra Sequeira, Maria Laura Fanani, Bruno Maggio, and Veronica I Dodero
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b00303 • Publication Date (Web): 12 Apr 2016
Downloaded from http://pubs.acs.org on April 21, 2016
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Development of a NonꢀIonic Azobenzene Amphiphile for Remote
Photocontrol of a Model Biomembrane.
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Luciano A. Benedini ‡, M. Alejandra Sequeira ‡, Maria Laura Fanani , Bruno Maggio ,
Verónica I. Dodero 1 † *.
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1
Instituto de Química del Sur (INQUISURꢀCONICET), Departamento de Química, Universidad Nacional del Sur,
8000FTN Bahía Blanca, Argentina.
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Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBICꢀCONICET), Departamento de Química
Biológica, Facultad de Ciencias Químicas, Universidad Nacional del Córdoba, Haya de la Torre y Medina Allende,
Ciudad Universitaria, X5000HUA Córdoba, Argentina
*Corresponding author:
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ABSTRACT:
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We report the synthesis and characterization of a simple nonꢀionic azoamphiphile, C12OazoE3OH
which behaves as an opticallyꢀcontrolled molecule alone and in a biomembraneꢀenvironment. First,
Langmuir monolayer and Brewster angle microscopy (BAM) experiments showed that pure
C12OazoE3OH enriched in the (E) isomer was able to form solidꢀlike mesophase even at low surface
pressure associated with supramolecular organization of the azobenzene derivative at the interface.
On the other hand, pure C12OazoE3OH enriched in the (Z) isomer formed a less solidꢀlike
monolayer due to the bent geometry around the azobenzene moiety. Second, C12OazoE3OH is wellꢀ
mixed in a biological membrane model, Lipoids75TM (up to 20 %mol), and photoisomerization
among the lipids proceeded smoothly depending on light conditions. It is proposed that the crossꢀ
sectional area of the hydroxyl triethylenglycol head of C12OazoE3OH inhibits azobenzenes Hꢀ
aggregation in the model membrane, thus the tails conformation change due to photoisomerization
is transferred efficiently to the lipid membrane. We showed that the lipid membrane effectively
senses the azobenzene geometrical change photoꢀmodulating some properties, like compressibility
modulus, transition temperature and morphology. In addition, photomodulation proceeds with a
color change from yellow to orange, providing the possibility to externally monitoring the system.
Finally, Gibbs monolayers showed that C12OazoE3OH is able to penetrate the highly packing
biomembrane model, thus C12OazoE3OH might be used as photoswitchable molecular probe in real
systems.
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INTRODUCTION:
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Molecular selfꢀassembly is the preferred tool of nature to build nanoꢀmachines like enzymatic
complexes, ribosomes and DNA transcription machinery, biomembranes among others. 1 By simple
observation of nature, it is clear that, it is possible to obtain molecular devices with exquisite
spatiotemporal control from molecules. This encourages chemists to design new molecules or use
nature building blocks like lipids in order to build devices in the nanoꢀ and microꢀrange. 2ꢀ4
In this context, π conjugated building blocks with amphiphilic features are employed because their
selfꢀassembly in water is accomplished by thermodynamically driven separation between the polar
medium and the rigid πꢀsurface. On the other hand, as π – π interactions are directional such selfꢀ
assembly can produce structurally precise nanoꢀstructures from small molecules.5. If the azobenzene
moiety is incorporated into an amphiphile structure, the inherent photoꢀresponsive capability of
azobenzene group might be transferred to the whole molecule, giving as a result a photoꢀresponsible
amphiphilic molecule.5 Under visible light, the azobenzene adopts its E (trans) configuration, and
the molecular geometry resembles a rodꢀtype; whereas upon UVꢀlight illumination, the molecular
geometry around the azobenzene moiety changes to bent configuration known as Z (cis). The
change in the length is really important, varying from 9 Å in E configuration to 5 Å in Z
configuration.6 Singleꢀtailed azobenzene amphiphiles are the simplest building blocks to develop
photoꢀtunable chemical systems, often combining guestꢀhost interactions or the presence of lipids.7ꢀ
12 The photoswitchable ability is predictable mainly in organic solvent;13 however, in the aggregate
state it will depend strongly on the interaction among molecules. If the azoamphiphile head is ionic
in character, the head effective size has a very strong effect on the system packing causing that, if
photoisomerization occurs, the molecular geometry change cannot be effectively sensed, especially
in mixtures with phospholipids.14ꢀ17 Recently, an ammonium salt head connected to a azobenzene
was reported to build up nonꢀphospholipid fluid liposomes with switchable photoꢀcontrolled
release.18
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Nonꢀionic azoamphiphiles are seldom investigated. A successful example was reported based on a
azobenzene with simple hydroxyl diethyleneglycol head able to form nanoꢀvesicles which, upon
photoisomerization, turned into bicontinuous phase.19 More recently, other azoamphiphile based on
dendritic glycerol head has emerged as suitable building block to obtain photoresponsible spherical
micelles.20, 21 Both systems showed efficient photoꢀcontrol of the water surface tension.22, 23 The
success of the responsiveness and the resulted functionality were due to the size of the head and the
nonꢀionic character which play an essential role in the packing parameter of both selfꢀassembled
systems.
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Our general research goals are the application of azoamphiphiles as membrane photoꢀswitches to
control spatioꢀtemporarily lipid membranes and cells. In this context, Langmuir and Gibbs
monolayers have offered great potential for the study of the organization of soft amphiphilic
materials24 and in particular of lipids to understand the packing of molecules based on their
chemical structure, intermolecular interactions, and interactions with the supporting subphase.25ꢀ27
In fact, Langmuir monolayers have been used to model azobenzenes selfꢀassembly at the interface
and processes occurring within them.28ꢀ30 However, their use to evaluate the behavior of
azobenzene/lipids mixtures was not reported.
The aim of the present work is to present a simple nonꢀionic photoswichtable azoamphiphile, 4ꢀ
dodecyloxiꢀ4’ꢀ(1ꢀhydroxy triethylenglycol) azobenzene (C12OazoE3OH) which behaves as photoꢀ
switchable probe alone and when it is mixed with a biological membrane mimetic, Lipoids 75TM.
The photoisomerization of C12OazoE3OH among the lipids induces interfacial, morphological and
thermal changes of the biomimetic membrane. Importantly, we showed that C12OazoE3OH is not
only well mixed in the biomembrane, but also it is able to penetrate it from the subphase.
Altogether, our findings show that C12OazoE3OH might be an efficient photoꢀresponsible probe to
remote control of biological membrane properties.
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EXPERIMENTAL SECTION
Material and Synthesis:
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All manipulations were carried out under an atmosphere of nitrogen. Acetonitrile was dried over
K2CO3 and distilled immediately before use.
1ꢀdodecyloxiꢀ4ꢀnitrobenzeno (1): To a solution containing 886.3 mg (6.38 mmol) of nitrophenol
and 5.28 g (38.28 mmol) K2CO3 in 20 ml acetonitrile under stirring was added 1.85 ml (7.66 mmol)
dodecyl bromide in 10 ml acetonitrile. The reaction mixture was refluxed for 27 hours and
subsequently filtered through Celite. The solvent was removed by reduced pressure and the crude
product was obtained as a solid. This solid was further purified by column chromatography on
neutral aluminum oxide (hexane: diethyl ether) and 1 was recovered as a white solid in 90% yield.
1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, 3H, J3= 6.9 Hz, CH3), 1.27ꢀ1.47(m, 18H, CH2), 1.82
(m, 2H, CH2), 4.05 (t, 2H, J3= 6.0 Hz, OCH2), 6.93 (d, 2H, J3= 9.0 Hz, ArꢀH), 8.19 (d, 2H, J3= 9.0
Hz, ArꢀH). 13CꢀNMR (CDCl3) δ (ppm): 14.07 (CH3), 22.67, 25.91, 28.98, 29.29, 29.32, 29.52,
29.56, 29.62, 29.63, 31.91 (CH2), 68.93 (OCH2), 114.41, 125.8, 141.4, 164.26 (ArꢀC).
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4ꢀdodecyloxiꢀaniline (2): To a solution of 300.0 mg (0.98 mmol) 1ꢀdodecyloxyꢀ4ꢀnitrobenzeno (1)
dissolved in 10 ml anhydrous CHCl3 was added 8.6 ml methanol. The reaction was stirred in the
presence of 50.0 mg of 10% Pd/C under hydrogen atmosphere for 4ꢀ6 hours at room temperature.
Then, it was filtered off and the solvent was removed under reduced pressure with quantitative
yield. The bright grey solid (2) was used without further purification in the following step.
4ꢀdodecyloxyꢀ4’ꢀhydroxyl azobenzene (3): To 271.5 mg (0.98 mmol) of (2) in 4.50 ml of 1:1
aqueous acetone was added 0.45 ml of hydrochloric acid concentrated. To this mixture 2.25 mL of
aqueous NaNO2 (93.15 mg, 1.35 mmol) was added and the solution was maintained at 0ºC for 1 h.
The resulting diazonium slurry was then added to the following mixture: phenol 131.6 mg (1.40
mmol), NaOH 52.9 mg (1.32 mmol), Na2CO3 229.0 mg (2.16 mmol) in 3.80 ml of MilliQ water
maintaining the temperature at 0ºC for an additional 1 h. Then, the mixture was stirred for further
16 h at room temperature. After this, it was neutralized with acetic acid and the precipitate
containing compound (3) was well washed with MilliQ water. Further purification was not
necessary, 40% yield (mp 96ꢀ97 ºC). 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.88 (t, 3H, J3= 6.9 Hz,
CH3), 1.27ꢀ1.47 (m, 18H, CH2), 1.81 (m, 2H, CH2), 4.03 (t, 2H, J3= 6.0 Hz, OCH2), 4.63 (s, 1H,
OH), 6.93 (d, 2H, J3 = 9 Hz, ArꢀH), 6.98 (d, 2H, J3 = 9 Hz, ArꢀH), 7.81 (d, 2H, J3 = 9 Hz, ArꢀH),
7.85 (d, 2H, J3 = 9 Hz, ArꢀH). 13CꢀNMR (CDCl3) δ (ppm): 14.07 (CH3), 22.67, 26.025, 29.22,
29.33, 29.38, 29.56, 29.58, 29.62, 29.65, 31.91 (CH2), 68.40 (OCH2), 114.72, 115.76, 124.33,
124.51, 146.91, 147.23, 161.27 (ArꢀC).
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4ꢀdodecyloxiꢀ4’ꢀ(1ꢀhydroxy triethylenglycol) azobenzene (4): To 75.0 mg (0.197 mmol) of 4ꢀ
dodecyloxyꢀ4’ꢀhydroxyl azobenzene (3) and 300 mg (2.17 mmol) K2CO3 suspension was added
63.0 mg (0.207 mmol) triethylene glycol pꢀtoluenesulfonate in 10 mL of dry acetonitrile The
reaction mixture was allowed to react at reflux for 80 hours and then filtered out. The solid was
further purified by column chromatography on neutral aluminum oxide (hexane: diethyl ether) and
pure (4) in 55% yield was obtained. C12OazoE3OH shows thermotropic behaivor: CrꢀSmC at 91.67°C
(ꢁH= 88.10 J g−1) SmCꢀPI at 107.28 °C (ꢁH= 33.77 J g−1). The LC behavior is not described here.
1H NMR (400 MHz; CDCl3): δ (ppm)= 0.88 (t, 3H, J3= 6.9 Hz, CH3), 1.27ꢀ1.47 (m, 18H, CH2),
1.81 (m, 2H, CH2), 2, 08 (s, 1Hꢀ, OH); 3,61ꢀ3,74 (m, 8H, CH2); 3,90 (t, 2H, J3= 6.0 Hz, OCH2);
4.03 (t, 2H, J3= 6.0 Hz, CH2), 4,22 (t, 2H, J3= 6.0 Hz, CH2); 6.99 (d, 2H, J3 = 9 Hz, ArꢀH), 7.00 (d,
2H, J3 = 9 Hz, ArꢀH), 7.86 (d, 2H, J3 = 9 Hz, ArꢀH), 7.87 (d, 2H, J3 = 9 Hz, ArꢀH). 13CꢀNMR
(CDCl3): δ (ppm) = 14.10 (CH3), 22.68, 26.00, 29.22, 29.33, 29.40, 29.50, 29.60, 29.63, 29.65,
31.91 (CH2), 61.8, 69.15, 69.71, 70.45, 70.59, 70.90, 72.50 (OCH2), 114.71, 114.85, 124.26,
124.32, 146.97, 147.32, 160.65, 161.275 (ArꢀC). EMꢀFAB: (m/z): 515, 40 (M+1H+) EMAR:
calculated for C30H46N2O5 515, 34 found. 515, 35.
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Synthesis of Vesicular Membranes
Lipoid s75TM containing 69ꢀ75% phosphatidylcholine was purchased from Lipoid Co. (Germany).
Other constituents of Lipoid s75TM are: 9.8% phosphatidylethanolamine and 2.1%,
lysophosphatidylcholine and had a fatty acid composition of palmitic (17%–20%), stearic (2%–
5%), oleic (8%–12%), linoleic (58%–65%) and linolenic (4%–6%) acid. 31 Chloroform was HPLC
grade. Water was purified by using a MilliꢀQ (Millipore, Billerica, MA) system, giving a product
with a resistivity of ~ 18.5 Mꢂ/cm.
TM
Mixtures of C12OazoE3OH: Lipoid s75
were conducted following the technique for obtaining a
thin film. The procedure involves the formation of a homogeneous organic film by vacuum
evaporation at room temperature of a chloroform solution of the C12OazoE3OH amphiphile (ZꢀE: 5ꢀ
95, 20 mol %) exposed to white light and Lipoid s75TM (80 mol %). To hydrate the film, MilliꢀQ
water at the lipid transition temperature (55ºC) is added to the round bottom flask, under slow
rotatory movement for 2 hrs. The next process involves vigorous shaking using a vortex, and then
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sonication of the sample to obtain the suspension of the film. Finally it is necessary at least two
days aging to achieve formation of the vesicular membranes, multilayers (approximately 0.5 mM).
The same procedure was performed to form pure Lipoid s75TM vesicular membranes.
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Methods:
UV-Vis Spectroscopy: Switching experiments were done with an 8 watts mercury arc lamp with
filter of 360 nm from Pleuger Antwerp Brussels and a white light bulb of 60 watts. UVꢀVis
spectroscopy data were recorded on a JASCO Vꢀ630BIO Spectrophotometer equipped with an
EHCSꢀ760 peltier.
Surface Pressure-Area Isotherms of Langmuir Monolayers: The first compression isotherms at 24
°C and 13 °C were performed for both C12OazoE3OH (4) and C12OazoOH (3) compounds. These
compounds were dissolved in a methanol/ chloroform (2:1) mixture to obtain a solution of 1 mg/ml
total concentration which was spread onto a 266 cm2 Teflon trough filled with 200 ml of subphase.
Concentration of 1 mg/ml of a mixture of Lipoid s75TM. The same protocol were used for mixed
monolayers of Lipoid s75TM: C12OazoE3OH (4) (80:20 mol%). The aqueous solution used as the
subphase was pH ~ 5.5 (NaCl 145 mM) for all the monolayer experiments. The film was allowed to
stand 5 min for solvent evaporation and monolayer relaxation at < 0.1 mN/m before being
subsequently compressed isometrically at a compression rate of 2 ± 1 Å2 moleculeꢀ1 minꢀ1.
The surface pressure was determined with a Pt plate using the Wilhelmy method, and the film total
area was continuously measured and recorded using a KSV Minitrough apparatus (KSV, Helsinski,
Finland) enclosed in an acrylic box which was continuously enriched with N2 gas in order to
prevent lipid oxidation. The subphase temperature (±0.5 ºC) was controlled by an external
circulation bath and kept at 25 ºC, unless indicated.
A representative experiment is shown in all figures from a set of three independent experiments that
differed in mean molecular area (MMA) and surface pressure measurements by less than 2 Å2 and
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0.2 mN/m, respectively. In order to analyze the film elastic behavior of the adsorbed molecules, the
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compressibility modulus Csꢀ1 was calculated from the isotherm data as, 32
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−1
d
π
Cs = −MMA
(
)
dMMA T
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where π represents the surface pressure. Measurements were carried out in darkness for
C12OazoE3OH (Z).
Brewster Angle Microscopy (BAM) Measurements: Monolayers of C12OazoE3OH (E), (Z) and
C12OazoOH (E) were prepared as described above using a KSV Minitrough apparatus (KSV,
Helsinski, Finland). The Langmuir apparatus was mounted on the stage of a Nanofilm EP3 imaging
elipsometer (Accurion, Goettingen, Germany) used in the BAM mode. Zero reflection was set with
a polarized 532 λ laser incident on the bare aqueous surfaces at the experimentally calibrated
Brewster angle (~53.1º). After monolayer formation and compression, the reflected light was
collected with a 20X objective. Under the required calibration, the gray level of each pixel of the
BAM images is directly related to the square of the film thickness and refraction index, in films that
do not show optical activity; 33 otherwise, the gray level also depends on the orientation of the 2D
crystal.
Adsorption of Azo-amphiphiles to Bare Air/Water Interface: Gibbs Monolayers. After injection of
25 µl of C12OazoE3OH (E) or 50 ꢀl of C12OazoE3OH (Z) to the aqueous subphase of a Teflon
trough under continuous stirring (trough volume: 15 ml; final subphase concentration 3.2 ꢃM for
C12OazoE3OH (E) and 6.39 ꢃM for C12OazoE3OH (Z), the changes in surface pressure and surface
potential at constant area were registered as a function of time, while a Gibbs monolayer was
established. Surface pressure was determined as described above. The equipment used was a
homemade circular Langmuir balance controlled by an electronic unit (Monofilmetter). All
experiments were performed at 23 ± 2 °C. Measurements were carried out in darkness for
C12OazoE3OH (Z).
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Penetration of Azo-amphiphiles to Phospholipid Monolayers: Penetration time curves were
performed by deposition of a chloroform solution of Lipoid s75TM at the air/water interface, in order
to form a monolayer previous to the injection of C12OazoE3OH (25µL of (Z) and 50 µL of (Z)) in
the subphase. Measurements were carried out in darkness for C12OazoE3OH (Z).
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Differential Scanning Calorimetry (DSC): Calorimetric measurements were performed with a Q20
Differential Scanning Calorimeter (TA Instruments). Samples were prepared using closed hermetic
aluminum pans which had been weighted with a ± 0.00001 g precision balance adding 10 mg of the
corresponding compound or water dispersion (2.8 mg/ml) to the pan: a) C12OazoE3OH (E) solid, b)
Lipoid s75TM multilamellar vesicular dispersion alone c) Mixtures of Lipoid s75 TM: C12OazoE3OH
(E) multilamellar vesicular dispersion and d) Mixtures of Lipoid s75 TM: C12OazoE3OH (Z)
multilamellar vesicular dispersion obtained by irradiating the Lipoid s75 TM: C12OazoE3OH (E) for
40 minutes with UV light (360 nm, 8 watts). The same procedure was performed to Lipoid s75 TM
vesicular membranes alone, showing no significant physical changes. The calorimetric
measurements were performed at a rate of 5°C/min.
Polarized Optical Microscopy (POM): A drop of the corresponding dispersion of 2.8 mg/ml
concentration was placed on glass and cover. POM were performed at 25°C using a Nikon Eclipse
E200 POL polarizing microscope.
RESULTS and DISCUSSION
Design, Synthesis and Photoisomerization. Regarding to potential applications, we designed a
structural simple amphiphile, C12OazoE3OH (4), by a simple and versatile methodology (Scheme
1). C12OazoE3OH was obtained directly from commercial 4ꢀnitrophenol in four steps, with an
overall yield of 86 %. The introduction of oxygen atoms as connectors between the azobenzene
moiety and the head and tail is synthetically simple and allows efficient photoisomerization with
slow thermal backꢀisomerization. Based on previous reports, 19ꢀ22 we hypothesized that the hydroxyl
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triethylenglycol head would increase the effective cross section area, thus favoring the molecular
motion of the tails in the aggregate due to light illumination. Finally, the incorporation of a C12
hydrophobic tail will favor bilayer formation and potential good mixing with natural lipids in
biological membranes.34
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Scheme 1: Synthetic pathway to C12OazoE3OH (4).
In chloroform, C12OazoE3OH (4) is a yellow solution composed of isomeric mixture of E: Z (95:5)
determinated by 1HꢀRMN which is named for simplicity as C12OazoE3OH (E) (see ESI). Upon UVꢀ
light illumination (360 nm, 8watts, 1 min) a photostationary state (pss) of E: Z (10:90) is reached,
named as C12OazoE3OH (Z) (orange solution). The photoconvertion ratio from E to Z isomer was
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evident from HꢀRMN experiments and UVꢀVis spectra (Figure 1). UVꢀVis spectrum of
C12OazoE3OH (E) showed a characteristic πꢀ π* transition centered in 358 nm and a small band at
450 nm corresponding to nꢀ π* transition. The Z→E isomerization was accelerated by illumination
with ordinary white light bulb of 60 watts (λ > 445 nm, 3 h, see ESI). The new πꢀ π* transition was
blueshifted to 311 nm and the band corresponding to nꢀ π* transition at 450 nm was more evident.
Thermal Z→E isomerization is slow enough allowing the evaluation of both pss separately (stable
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18 h in darkness). This experiment suggests that C12OazoE3OH could be used as optically
controlled probe.
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Figure 1. UVꢀVis spectra of C12OazoE3OH before (●) and after (△) UVꢀlight illumination in
chloroform (7, 5 ꢃM). Optical stability was achieved up to five cycles.
Langmuir Monolayers. First compression curves of Langmuir monolayers at 24 °C shown in
Figure 2a exhibited higher values of collapse pressure for C12OazoE3OH (E) (~53mN/m) than
C12OazoE3OH (Z) (42 mN/m). The mean molecular area (MMA) at such high pressures for both
C12OazoE3OH (E) and C12OazoE3OH (Z) are similar (~23 ± 2 Å2/molecule), showing that the
azobenzene group is perpendicular to the surface plane at collapse pressure.35 This value is in
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agreement with those reported previously for a compound with a hydroxyl diethylenglycol head
and different linkers between the azo moiety and the hydroxyl head, (C4azoOLinkerE2OH).
Moreover, it is close to the crossꢀsection area of hydrocarbon chain ~18 Å2/molecule. 32
The compression isotherms of compound 3 C12OazoOH (E) (E: Z (90:10)) were included to
compare the influence of the head length. Both C12OazoE3OH (E) (▲) and C12OazoOH (E) (●)
monolayers were similar in saline solution pHb~ 5.5, showing a phase transition at low surface
pressure (π ≤ 5 mN/ m) and a collapse at ~ 55 and 60 mN/ m, respectively. However, C12OazoOH
(E) monolayers showed no sign of a pressure decrease after collapse, showing high stability of the
film at 24 °C (not shown). This effect has been reported previously in monolayers of azobenzeneꢀ
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polyvinyl alcohol and for ureaꢀazobenzene derivatives
and it was associated with strong
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intermolecular interactions. Here, the πꢀπ stacking of the aromatic rings of C12OazoOH (E) may be
maximal due to the minor steric repulsion of the small OH head. The phase transition of
C12OazoE3OH (E) occurred at 50 Å2/molecules, whereas for C12OazoOH (E) it occurred at 40
Å2/molecules. In this case, the larger hydroxyl triethylenglycol group in C12OazoE3OH (E) had to
exert stronger head group repulsion than the hydroxyl head in C12OazoOH (E), resulting in a higher
MMA when phase transition occurs. C12OazoOH (3) showed a fast thermal ZꢀE isomerization rate
after UV illumination, thus the Z enriched pss was not measured at the air/water interface.
Langmuir monolayer compression curves of C12OazoE3OH (Z) showed a smooth slope with no
transition until the collapse. The bent geometry around the azobenzenes of C12OazoE3OH (Z) could
be the reason of such behavior. Similar results for E and Z isomers were described previously for
other azobenzenes amphiphiles and it was explained considering supramolecular organization at the
air/water interface.36, 37
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For C12OazoE3OH (E), C12OazoE3OH (Z) and C12OazoOH (E) films, the solubility into the
subphase was evaluated by assessing the rate of monolayer area loss when kept at the constant
surface pressure of 35 mN/m (ESI, Fig. S7). The films composed by C12OazoE3OH (E) and
C12OazoOH (E) show a rate of area loss of 13 % and 5% after 23 min, respectively, being aprox.
28% for C12OazoE3OH (Z) monolayers. This experiment evidenced a higher solubility of the isomer
(Z) at a constant high constant pressure compared with the (E) isomers, which can be explained by
the increase of dipole moment of the Z isomer. 6
Monolayer curves of C12OazoE3OH (E) at 13 °C displayed essentially the same collapse
characteristics (MMA and surface pressure) than those at 24 °C; however, at 13 °C the phase
transition at 50 Å2/molecule was not observed (ESI, Fig. 6). Nevertheless, the monolayers at 13 °C
were more suitable for BAM experiments due to their reproducibility.
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The C12OazoOH (E) film shows a condensed or solid character as evidenced by a relatively high Csꢀ
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values (300 mN/m, Figure 2b), above 5 mN/m.37 For C12OazoE3OH (E) monolayers, it was
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observed a less compact behavior with Csꢀ1 ~ 100 mN/m, suggesting LC state at π > 5 mN/m. The
difference of Csꢀ1 values between C12OazoE3OH (E) and C12OazoOH (E) reinforces the idea that
C12OazoOH (E) forms highly condensed monolayers due to strong intermolecular interaction which
did not operate neither in C12OazoE3OH (E) monolayers nor in the monolayers of C12OazoE3OH
(Z) (Csꢀ1 ~60 mN/m).
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Figure 2. A) Langmuir isotherms at 24°C, compression rate of 2±1 Å2 molꢀ1minꢀ1: C12OazoE3OH
(E) (▲), C12OazoE3OH (Z) (ꢀ) and C12OazoOH (E) (●). B) Compressibility modulus of
C12OazoE3OH (E) (▲), C12OazoE3OH (Z) (ꢀ) and C12OazoOH (E) (●). Arrows indicate the
beginning of the phase transition. The curves shown are chosen from a set of duplicates that
differed by less than 2 Å2/molecule.
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Brewster Angle Microscopy. Figure 3A shows that C12OazoOH (E) displays a solidꢀlike
character, detecting positive spherulites at low surface pressure (<1 mN/m) characteristic of a
Hexatic I mesophase 38 which it was lost above 5 mN/m. On the other hand, C12OazoE3OH (E) and
C12OazoE3OH (Z) monolayers are composed of highly reflective and microꢀheterogeneous phases,
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which resemble a condensed phase. Pedrosa et. al. described the same behavior for a carboxylic
azobenzene derivative, they attributed the features to the tendency of E azobenzene hydrophobic
tails to form face to face Hꢀ aggregates at the air/water interface. In particular, C12OazoE3OH (E)
shows below 3 mN/m islands of expanded phase which further increase of the surface pressure lead
to a uniform solidꢀlike mesophase similar to those observed for C12OazoOH (E) (compared Figure
3 A and B near the collapse). These aggregates were able to form mesoscopicꢀsized supramolecular
assemblies having a regular molecular alignment with strong birefringence; hence, their aggregates
may be detected directly using polarized light microscopy. It is known that E azobenzene
derivatives have a high tendency to form faceꢀtoꢀface Hꢀaggregates, both in bulk crystalline
structures and in monolayers, as it has been demonstrated using a range of experimental
techniques.36, 37, 39ꢀ41 Interestingly, BAM experiments of C12OazoE3OH (Z) (Figure 3C), showed
wormꢀlike shapes as surface pressure was increased.
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Figure 3. BAM micropictures at 13 ºC of: A) C12OazoOH (E), B) C12OazoE3OH (E), C)
C12OazoE3OH (Z). Surface pressures are indicated in each picture (the complete sequence is
presented in ESI: Fig S8, S9, S10, respectively). Scale bars correspond to 20 µm
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These shapes were bright roads in vertical position and dark in horizontal position as a consequence
of its differential interaction with polarized light. Therefore, it might be the same structure but at
different polarization planes. In general, monolayer phases with tilted molecules often show long
range tilt orientation order giving rise to optical anisotropy in the plane. The observed bright BAM
reflectivity upon compression could be a result of the high tilted geometry of C12OazoE3OH (Z)
which is maintained near collapse. (Figure 4).
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Figure 4. Proposed models near collapse of a) C12OazoE3OH (E), b) C12OazoE3OH (Z) and c)
C12OazoOH (E) molecules at the interfase air/water. Atoms reference: red (Oxygen), light blue
(Carbon), blue (Nitrogen), white (Hydrogen)
Photoisomerization at the interface. First, monolayers of C12OazoE3OH (E) were kept at a surface
pressure of 2 mN/m and 10 mN/m in independent experiments and then, both systems were allowed
to equilibrate for several minutes. After that, the monolayers were illuminated by UVꢀlight for 40
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min. When UVꢀlight irradiation was stopped, both systems were kept in darkness. A decrease in the
MMA of 8 ± 1 and 6 ± 1 was detected, respectively (ESI, Fig. S11). The observed behavior has
been previously reported for other azoamphiphiles. 29, 30 It has been explained considering that in
general molecules of E azobenzenes possess a zero dipolar moment meanwhile Z isomers display
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an increase of around 4 Debye in many cases. Thus, the increase of the molecular dipole moment
in the Z configuration should increase their solubility in the subphase (aqueous solvent) and the loss
of C12OazoE3OH (Z) molecules would be reflected by a reduction of the monolayer area.
Nevertheless this experiment showed that C12OazoE3OH (E) monolayers could be disturbed by UVꢀ
light illumination.
Langmuir Monolayers of a Biomimetic Lipid Membrane and C12OazoE3OH. Lipoid s75TM
composed of 75% of phosphatidylcholine could be a suitable model of biological membrane
because of its complexity.31,42 For this reason, we envisaged that the evaluation of C12OazoE3OH
integrated in this membrane could be used as a proof of concept of the utility of C12OazoE3OH as
photoswitchable probe in real biomembranes. When Lipoid s75TM was spread at the air/water
interface, with or without 20 mol% of C12OazoE3OH (E) or C12OazoE3OH (Z), the compression
isotherm showed a smooth slope according to a LE behavior at low surface pressures (see inset in
Figure 5). The beginning of a LEꢀLC transition is observed only for C12OazoE3OH (Z) at about 12
mN/m. The compressibility modulus for pure Lipoid s75TM was lower (~45 mN/m) than the values
reached by monolayers formed by mixtures of Lipoid s75TM and C12OazoE3OH (E) or (Z) which
were both similar (~70 mN/m) at high pressures (Figure 5). This increase showed that
C12OazoE3OH conferred to the membrane a more condensed character which was expected
considering the molecular rigidity of the azoamphiphile and the solidꢀ like character at the interface.
Interestingly, below 20 mol % of C12OazoE3OH, the lipid did not show a significant variation of the
interfacial behavior. Considering the complexity of the system, we decided to further evaluate
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Lipoid s75TM /C12OazoE3OH in bulk in order to detect other changes on Lipoid s75TM membrane
promoted by the presence of C12OazoE3OH.
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Figure 5. Compressibility modulus of a representative monolayer composed of Lipoid s75TM:
C12OazoE3OH (E) (80:20 mol%) (▲), Lipoid s75TM: C12OazoE3OH (Z) (80:20 mol%) (ꢀ) and
pure Lipoid s75TM (■) as a function of surface molecular packing. The inset shows compression
isotherms which were used to calculate compressibility modulus. The arrows indicate the beginning
of the more condensed phase. The curves shown are chosen from a set of duplicates that differed by
less than 2 Å2/molecule.
Photoisomerization of Vesicular Membranes. Vesicular membranes of Lipoid s75TM alone and
with C12OazoE3 (E) were obtained by evaporationꢀhydration method.43, 44 After UV illumination of
Lipoid s75TM: C12OazoE3OH (E) mixture in water, a color change from yellow to orange was
observed (as detected when pure C12OazoE3OH (E) was illuminated in chloroform).
Photoisomerization of Lipoid s75TM: C12OazoE3OH proceed smoothly as observed for pure
C12OazoE3OH in chloroform (compare Figures 1 and 6). The position of the absorption maxima of
πꢀ π* transition of C12OazoE3OH (E) centered in 358 nm is characteristic of (E) monomer, showing
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that the azoamphiphile is well mixed among the lipids, inhibiting the formation of H aggregates in
the biomembrane and allowing complete photoisomerization.14, 45
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After UV illumination πꢀ π* transition maxima is centered in 323 nm and the nꢀ π*transition at 450
nm increases which corresponds to (Z) isomer. It is possible to estimate a 10:90 pss, which is much
higher than those reported previously for a ionic pseudoglyceryl single chain azobenzene lipid
integrated in a phospholipid vesicular membrane (35:65 pss).46
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Figure 6. A) UVꢀVis spectra of C12OazoE3OH before (●) and after (△) UVꢀlight illumination in the
vesicular membrane. B) Schematic representation of C12OazoE3OH (E) in yellow and
C12OazoE3OH (Z) in orange integrated in the biomembrane.
Physical changes of a biomimetic membrane promoted by C12OazoE3OH. The Mutilamellar
vesicular dispersions described above were observed in a polarizer optical microscope (POM) under
crossed polarizers. Because of the complexity of the whole system, all POM experiments were
performed at 25°C where lamellar phases of PC are favored.47, 48 In fact, dispersion of Lipoid s75TM
pure in water formed lyotropic myelin figures, which are multilamellar tubular microstructures
(Figure 7A).49 On the other hand, mixtures of C12OazoE3OH either in (E) or (Z) and Lipoid s75TM
showed two different but very complex lyotropic pattern, which resembled laminar phase.
Importantly, the presence of C12OazoE3OH in the mixture of Lipoid s75TM inhibited the formation
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of myelin figures (Figure 7B and 7C).50 It seems that the incorporation of C12OazoE3OH among
Lipoid s75TM breaks the tubular myelin structure towards the formation of little folded sheets called
lamellae, which in some cases twist themselves into a circular arrangement called spherulites.51, 52
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Figure 7. POM micrograph (crossed polarizers) of vesicular membrane dispersions at 25°C of A)
Lipoid s75 TM, B) mixture of Lipoid s75 TM / C12OazoE3OH (E) (80:20 mol%), C) mixture of Lipoid
s75 TM / C12OazoE3OH (Z) (80:20 mol%). The concentration of all dispersions was 2.8 mg/ml.
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Differential scanning calorimetry (DSC) experiments of multilamellar vesicular membranes of
Lipoid s75TM alone showed a nonꢀcooperative transition temperature (Tm) of ~ 58°C (Figure 8A)
which in the presence of 20 mol % C12OazoE3OH (E), increased to 100 °C (Figure 8C), showing
that C12OazoE3OH (E) transferred its structural rigidity to the lipid environment. After UVꢀ
illumination Tm shifted to ~ 80 °C (Figure 8D), showing that C12OazoE3OH (Z) induces a decrease
on the thermal transition temperature because of its bent molecular structure. Probably, both
C12OazoE3OH isomers are inserted with its large hydroxyl head close to the polar head of the lipids
and partly immobilize those regions of the hydrocarbon chains closest to the polar head groups,
similar to the known cholesterol interaction with lipids. In addition, the broad DSC peaks does not
allow an accurate calculation of thermodynamics parameters, however they evidenced that the
transition is nonꢀcooperative, which it was expected for complex systems like Lipoid biomimetic
membranes.53 In addition, recently it was described that in the tilted gel phase, complex shapes can
form spontaneously even in a membrane containing only a single lipid component leading nonꢀ
cooperative transitions.54
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Figure 8. From bottom to top: DSC thermograms are shown A) solid C12OazoE3OH*, B)
multilamellar vesicular dispersion of Lipoid s75 TM; C) multilamellar vesicular dispersion of Lipoid
s75TM: C12OazoE3OH (E) mixture (80:20 mol%); D) multilamellar vesicular dispersion of Lipoid
s75 TM: C12OazoE3OH (Z) mixture (80:20 mol%). Concentration of all dispersion was 2.8 mg/ml.*
CrꢀSmC at 91.67°C (ꢁH= 88.10 J g−1) SmCꢀPI at 107.28 °C (ꢁH= 33.77 J g−1). The thermotropic behavior
of pure C12OazoE3OH is not described here.
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Gibbs Monolayers. If C12OazoE3OH will be used as remote photoꢀcontrol molecule in real
membranes, it would be capable to penetrate a highly compact monolayer. To this aim, Gibbs
monolayers of (E) and (Z) C12OazoE3OH were obtained on a bare surface to evaluate the increase
on surface pressure when the corresponding C12OazoE3OH isomer was injected under the subphase.
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The pressures reached were ~ 25 mN/m for C12OazoE3OH (E) and 20 mN/m for C12OazoE3OH
(Z) at concentration of 3.20 µM and 6.39 µM, respectively (Figure 9A). Then, the penetration
behavior of C12OazoE3OH (E) and C12OazoE3OH (Z) into a preformed Lipoid s75TM monolayer
showed that both isomers were able to penetrate the lipid monolayer for all initial surface pressures
tested (figure 9B). The values plotted are for separated monolayers of different initial surface
pressures obtained by the addition of C12OazoE3OH (E) and C12OazoE3OH (Z), respectively. Marsh
has shown that, the best correspondence with various bilayer properties of pure lipids and biological
membranes is obtained for monolayers at surface pressure of 30ꢀ35 mN/m.55 More recently, a
correspondence between bilayers and monolayers of pure lipids were obtain by multiphoton
excitation fluorescence microscopy.56 These authors reported that monolayerꢀbilayer
correspondence occurs when the lateral pressure of the monolayer is 26ꢀ28 mN/m and 28ꢀ31 mN/m
for DOPC and DPPC, respectively. Our experiments showed that, C12OazoE3OH (E) penetrates the
lipid monolayer to an extrapolated maximal pressure of ~ 37 mN/m, and C12OazoE3OH (Z) reaches
the highest cut off value at ~ 43 mN/m. The obtained extrapolated maximal values are in the range
of other penetrating molecules, like δꢀlysin (33 mN/m) and melitin (43 mN/m) and their interaction
with real reconstituted biomembrane like sheep erythrocyte lipids.57
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Figure 9: A) Gibbs monolayers of C12OazoE3OH (E) (▲) and C12OazoE3OH (Z) (ꢀ) formed by
adsorption to a bare air/saline solution subphase. The curves are representative of three experiments
developed in independent runs. The arrows indicate the time of C12OazoE3OH (E) (black) and
C12OazoE3OH (Z) (grey) injection into the subphase, respectively B) Penetration cutoff curves for
C12OazoE3OH (E) (▲) and C12OazoE3OH (Z) (ꢀ)into Lipoid s75TM monolayers at different initial
surface pressure (mN/m). Results represent the average of two independent experiments. Final
subphase concentration was 3.20 µM C12OazoE3OH (E) for and 6.39 µM for C12OazoE3OH (Z).
It had been previously reported that the penetration ability of the amphiphiles depends on the
aggregate size.58, 59 Dynamic light scattering (DLS) experiments of C12OazoE3OH (Z) in water
showed that it was composed of one population of around 110 ± 50 nm of diameter, meanwhile
C12OazoE3OH (E) was composed of two population one of around 110 ± 20 nm and a second
around 450 ±50 nm at room temperature (ESI, Fig. S12). This experiments suggest that
C12OazoE3OH (Z) molecules forms aggregates smaller than those of the C12OazoE3OH (E) at the
concentration tested and, thus, the subphaseꢀsurface equilibrium may be improved. The high cut off
values obtained showed that C12OazoE3OH might potentially penetrate a cell membrane.
CONCLUSION:
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In conclusion, we provided experimental evidence that C12OazoE3OH is a suitable optical
controlled molecule. The supramolecular organization of pure C12OazoE3OH at the interface is
complex and it will be further studied by LangmuirꢀBlodgett films. C12OazoE3OH was well
integrated in a complex biomembrane model, like Lipoid s75TM. The collapse surface pressure was
similar (40 mN/m) for the biomembrane alone or mixtures with both C12OazoE3OH isomers (20
mol%) however, a 25 mN/m increase of Csꢀ1 was detected in the presence of C12OazoE3OH,
showing that the lipid membrane exhibits a more condensed character. Importantly, in the complex
lipid mixture E→Z photoisomerization of C12OazoE3OH proceeded smoothly depending on light
conditions. It appears that the crossꢀsectional area of the hydroxyl triethylenglycol head of
C12OazoE3OH may inhibit azobenzene H aggregates formation in the model biomembrane. Because
of that, the tails conformation change due to photoisomerization is transferred efficiently to the lipid
membrane, photoꢀmodulating the lipid physical properties. DSC and POM experiments showed that
the E→Z photoisomerization in the lipid mixture induced thermal and morphological changes
towards a more rigid system depending on light illumination, as observed by Langmuir monolayers.
Probably, both C12OazoE3OH isomers are inserted with its large hydroxyl head close to the polar
head of the lipids and partly immobilize those regions of the hydrocarbon chains closest to the polar
head groups, similar to cholesterol. A better understanding of this behavior will require the
evaluation of C12OazoE3OH with pure lipids. Finally, both C12OazoE3OH (E) and (Z) isomers were
able to penetrate a highly packing biomembrane, showing their potential as external probes in real
systems. Further experiments are currently under progress to translate the use of C12OazoE3OH to
photoꢀcontrol the translocation of other molecules through lipid membranes. 60
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: veronica.dodero@uns.edu.ar
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Present Addresses
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†
Universität Bielefeld, Fakultät für Chemie, Organishe Chemie, Univeristätsstr. 25, 33615 Bielefeld, Germany, eꢀmail:
veronica.dodero@uniꢀbielefeld.de
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. ‡These authors contributed equally.
Acknowledgments
This work was supported mainly by UNSꢀPGI (Universidad Nacional del Sur), Secretary of Science
and Technology of Universidad Nacional de Córdoba (SECyTꢀUNC), CONICET PIP 2014ꢀ2016
(National Scientific and Technical Research Council) and ANCyPT PICTꢀPRH 2010ꢀ2013 and
PICT 2014ꢀ1627 (National Agency for Promotion of Science and Technology) Argentinian
Research grants. M. A. S and L. A: B. are also grateful for their CONICET fellowship. B.M. and
M.L.F. are Career Investigators of CONICETꢀUNC. V. I. D. and L. A. B. thank Prof. H Ritacco for
monolayers reproducibility experiments. V. I. D. thanks Prof. N. Sewald from Fakultät Chemie,
Universität Bielefeld for his support and J. L. Mascareñas for Mass Analysis. V.I.D acknowledges
her Alexander von Humboldt Foundation fellowship (HERMES).
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SUPPORTING INFORMATION Structural Characterization of C12OazoE3OH: Hꢀ,
CꢀNMR
Spectra, FAB spectra, Elemental Composition, Photoꢀconversion spectra, additional Langmuir
monolayers and BAM experiments of C12OazoE3OH, DLS experiments of C12OazoE3OH.
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