RESEARCH ARTICLE
Interactions of Aqueous Imidazolium-Based
Ionic Liquid Mixtures with Solid-Supported
Phospholipid Vesicles
Patricia Losada-Pe
´
rez
1
*, Mehran Khorshid
1
, Frank Uwe Renner
1,2
1 Institute for Materials Research IMO, Hasselt University, Diepenbeek, Belgium, 2 IMEC vzw, Associated
lab IMOMEC, Diepenbeek, Belgium
*
patricia.losadaperez@uhasselt.be
Abstract
Despite the environmentally friendly reputation of ionic liquids (ILs), their safety has been
recently questioned given their potential as cytotoxic agents. The fundamental mecha-
nisms underlying the interactions between ILs and cells are less studied and by far not
completely understood. Biomimetic films are here important biophysical model systems to
elucidate fundamental aspects and mechanisms relevant for a large range of biological
interaction ranging from signaling to drug reception or toxicity. Here we use dissipative
quartz crystal microbalance QCM-D to examine the effect of aqueous imidazolium-based
ionic liquid mixtures on solid-supported biomimetic membranes. Specifically, we assess in
real time the effect of the cation chain length and the anion nature on a supported vesicle
layer of the model phospholipid DMPC. Results indicate that interactions are mainly driven
by the hydrophobic components of the IL, which significantly distort the layer and promote
vesicle rupture. Our analyses evidence the gradual decrease of the main phase transition
temperature upon increasing IL concentration, reflecting increased disorder by weakening
of lipid chain interactions. The degree of rupture is significant for ILs with long hydrophobic
cation chains and large hydrophobic anions whose behavior is reminiscent of that of antimi-
crobial peptides.
Introduction
Ionic liquids (ILs) are a large class of ionic compounds usually composed of an organic cation
and an organic or inorganic anion. They present unique physical and chemical properties such
as an extremely low vapor pressure, high ionic conductivity, very good chemical and thermal
stability, and a broad liquid temperature range (their main phase transition temperature falls
below 100°C) [
1–3]. Ionic liquids are often referred to as “Green solvents” due to their negligi-
ble vapor pressure, which minimizes their release into the atmosphere and renders them non-
flammable. All these characteristics provide ILs with great potential for applications in a variety
of fields, such as lubricants, electrolytes or bioprocessing, and make them eventually more envi-
ronmental-friendly and safer substitutes to the traditional organic solvents in many chemical
PLOS ONE | DOI:10.1371/journal.pone.0163518 September 29, 2016 1 / 15
a11111
OPEN ACCESS
Citation: Losada-Pe
´
rez P, Khorshid M, Renner FU
(2016) Interactions of Aqueous Imidazolium-Based
Ionic Liquid Mixtures with Solid-Supported
Phospholipid Vesicles. PLoS ONE 11(9):
e0163518. doi:10.1371/journal.pone.016351 8
Editor: Christof Markus Aegerter, Universitat
Zurich, SWITZERLAND
Received: January 26, 2016
Accepted: September 9, 2016
Published: September 29, 2016
Copyright: © 2016 Losada-Pe
´
rez et al. This is an
open access article distributed under the terms of
the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: Financial support was provided by Fonds
Wetenschappelijk Onderzoek, Odysseus program
under G0D0115N.
Competing Interests: The authors have declared
that no competing interests exist.
industry processes [4–8]. As a matter of fact, their reputed character as environmentally
friendly solvents has motivated a vast number of studies over the last decade [
1–8]. Yet, their
low volatility renders them odorless and thus hard to detect in the event of a leak to aquatic
ecosystems. Their stability and related low biodegradability make their environmental fate a
potential problem [9]. In this respect, research on IL-induced toxicity is very scarce compared
to that carried out for the physicochemical characterization and their use as solvents in many
industries. The toxicity has been studied to date for cell cultures [
10, 11] and microorganisms
[
12]. The fundamental mechanisms behind IL interactions with biological systems and their
related toxicity have been rarely studied and are not well understood also, in part, due to the
complexity of the systems studied so far. Biomimetic films are important biophysical model
systems to elucidate fundamental aspects and mechanisms relevant for a large range of biologi-
cal interaction ranging from biosignaling to drug reception or toxicity.
Among the different kinds of biomolecules, the interaction of ILs with lipid-based structures
is of considerable relevance, since lipids and in particular phospholipids constitute the building
blocks of the cell membrane, i.e. the protective barrier separating the contact of ILs with a living
cell. Fundamental research has focused on MD simulations [
13–15] and on few experimental
studies for model lipid systems, such as large unilamellar vesicles (LUVs) using mainly lumi-
nescence, differential scanning calorimetry and neutron reflectometry [
16–20]. The study of
ILs interactions with solid-supported lipid layers is mainly limited to the stability of 2D-sup-
ported lipid bilayers (SLBs) exposed to very high concentrated aqueous mixtures of ILs (much
higher than their CAC) by using atomic force microscopy and quartz crystal microbalance [
21,
22]. The study of the effect of diluted aqueous mixtures of ILs on solid-supported membranes
is so far restricted to phosphonium based ILs [23]. Further studies of different supported layer
geometries and systems are thus necessary to get a deeper understanding in a wider concentra-
tion range. Being 3D-supported systems, supported lipid vesicles (SLV) layers offer specific
advantages and represent a geometry in between extended flat layers and real cells. SLV are
thus interesting layers for both fundamental biophysical studies and pharmaceutical screening
applications [
24–28].
In this work, we have used quartz crystal microbalance with dissipation monitoring
(QCM-D) to examine the effect of imidazolium-based ILs on SLVs varying two parameters:
the length of the cation and the nature of the anion in the low concentration regime (below the
CAC for most of the studied systems). Advantages of QCM-D are its compact set-up with a
small quantity of sample solution needed, a low temperature equilibration time, and no need
for labelling molecules. Traditionally, QCM-D has been mainly used to characterize vesicle
adsorption kinetics and formation of SLBs and bilayer-protein interactions, see, for instance
[
29–32]. Only recently, it was proposed as a method to study structural layer changes upon the
main phase transition of phospholipid layers [
33–36]. Apart from looking at the stability of the
supported vesicle layer upon exposure to the aqueous IL mixture, we will make use of a recently
developed strategy to examine the resulting changes in lipid phase behaviour [
37]. Specifically,
we calculate the temperature derivative of the directly measured frequency shift response on
layers of lipid + IL and observe extrema which stand as a token of the lipid main phase transi-
tion. We have chosen a saturated phospholipid, dimyristoilphosphatidylcholine (DMPC), and
ILs with imidazolium-based cations with hydrophilic and hydrophobic anions. In addition the
effective shear viscosity temperature profile have been obtained by means of a Voigt-based vis-
coelastic model to analyze its changes upon the main phase transition. Both approaches yield
consistent information on the IL-induced changes on the phase transition and viscoelastic
properties of the lipid layers under study. Our results can be explained as a function of the cat-
ion length and the degree of hydrophobicity of the anion.
Imidazolium-Based Ionic Liquid Interactions with Solid-Supported Biomimetic Membranes
PLOS ONE | DOI:10.1371/journal.pone.0163518 September 29, 2016 2 / 15
Materials and Methods
DMPC was purchased from Avanti Polar Lipids (Alabaster, AL). 1-butyl-3-methylimidazolium
chloride [C
4
mim]Cl, assay 98%, 1-octyl-3-methylimidazolium chloride [C
8
mim]Cl,
assay 97%, 1-decyl-3-methylimidazoliumchloride [C
10
mim]Cl, assay 96%, 1-Butyl-
3-methylimidazolium tetrafluoroborate [C
4
mim][BF
4
], assay 97%, 1-Butyl-3-methylimida-
zolium bis(trifluoromethylsulfonyl)imide [C
4
mim][Tf
2
N], assay 98%, were purchased from
Sigma-Aldrich (Diegem, Belgium). A schematic of the chemicals used is given in Fig 1. A list
including the CACs of the ionic liquids used can be found in
S1 Table). Spectroscopic grade
chloroform assay 99.3% (stabilized with about 0.6% ethanol) was obtained from Analar (Nor-
mapur). HEPES buffer (pH 7.4) consisting of 10 mM HEPES from Fisher Scientific (assay
99%) and 150 mM NaCl-from Sigma-Aldrich (assay 99.5%) was used for hydration of the
dried lipids. The quantities of lipids to reach the desired mixture concentrations were deter-
mined gravimetrically using a Sartorius balance yielding a maximal mole fraction uncertainty
of ± 0.002.
Vesicle preparation
The lipid powder was first dissolved in spectroscopic grade chloroform and the solvent was
then evaporated under a mild flow of nitrogen in a round bottomed flask. The resulting lipid
film was kept under vacuum overnight to remove residual solvent. Then, the lipid was
hydrated with HEPES buffer. Hydration to 0.5 mg/ml was carried out under continuous stir-
ring in a temperature-controlled water bath at 45°C, well above the main phase transition
temperature of DMPC (T
m
~ 24°C). Small unilamellar vesicles (SUVs) were formed by extru-
sion through a filter support (Avanti Polar Lipids) with a pore size of 100 nm for 25 times.
Vesicle effective sizes and polydispersities were determined by dynamic light scattering (Zeta
Pals, Brookhaven Instruments Corporation). The vesicle dispersions were stored at 4°C and
used within 2 days.
Fig 1. Schematic chemical structure of the lipid and ionic liquids studied in this work.
doi:10.1371/journal.pone.0163518.g001
Imidazolium-Based Ionic Liquid Interactions with Solid-Supported Biomimetic Membranes
PLOS ONE | DOI:10.1371/journal.pone.0163518 September 29, 2016 3 / 15
Quartz crystal microbalance with dissipation
Quartz crystal microbalance with dissipation monitoring (QCM-D) is an acoustic surface-sen-
sitive technique based on the inverse piezoelectric effect. The application of an AC voltage over
the sensor electrodes causes the piezoelectric quartz crystal to oscillate at its acoustic resonance
frequency. As a result, a transverse acoustic wave propagates across the crystal, reflecting back
into the crystal at the surface. When the AC voltage is turned off, the oscillation amplitude
decays exponentially, this decay is recorded and the frequency (f) and the energy dissipation
factor (D) of different overtones are extracted [
24]. The dissipation D is the ratio between the
dissipated energy during one vibration cycle and the total kinetic and potential energy of the
crystal at that moment and can reveal insight in the viscoelastic behavior of surface films.
QCM-D uses a compact sample and crystal compartment attached to a micro-fluidic sys-
tem. When molecules adsorb to an oscillating quartz crystal, water (or buffer) couples to the
adsorbed material as an additional dynamic mass via direct hydration and/or entrapment
within the adsorbed film. The layer is sensed as a viscoelastic hydrogel composed of the mole-
cules and the coupled water together. The adsorbed layer is described by a frequency-depen-
dent complex shear modulus, defined as [
38,39]:
G ¼ G
0
þ iG@ ¼ m
f
þ 2pif Z
f
¼ m
f
ð1 þ 2piwÞ; ð1Þ
where G’ and G” stand for energy storage and dissipation, respectively, f is the oscillation fre-
quency, μ
f
is the elastic shear storage modulus, η
f
is the shear viscosity, and χ = η
f
/μ
f
, is the
relaxation time of the layer.
For the current measurements, we have used QCM-D on a Q-sense E4 instrument (Gothen-
borg, Sweden) monitoring the frequency shift Δf and the dissipation change ΔD. Q-sense E4
also enables heating or cooling temperature scans from 15°C to 50°C. AT-cut quartz crystals
with Au coating (diameter 14 mm, thickness 0.3 mm, surface roughness 3 nm and resonant fre-
quency 4.95 MHz) were used. The Au-coated quartz sensors were cleaned with a 5:1:1 mixture
of Milli-Q water (conductivity of 0.055 S cm
–1
at 25°C), ammonia and hydrogen peroxide, and
were UV-ozone treated with a Digital PSD series UV-ozone system from Novascan for 15 min,
followed by rinsing in milli-Q water and drying with N
2
. The changes in Δf/n and in ΔD were
monitored at five different overtones (from 3
rd
to 11
th
, the fundamental frequency is rather
unstable reaching the farthest out to the edge of the sensor and likely affected by the O-ring).
The temperature stability at constant temperature was ± 0.02°C. For the phase behavior study,
temperature scans with alternating heating and cooling were performed at a rate of 0.4°C/min,
maintaining 60 minutes of stabilization between successive ramps. For each sample, experi-
ments were carried out twice in independent runs in order to test the repeatability of the
measurements.
Results and Discussion
Stability of DMPC vesicle layers exposed to imidazolium-based ionic
liquids
The experiments were carried out at a temperature of 30°C using commercial gold-coated
QCM-D substrates. This system is known to favor intact vesicle adsorption and results in a sta-
ble SLV [
29,40]. Here, the effect of aqueous IL mixtures on a DMPC SLV has been studied for
systems with first, common anion Cl
-
and increasing cation length and second, systems with
common cations [C
4
mim] and different anion nature. After obtaining a stable baseline in the
QCM-D measurement in HEPES buffer, the 0.5 mg/mL DMPC vesicle dispersions were intro-
duced at a rate of 50 μL/min for 20 minutes until a clear signature of a vesicle layer formation
Imidazolium-Based Ionic Liquid Interactions with Solid-Supported Biomimetic Membranes
PLOS ONE | DOI:10.1371/journal.pone.0163518 September 29, 2016 4 / 15
was observed. The large frequency shift Δf and dissipation ΔD values observed and the fact that
the different overtones do not overlap denotes the deposition of an acoustically non-rigid vesi-
cle layer. Then, the IL aqueous mixture (mostly) with a fixed concentration of 50 mM was
injected for 15 minutes and the pump was stopped. For [C
4
mim]Cl, [C
4
mim][BF
4
] and
[C
8
mim]Cl this concentration lies clearly below their respective CACs [41, 42]. At a 50 mM
concentration of [C
10
mim]Cl- the presence of aggregates cannot be ruled out judging from the
literature concentration interval where the CAC takes place (45–60 mM) [43]. For [C
4
mim]
[Tf
2
N], its solubility limit is 20 mM. For concentrations larger than 20 mM no visible precipita-
tion was observed before introducing the ionic liquid in the measuring cell [
44, 45]. Fig 2 illus-
trates the effect of common anion systems with [C
N
mim] cation lengths N = 4, 8, and 10. As it
can be observed, the interaction of [C
4
mim]Cl with the DMPC vesicle layer is reflected only in
a slight decrease of Δf and increase of ΔD in
Fig 2A, indicating a small, partial insertion of the
ionic liquid molecules into the adsorbed vesicle layer. Though not shown, further rinsing with
HEPES buffer resulted in none or only a very slight decrease of Δf and increase of ΔD, leaving
the layer practically unaltered (see
S1 Fig). Upon addition of long-chain length IL molecules
Fig 2. Time evolution of Δ f/n (solid lines) and ΔD (dashed lines) during a QCM-D experiment of DMPC vesicle
adsorption exposed to (A) [C
4
mim]Cl, (B) [C
8
mim]Cl and (C) [C
10
mim]Cl at a concentration of 50 mM. (D) ΔD vs Δf/
n plot for all three mixtures. Black arrows indicate the time when a given sample was added. (E) Schematic
representation of the action of short chain and long chain ionic liquids on DMPC SLVs.
doi:10.1371/journal.pone.0163518.g002
Imidazolium-Based Ionic Liquid Interactions with Solid-Supported Biomimetic Membranes
PLOS ONE | DOI:10.1371/journal.pone.0163518 September 29, 2016 5 / 15