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Graphene Oxide and Lipid Membranes: Interactions
andNanocomposite StructuresRickard Frost, Gustav Edman Jönsson,
Dinko Chakarov, Sofia Svedhem,* and Bengt Kasemo
Department of Applied Physics, Chalmers University of
Technology, SE-412 96 Göteborg, Sweden
*S Supporting Information
ABSTRACT: We have investigated the interaction betweengraphene
oxide and lipid membranes, using both supportedlipid membranes and
supported liposomes. Also, the reversesituation, where a surface
coated with graphene oxide wasexposed to liposomes in solution, was
studied. We discoveredgraphene oxide-induced rupture of preadsorbed
liposomes andthe formation of a nanocomposite, bio-nonbio
multilayerstructure, consisting of alternating graphene oxide
monolayersand lipid membranes. The assembly process was monitored
inreal time by two complementary surface analytical techniques (the
quartz crystal microbalance with dissipation monitoringtechnique
(QCM-D) and dual polarization interferometry (DPI)), and the formed
structures were imaged with atomic forcemicroscopy (AFM). From a
basic science point of view, the results point toward the
importance of electrostatic interactionsbetween graphene oxide and
lipid headgroups. Implications from a more practical point of view
concern structure−activityrelationship for biological health/safety
aspects of graphene oxide and the potential of the nanocomposite,
multilayer structure asscaffolds for advanced biomolecular
functions and sensing applications.
KEYWORDS: Graphene oxide, lipid membrane, QCM-D, DPI, AFM,
layer-by-layer
Graphene-based materials are intensely explored for
variousapplications,1 and a novel direction in this field
isrepresented by nanocomposite materials of graphene or
carbonnanotubes with lipid membranes. Two significant examples
ofthis development are the use of graphene flakes in
miniaturizedbioelectronic devices,2 and of carbon nanotubes as
scaffolds forphotoelectrochemical processes intended for light
harvesting.3
The advancement of these and similar applications will
bedependent on control over the assembly processes of graphene,or
graphene derivatives, and lipid membranes. Toward this end,the aim
of the present study was to explore the interaction ofgraphene
oxide (graphene with OH and COOH functionalgroups)4 with lipid
membranes of different compositions. It wasof particular interest
to investigate if the expected electrostaticattraction between
graphene oxide and oppositely chargedmembranes would dominate over
other interactions and lead to(i) flat deposition of graphene oxide
onto membranes and (ii)very different interactions with positively
and negativelycharged lipid membranes. Possible interference from
hydro-phobic interactions between graphite-like areas of
grapheneoxide and the interior (hydrophobic) lipid tails of
themembrane, or van der Waals interactions, which might besizable
in view of the large number of atoms in a large grapheneoxide
flake, were hypothesized to potentially lead to moredisordered
structures, than expected on purely electrostaticgrounds. These
ideas were tested experimentally and the resultswere applied to
assemble multilayer structures of alternatinggraphene oxide sheets
and lipid membranes in a layer-by-layer(LbL) fashion. Despite the
broad application of the versatile
and inexpensive LbL deposition methodology,5,6
graphenederivatives as well as lipid membranes are still rarely
used inLbL-assemblies. Hitherto, the most common materials used
inthese systems are synthetic polymers,7 biopolymers,8 andinorganic
substances.9 A recent study reports on the LbL-assembly of graphene
oxide and block copolymer micelles.10
In this Letter, we report for the first time on the assembly ofa
multilayered structure of lipid membranes, which arebiologically
relevant supramolecular structures, and grapheneoxide. These novel
nanocomposites might, for example, pavethe way for intercalation of
(conducting) graphene-derivedsheets between (insulating) lipid
membranes and relatedstructures. Another aspect relating to the
present study is thepotential health/safety risk with graphene and
graphene oxidein biological systems, and the development of
structure−activity relationships to predict nanotoxicity. Although
far fromthe complexity of biological systems, we believe that
studies ofsimple model systems are an important complement
tostandard toxicity assays. We, and others, have
earlierdemonstrated the usefulness of early stage screening
ofnanoparticles, by using lipid membranes as mimics of
cellmembranes.11,12
Interaction of Graphene Oxide with Positively andNegatively
Charged Lipid Membranes. Differently chargedsupported lipid
membranes were prepared on SiO2 surfaces
Received: September 7, 2011Revised: May 2, 2012Published: June
1, 2012
Letter
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© 2012 American Chemical Society 3356
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using extruded liposomes (mean diameter of 80−90 nm)composed of
different lipids. 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine
(POPC), 1-palmitoyl-2-oleyl-sn-glycero-3-phospho-L-serine (POPS),
and 1-palmitoyl-2-oleyl-sn-glycero-3-ethylphosphocholine (POEPC)
were used to preparenegatively charged POPC/POPS (3:1) liposomes
(ζ-potential:−26 ± 1.2 mV) and positively charged POPC/POEPC
(3:1)liposomes (ζ-potential: 22 ± 0.8 mV), respectively.13
Underappropriate conditions, these liposomes adsorb to the
SiO2surface and spontaneously rupture to form fluid supported
lipidmembranes containing about 25% of the charged lipid,according
to a well-established protocol and mechanisticscenario.14,15
The formation of supported lipid membranes, as well as
thesubsequent exposure of these model membranes to an
aqueoussuspension of graphene oxide (0.5−5 μm sheets with an
oxygencontent of 20%, according to specifications), were
monitoredby the quartz crystal microbalance with dissipation
monitoringtechnique (QCM-D) (experimental details are provided in
theSupporting Information). In a QCM-D experiment, thefrequency
shift, Δf, efficiently measures the accumulation ofmass onto the
sensor surface and the D factor (energydissipation or damping of
the shear oscillation of the QCM-Dsensor), represented as ΔD,
reflects the structural/viscoelasticproperties of the adlayer.The
QCM-D signals, resulting from exposure of the lipid
membranes to graphene oxide, are shown in Figure 1. When
the negatively charged POPC/POPS (3:1) membrane surface
isexposed to graphene oxide, the frequency and the
dissipationresponses (Δf and ΔD) show that no adsorption of
grapheneoxide occurs, which we attribute to the electrostatic
repulsionbetween the lipid membrane headgroups and graphene
oxide.This is in accordance with the measured ζ-potential of
theaqueous dispersion of graphene oxide (−56 ± 1.1 mV, pH 4),which
is similar to previously reported values.16 In contrast, onthe
positively charged POPC/POEPC (3:1) membrane, thereis a clear mass
uptake signaled by a monotonic decrease of thefrequency, which
saturates after about 10 min at a frequencyshift of around -6 Hz.
The magnitude of this shift and the factthat no dissipation shift
is observed suggest that the grapheneoxide flakes adsorbs flat on
the membrane and covers most ofthe surface. The absence of any ΔD
response indicates that theflakes are sufficiently rigidly attached
not to slip on the lipidsurface during the oscillatory motion of
the sensor surface. Theinteraction is irreversible upon rinsing
with buffer, and in
separate control experiments it is shown that the grapheneoxide
did not adsorb to the bare SiO2 substrate, which is anexpected
result since it is negatively charged under the presentexperimental
conditions (see Supporting Information).Because of the seemingly
rigid attachment of the graphene
oxide to the lipid membrane (low dissipation and similar QCM-D
responses for all harmonics), the Sauerbrey equation wasapplied to
estimate the amount of deposited mass (macoustic =C*Δfz where C =
−17.7 ng/(cm2Hz) and Δfz is the normalizedfrequency shift at the
zth harmonic (here z = 7)).17
The adsorbed mass of graphene oxide at saturation (Figure3) was
found to be 103 ± 11 ng/cm2. This number is close tothe theoretical
mass of a flat monolayer of graphene oxide,which is 101 ng/cm2.
Nevertheless, a certain degree ofnonideality in the graphene oxide
layers can be expected dueto, for example, incomplete coverage, or
partly overlapping ofgraphene oxide flakes prior to (according to
specifications>80% of the graphene oxide are single monolayers
insuspension) or caused upon adsorption to the surface, despitethe
electrostatic repulsion between them. Given that the oxygencontent
of graphene oxide is about 20%, hydration effects mayalso
significantly alter the mass detected by QCM-D, whichincludes both
the actual graphene oxide mass and any liquidacoustically coupled
to adsorbed flakes (for example, at the rimof the flakes, or
trapped in between the flakes and themembrane).The interpretation
of the QCM-D results as discussed above
was strengthened by data acquired by two
complementarytechniques. Atomic force microscopy (AFM) results (see
textand Figure 6 below) showed that large parts of the POPC/POEPC
(3:1) lipid membrane surface was covered with amonolayer of
graphene oxide but it also revealed gaps inbetween adjacent
graphene oxide flakes and overlapping flakes.The properties of this
system were further studied by an opticalsurface analytical
technique to obtain fundamentally differentinformation about the
material compared to the viscoelastic(nanomechanical)
characterization by QCM-D. Commonoptical techniques, which are
often used for such studies,include surface plasmon resonance
(SPR)-based techniques,reflectometry, and, more recently, the dual
polarizationinterferometry (DPI). Here DPI experiments were
performed,as this technique offers the possibility to explore the
anisotropicproperties of the studied materials. In particular DPI
is usefulfor studies of lipid membranes,18 and an attractive
approach toaddress the nonrandom orientation of both the lipid
moleculesand the graphene oxide flakes in our study. The DPI
techniqueis based on two parallel waveguides, where the upper side
of thetop waveguide is the sensor surface (silicon
oxinitride).Changes in refractive index (due to the polarizability
ofadsorbed molecules) close to this surface are measured aschanges
in the position of the fringes in the interference patterngenerated
beyond the end of the waveguides, using twoorthogonal polarizations
of the incident light (transverseelectric (TE) and transverse
magnetic (TM)). DPI haspreviously been used to study optical
anisotropy of lipidmembranes.18 For isotropic materials, the TM
response isalways larger than the TE response due to the
differences infield intensity profiles. In Figure 2, DPI data for
the formationof a POPC/POEPC (3:1) membrane and subsequent
additionof graphene oxide are shown (see Supporting Information
forexperimental details). In the figure, the anisotropic properties
ofboth the lipid membrane and the layer of graphene oxide
areevident through the difference between the TM and TE
Figure 1. QCM-D data recorded during exposure of supported
lipidmembranes, POPC/POPS (3:1) and POPC/POEPC (3:1), to agraphene
oxide suspension. Blue lines represent frequency shifts, Δf,(mass
uptake) and red lines represent dissipation shifts, ΔD.
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responses. The lipid membrane generates an enhanced TMresponse
relative to the TE response compared to the expectedresults for an
isotropic lipid film due to the larger polarizabilityof the lipid
molecules in the membrane in the direction normalto the sensor
surface, similarly to results obtained in previousstudies.18 In
contrast, the graphene oxide generates anenhanced TE response
relative to the TM response, which isa quite uncommon situation.
This result supports theconclusion that the graphene oxide adsorbs
flat onto the lipidmembrane.From the DPI data, the mass and the
birefringence (that is,
the difference between nTM and nTE) of the POPC/POEPC(3:1)
membrane and the layer of graphene oxide werecalculated (Figure 3).
Two different approaches were used tocalculate the mass and
birefringence of the latter layer, (i) the
refractive index (n) and the refractive index increment
(dn/dc)of graphene oxide were estimated from literature data
(seeSupporting Information for details), or (ii) the thickness of
thegraphene oxide layer was determined from the QCM-D datausing
Voight-based modeling.19 The two approaches yieldsimilar results.
In Figure 3A, it is evident that the calculatedmasses of a
POPC/POEPC (3:1) membrane from DPI andQCM-D data are very similar.
However, the mass of thegraphene oxide layer as determined by QCM-D
(103 ± 11 ng/cm2) is larger than the corresponding mass extracted
from theDPI data (70 ± 7 ng/cm2), leading to an estimation of
thewater content of the graphene oxide layer of around 32%. Sucha
difference in mass is commonly seen when comparing QCM-D data with
optical data, for example, for biomolecules (thewater content of a
dense protein layer is typically 55%20) due tothe difference in
sensing principle. In contrast to QCM-D,which is an acoustic method
sensitive not only to the adsorbedmaterial but also to acoustically
coupled solvent (water), DPI isan optical technique where the
solvent associated with thesensor surface does not generate a
response (the same holds forSPR, ellipsometry, and reflectrometry).
The present resultshows that the layer of graphene oxide is more
hydrated thanthe lipid membrane. Since the optical mass is smaller
than thetheoretical mass of a monolayer of graphene oxide, this
resultsupports the above interpretation of the QCM-D results,
thatthere are gaps between adjacent flakes of graphene oxide.The
birefringence of the lipid membrane and the layer of
graphene oxide are shown in Figure 3B. It is evident that
thelipid membrane has a larger polarizability perpendicular to
thesensor surface as opposed to the layer of graphene oxide.
Thisdata is interpreted as that the graphene oxide is ordered
inplane with (parallel to) the sensor surface.
LbL Structures of Graphene Oxide and LipidMembranes. The
previous section demonstrated preparationof a structure consisting
of a positively charged POPC/POEPC(3:1) lipid membrane on the SiO2
substrate, covered on theliquid side by a saturation layer of
graphene oxide, as shown inFigures 1 and 2. Further sequential
additions of POPC/POEPC(3:1) liposomes and graphene oxide to the
initial layers of lipidmembrane and graphene oxide generates an
interestingmultilayered structure, as described in detail below and
inFigure 4.
QCM-D Observations. The QCM-D data for the sequentialbuild-up of
the multilayered graphene oxide−lipid membranestructure are
presented in Figure 5. At the second injection ofliposomes,
starting at step IV in Figure 5, there is a largenegative shift in
the frequency, signaling a quite large massuptake, and a large
increase in dissipation, corresponding to theformation of a softer
layer on the QCM-D sensor surface. Weattribute these results to the
formation of about one monolayerof intact liposomes on the
previously formed graphene oxidelayer. This is similar to what has
been observed previously forintact liposome adsorption on inorganic
surfaces like TiO2
21
and Au,22 where the surfaces interact strongly enough with
theliposomes to adsorb them irreversibly but not strongly enoughto
induce rupture. In this respect, the liposome interaction
withgraphene oxide is qualitatively similar to TiO2 and Au.
Thereason for the large changes in Δf (−50 ± 2 Hz) and ΔD (9 ±2 ×
10−6) is that the intact liposomes carry water in theirinterior,
causing a large mass adsorption, which is the sum ofthe lipid and
water masses. The “soft” structure of adsorbedliposomes, much
softer than a lipid bilayer, is responsible for
Figure 2. DPI data obtained (I−II) during the formation of
asupported POPC/POEPC (3:1) membrane onto the waveguidesensor
surface, (*) the subsequent liquid exchange from PBS to water,and
(III) the addition of the graphene oxide suspension.
Figure 3. (A) Calculated masses of a POPC/POEPC (3:1)
membraneand the subsequently adsorbed layer of graphene oxide based
onQCM-D and DPI data. Calculations were performed according
toapproach (i) as discussed above. Similar results were obtained
byassuming a layer thicknesses based on modeling of the QCM-D
data(approach (ii)). (B) Birefringence of the lipid membrane and
the layerof graphene oxide, determined by DPI. Experiments were
performedin three (QCM-D) or four (DPI) replicates.
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the large increase in dissipation, as previously shown
bycombined QCM-D and AFM studies.22
The subsequent addition of graphene oxide to the liposomecovered
graphene oxide surface, step V in Figure 5, generates asurprising
result, namely an increase in frequency, that is, adecrease in Δf
and thus a net mass loss, and a simultaneouslarge decrease of the
dissipation (negative ΔD change). Thesetwo observations constitute
strong evidence that the grapheneoxide induces rupture of the
preadsorbed liposomes. It shouldbe noted that the average size of
the graphene oxide flakes ismuch larger than the liposome diameter
and results obtainedwith smaller flakes might be different. In the
liposome ruptureprocess, the water inside the liposomes is released
to the bulk
liquid (causing a mass loss that by far exceeds the added massof
the rupture-inducing adsorbed graphene oxide) and a secondlipid
membrane is formed. The high dissipation value returns toa low
value, since there are no longer soft, intact liposomes onthe
surface, after they have ruptured and fused to a membrane.A similar
process occurs for some liposomes (for example,POPC) when they
reach a critical coverage on, for example,SiO2 surfaces, but in
this case a one-sided surface interaction isenough to induce
rupture.14,15
A question that arises is where the graphene oxide flakes
thatinduce the liposome rupture go after the ruptures havehappened.
Most likely they form a second adsorbed grapheneoxide layer on the
second lipid membrane (originating from therupturing liposomes),
because the situation is very similar tothe first adsorption step
of graphene oxide on the first lipidmembrane. However, in this case
liposome rupture andgraphene oxide adsorption occur simultaneously
rather thansequentially.The frequency and dissipation responses due
to the sum of
the second lipid membrane and the second layer of grapheneoxide
(Δf and ΔD between steps I and IV in Figure 5) arelarger compared
to the corresponding values for the first twolayers (the Δf and ΔD
values between steps IV and VI in Figure5). There are several
possible explanations for these largervalues. The three most likely
reasons are the following: (i) Thesecond lipid membrane plus its
adsorbed layer of grapheneoxide is a more hydrated and softer
structure. If this were thecase the larger Δf and ΔD responses
would be due to moreassociated/trapped solvent than in the first
two layers (a firstlipid membrane and a first graphene oxide
layer). (ii) It is notunlikely that the second lipid membrane is
less perfect than thefirst one, since the rupture of liposomes,
induced by thegraphene oxide, may be incomplete. Furthermore, we
suspectthat there are areas of the first membrane that are not
coveredby graphene oxide, as discussed above. In this case, the
largerΔf and ΔD responses would be due to some intact
liposomescoexisting with the second membrane. (iii) When
grapheneoxide flakes land on the preadsorbed monolayer of
intactliposomes and induce their rupture, excess lipid material
willreturn to the bulk phase or stay around as excess lipidmembrane
patches. This lipid material can adsorb on top of thesecond layer
of graphene oxide, that is, on its side facing thebulk liquid,
yielding a larger mass uptake than for just onesequence of lipid
membrane and graphene oxide. The latterwould actually correspond to
the beginning of the buildup ofthe third lipid membrane, which in
turn would enable somefurther adsorption of graphene oxide and so
on. The net effectwould be a mass uptake, which is larger than for
formation ofjust a second lipid membrane + graphene oxide layer, as
wasalso observed in the experiment. Note that this process was
notpossible upon the first addition of graphene oxide, since
thestarting point in that case was a completed lipid membrane,with
no intact liposomes and no surplus lipid mass.The relative
importance of these three possible mechanisms
was elucidated by AFM analyses. AFM data showed that thereare
multiple layers on the surface and that the number of layersexceeds
the sum of the number of cycles of added liposomesand graphene
oxide, that is, scenario (iii) is supported by theAFM data as at
least one contributing mechanism. These dataare presented in Figure
6C and described in more detail in therelated text.When continuing
the experiment by again adding liposomes
of the same lipid composition as before, followed by addition
of
Figure 4. Schematic representation of the formation of a
POPC/POEPC (3:1) lipid membrane and subsequent adsorption of
grapheneoxide. Further addition of POPC/POEPC (3:1) liposomes
andgraphene oxide results in a multilayered structure of these
materials, asdeduced from the results shown in Figure 5. The figure
is not drawn toscale.
Figure 5. QCM-D data showing the sequential build-up of
amultilayered structure of POPC/POEPC (3:1) membranes andgraphene
oxide, starting from (I) the adsorption of liposomes to asilica
surface and (II) the spontaneous formation of a supported
lipidmembrane. Next, (III) graphene oxide is added, followed by (IV
andVI) additional injections of liposomes and (V and VII) of
grapheneoxide. The additions of graphene oxide and liposomes are
preceded bya (*) liquid exchange to water and PBS respectively.
These eventsgenerate responses in both Δf and ΔD due to the change
in liquid bulkcomposition. (These liquid exchanges (marked *)
generated shifts inboth Δf and ΔD due to the change of bulk
composition. However,when replacing the PBS with water after
adsorption of liposomes, anosmotic gradient is formed across the
membrane of the liposomes.This gradient may cause transient
variations in the amount of coupledwater, processes that also
generate responses in Δf and ΔD.)
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graphene oxide, the result is qualitatively similar to the
secondcycle. Liposomes adsorb intact to the surface and rupture
uponthe subsequent addition of graphene oxide. However, in
thiscycle the QCM-D responses are even larger than before,
bothafter adsorption of liposomes and after addition of
grapheneoxide. Again, we attribute these large responses to
scenario (iii)as described above. These results prove that that the
formedmultilayered structure is terminated by graphene oxide, that
is,graphene oxide stays adsorbed to the lipid membrane
afterliposome rupture. In separate experiments, the
sequentialassembly of alternating lipid membrane and graphene
oxidestructures was studied by DPI. The analysis of these
datasupports the interpretation of the QCM-D data (DPI data
aregiven as Supporting Information).AFM Observations. As described
above, graphene oxide
selectively adsorbs to the positively charged POPC/POEPC(3:1)
membrane. The adsorbed material on this type ofmembrane, that is,
after stage III in Figure 5, was visualized byAFM. In such an AFM
image, a partial overlap of somegraphene oxide sheets is visible
(Figure 6A). For the grapheneoxide terminated structures, it was
suggested by the surfacewettability properties that the multilayers
remained on thesurface as the samples were dried (see further
below). TheAFM image of a sample obtained after three cycles of
liposomesand graphene oxide had been added, that is, after stage
VII inFigure 5, followed by evaporation of the solvent (water)
atroom temperature, is shown in Figure 6B. This image wasrecorded
on a QCM-D sensor that was demounted after thebuild-up of the
nanocomposite structure on the sensor surface.Similar images were
obtained in liquid (requiring a manualsample preparation procedure)
by AFM and in air by scanning
electron microscopy (not shown). Interestingly, a
topographyinvestigation of the image in Figure 6B reveals the
formation oflamellar structures with steps of different heights.
This isvisualized in the histogram in Figure 6C that shows that
someheights on the surface occur more frequently than others.
Thehistogram shows at least six peaks, that is, six different
heightsthat are frequent and well separated from each other.
Weattribute these specific heights to multiples of the thickness of
alipid membrane with an adsorbed graphene oxide layer.
Forcomparison, a lipid membrane is around 5 nm thick, while
thethickness of a mono-, bi-, and trilayer of graphene oxide is
1.6,2.6, and 3.6 nm, respectively.23 In contrast to
graphiteintercalation compounds24 in which the planes of
carbonatoms are separated by a few angstroms, the graphene flakes
inour multilamellar structures are separated at the nanometerscale.
The fact that there are more layers detected in thehistogram than
cycles of added liposomes and graphene oxidein the experiment was
explained above as an overspill of lipidsto adjacent sheets of
graphene oxide upon liposome rupture(mechanism (iii)), a process
that enables further adsorption ofgraphene oxide. In this way, each
cycle of added liposomes andgraphene oxide gives on average rise to
more than a single pairof layers of these materials.
Additional Comments on the Interaction betweenGraphene Oxide and
Lipid Membranes. The present studyprovides insight into the nature
of the interaction between lipidmembranes and graphene oxide. Under
the given experimentalconditions, the driving force for adsorption
of negativelycharged flakes of graphene oxide to positively charged
lipidmembranes seems to be governed primarily by electrostatics.We
cannot exclude a significant contribution from other factorslike
H-bonding to functional groups (for example, OH orCOOH groups) on
graphene oxide, or van der Waalsinteractions. However, the
electrostatic interactions are likelydominant, based on the absence
of adsorption of grapheneoxide on negatively charged lipid
membranes. An interestingextension of the present experiments would
be to graduallydecrease the positive charge of the lipid membrane
toinvestigate if this would alter the way the graphene oxideflakes
bind to the membrane, for example, if there is some lowlevel of
positively charged lipids at which the adsorptionbecomes reversible
and/or where the multilayer LbL structurewould be built by
alternating layers of adsorbed intact vesiclesand graphene oxide
rather than with lipid membranes inbetween the graphene oxide
flakes. An experiment in thisdirection, using liposomes containing
only 10% POEPC, ispresented as Supporting Information. Briefly, the
resultsindicate that too low POEPC-content in the liposomes leadto
incomplete rupture when exposed to graphene oxide.There are two
additional features in the present work worth
highlighting. First, it was observed that graphene oxide
inducesrupture of preadsorbed liposomes on a graphene surface.
Thisobservation can most likely be generalized to the possibility
ofpreparation of supported lipid membranes from liposomes onother
surfaces than those that do not spontaneously promotesuch
formation. Indeed, in a separate experiment, grapheneoxide was seen
to also induce rupture of intact POPC/POEPC(3:1) liposomes adsorbed
on a gold substrate (see SupportingInformation). In addition,
graphene oxide may open up a wayto form lipid membranes of other
lipid compositions, thanthose where the liposomes spontaneously
rupture and fuse intoa supported lipid membrane. Today, research
within this area isusually focused on identifying fusogenic agents
such as peptides
Figure 6. (A) AFM image of graphene oxide adsorbed on a
supportedPOPC/POEPC (3:1) membrane in water (z-range: 29 nm).
(B)Multilayered structure of graphene oxide and POPC/POEPC
(3:1)membranes on a QCM-D crystal that was dried and imaged after
stepVII in Figure 3 (z-range: 81 nm). (C) Histogram of the height
profilein image (B). The zero level in the histogram corresponds to
thelowest point in the image. The z-range in image A and B refers
to thetotal range in the z-dimension, that is, the height
difference betweenthe lowest (darkest) and the highest (brightest)
point in the image.
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that can induce rupture and fusion.25 The present
observationsidentify graphene oxide as an interesting “fusogenic”
alternative.Mechanistically, it must be the additional deformation
of theliposomes, when they have a graphene oxide surface on
tworather than on only one side, which induces the rupture. Insome
applications, removal of the graphene oxide after themembrane has
been formed would be desirable. However, wehave found that
increasing the ionic strength or lowering thepH does not easily
remove the graphene oxide. Optimization ofthe interaction forces is
likely needed in order to establish aprotocol for the removal of
the adsorbed graphene oxide.Second, it was noted that the prepared
samples did not
dewet when exposed to air, which is otherwise commonlyobserved
for plain lipid membranes. Accordingly, AFM analysesrevealed a well
organized multilayered structure both in liquidand on a dried QCM-D
crystal (Figure 6), that is, themultilayered structure was largely
maintained even after drying.These results indicate that the
presence of graphene oxidepreserves the lipid membrane upon drying.
Previous work inthis area has suggested that covering a lipid
membrane withpolyethylene glycol26 or proteins27 will induce
resistance to theair−water interface. In a different approach, the
lipids in themembrane have been cross-linked to enhance its
resistance todetergent.28
It should be noted that the LbL growth of alternatingsupported
lipid membrane and graphene oxide layers was notperfect and that
some intact liposomes and some more thandouble layer structures are
formed for each pair of liposomeand graphene oxide exposures.
However, the process was alsonot optimized. Improvements
approaching a more ideal LbLgrowth seem probable. One such step
would involve adsorbingless than a saturation layer of intact
liposomes on the previouslyformed graphene oxide layer, since this
would reduce the excessamount of lipid material, beyond what is
needed for just a lipidmembrane. We also speculate that it might be
possible tochemically reduce the graphene oxide in order to restore
theconductive properties of graphene, after adsorption on
themembrane. If this were possible one could produce
conductinggraphene layers intercalated between insulating lipid
mem-branes. In this context, we note that a recent theoretical
studysuggests that graphene flakes would prefer to be
incorporatedinside the membrane, rather than adsorbed to the
membranesurface.29 A second possible extension of the current work
is tofirst cover graphene with lipids before suspending it in
anaqueous medium and depositing the lipid-graphene assemblieson a
surface.Concluding Remarks. In conclusion, this letter presents
sequential build-up of multilayered lipid membranes separatedby
graphene oxide. It is shown that anionic graphene oxideadsorbs
selectively to positively charged lipid membranes,which
subsequently enables further adsorption of intactliposomes.
Furthermore, graphene oxide induces rupture ofadsorbed liposomes on
graphene oxide (and on gold) andforms additional stacked lipid
membranes on the formersurface. The final surface assembly is
composed of several layersof graphene oxide with lipid membranes on
each side. Afterdrying this assembly, where the last exposure was
made tographene oxide, it was shown that a well-organized
structureremained at the surface. The presented results open up for
newpreparation procedures to intercalate structures of
grapheneoxide, and potentially of graphene, between lipid
membranes.
■ ASSOCIATED CONTENT*S Supporting InformationExperimental
methods and additional QCM-D and DPI dataare available. This
material is available free of charge via theInternet at
http://pubs.acs.org.
■ AUTHOR INFORMATIONCorresponding Author*E-mail:
[email protected]. Telephone: +46 (0)31772 34 28. Fax: +46
(0)31 772 31 34.
NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSThis work was financially supported by the
Swedish Environ-mental Research Council FORMAS (NanoSphere,
ProjectNumber 2009-1696), the Swedish Foundation for
StrategicResearch (SSF, RAM08-0109, methamaterials), and theSwedish
Research Council (VR, Project Number 621-2007-4375). The Farfield
Group Ltd. (Manchester, U.K.) is gratefullyacknowledged for the
access to a DPI instrument and especiallyDr. Marcus Swann for
supporting the DPI data analyses.
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