Graphene Oxide and Lipid Membranes: Size-Dependent Interactions

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Graphene oxide and lipid membranes: Size-dependent interactions Rickard Frost, Sofia Svedhem, Christoph Langhammer, and Bengt Kasemo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b03239 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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Graphene oxide and lipid membranes: Size-dependent interactions

Rickard Frost1, *, Sofia Svedhem2, Christoph Langhammer2, and Bengt Kasemo2, *

1

Department of Energy and Environment, Chalmers University of Technology, SE-

412 96 Göteborg Sweden 2

Department of Applied Physics, Chalmers University of Technology, SE-412 96

Göteborg Sweden

*Corresponding Authors:

Rickard Frost, e-mail: [email protected] Bengt Kasemo, e-mail: [email protected]

Abstract We have investigated the interaction of graphene oxide (GO) sheets with supported lipid membranes with focus on how the interaction depends on GO sheet size (three samples in the range of 90 – 5000 nm) and how it differs between small and large liposomes. The layer-by-layer assembly of these materials into multilamellar structures, as discovered in our previous research, is now further explored. The interaction processes were monitored by two complementary, real time, surface-sensitive analytical techniques; quartz crystal microbalance with dissipation monitoring (QCM-D, electro-

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acoustic sensing) and indirect nanoplasmonic sensing (INPS, optical sensing). The results show that the sizes of each of the two components, graphene oxide and liposomes, are important parameters affecting the resulting multilayer structures. Spontaneous liposome rupture onto graphene oxide is obtained for large lateral dimensions of the graphene oxide sheets. Keywords: Graphene oxide, lipid membranes, liposomes, QCM-D, INPS

Introduction A very active subfield of research following the discovery/development of graphene is its interfacing with biological systems.1 Biomedical applications in analytical or diagnostic devices and therapies, as well as the dose-dependent toxicity to cells and organisms are being explored.2, 3 (The number of publications found by combining graphene and bio* (Thomson Reuters WoS) has increased by a factor of about 12 from 2010 to 2014, which is almost three times faster than for graphene generally.) In such biologically and biomedically oriented studies, suspensions of graphene oxide (GO) are commonly used.4 GO is hydrophilic and, in contrast to pristine graphene, readily dispersed in water due to the presence of epoxy, hydroxyl and carboxyl functional groups.5, 6, 7 Promising application areas where GO has been applied in biological systems include drug delivery vehicles8, 9, 10, analytical/sensing devices11 and GO-based nanocomposite scaffolds for tissue engineering12,

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In

addition to applications, GO is also being investigated from a nanosafety/nanorisk perspective.14, 15 In almost all biological or biomedical applications a fundamental question that needs to be addressed is how GO interacts with lipid membranes.16, 17


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In such research, two types of model systems complement each other; (i) native cell membranes in cell cultures in vitro or in vivo, and (ii) model lipid membranes or liposomes in vitro. A subclass of the latter is membranes (bilayers) and liposomes supported on a surface, allowing use of surface sensitive techniques to study GOmembrane interactions.18 For supported lipid membranes the underlying solid support (commonly SiO2), and its properties, is of critical importance.19 In this regard, GO (and related materials) has recently become of interest as a substrate for the formation of lipid membranes.20, 21 Thus, there are three main reasons for studying GO-lipid membrane interactions; (i) To characterize the biological interactions of GO from the point of view of understanding biological effects of GOderived materials, relevant in, e.g., nanosafety research; (ii) To evaluate GO as a substrate material for the formation of supported lipid membranes, with the aim to characterize membrane properties or processes therein, towards engineering of advanced nanodevices; (iii) to build hybrid structures (nanocomposites) of GO and other materials (in this case GO – lipid composites).

In a previous paper, we reported on interactions between GO and lipid structures and found that GO sheets adsorbed onto positively charged lipid membranes supported by a silica surface.17 In a subsequent step, intact liposomes (with a diameter in the range 80 - 90 nm, and of the same lipid composition) adsorbed onto the GO sheets, which resided on the supported lipid membrane. Further addition of GO (lateral dimension 0.5 – 5 μm) induced rupture of the liposomes and resulted in the formation of a multilayered composite of lipid membranes and GO (Figure 1).


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Figure 1. Schematic representation of the sequential buildup of a multilayered GO/lipid membrane structure. The figure is not drawn to scale. Reproduced from reference [17].

Based on these findings, several questions emerge. Of particular interest is how the GO - liposome interactions depend on GO sheet size and liposome size. How does, e.g., GO-induced liposome rupture depend on the size of GO sheets adsorbing to the liposomes? In the light of our previous findings, we hypothesize that liposome rupture occurs when liposomes are exposed to two GO sheets, one on each side of the liposome, where the GO sheets have the same size as, or are larger than, the cross-sectional area of the liposome. This hypothesis is based on our previous finding that although liposomes do not rupture when adsorbing to GO, liposome rupture occurs when GO sheets are subsequently adsorbing on top of the adsorbed liposomes.17 In these experiments, the GO sheets were much larger than the liposomes. The hypothesis implies, e.g., that a liposome exposed to GO sheets smaller than the liposome will not rupture but rather create a GO-functionalized (coated) surface. Thus, in the present study, in order to test this prediction, we explore experimentally the interactions of three different GO samples, of different


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lateral size, with both small and large unilamellar vesicles (SUVs and LUVs). As before, GO is adsorbed to a preformed lipid membrane, subsequently exposed to liposomes, and finally to GO. The presentation of the obtained results follows the same structure, preceded by data on sample characterization. Two complementary surface-based analytical techniques are applied to characterize the GO - lipid membrane interactions, namely the quartz crystal microbalance with dissipation monitoring (QCM-D) and the indirect nanoplasmonic sensing (INPS) technique. QCM-D is a technique that pioneered the understanding of how supported lipid membranes form on solid surfaces from unilamellar vesicles in solution.22, 23, 24 INPS is an optical sensing method, utilizing the strong electromagnetic field amplification around optically excited localized surface plasmon resonances (LSPR) in metallic nanoparticles.25,

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The method monitors how these resonances shift when the

dielectric environment near the sensing nanoparticles changes, e.g. due to changes in optical mass or optical properties. The present study shows that the acoustic (QCM-D) and the optical (INPS) sensing techniques constitute a powerful combination to characterize nanomaterial interactions with lipid structures.

Material and Methods Chemicals were obtained from commercial sources and used without further purification. Deionized water was obtained from a Milli-Q water purification system (Millipore, France). Phosphate buffered saline (PBS) was prepared from tablets (0.0015 M potassium dihydrogen phosphate, 0.0081 M disodium hydrogen phosphate, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4).


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Liposomes were prepared from 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-ethylphosphocholine (POEPC) (Avanti Polar Lipids Inc., USA). Aqueous suspensions of graphene oxide were obtained from graphene-supermarket.com.

Liposome preparation Small and large unilamellar vesicles (SUVs and LUVs) were prepared by the extrusion method. Both types of liposomes had the same lipid composition, POPC:POEPC (3:1). For each batch of liposomes, a total of 6 mg of lipids dissolved in chloroform was added to a round-bottomed flask, and the solvent was evaporated under a flow of nitrogen to give a lipid film distributed around the inner wall of the flask. Residual solvent was removed by applying a low pressure for at least one hour. Subsequently, the lipids were rehydrated in 1.2 mL of PBS (final concentration of 5 mg/mL). To avoid spontaneous generation of small lipid structures during the rehydration, this step was performed under slow rotation of the round-bottomed flask for about one hour. The slow hydration was only performed during the preparation of LUVs. To produce SUVs, the lipid suspension was extruded 11 times through a 100 nm polycarbonate membrane and another 11 times through a 30 nm polycarbonate membrane using a mini extruder (Avanti Polar Lipids Inc., USA). To produce LUVs, the lipid suspension was extruded 21 times through a 400 nm polycarbonate membrane. The liposome solutions were stored at 4°C.

Dynamic and electrophoretic light scattering


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The size distribution and ζ-potential of the prepared liposomes were measured using a Zetasizer Nano (Malvern Instruments Ltd., UK) after each extrusion. Prior to measurements, the liposomes were diluted in PBS to a final concentration of 0.05 mg/mL. The measurements were performed in triplicates at a temperature of 22°C.

X-ray photoelectron spectroscopy (XPS) Samples surfaces were prepared by drop casting concentrated GO suspensions (GO 1, GO 2, GO 3) on Si wafers with a native oxide surface. The samples were analyzed employing monochromatic Al-Kα radiation 45° take-off angle (Perkin Elmer PHI 5000C ESCA system). The C1s peaks of the different samples were analyzed using the software XPS Peak 41.

Scanning electron microscopy (SEM) SEM images were obtained using a Supra 60 VP microscope (Zeiss, Germany). An acceleration voltage of 3 keV was applied to record images up to 60 000 times magnification. The GO samples were deposited on Si-wafers with a native oxide surface.

Quartz crystal microbalance with dissipation monitoring (QCM-D) QCM-D experiments were performed under constant flow (100 μL/min) at 22°C in an E4-instrument (Biolin Scientific, Sweden). The measurements were performed at several harmonics (z = 3, 5, 7, 9, 11, and 13). All presented QCM-D data were recorded at the 7th harmonic and frequency shifts were normalized by division with


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7. Prior to analysis, SiO2-coated QCM-D sensors (Q-Sense, Sweden) were treated in UV-ozone for >30 min, rinsed with water and dried. In the QCM-D experiments, supported lipid membranes were formed by adding liposomes (diluted in PBS to 0.1 mg/mL) to the clean SiO2-surfaces. After the membrane formation, the continuous flow of PBS was replaced with water and an aqueous solution of graphene oxide (diluted in water to 25 μg/mL) was added. Before further addition of liposomes the liquid was exchanged from water to PBS. The Q-Tools data evaluation software (Biolin Scientific, Sweden) was used to estimate the mass adsorbed in different experiments. Presented calculated masses were based on three experiments.

Indirect nanoplasmonic sensing (INPS) INPS experiments were performed under constant flow (100 μL/min) at room temperature in a X-nano instrument (Insplorion, Sweden). The instrument was run by a custom made MATLAB program that recorded the full extinction spectra at each time point, and monitored the wavelength of the extinction peak maximum. The sensors (Insplorion, Sweden), with a SiO2-spacer layer, were treated in UVozone for >30 min, rinsed with water and dried just prior to use. The sequence of events during the experiments were performed in the same way as for the QCM-D experiments, see the previous paragraph. In some of the performed experiments a drift in the response was observed. In these cases, the drift was assumed to be constant over the presented timeframe and compensated for accordingly during the data evaluation.


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Results and discussion Characterization of graphene oxide samples Three commercially available GO samples (named GO 1, GO 2, and GO 3) with sizes within the range of 90-5000 nm, as specified by the supplier, were used in this study. To characterize each sample with respect to their lateral size, the GO sheets were adsorbed to a planar Si substrate (with an outer layer of silica) and imaged using SEM. Representative images of all samples are presented in Figure 2A-C. The SEM analyses show that sample 1 (GO 1) contained the smallest GO sheets, in accordance with the specified size of 90±15 nm, no large sheets (in the μm size range) were observed. For sample 2 (GO 2), with a specified size range of 500-5000 nm, the dominating fraction of the GO sheets had a lateral dimension in the lower end of the quoted size range. Larger, μm-sized, GO sheets were also present but only to a minor extent. In sample 3 (GO 3), a significant fraction of the GO sheets had a lateral dimension >1 μm and only a minor fraction within the specified size range of 300-700 nm. Thus, the average lateral size (d) of the GO samples was dGO1 < dGO2 < dGO3. Apart from the size of the GO sheets, their chemical composition is an important determinant of their interactions with lipid structures. For this purpose, the GO samples were further characterized using x-ray photoelectron spectrometry (XPS). In Figure 2D-F the C1s spectra of the three GO samples are presented. The shapes of the spectra are in good agreement with earlier XPS analysis of GO.27, 28 The spectra were fitted using four components present in the GO samples: (I) O-C=O, (II) C=O, (III) C-O and (IV) C-C. A fifth component (V), attributed to hydrocarbon contamination, was included in GO 2 due to the large width of Peak IV in this


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sample. Most likely, hydrocarbon contaminations were also present in the other samples, although to a lower degree. It should be noted that the observed absolute binding energies are shifted towards lower energies compared to literature values. This is likely due to charging effects in our analysis of the poorly conductive GO samples. Despite the shift in binding energy of the C1s spectra the relative shifts between the different components (functional groups) are in agreement with earlier studies. The C/O ratio calculated as the ratio of the total area of the C1s spectra (disregarding peak V in GO 2) and the sum of the oxygen containing components, weighted by their stoichiometric ratio (i.e. 2, 1, and 1 for O-C=O, C=O, and C-O respectively), are 1.5 ± 0.1 for the three GO samples. Thus the degree of oxidation is similar for all samples. Table S1 (supporting information) gives more detailed information regarding the binding energies and the atomic percentage of each functional group present in the GO samples.


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Figure 2. (A-C) SEM images of the GO 1-3 samples and (D-F) the corresponding C1s XPS spectra. The C1s spectra have been fitted by four components of GO (I: O-C=O, II: C=O, III: C-O and IV: C-C). A fifth component (V), attributed to carbon contaminations, is present in one of the samples (GO 2).

Adsorption of graphene oxide sheets of different size to supported lipid membranes As a first step, supported lipid membranes were formed on the SiO2 sensor surfaces from extruded liposomes, a well-established process13-17 following a specific mechanistic scenario that is described in some detail later on. The liposomes were composed of a 3:1 mixture of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-ethylphosphocholine (POEPC). POPC is a zwitterionic lipid with a net neutral charge while POEPC is positively charged. The structures of the two types of lipids are similar but the presence of an ethyl group in the lipid head group of POEPC eliminates the negative charge of POPC. This binary lipid composition yields liposomes with lipid head groups having a net positive charge (ζ-potential 21±4 mV) at pH 7.4. It was assumed that the supported lipid


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membranes formed on the sensor surfaces had the same surface charge as the corresponding liposomes in bulk solution. The supported lipid membranes were exposed to aqueous suspensions of GO sheets of different average lateral size. The adsorption of GO to POPC:POEPC (3:1) lipid membranes was monitored using QCMD (Figure 3A). This technique generates two measured responses, the shift in resonance frequency of the piezoelectric sensor (Δf), related to the mass change upon adsorption, and the shift in (energy) dissipation (ΔD), which originates from the damping of the sensor oscillation, caused by the deposit.29 The Δf response relates to the hydrated mass coupled to the oscillatory motion of the sensor, i.e. the GO mass and any GO associated solvent, while the ΔD response relates to the viscoelastic properties of the surface deposit. From the result in Figure 3A (solid blue line) it is evident that only a small amount of GO 1 (nano-GO) adsorbed to the membrane (Δf = -1.6±0.2 Hz corresponding to about 20% of a coherent graphene sheet over the whole sensor surface). The most likely cause for this quite low coverage is that although the sheets are electrostatically attracted to the positively charged bilayer, there is also mutual electrostatic repulsion between adjacent GOsheets, since they are terminated with hydroxyl and carboxyl functional groups. This interpretation is based on the most widely adopted chemical structure of GO, the Lerf-Klinowski model.30 Depletion of the laterally mobile, positive lipids from uncovered areas of the lipid membrane, when GO sheets bind to the positive head groups of POEPC, could in principle also contribute to a low GO coverage. However, such charge depletion cannot occur in the present experiments, as discussed later on.


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Upon adsorption of GO 1, no significant ΔD was observed suggesting that the GO sheets adsorbed flat and firmly fixed onto the membrane. The larger sizes of GO, that is GO 2 and GO 3, generated larger frequency shift responses, Δf = -6.9±0.5 and 9.5±1.0 Hz, respectively. The adsorption of these two GO samples was also associated with a slight increase in ΔD, which may indicate that the GO-sheets did not perfectly align parallel onto the lipid membrane, possibly due to partial aggregation or folding. This may indicate that some water is “trapped” in the structure and adding to the mass, generating the observed Δf. From the results it is also evident that the GO 3 sample has markedly slower adsorption kinetics than the other two samples, which we attribute to the larger size of the GO sheets in this sample. However, the kinetics is not a prime topic of this paper, instead our focus is on the (irreversible) saturation coverage for the different graphene oxide samples. In Figure 3B, the total (hydrated) masses of the GO adlayers have been calculated according to the Sauerbrey equation (macoustic = C*Δfz where C = -17.7 ng/cm2Hz and Δfz is the normalized frequency shift at the zth harmonic (here z =7)). The Sauerbrey equation was assumed to be valid since the ΔD values were small compared to the corresponding Δf responses and in a range usually consistent with the requirements for applying the Sauerbery equation, i.e. the GO was quite rigidly attached to the lipid membrane. In our previous study, we determined the degree of hydration of the GO adlayer to 32% by relating its hydrated mass (determined by QCM-D) to its “dry” mass (determined by dual polarization interferometry (DPI)).17 The theoretical dry mass of a complete GO monolayer was calculated to 101 ng/cm2 and


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by accounting for the experimentally derived hydration level of 32% the hydrated mass will be 133 ng/cm2. When comparing the calculated (hydrated) masses from the experimental data with the corresponding mass of a theoretical GO monolayer (133 ng/cm2), it is evident that GO 1 coverage is well below a full monolayer. The larger sizes of GO give rise to hydrated masses similar to, and above, that of a GO monolayer. A mass above a full GO monolayer indicates that the adsorbed GO sheets overlap to a certain extent. It is interesting to note that the GO used in our previous study (GO 2), from the same source, did not generate a shift in dissipation. The observed change in material properties might be due to a difference in the manufacturing protocol during the last few years (or a batch-to-batch variation). For the purpose of this study, this difference is not of significance, as it was possible to accurately reproduce the formation of a multilayered GO/lipid membrane structure also using the new GO sample of the same size.


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Figure 3. (A) QCM-D data for the adsorption of GO of different size on POPC:POEPC (3:1) supported lipid membrane. The formation of the lipid membranes is omitted from the plot. (B) Calculated masses of the adsorbed GO layers. The dashed lines represent the theoretical mass corresponding to an ideal layer of GO (101 ng/cm2) and the hydrated mass of the same layer (133 ng/cm2).

The QCM-D results were complemented by INPS studies of the optical properties of the adlayers. Changes in refractive index (RI), within the penetration depth of the induced electromagnetic field of the sensor particles in INPS, manifests itself as a shift of the extinction peak maximum of the plasmon resonance (Δλ). An increase in RI will generate a red shift of the peak position. For this reason, INPS is not sensitive to associated solvent (unless its composition is changed). Sometimes this is referred to as a technique that similar to SPR, measures “dry” mass. When studying GO


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adsorption to POPC:POEPC (3:1) lipid membranes using INPS, it became evident that not only the dry mass adsorption process gave rise to the recorded peak shift. Upon GO adsorption, an initial negative Δλ was observed that reached a minimum and later became positive to eventually reach a final net positive response. The initial negative Δλ is likely due to the wavelength dependent light scattering by GO in suspension, since its absorption is greater at the lower end of the visible spectrum (not shown). A characteristic plot of the INPS response of the GO adsorption process is presented as supporting information (Figure S1). Similarly, the peak shift recorded during formation of the lipid membranes decrease slightly before the response increases (Figure S3). This feature has also been observed by others.31, 32

Investigating the dependency of size on liposome/graphene oxide interactions In the next step, SUVs and LUVs were adsorbed onto GO sheets of different sizes, which had been preadsorbed to the supported lipid membranes. The average sizes of the liposomes were determined by dynamic light scattering to be 84 nm (PDI 0.1) and 315 nm (PDI 0.2) for the SUVs and LUVs respectively. Examples of measured size distributions are given as supporting information (Figure S2). Thus, the size of the smallest GO sheets (GO 1) is in the same size range as the diameter of the SUVs while it is well below the average diameter of the LUVs. In contrast, the largest GO sheet size of several micrometers (mainly present in GO 3, but also to some extent in GO 2) is much larger than both types of liposomes. In other words, many liposomes can adsorb on the larger GO sheets, but on the smallest sheets (GO 1) only one


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liposome can adsorb. Intermediate GO sizes, predominantly present in GO 2, is clearly larger than the SUVs but close to the size of the LUVs. Note that the lamellarity of the liposomes has not been analyzed in the present study. Previous studies have indicated that liposomes extruded through 100 nm pores are predominantly unilamellar.33 However, liposomes extruded through larger pore sizes may generate a greater fraction of liposomes with multilamellar character. This fraction may be reduced by a freeze-thaw pretreatment process upon preparation.34

In Figure 4, QCM-D data of the SUV and LUV adsorption to the differently sized GOsamples are shown. The results are presented as Df-plots. This type of plot visualizes the data in a compact manner and aids the identification of different regimes in an adsorption process. The main drawback is that the time is eliminated as explicit parameter and for this reason it is not possible to judge if processes have reached equilibrium. However, the Δf and ΔD responses versus time, for the full QCM-D experiments, are presented later in Figure 6. For a comparative purpose, the kinetics resulting upon addition of liposomes to SiO2-surfaces (forming supported lipid membranes) is included in Figure 4. Briefly, the mechanistic scenario when forming a supported lipid membrane from liposomes on a SiO2-surface is as follows:22, 23, 24, 35 First, liposomes adsorb intact to the surface generating a negative shift in frequency (mass adsorption) and a positive shift in dissipation (viscoelastic structure), with an almost linear ∆D-∆f relation. When a certain surface coverage is reached the liposomes rupture and a


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planar lipid membrane is formed on the support. During this process the solvent in the interior of the liposomes is released to the surroundings (causing a mass loss and positive shift in frequency) and a more rigid structure is formed (negative shift in dissipation). This adsorption-rupture-formation process gives rise to a cusp in the Df-plot that is best illustrated by SUVs (Figure 4A). A good quality lipid membrane has a final Δf of about -26 Hz and a ΔD close to zero.22

Figure 4. Df-plots of (A) SUV and (B) LUV adsorption to SiO2 (SLB formation) and to preadsorbed graphene oxide sheets of different size. The inset in (A) shows the same data at a different scale.

If the liposomes do not rupture upon adsorption, as is e.g., the case for SUVs on TiO236 and Au37, the resulting Df-plots are essentially linear throughout the


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adsorption process. In Figure 4, it is evident that adsorption of SUVs and LUVs to GO 1 results in a close to linear relationship between Δf and ΔD, i.e., all liposomes adsorb intact and contribute equally to the Δf and ΔD changes independent of when the adsorption occurs. Notably, the slope for LUV adsorption is greater compared to the same process for SUVs since large liposomes generate a more viscoelastic structure at the surface (more dissipation per unit frequency (mass) shift. The fact that the maximum Δf response is larger for SUVs compared to LUVs, when adsorbed to GO 1, is mainly related to the slow kinetics for LUV adsorption, which is not revealed by the Df-plot. If a longer adsorption time had been allowed, the situation would have reversed (see Figure 6 for time resolved data). Considering the low coverage of GO 1 on the surface and their small size, it is reasonable to conclude that the adsorbed liposomes are well separated from each other and that the liposome/GO ratio is 1:1. Previously reported data show that a full monolayer of 110 nm liposomes generate a frequency shift of about -240 Hz36, i.e. more than three times the present response from SUV adsorption to GO 1. This difference is mainly explained by the low surface coverage of GO, which above was quoted to be around 20% of a full monolayer of GO.

Compared to the QCM-D responses of SUV and LUV adsorption to predeposited GO 1, less adsorbed mass was detected during their adsorption to predeposited GO 2. The smaller mass adsorbed to GO 2 indicate that some of the adsorbed liposomes rupture, i.e. less water is associated with the surface. However, for both liposome sizes, the recorded responses were larger than expected if the all of the adsorbing


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liposomes would rupture. The slopes of the Df-plots for the recordings of liposome adsorption to GO 2 are not as linear (low initial slope) as in the experiments with preadsorbed GO 1 (Figure 4). One mechanistic scenario that explains the results is the following: At first, when the liposomes start to adsorb to the surface of the GO sheets present in the GO 2 sample, some liposomes rupture. This process gives rise to an initially low ∆D-response and forms patches of planar lipid membranes at preferred sites (e.g., on the large GO sheets present in this sample). After some time has elapsed (coverage has increased), liposomes remain intact upon adsorption, leading to a higher value of the Df-slope (similar to experiments with GO 1). A possible cause for the observed adsorption scenario is the heterogeneous chemical structure of GO, e.g. the liposomes adsorb differently to different areas of the GO surface.

When looking at the results of liposome adsorption to predeposited GO 3 it is notably different from the experiments where liposomes adsorb to predeposited GO 1 or GO 2. The QCM-D recording for SUVs adsorbing to predeposited GO 3, is similar to what is found for SUV adsorption to SiO2. In particular, the characteristic peak in ΔD (associated with liposome rupture) is present and the final ΔD is low. This result indicates that SUVs spontaneously adsorb and rupture on the GO sheets present in the GO 3 sample (large fraction of micrometer sized GO sheets). The corresponding data for LUVs show a plateau when the frequency decreases (mass adsorption) without a simultaneous increase in dissipation. This result indicates that also LUVs adsorb and rupture on sheets in the GO 3 sample. However, at a certain point, LUVs


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adsorbed intact giving rise to a steeper slope in the latter part of the Df-plot. This second regime, i.e. when liposomes remain intact upon adsorption, is much less pronounced for the small liposomes.

If the liposomes rupture or not upon adsorption is related to both the contact area between liposomes and GO sheets, and the chemical structure of GO. It is, based on trends in previous work on liposome adsorption on different surfaces, reasonable to assume that the liposomes will rupture if the GO - liposome interaction is sufficiently strong, i.e. if the liposomes adsorb to a highly charged area of the GO sheets and a sufficient contact area is allowed. In a previous cryo-TEM study, it has been observed that DOPC liposomes preferably associate with the edges of large GOsheets.38 For this reason, the edges may limit the allowed contact area and prevent liposome rupture. In accordance with this reasoning, our results suggest that liposome rupture occurs predominantly on large GO sheets. A tentative explanation is the low ratio between edge length and surface area, i.e., liposome rupture may occur onto the GO surface away from the edges, without a major influence of the edge. Edge bound liposomes may then still rupture via the previously proposed autocatalytic effect,39 where rupturing liposomes induce rupture of adjacent liposomes.

When the coverage of GO at the membrane surface is high, as in the cases for GO 2 and GO 3, a single liposome may adsorb to two or more adjacent GO sheets, possibly separated by a small gap exposing the underlying lipid membrane. In this complex


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case, the fate of the liposome (i.e. if the liposome ruptures or not) could be different compared to if the liposome adsorbed to the edge of only one GO sheet. In addition, the adsorbed liposome interacts both with GO and the underlying lipid bilayer, the latter opening for lipid exchange between the bilayer and the liposome (and for a change of net charge of both entities).26

Due to the lateral mobility of lipids in the membrane, positively charged lipids (POEPC) are likely accumulated underneath adsorbed GO sheets, reducing the surface charge in uncovered areas of the lipid membrane. If the uncovered areas were depleted from POEPC lipids, addition of POPC:POEPC (3:1) liposomes would not specifically target GO but may also adsorb to the remaining POPC membrane, although their interaction would be transient due to exchange of lipids.40 To prove the excess of POEPC lipids, compared to what the GO flakes could occupy/consume, titration type control experiments were performed (data not shown). In these experiments, negatively charged liposomes (POPC:POPS (3:1)) were found to adsorb to the POPC:POEPC membrane after adsorption of GO, revealing presence of a net positive charge at uncovered areas of the membrane. Thus, the presented data of SUV and LUV adsorption is specifically targeted to adsorbed GO-sheets.

Additional INPS measurements were then performed to further characterize the liposomes adsorbed to GO sheets. Figure 5 shows INPS-data of SUV and LUV adsorption to preadsorbed GO on POPC:POEPC (3:1) lipid membranes, i.e. the same situation as for the previously shown QCM-D data. The results showed that SUV and


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LUV adsorption to GO 1 (Figure 5A) gave similar final responses although the rate of liposome adsorption was different. The different kinetics is explained by the slower diffusion rate of LUVs compared to SUVs. At first, the similar final responses might seem surprising when the liposome:GO ratio is 1:1. However, considering the limiting penetration depth of the sensing field and that it is likely that the small size of the GO sheets limits the deformation of the liposomes (specifically for LUVs), it is reasonable to believe that one LUV gives rise to about the same response as one SUV, as the data suggests. It should be noted that it has previously been observed that the peak shift at saturation coverage for adsorption of POPC liposomes to a TiO2 surface was similar for diameters of 80-160 nm (Δλmax of 3.26±0.06 nm).31 Note however that for non-deformable liposomes a peak shift at saturation coverage according to Δλmax ∝ 1/ρ (where ρ is the radius of the liposome) is expected.31 One remark that is of relevance at this point is that if the liposomes rupture and form a planar lipid membrane at the surface of GO the observed Δλ would naturally be the same for both SUVs and LUVs. See supporting information for INPS data of supported lipid membrane formation using SUVs and LUVs on SiO2 (Figure S3). The INPS-data for SUV and LUV adsorption on GO 2 (Figure 5B) SUVs generated about double the response of LUVs. In line with the QCM-D data, it is noted that the liposomes do not rupture to a large extent upon adsorption to this GO sample (since the responses in that case would have been similar). Since the majority of the GO sheets are larger than the size of the liposomes, in contrast to GO 1, there is no restriction regarding to which extent the liposomes may be deformed upon adsorption. Assuming non-deformable liposomes, the SUVs would generate


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approximately a four times greater response than the LUVs (according to Δλmax ∝ 1/ρ). However, the fact that SUVs only generated twice the response for the LUVs indicates that the LUVs deform, or ultimately rupture, at the GO surface.

In the last case, liposome adsorption to GO 3 (Figure 5C) showed a similar response (Δλ ≈ 1.7-1.8 nm) for the two types of liposomes. As already concluded, the two types of liposomes may yield similar Δλ, whether they adsorb intact or rupture to form a planar membrane. However, in light of the QCM-D data it could be concluded that the liposomes indeed rupture and form a lipid membrane on top of the GO sheets. The absolute response is slightly smaller than the response recorded during formation of the first lipid membrane (Δλ ≈ 2.3 nm, Figure S3). The two main reasons for the smaller response is that the second membrane is located further away from the plasmonic Au particles and that the GO layer is not perfectly homogenous compared to the underlying SiO2 substrate.


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Figure 5. INPS data of the addition of SUVs and LUVs to (A) GO 1, (B) GO 2, and (C) GO 3. The preceding lipid membrane formation and adsorption of GO have been omitted from the plots.

Graphene oxide induced liposome rupture and the formation of LbL-structures In our previous study, using SUVs and large GO-sheets (0.5 – 5 μm), it was shown that preadsorbed liposomes on GO ruptured upon exposure to new GO flakes, forming a multilamellar structure of GO and lipid membranes.17 To explore how the fate of both SUVs and LUVs depend on the size of the GO sheets, the already adsorbed liposomes were exposed to GO of the same size as they were initially


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adsorbed on. In Figure 6, data from the complete QCM-D experiments using SUVs and LUVs together with two GO samples (GO 1 and GO 2) is presented. In these four cases, it has already been concluded that a large fraction of the liposomes adsorbed intact at the GO surface (Figure 4). The complete experiments include formation of a POPC:POEPC (3:1) supported lipid membrane (I-II), adsorption of GO (III), adsorption of POPC:POEPC (3:1) liposomes (IV), and addition of GO (V). Data on steps I-IV have already been presented and discussed and for this reason these events are shaded in Figure 6. However, here the Δf and ΔD responses are plotted versus time, which are new data in the sense that the time evolution is displayed, and the previously mentioned difference in kinetics between SUVs and LUVs is here clearly evident. In the last step (V), liposome rupture will be indicated by a decrease in ΔD (final value close to zero) since a rigid structure, i.e. the SLB, will be formed. Considering Δf of the same process it is a sum of two competing events, an increase in mass (negative Δf) due to adsorption of GO mass and a decrease in mass (positive Δf) due to release of the liquid interior of the rupturing liposomes. Stabilization of the intact liposome layer by the GO sheets, without any other mass change than the added GO mass, may also be a possible outcome generating a negative ΔD, if the structure becomes more rigid. Together, the two responses (ΔD and Δf) give a good indication of the fate of the liposomes.

When preadsorbed SUVs were exposed to GO 1 (Figure 6A, step V) ΔD decreased somewhat indicating partial liposome rupture or stabilization of intact liposomes (liposomes become more rigid). For the completeness of the study, the previously


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reported case using SUVs and larger GO sheets is reproduced (Figure 6B). As before17, the final low ΔD and positive Δf, indicate rupture of the liposomes upon GO exposure (step V). When considering the result of LUVs, it is evident that the liposomes remained intact upon addition of GO 1 (Figure 6C) a conclusion drawn based on the large final ΔD. Furthermore, the significant negative Δf during addition of GO 1 (step V) indicate that the small GO sheets largely adsorbed to the surface of the LUVs. The corresponding experiment using larger GO sheets (Figure 6D) showed that the LUVs remained intact upon addition of GO.

Figure 6. QCM-D data showing the full sequence of (I) addition of POPC:POEPC (3:1) liposomes spontaneously forming a (II) supported lipid bilayer followed by addition of (III) graphene oxide, (IV) POPC:POEPC (3:1) SUVs or LUVs, and (V) graphene oxide. The additions of graphene oxide and liposomes are preceded by a (*) liquid exchange to water and PBS respectively. The plots show data


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for both GO 1, together with (A) SUVs and (C) LUVs, and for GO 2, together with (B) SUVs and (D) LUVs. The shaded areas (steps I-IV) correspond to events presented earlier in the article.

The interpretation of the data presented is shown schematically in Figure 7. At the top of the figure, a supported lipid membrane was formed by liposome rupture onto a SiO2-substrate (sensor surface). This process was followed by adsorption of GO, and subsequent addition of SUVs/LUVs that were shown to adsorb intact. In the last step, the preadsorbed liposomes were exposed to GO of the same size as they were initially adsorbed on. The result showed that complete rupture occurred only when SUVs were exposed to large GO sheets GO 2. In the other cases GO adsorbed onto intact liposomes, either to the surface of single liposomes (GO 1) or spanning several liposomes (GO 2). In Figure 7 all liposomes are represented as unilamellar. As mentioned earlier in the article this representation is true for the smaller liposomes but the larger may contain a more significant fraction of liposomes with a multilamellar structure. Compared to unilamellar liposomes, properties of multilamellar liposomes of similar size that may influence the QCM-D and INPS responses are the mass and bending rigidity.


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Figure 7. Schematic representation of the GO/lipid membrane (SLB and liposome) interactions, deduced from the data presented in Figure 6. The following series of events is illustrated (from the top); (I) addition of liposomes, (II) spontaneous formation of a supported lipid membrane, (III) addition of GO 1 (90 nm) and GO 2 (500-5000 nm, GO sheet sizes in the lower end of this range are dominating), (IV) addition of SUVs and LUVs to adsorbed GO 1 and GO 2, (V) addition of GO 1 and GO 2 to the adsorbed liposomes. Not drawn to scale.

Concluding remarks In this study, the use of an electro-acoustic (QCM-D) and an optical (INPS) analytical technique has been proven to be a prosperous combination to probe interactions at the nano-bio interface. By this combination it was possible to, in some detail, elucidate mechanistic scenarios of the GO interaction with supramolecular lipid structures (surface supported membranes and liposomes). The results open up for fabrication of interesting GO/lipid membrane assemblies by using GO sheet size and


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SUV/LUV sizes as parameters. For example, functionalization of liposome surfaces with nano-GO may be further explored as a device for multiplexed drug delivery. In addition, it has been proven possible to intercalate not only lipid membranes but also liposomes between stacked GO-sheets with large lateral dimensions. Another intriguing finding in the present study is the spontaneous rupture of SUVs (and partly LUVs) at the surface of large GO sheets (GO 3). This result is likely to allow for a better control of the layer-by-layer assembly of these materials. With all the presented data at hand, it is evident that our initial hypothesis (that liposome rupture occurs when liposomes are exposed to two GO sheets, one on each side of the liposome, where the GO sheets have the same size as, or are larger than, the cross-sectional area of the liposome) does not seem to be true in all cases. For example, it has been shown that not all adsorbed SUVs exposed to GO of the same size rupture. Furthermore, LUVs exposed to GO sheets, with lateral dimensions larger than the LUV diameter, do not rupture.

The present study may form a basis for continued research regarding in vitro characterization of GO/lipid membrane interactions including, e.g., the effect of charge density in the model membrane (lipid composition) and the GO (degree of oxidation (e.g. using partly reduced GO)). Specifically, by tuning the lipid composition of the supported membrane (relative amounts of GO binding and nonbinding lipids) and by choosing specific lateral dimensions of the GO-sheets it is likely possible to form well separated GO sheets (rafts) with a lipid bilayer or liposomes on top.


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Acknowledgements This work was financially supported by the Chalmers Area of Advance in Nanoscience and Nanotechnology. Lars Ilver is gratefully acknowledged for performing the XPS measurements and for fruitful discussions regarding the interpretation of the results.

Supporting Information INPS data of GO adsorption on POPC:POEPC (3:1) membrane; Size distributions of SUVs and LUVs determined by DLS; QCM-D and INPS data of the lipid membrane formation process using SUVs and LUVs

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