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Interaction of silver nanoparticles with tethered bilayer lipid membranes Renee V Goreham, Vanessa C Thompson, Yuya Samura, Christopher T. Gibson, Joseph G. Shapter, and Ingo Köper Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b00586 • Publication Date (Web): 07 May 2015 Downloaded from http://pubs.acs.org on May 11, 2015

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Interaction of silver nanoparticles with tethered bilayer lipid membranes Renee V Goreham‡, Vanessa C Thompson‡, Yuya Samura, Christopher T Gibson, Joseph G Shapter, Ingo Köper* Flinders Centre for NanoScale Science and Technology and School of Chemical and Physical Sciences, Flinders University, Bedford Park, SA, Australia. 5042. KEYWORDS Silver nanoparticles, tethered bilayer lipid membranes, nanoparticles, electrical impedance spectroscopy, atomic force microscopy ABSTRACT

Silver nanoparticles are well known for their antibacterial properties. However, the detailed mechanism describing the interaction between the nanoparticles and a cell membrane is not fully understood, which can impede the use of the particles in biomedical applications. Here, a tethered bilayer lipid membrane has been used as a model system to mimic a natural membrane and to study the effect of exposure to small silver nanoparticles with diameters of about 2 nm. The solid supported membrane architecture allowed for the application of surface analytical techniques such as electrochemical impedance spectroscopy and atomic force microscopy.

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Exposure of the membrane to solutions of the silver nanoparticles led to a small but completely reversible perturbation of the lipid bilayer.

Introduction Engineered nanomaterials have attractive properties for biomedical applications and are increasingly being incorporated into commercial products. In particular, the use of engineered silver nanoparticles (AgNPs) in biomedical applications has increased rapidly due to their desirable antibacterial,1, 2, 3 electronic,4 catalytic5 and photonic4, 6 properties. AgNPs have been used for more than 100 years and it is estimated that 320 tons of AgNPs are manufactured each year.7 Current uses for AgNPs include nanomedical devices, environmental remediation, cosmetics, household products, room sprays and food products.8,

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More recently, AgNPs are

more frequently being developed for use in medical imaging and biosensing. While the use of AgNPs and the risk of exposure to AgNPs increases, little is known about the mechanism by which AgNPs interact with mammalian cells and these mechanisms have not been rigorously studied. AgNPs can be produced in a variety of sizes and shapes2,

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and usually are coated with a

capping or stabilising agent. Many nanoparticle toxicity studies have been performed with a high variation in results, which has largely been attributed to differences in capping agents and nanoparticle size. When capping agents are kept constant, smaller nanoparticles are more readily taken up by human cells than larger nanoparticles.9, 11 Capping agents have a dramatic role on nanoparticle function in the body, with zeta-potential of the capped nanoparticle being perhaps more important than the underlying nanoparticle itself.12 This variability of activity with subtle


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changes in size and chemistry highlights the importance of clearly understanding the mechanism of action of the particles. AgNPs interact with mammalian cells to cause inflammation, denaturation of proteins, oxidative stress and disruption of the cell membrane. Therefore, current methods to assess the cytotoxicity of nanoparticles are naturally complicated in an attempt to describe complex cellular responses.8, 13 Interaction with the membrane alone can cause cytotoxicity through a variety of mechanisms, such as adherence of the nanoparticle to the membrane, aggregation around the membrane, removal of lipids from the membrane or stably embedding into the membrane.14 Cellular membranes are naturally very complex, composed of a mixture of proteins, lipids, and carbohydrates. Silver has a tendency to interact with each of these molecules,15,

16, 17

but

predominantly with lipid and proteins. A model lipid bilayer system is useful to understand the interaction of AgNPs specifically with the lipid portion of the cellular membrane. Several lipid membrane models have been used to understand specific nanoparticle-membrane interactions, including black lipid membranes,18 adjoining lipid vesicles studied by electrophysiological measurements,19 mercury droplet membranes,20,

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giant unilamellar vesicles,20,

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and supported lipid bilayers on flat surfaces23

and nanoparticle surfaces.24 Tethered bilayer lipid membranes (tBLMs) are model lipid membranes which consist of a lipid bilayer that is attached to a solid support via a short tether group.25 tBLMs do not have residual organic solvents incorporated as a result of their production, making them more comparable to cellular membranes. Additionally they offer an increased stability compared with other model systems, making systematic investigations of membrane related processes feasible.26 tBLMs are useful as their electrical properties can be readily measured using electrical impedance spectroscopy (EIS) due to their excellent sealing properties


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while atomic force microscopy (AFM) in fluid can be used to investigate the morphological and mechanical changes of the tBLM.27 It has been shown that nanoparticles, depending on their size, are able to penetrate cell membranes without any cellular machinery20,

24, 28, 29

. The charge of the nanoparticles is also

important; anionic nanoparticles are able to cause localised gelling of zwitterionic lipids, while cationic nanoparticles cause localised fluidity of otherwise gelled lipids22. Although there have been studies on how nanoparticles interact with model lipid bilayers,6, 22, 24, 28, 30, 31 limited work has been conducted on AgNP-membrane interactions. Polymer-capped AgNPs have been used, where the capping potentially can mask silver-specific effects of AgNPs.9, 32 Here the interactions of citrate coated AgNPs with tBLMs have been investigated. The tBLM used was a well-characterised system with a DPhyTL inner and a DPhyPC outer leaflet,27 which provides a robust model platform to analyse membrane related processes using a wide range of surface analytical techniques.

Results and Discussion AgNPs were synthesised using a two-step seed-mediated method and showed a characteristic absorbance peak at 417 nm (Figure 1 a). AgNPs were approximately 2 nm in diameter, determined using DLS and TEM (Figure 1 b, c), and spherical in shape (Figure 1 c), with a zetapotential of -28 mV. The particles were stable over time when stored in pure water; however, they aggregated when suspended in 100 mM NaCl solution (Figure 1 d). Over time, the formation of two distinct populations of differing size with diameters of 83.5 ± 24.2 nm (present in 3 of 4 samples) and diameters of 151.1 ± 35.0 nm (present in 4 of 4 samples) could be observed (Figure 1 d), probably due to a complex aggregation mechanism of silver nanoparticles.


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To investigate the interaction of AgNPs with lipid bilayer membranes, tBLMs were exposed to AgNP solutions (Scheme 1). The electrical properties of the tBLM were monitored using EIS (Figure 2). EIS is a well-established technique to study the electrical properties of membranes and changes in these properties due to interactions with foreign material. The EIS data can be represented in Bode plots, where the absolute impedance (|Z|) and phase angle (θ) are plotted as a function of frequency. For this type of representation, an ideal capacitor has a slope of |Z| = -1 and a phase shift of θ = -90° whereas ideal resistors display a slope of |Z| = 0 and a phase shift of θ = 0°. The experimental data was fitted using an equivalent circuit (inset, Figure 2), where different resistors and capacitors represent the different parts of the membrane architecture. The electrolyte has purely ohmic behaviour (Rel), whereas the lipid bilayer can be described as an RC-element (RmCm). The sub-membrane spacer region combined with the electrode interface can be represented by a single capacitor (Cs). This R(RC)C equivalent circuit has been shown to give a good representation of the electrical properties of a tBLM.27 After tBLM formation, 10 or 20 µL of AgNPs (14.8 ng/µL Ag) were added to the membrane. Twenty-four hour exposure of the membrane to the nanoparticle suspension led to significant changes in the electrical properties of the membrane (Table 1, Figure 2). For both cases, the membrane resistance decreased, indicating a perturbation of the bilayer architecture or the formation of small defects. The concomitant small decrease in membrane capacitance (Cm) indicates that the overall bilayer structure remained intact and can also possibly indicate a slight increase in membrane thickness due to the adsorption of nanoparticles at the membraneelectrolyte interface. The results in Table 1 are for a representative experiment. Since the electrical parameters of the tBLM fluctuate in between preparations, it is difficult to give average


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values of the absolute resistance and capacitance values, and instead, averages of the changes are reported.33 The experiments have been reproduced at least three times, and the electrical resistance of the membrane decreases in average by 50 ± 20% and the capacitance changed by 80 ± 30%. The changes in the spacer capacity are very large (500 ± 500 %), however this element of the electrical model circuit can only be seen at very low frequencies, resulting in the high uncertainties. The capacity of a plate capacitor can be expressed as ஺

‫ߝߝ = ܥ‬௥ , ௗ

with ε and εr being the vacuum permittivity and the dielectric constant of the capacitor, respectively, and A and d being the area and thickness of the capacitor, respectively. The capacitance describing the electric double layer at the gold layer and spacer region increased dramatically upon nanoparticle exposure. It is unlikely that this region changed drastically in size, thus any changes were predominantly due to changes in the dielectric constant. The increase in capacitance could be due to an influx of water into the spacer region, triggered by the perturbation in the bilayer architecture. The membrane resistance and the spacer capacitance are visible at very low frequencies only, which means the exact magnitude of change in these electrical parameters is prone to large errors. Despite these large changes in spacer capacitance, all effects were completely reversible upon rinsing, indicating that it is unlikely that the AgNPs penetrated through the bilayer and accumulated in the sub-membrane space. In a control experiment, assessing the influence of the citrate as capping agent on the membrane, no changes in the electrical properties have been observed in the absence of AgNPs. (data not shown)


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To further investigate whether the AgNPs were adsorbed to the membrane or permanently integrated within it, the soluble contents of the EIS measurement cell were analysed by ICP-MS (Supplementary information, Table 1). A total of 296 ng of silver was added to the tBLM at the beginning of the EIS experiment. After 24 h, the solution above the membrane still contained 269 ng of silver, indicating that 9.12 % of the silver had interacted with the membrane. However, this amount could be almost completely (a total of 99.6%) recovered in subsequent rinsing steps, in very good agreement with the completely reversible impedance data. These results also agree with the amount of Au recovered from anionic AuNP-supported lipid bilayer interaction studies where >60% of the Au remained in solution, and 5% was recovered after washing.30 This is despite differences in the lipid model used as Hou et al.30 used a supported lipid bilayer (egg phosphatidyl choline) on a SiO2 nanoparticle support. In order to determine whether the AgNPs caused any morphological or mechanical changes to the tBLM, the bilayers were analysed by AFM (Figure 3).

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Similar to the studies with EIS,

AFM analysis was done on the tBLMs before exposure to AgNPs (20 µL), immediately following the addition of the AgNPs and after 24 h exposure. The bare tBLM was essentially flat and featureless, (Figure 3 a, d) in good agreement with previous experiments.27 Immediately after addition of the AgNPs, small nanoparticles of 2-3 nm were present on the bilayer surface (Figure 3 b, e). After 24 h, larger particles of about 15 nm diameter were visible. (Figure 3 c, f). This is in contrast to the agglomeration of AgNPs to 151.1 nm in 100 mM NaCl solution alone (Figure 1 d) suggesting stability is offered to the AgNPs by the zwitterionic headgroups of the DPhyPC molecules which comprise the outer leaflet of the tBLM.36 The mechanical properties of the membrane were investigated by recording force curves before and after the exposure of tBLMs to AgNPs (Figure 4) on various positions across the


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surface. Probe deflection provides a force distance curve in which thickness and Young’s modulus of the bilayer can be measured.34, 35 Incubation of the tBLM with AgNPs caused little difference in the properties observed by AFM. The membrane thickness (4.7 ± 1 nm), as measured by the distance between contact and breakthrough point in the approach force distance curves,37, 38 remained unchanged as did the Young’s modulus (400 ± 100 MPa), calculated from force curves of the tBLM recorded before and 24 hours after AgNP exposure. The numerical value is in good agreement with previously published results of 285 ± 113 MPa for DPPC supported lipid bilayers.39 The AFM results are also in good agreement with the EIS data, indicating that the AgNPs interacted with or adsorbed to the lipid bilayer, but only caused small and reversible perturbation of the bilayer structure. The results are also consistent with sum frequency generation and ATR-FTIR experiments investigating AuNP-lipid bilayer interactions, which showed no defects in the bilayer for 5 nm AuNPs at low concentration. 40 Similarly, in primary EIS studies of DOPC monolayers on a Hg electrode, silica nanoparticles did not change the monolayer capacitance.20 The exact mechanism by which AgNPs transiently disrupt the membrane is unclear, but may be similar to that seen for other anionic nanoparticles. The AgNPs used in the current study are encapsulated with carboxylic acid groups and have a zeta-potential of -28 mV, so they may interact with the positively charged surface amine groups of DPhyPC.41 The increase in bilayer permeability in response to nanoparticles is similar to that for SiO2 nanoparticles (50 and 500 nm in diameter) using electrophysiological measurements on a DOPC bilayer,19 and has been associated with localised gelling of lipids in vesicles with PC head groups by SiO2 NPs.20, 22, 42 AgNPs may cause localised, reversible reduction in fluidity of the membrane in the area immediately surrounding the AgNP, with a resultant increase in permeability. Nanoparticles can


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interact with lipid vesicles to cause disruption of the vesicle or become engulfed by a lipid bilayer.20, 29, 42

Conclusion The interactions of small silver nanoparticles with a model membrane platform have been investigated. By using complementary surface analytical tools such as EIS and AFM, only a slight reversible perturbation of the bilayer membrane could be observed. The electrical properties of the bilayer were affected, whereas the mechanical properties of the membrane did not change due to the presence of AgNPs. The changes in the electrical properties were reversed after rinsing of the bilayer, indicating that the nanoparticles only weakly bound to the bilayer surface, and did not deeply penetrated into the membrane, as also shown by AFM imaging. Further studies on the effect of AgNP size and functionalization are needed to fully understand the mechanism of the AgNP and tBLM interaction.

Experimental Section Synthesis of AgNPs AgNPs were synthesized following a two-step seed-mediated process adapted from Pal et al.2 Briefly, 0.5 mL of 10 mM NaBH4 (5 x 10-6 mol; 99%, Sigma-Aldrich) was added to a solution of 0.5 mL of 0.01 M AgNO3 (5 x 10-6 mol; Sigma-Aldrich) and 20 mL of 0.001 M sodium citrate (20 x 10-6 mol; Univar), while stirring. This was stirred for 5 min, then aged for 1.5 h. Three mL of the resultant seeds were then added to 100 mL of boiling 0.001 M AgNO3 (1 x 10-4 mol), with 15 mL sodium citrate (final concentration of 0.001 M) and 45 mL Milli-Q H2O in 150 mL final volume.

The reaction was heated with stirring until the solution turned pale yellow,


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approximately 30 sec after addition of sodium citrate. This was then cooled at room temperature for 30 min, and refrigerated for future use. AgNPs were washed once with Milli-Q H2O (18.2 MΩcm) prior to use by centrifuging in Teflon tubes at 3000 g for 50 min (Allegra X-22, F1010 rotor, Beckman Coulter), removing supernatant, adding Milli-Q H2O, centrifuging again at 3000 g for 50 min, removing supernatant and retaining pellet. AgNPs were characterised by UVvisible spectroscopy (Cary 50, Varian), dynamic light scattering (DLS; HPPS, Malvern), zetapotential (Zetasizer Nano ZSP, Malvern) and transmission electron microscopy (TEM; Philips CM200, Adelaide Microscopy). Particle size was determined by DLS using the software package on the instrument and standard values (material RI: 0.2, Dispersant RI: 1.33, Sample viscosity: 0.8872, Absorption index: 0.01). The attenuation index, measurement duration, and measurement position were all determined by the instrument software. Average particle size was determined from three or more replicates, and standard error of the mean calculated. TEM samples were prepared by air drying a droplet of colloidal suspension on a copper TEM grid. Particle size determined from TEM images utilised Image J software (NIH, USA), with a minimum of 500 particles measured.

Membrane formation Ultra flat gold substrates were prepared using a template-stripping process,43 and coated with a monolayer of 2,3-di-O-phytanyl-sn-glycerin-1-tetraethylenglycollipoic acid ester (DPhyTL; 0.2 mg mL-1 in ethanol; synthesised as described previously44) by self-assembly for a minimum of 48 h at 4 °C. The monolayer was then fused with small DPhyPC vesicles (2 mg ml-1; Avanti Polar Lipids, Alabaster, AL), extruded through a 50 nm membrane, to form a tBLM. A detailed description of the membrane formation process can be found elsewhere.27


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Electrical Impedance Spectroscopy EIS measurements were conducted using an Autolab PGSTAT 30 impedance spectrometer. Three electrode measurements were performed in customised Teflon cells (1 mL) with the substrates as the working electrode (electrochemically active area of 0.28 cm2), a coiled platinum wire as the counter electrode and ET072 leakless miniature Ag/AgCl reference electrode (eDAQ Pty Ltd). Spectra were recorded for frequencies between 2 mHz and 20 kHz at a 0V bias potential with an AC modulation amplitude of 10 mV. Raw data was analysed using NOVA 1.10 (Metrohm Autolab B. V.) and fitted to an R(RC)C equivalent circuit of capacitors and resistors. Final values were normalized to the electrode surface area. Each experiment was repeated a minimum of three times. While the electrical properties of tBLMs typically vary between experiments, the changes in resistance and capacitance were reproducible. Representative results are presented.

Inductively coupled plasma mass spectrometry analysis Contents of the impedance cell were analysed for their silver contents using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500). Prior to ICP-MS measurements, samples were dissolved in 5 % nitric acid.

Atomic force microscopy (AFM) All AFM measurements were acquired using a Bruker Multimode VIII AFM with NanoScope V controller and silicon nitride ScanAsyst-Air probes. AFM fluid cell was sonicated in ethanol for 5 min before use, immediately followed by extensive rinsing with ethanol and drying under a


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stream N2 gas. Freshly prepared template stripped gold was placed into an AFM fluid cell and 10 mM NaCl solution was introduced into the cell and left to equilibrate for an hour prior to imaging. The template stripped gold was then replaced with a DPhyTL-coated gold slide, and images taken at each step. Images were taken 24 hours after the addition of extruded DPhyPC vesicles (50 µL.mL-1), upon addition of 20 µL AgNPs and 24 hours after AgNP addition. AFM images were acquired in peak-force tapping mode with all the parameters including set-point, scan rate and feedback gains adjusted to optimise image quality and minimise imaging force. All force distance curves were acquired at a constant rate of 1 Hz using a cantilever deflection of 100 nm and a z-scanner ramp size of 200 nm. The cantilever deflection sensitivity was calibrated from the slope of the contact regions of the force curves after lipid bilayer breakthrough. Sets of at least 100 force curves over different areas of the sample were acquired and repeated on three different samples with three different tips to ensure reproducibility. Spring constant calibration was performed using the reference cantilever method45 and the simplified Sader method for cantilevers of arbitrary shape.46 The spring constant for all cantilevers was determined to be between 0.44 to 0.46 N.m-1. The Young’s modulus of the lipid bilayers was calculated by fitting the approach curve of the force distance curves using the Hertz model (Bruker NanoScope Analysis software, version 1.40).47

The scanners were calibrated in the x, y and z using silicon calibration grids (Bruker

model numbers PG: 1 µm pitch, 100 nm depth and VGRP: 10 µm pitch, 180 nm depth). Height images were analysed using Bruker NanoScope Analysis software (version 1.40).


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Figure 1. Characterisation of AgNPs: (a) UV-visual spectroscopy indicated spherical shape; (b) DLS indicated that more than 98% of AgNPs were 1.8 ± 0.2 nm; (c) TEM imaging indicated that the AgNPs were in average 2.8 ± 0.9 nm. Arrows indicate examples of small nanoparticles. (d) DLS of AgNPs in 100 mM NaCl over 24 h. Quadruplicate results presented. Open symbols, peak 1, closed symbols, peak 2. Diamonds, sample 1, squares, sample 2, circles, sample 3, triangles sample 4.


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Figure 2. Bode plot of tBLM before (squares), or after 24 h AgNP exposure (circles), and 9 mL of washing with 100 mM NaCl (triangles) at a bias of 0 volts. (a) 10 µL or (b) 20 µL AgNPs were added. The equivalent circuit used is shown as an inset in (a). Results shown are representative of at least three replicate experiments.


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Figure 3 AFM height images (a-c, 1x1 µm, z-scale -4.5 nm – 10 nm) and particle analysis crosssections (d-f) of the tBLM (a, d) and the tBLM membrane exposed to AgNPs at 0 h (b, e) and 24 h (c, f). Images are representative of multiple scans.


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Figure 4. AFM force curves show no change in bilayer characteristics after treatment with AgNPs. Dotted lines are approach curves, while solid lines are retract curves. AFM force curves of a pristine tBLM (a) after 24 h incubation with 20 µL AgNPs (b).

Scheme 1. Schematic representation of the strategy for investigating changes to tBLMs upon AgNP exposure


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Table 1. Resistance and capacitance values for tBLMs before and after exposure to 10 or 20 µL AgNPs. Results shown are representative of at least three replicate experiments.

Membrane Resistance (Rm) / MΩ cm2

Membrane Capacitance (Cm) / µF cm-2

Spacer Capacitance (Cs) / µF cm-2

1.0

1.8

10 µL 31 AgNPs

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20 µL 11 AgNPs

0.59

28

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31


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AUTHOR INFORMATION Corresponding Author *[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENT Flinders Analytical is acknowledged for support during the ICP-MS experiments, TEM and AFM experiments were possible by using the AMMRF facilities. Dr Christopher McDevitt, Research Centre for Infectious Diseases, University of Adelaide is acknowledged for the use of their Zetasizer instrument. Partial funding was provided by the ARC discovery grants DP110103032 and DP120100900. SUPPORTING INFORMATION AVAILABLE Supporting information is available for this manuscript. This information is available free of charge via the Internet at http://pubs.acs.org/.

ABBREVIATIONS AgNPs, silver nanoparticles; AFM, atomic force microscopy; Dynamic Light Scattering, DLS; EIS, electrical Impedance Spectroscopy; ICP-MS, inductively coupled plasma mass spectrometry; tBLM, tethered bilayer lipid membranes; TEM, transmission electron microscopy.


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