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  • Single Plasmonic Nanoparticle Tracking Studies of Solid SupportedBilayers with Ganglioside LipidsLaura B. Sagle, Laura K. Ruvuna, Julia M. Bingham, Chunming Liu, Paul S. Cremer,

    and Richard P. Van Duyne*,

    Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United StatesDepartment of Chemistry, Texas A&M University, College Station, Texas 77843, United StatesDepartment of Chemistry, Saint Xavier University, Chicago, Illinois 60655, United States

    *S Supporting Information

    ABSTRACT: Single-particle tracking experiments were car-ried out with gold nanoparticle-labeled solid supported lipidbilayers (SLBs) containing increasing concentrations ofganglioside (GM1). The negatively charged nanoparticleselectrostatically associate with a small percentage of positivelycharged lipids (ethyl phosphatidylcholine) in the bilayers. Thesamples containing no GM1 show random diffusion in 92% ofthe particles examined with a diffusion constant of 4.3(4.5) 109 cm2/s. In contrast, samples containing 14% GM1 showeda mixture of particles displaying both random and confined diffusion, with the majority of particles, 62%, showing confineddiffusion. Control experiments support the notion that the nanoparticles are not associating with the GM1 moieties but insteadmost likely confined to regions in between the GM1 clusters. Analysis of the root-mean-squared displacement plots for all of thedata reveals decreasing trends in the confined diffusion constant and diameter of the confining region versus increasing GM1concentration. In addition, a linearly decreasing trend is observed for the percentage of randomly diffusing particles versus GM1concentration, which offers a simple, direct way to measure the percolation threshold for this system, which has not previouslybeen measured. The percolation threshold is found to be 22% GM1 and the confining diameter at the percolation threshold only50 nm.

    INTRODUCTIONSingle-particle tracking experiments utilizing plasmonic nano-particles have gained popularity in recent years for studyingbiological phenomena ranging from the motion of motorproteins to membrane fusion in live cells.1,2 Unlike fluorescencedyes, plasmonic nanoparticles have large scattering crosssections and do not photobleach or blink, which allows formeasurements over long time periods.3 Single-particle trackinghas proven particularly useful for studying cellular membranes,since it enables direct characterization of the type of motion ofdifferent components in the membrane.4,5 For example, single-particle tracking of lipids in cellular membranes have greatlyincreased our understanding of the steps involved in membranefusion as well as lipidcytoskeleton interactions.6,7 In addition,single-particle tracking has been used to study proteinproteininteractions in membrane environments.8 These measurementshave allowed for a better understanding of the compartmen-talization of proteins in the cell membrane.9,10

    One of the more prominent ganglioside lipids present in theplasma membranes of mammalian cells, GM1 is a receptor forboth cholera toxin and Escherichia coli heat-labile enter-otoxin.11,12 The binding of cholera toxin involves all five Bsubunits of the protein binding GM1 and has been extensivelystudied as a multivalent binding system in cellular mem-

    branes.1315 Interestingly, unlike other multivalent bindingsystems in which an increase in binding strength is observed(the Kd value is smaller) with an increase in ligandconcentration,16 the cholera toxinGM1 system shows theopposite effect. Mainly, the binding of cholera toxin actuallydecreases with increasing GM1 concentration.

    17,18 A systematicstudy of cholera toxin binding to GM1 was recently carried outusing solid supported lipid bilayers (SLBs), a cell membranemimetic.19 A model was introduced in which the higher GM1concentrations induce clustering of the gangliosides in thebilayer. This clustering of GM1 within the SLBs is believed toinhibit the binding of cholera toxin. Additional studies usingAFM and fluorescence imaging also lend substantial weight tothe idea of GM1 clustering.


    In addition to the recruitment of bacterial and viral toxins,GM1 has also been implicated as one of the key componentspresent in lipid rafts domains of cellular membranes, whichhave a profound influence on cell signaling and trafficking.2426

    Clusters rich in GM1 that form in membranes are oftenmodeled as liquid ordered phases, with substantially reducedmobility of their constituent lipids.27 In addition, the clusters

    Received: June 4, 2012Published: August 31, 2012


    2012 American Chemical Society 15832 | J. Am. Chem. Soc. 2012, 134, 1583215839

  • promote lipid segregation, with little exchange of lipids betweenphases.28 Thus, increasing the GM1 concentration in amembrane will eventually lead to a percolation threshold, inwhich the GM1 clusters are the major component and the fluidphase becomes discontinuous. This has a significant effect onthe cells ability to interact and respond to its environment,since protein components in the fluid part of the membrane arethen isolated from each other. Phase behavior of many differenttypes of mixed lipid bilayer systems, such as the liquid/gelphase DMPC/DSPC,29 liquid/solid phase DPPC/LigGalCer,30

    and DMPC/cholesterol31,32 have been carried out usingtechniques such as NMR, ESR, and DSC. Recently,fluorescence recovery after photobleaching (FRAP) has beenused to study the connectivity of different components in amixed lipid bilayer and provided a measurement of thepercolation threshold of these systems.33,34 Unfortunately,trends observed using FRAP are often a result of manyunderlying mechanisms and further modeling is often requiredto understand the percolation behavior of the system.Alternatively, a more direct way of measuring a percolationthreshold in a mixed lipid system is to carry out single-particletracking studies, in which the molecule being tracked is onlysoluble in one component of the mixture.Herein, we revisit fundamental questions concerning the

    percolation threshold in mixed lipid bilayers containingincreasing concentrations of GM1 using a single nanoparticletracking system which can simultaneously measure the positionof the nanoparticle (with 7 nm precision) as well as theplasmonic spectrum. To our knowledge, a fundamental study ofpercolation and phase behavior of bilayers containing GM1using single-particle tracking has not been carried out. TheSLBs used here are composed of 0.1% ethyl phosphatidylcho-line (EPC), a positively charged lipid that attracts the negativelycharged citrate-capped gold nanoparticles. Since particletracking experiments are able to characterize the type ofmotion present, it was expected that GM1 clustering wouldproduce differences in the observed diffusion of these positivelycharged lipids. In agreement with the GM1 clustering model,most of the particles observed in the SLBs containing more

    than 5% GM1 showed confined motion, in stark contrast to theparticles examined in SLBs with no GM1. As depicted in Figure1, the data presented herein indicate that the motion of thelipids in bilayers containing >5% GM1 is confined by the GM1clusters themselves. A plot of percent confined particles vs GM1concentration shows a linear trend and offers a directmeasurement of percolation threshold. This percolationthreshold is defined as the GM1 concentration needed tohave 100% confined particles. A plot of percent confinedparticles vs GM1 concentration shows this value at 22%. Inaddition, a linear trend in the diameter of the confining regionvs GM1 concentration yields a diameter of only 50 nm at thepercolation threshold, which is 25 times the size of a typicalmembrane protein. Lastly, a decreasing trend observed for thediffusion constants of the confined particles with increasingGM1 concentration agrees with the notion that diffusiondecreases at barriers to the GM1 clusters.

    EXPERIMENTAL METHODSSmall Unilamellar Vesicles and Gold Nanoparticle-Labeled

    Solid Supported Bilayers. Small unilamellar vesicles (SUVs) weremade from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC), 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-amino]dodecanoyl)-sn-glycero-3-phosphocholine (NBDPE), and 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (EPC), which were allpurchased from Avanti Polar Lipids (Alabaster, AL), and gangliosideGM1, which was purchased from Matreya LLC (Gap, PA). SUVs wereprepared by vesicle extrusion, which has been described in detailelsewhere.35,36 Briefly, this process involved the evaporation of a lipid-chloroform solution under a stream of nitrogen followed by vacuumdesiccation for 4 h. The lipids were then rehydrated in phosphatebuffered saline (PBS) solution, which consisted of 10 mM sodiumphosphate, 150 mM NaCl, and 0.2 mM sodium azide. The pH of thePBS solution was adjusted to 7.4 with small amounts of 1 M HCl. Thetotal concentration of the lipids in solution was 1.0 mg/mL. Afterseveral freezethaw cycles, the solutions were extruded through apolycarbonate filter (Whatman) with 100 nm pores. The size of theresulting SUVs was confirmed to be 7080 nm by dynamic lightscattering using a Brookhaven Instruments 90Plus particle sizeanalyzer.

    Figure 1. Schematic of how nanoparticle movement changes in bilayers composed of no GM1 lipids (A) vs bilayers containing >5% GM1 lipids (B).Nanoparticles electrostatically bound to bilayers containing >5% GM1 display confined motion that is restricted to the distance between GM1clusters. The bilayers are composed of phosphocholine (POPC), ethyl phosphatidylcholine (EPC), nitrobenzoxadiazole phosphoserine (NBDPE),and ganglioside (GM1). For simplicity, GM1 molecules in the lower leaflet are not drawn.

    Journal of the American


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