13534 DOI: 10.1021/la902039g Langmuir 2009, 25(23), 13534–13539Published on Web 09/09/2009

pubs.acs.org/Langmuir

© 2009 American Chemical Society

Liposomes Tethered to Omega-Functional PEG Brushes and Induced

Formation of PEG Brush Supported Planar Lipid Bilayers

Qiong Ye, Rupert Konradi,† Marcus Textor, and Erik Reimhult*

Swiss Federal Institute of Technology, Laboratory for Surface Science andTechnology,Wolfgang-Pauli-Strasse10, CH-8093 Z€urich, Switzerland. †Current address: BASF SA, Fine Chem & Biocatalysis Res, D-67056

Ludwigshafen, Germany.

Received June 6, 2009. Revised Manuscript Received August 18, 2009

Self-assembly of planar supported lipid bilayers on top of hydrophilic polymer brushes is a desirable alternative tosolid supported lipid bilayers and covalently tethered lipid bilayers for applications like sensing on transmembraneproteins which require a large aqueous volume between membrane and substrate. We present a simple dip-and-rinsemethod to produce poly(ethylene glycol) (PEG) brushes with sparse positively charged hydrophobic tethers, usingpoly(L-lysine)-graft-poly(ethylene glycol)-quaternary ammonium compound copolymers. The interaction of suchpolymer coatings with liposomes of different compositions and the conditions for formation of planar lipid bilayersof extraordinarily high fluidity on top of the >10 nm thick reservoir by liposome self-assembly and sequentiallytriggered rupture are investigated.

1. Introduction

The cell membranes contain a variety of components which areassociated with life-sustaining functions and medical disorders.Given the complexity of real cell membranes and the organismthey surround there is an ongoing search for simpler modelsystems where their properties and the properties of their con-stituents can be investigated under controlled conditions withquantitative biosensor techniques.

Two important examples of biomembrane model systems areunilamellar phospholipid vesicles (liposomes) and supported lipidbilayers (SLB).1 In the former, a bilayer of amphiphilic phospho-lipid molecules forms a spherical shell, separating an “intra-cellular” liquid volume from the “extra-cellular” space, whileSLBs are planar, two-dimensional, extended bilayers adsorbed ona substrate. SLBs are preferably prepared by a method pioneeredby McConnell et al.2 in which liposomes adsorb on a suitablesurface. The surface interaction induces rupture and fusion of thevesicles to a continuous planar bilayer. The method produces;when successful;solvent-free fluid lipid bilayers spanning evenmacroscopic surface areas with few defects.

For solid supportedmembranes the water reservoir between themembrane and the underlying solid substrate is typically only∼1 nm thick.3 The small resulting volumedoes not prevent insertedtransmembrane proteins from coming into contact with the under-lying substrate, and this results in pinning and loss of function.4

A few SLB platforms have been suggested to remedy thisproblem. This has so far entailed assembling so-called tetheredlipid bilayers using amono- or submonolayer of covalently boundtethered lipids with a short hydrophilic spacer of typically a few

ethylene glycol units between the lipid and the support5-9 or thickhydrophilic polymer cushions onto which membranes can bespread.10 Of these platforms the former requires rigorous surfacepreparation protocols for successful application11 and does notprovide enough space for large membrane proteins like e.g.integrins which can have hydrophilic domains extending furtherthan a couple of nanometers.4 Tanaka and co-workers made useof thicker polymer cushions predeposited on the substrate ofinterest onto which membranes could be spread.10 The techniquehas mainly been successful using cellulose polymer cushions, andon this platform a higher fraction ofmobile large transmembraneproteins has been demonstrated.4,12 Despite the promise of thissystem, it has not been implemented byother groups, whichmighttestify to a delicate process which is also difficult to implement in anonexpert laboratory.

We present here the formation of poly(ethylene glycol) (PEG)supported lipid bilayers on an easily synthesized graft copolymer,poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG). PLL-g-PEG can be self-assembled as monolayer by simple “dip-and-rinse” processing on any substrate with negative surface poten-tial.13 With a molecular weight of 2-3.4 kDa per PEG chain andoptimized grafting density, there is approximately >10 nmof hydrophilic space provided between membrane and substrate,as estimated by Spencer and co-workers from force-volumespectroscopy of similar PLL-g-PEG films,14,15 mainly depending

*To whom correspondence should be addressed. E-mail: erik.reimhult@mat.ethz.ch.(1) Sackmann, E. Science 1996, 271, 43-48.(2) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim.

Biophys. Acta 1986, 864, 95-106.(3) Bayerl, T. M.; Bloom, M. Biophys. J. 1990, 58 (2), 357-362.(4) Tanaka, M.; Sackmann, E. Nature 2005, 437 (7059), 656-663.(5) Janshoff, A.; Steinem, C. Anal. Bioanal. Chem. 2006, 385 (3), 433-451.(6) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10 (1), 197-210.(7) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew.

Chem., Int. Ed. 2003, 42 (2), 208.

(8) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D.J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14 (3), 648-659.

(9) Cheng, Y. L.; Boden, N.; Bushby, R. J.; Clarkson, S.; Evans, S. D.; Knowles,P. F.; Marsh, A.; Miles, R. E. Langmuir 1998, 14 (4), 839-844.

(10) Tanaka, M.; Rossetti, F. F.; Kaufmann, S. Biointerphases 2008, 3 (2),FA12-FA16.

(11) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.;Karcher, I.; Koper, I.; Lubben, J.; Vasilev, K.; Knoll, W. Langmuir 2003, 19 (13),5435-5443.

(12) Goennenwein, S.; Tanaka, M.; Hu, B.; Moroder, L.; Sackmann, E.Biophys. J. 2003, 85 (1), 646-655.

(13) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N. P.; Hofer, R.; Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104(14), 3298-3309.

(14) Drobek, T.; Spencer, N. D.; Heuberger, M. Macromolecules 2005, 38 (12),5254-5259.

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on how much osmotic pressure is experienced by the lipidmembrane at the slightly attractive interface.14,16 To avoid costlysynthesis of lipomimetic tethers, we investigated the effect of usinga simple quaternary ammoniumcompound (QAC) consistingof apositively charged quaternary ammonium salt with a C12H25-chain end-functionalized to the PEG as themembrane interacting

functional group (Figure 1a). QACs are thought to bemembrane-active antimicrobial compounds17-25 and could thus be used tostrongly interact with negatively charged liposome membranes atlow surface densities. Instead of direct rupture and formation ofthe SLB on a rather low surface energy interface with a highdensity of hydrophobic moieties, we choose to investigate astrategy of sequential capture of liposomes (Figure 1b) andcontrolled deformation of liposomes into a high-energy state bytuning of the number of QAC groups (Figure 1c), followed bytriggered fusion into a planar lipid bilayer (Figure 1d). Acombination of techniques is explored to use this platform as away to probe the interaction strength of ligands with differentliposomal membranes on a pure nonfouling background. Theplatform is suitable for combination with a large range ofbiosensing techniques due to the well-defined proximity to thesubstrate. Finally, we demonstrate that planar lipid bilayers canbe formed by tuning the lipid composition and number ofpresented QACs and using PEG induced fusion, as suggestedby Lentz et al.26 and Berquand et al.27 Thanks to the largerhydrophilic space and lower tether density compared to pre-viously reported tethered membranes the formed membranes areshown to possess much higher fluidity.

2. Materials and Methods

All lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho-line (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), L-R-phosphocholine (egg, chicken) (egg-PC), phosphatidylcholine-NBD (NBD-PC), cholesterol (CH),sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA)were purchased from Avanti Polar Lipids. All samples weresuspended in 10 mM HEPES (Fluka, Switzerland) and 150 mMNaCl (Fluka, Switzerland) buffer prepared from ultrapure Milli-Q water (Millipore) and adjusted to pH 7.4 using concentratedNaOH (Merck, Switzerland). Unilamellar liposomes were pre-pared by bath sonication (Branson Ultrasonics Corp.) at5 mg/mL following a protocol described previously.28 In brief,the desired lipid composition was mixed in a round-bottom flaskfrom stock solutions in chloroform. The chloroform was com-pletely removed by drying the lipids to a thin film under nitrogenflow and the lipids then resuspended in buffer for sonication at5 mg/mL. For the study of liposomes adsorbed to differentdensities of QACs, bath sonication was replaced by extrusionusing a hand-held extruder and extruding the rehydrated lipidsolution 31 times through double-stacked 100 nm pore sizepolycarbonate membranes (Avestin, Canada). Average vesiclesize was determined by dynamic light scattering (Malvern3000 Has, Malvern Instruments, UK) with the following averageintensity-weighted hydrodynamic diameters recorded for bathsonicated liposomes: POPC:POPA (98/2 mol/mol) 52 nm,POPC:POPA (70/30 mol/mol) 45 nm, POPC:POPG (80/20 mol/mol) 33nm, egg-PC:DOPE (65:35) 74nm,DOPC:DOPE:SM:CH(35/30/15/20 mol/mol/mol/mol) 170 nm, and extruded POPC:POPA (98/2mol/mol) 114 nm. Liposome solutions were used at adiluted concentration of 0.1 mg/mL.

Figure 1. Schematics of the PLL-g-PEG-QAC platform for lipo-some capture and induced rupture to form SLB. (a) Self-assemblyof poly(L-lysine)-graft-poly(ethylene glycol)-Nþ(CH3)2(C12H25)using dip and rinse. (b) Adsorption of liposomes onto PLL-g-PEG-QAC at low QAC density. (c) Increased deformation ofliposomes adsorbed onto PLL-g-PEG-QAC at higher QAC den-sity. (d) PEG(8 kDa)-induced rupture of deformed liposomes intoa polymer supported planar lipid bilayer.

(15) Pasche, S.; Textor, M.; Meagher, L.; Spencer, N. D.; Griesser, H. J.Langmuir 2005, 21 (14), 6508-6520.(16) Konradi, R.; Textor, M.; Reimhult, E. In Surface Analysis and Techniques

in Biology; Smentkowski, V. S., Ed.; Springer Science: Berlin, 2009.(17) Isquith, A. J.; Abbott, E. A.; Walters, P. A. Appl. Microbiol. 1972, 24 (6),

859-63.(18) Nakagawa, Y.; Hayashi, H.; Tawaratani, T.; Kourai, H.; Horie, T.;

Shibasaki, I. Appl. Environ. Microbiol. 1984, 47 (3), 513-18.(19) Tashiro, T. Macromol. Mater. Eng. 2001, 286 (2), 63-87.(20) McCubbin, P. J.; Forbes, E.; Gow, M. M.; Gorham, S. D. J. Appl. Polym.

Sci. 2006, 100 (1), 381-389.(21) El-Hayek, R. F.; Dye, K.; Warner, J. C. J. Biomed. Mater. Res., Part A

2006, 79A (4), 874-881.

(22) Cen, L.; Neoh, K. G.; Kang, E. T. Langmuir 2003, 19 (24), 10295-10303.(23) Murata, H.; Koepsel, R. R.; Matyjaszewski, K.; Russell, A. J. Biomaterials

2007, 28 (32), 4870-4879.(24) Lewis, K.; Klibanov, A. M. Trends Biotechnol. 2005, 23 (7), 343-348.(25) Oosterhof, J. J. H.; Buijssen, K.; Busscher, H. J.; van der Laan, B.; van der

Mei, H. C. Appl. Environ. Microbiol. 2006, 72 (5), 3673-3677.(26) Lentz, B. R.; Lee, J. K. Mol. Membr. Biol. 1999, 16 (4), 279-296.(27) Berquand, A.; Mazeran, P. E.; Pantigny, J.; Proux-Delrouyre, V.; Laval,

J. M.; Bourdillon, C. Langmuir 2003, 19 (5), 1700-1707.(28) Merz, C.; Knoll, W.; Textor, M.; Reimhult, E. Biointerphases 2008, 3 (2),

FA41-50.

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The synthesis of poly(L-lysine)(20 kDa)-graft[7.4]-poly-(ethylene glycol)(3.4 kDa)-Nþ(CH3)2(C12H25) (PLL-g-PEG-QAC) and the interaction of formed monolayers with serumproteins and bacteria will be published elsewhere.29 PLL-(20 kDa)-g[3.4]-PEG(2 kDa) was purchased from SuSoS(Switzerland). Poly(ethylene glycol) MW 8000 Da (PEG(8 kDa))was purchased from Sigma-Aldrich (Switzerland) and used at30% w/v. All sensor substrates were coated by magnetron sput-teringwith 12 and 50 nmofTiO2 onOWLSwaveguides and coverglass and QCM-D crystals, respectively. Before adsorption ofPLL-g-PEG/PLL-g-PEG-QAC to a substrate the magnetronsputtered TiO2-coated QCM crystal (Q-Sense, Sweden), OWLSwaveguide (MicroVacuum, Hungary) or cover glass was cleanedby soaking in SDS, rinsing with Milli-Q water and 30 minexposure to UV/ozone (Boekel UV Clean model 135500, BoekelIndustries).

Quartz crystal microbalance with dissipation monitoring(QCM-D)30 experiments were conducted on a Q-Sense E4 instru-ment (Q-Sense, Sweden). Measurements were carried out at50 μL/min buffer flow and 24 �C. After stabilization of thebaseline in buffer the adsorption of PLL-g-PEG-QAC would bemonitored until saturation as described below. After repeatedrinsing of the flow cell with buffer liposome solution was addedand the adsorption monitored for changes in resonant frequencyΔf and energy dissipation ΔD using overtones 3-13 (15-65MHz). The mass of the adlayer (including trapped water) isroughly proportional to the change in Δf according to theSauerbrey relation, Δm=-kΔf (k=17.7/n ng/(cm2 Hz), wheren=overtonenumber 1, 3, ...), but increasinglyunderestimating thetotal coupled mass for increasing liposome size.31

PLL-g-PEG-QAC followed by liposomes were injected usingsyringes into a liquid cell for Optical Waveguide LightmodeSpectroscopy (OWLS, MicroVacuum, Hungary)32 following theprotocol described for QCM-D. OWLS determines the adsorbedlipid mass at the interface by probing changes in the refractiveindexwithin the evanescent field at the interface of thewaveguide.The change in refractive index is related to the mass of adsorbedmolecules by deFeijter’s formula,m=dA(nA- nC)/(dn/dc), wherem is the adsorbed mass, dA the layer thickness, nA the refractiveindex of the adsorbed layer, nC the refractive index of themedium,and dn/dc the refractive index increment.33 The value of dn/dc isnot well established for lipids, and calculation of the adsorbedmass is further complicated by the large optical anisotropy of thelipidmembrane and changes in average distance of the lipids fromthe waveguide surface.28,34-37 On the basis of a survey of valuesfor lipid refractive index and density used in the literature, dn/dc=0.135 cm3/g was assumed for lipid the adsorbed lipid layers andused for relative comparisons.34 While relative comparisons areaccurate, the calculated masses should not be taken as exactestimates of the adsorbed lipid mass.

Fluorescence recovery after photobleaching (FRAP) was con-ducted using a confocal laser scanning microscope (Zeiss LSM510, Zeiss, Germany) to verify formation of planar lipid mem-branes or liposome films. 1% NBD-PC was used as fluorescentlipids with all lipid mixtures. After vesicle adsorption on a coatedcover glass in an open cell the vesicle solution was exchanged for

pure buffer. A focused circular laser pulse (488 nm, 8.9 μm indiameter, 100% intensity) was used to bleach a spot in themembrane. The fluidity of the membrane was measured by therate and percentage recovery of fluorescence intensity of thebleached spot. The calculation of the diffusion coefficient wasdone based on the evaluation ofLopez et al.38 andAxelrod et al.39

3. Results and Discussion

3.1. Formation of the PLL-g-PEG-QAC Coating. ThePEG coatings with different QAC densities were obtained bymixing functionalized PLL-g-PEG-QAC with nonfunctionalizedPLL-g-PEG at the molar ratios 10, 50, and 100%29 and exposingthe required substrate cleaned according to above to the polymermix dissolved inHEPES buffer. The incubationwasmonitored insitu for QCM-D and OWLS measurements and a saturatedmonolayer was observed to form within 10 min guided by theelectrostatic interaction of the positively charged PLL backboneand the negatively charged TiO2 coated substrate,13 using apolymer concentration of 0.1 mg/mL. The substrate was thor-oughly rinsed with buffer after 30 min incubation and a newbaseline established for QCM-D and OWLS measurements. Nosignificant mass loss was recorded during rinsing and so-preparedPLL-g-PEG-QAC-coated substrates were completely stable formeasurements up to 10 h, which was the longest time recorded.

Typical frequency and dissipation shifts obtained by QCM-Dfor the polymer adlayers wereΔf≈ 35Hz andΔD≈ 3� 10-6 formixed PLL-g-PEG-QAC/PLL-g-PEG films, andΔf≈ 40 Hz andΔD ≈ 4 � 10-6 for 100% PLL-g-PEG-QAC films. For the sameconditions the adsorbed mass was recorded as ∼145 ng/cm2 forpure PLL-g-PEG and 170 ng/cm2, using dn/dc=0.139,40 for100%PLL-g-PEG-QAC, in agreement with the higher molecularweight of the latter.3.2. Influence of QAC Density. Liposomes were first ex-

posed to the PLL-g-PEG-QAC with different densities of QACligands in order to probe the capture efficiency and the effect onliposome deformation, i.e., interaction strength. Table 1 showsthe results for extruded POPC:POPA (98/2 w/w) liposomes whenthe QAC density was varied between 10, 50, and 100% of thegrafted PEG, corresponding to an approximate surface density of0.04, 0.2, and 0.4 QACs per nm2, respectively.15,41 The surfaceQAC density was assumed to correspond to the molar ratio ofPLL-g-PEG-QAC:PLL-g-PEG in solution due to their similarmolecular weights. The ratio of change in dissipation to change infrequency (-ΔD/Δf) obtained after saturated adsorption and

Table 1. QCM-D Response with Standard Errors of the Mean for PO-

PC:POPA (98:2 w/w) Liposome Adsorption on 10% (Three Indepen-dent Measurements), 50% (Three Independent Measurements), and

100% (Two Independent Measurements) Mixtures of PLL-g-PEG-Q-

AC/PLL-g-PEGa

10% QAC 50% QAC 100% QAC

-Δf (Hz) 91 ( 15 190 ( 19 163 ( 19ΔD (10-6) 13 ( 1 20 ( 6 11 ( 2-ΔD/Δf (1/GHz) 183 ( 31 102 ( 19 66 ( 9

aLiposome average diameter is 114 ( 30 nm. The same trend ofdecreasing-ΔD/Δf ratio with increased QAC density was observed forother lipid mixtures and liposome diameters.

(29) M€oller, J.; Rismantojo, E.; Vogel, V.; Nyfeler, E.; M€uhlebach, A.;Reimhult, E.; Textor, M.; Konradi, R., manuscript in preparation.(30) Rodahl, M.; Kasemo, B. Rev. Sci. Instrum. 1996, 67, 3238-3241.(31) Reimhult, E.; Hook, F.; Kasemo, B. J. Chem. Phys. 2002, 117 (16),

7401-7404.(32) Voros, J.; Ramsden, J. J.; Csucs, G.; Szendro, I.; De Paul, S. M.; Textor,

M.; Spencer, N. D. Biomaterials 2002, 23 (17), 3699-3710.(33) de Feijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17 (7),

1759-1772.(34) Mashaghi, A.; Swann, M.; Popplewell, J.; Textor, M.; Reimhult, E. Anal.

Chem. 2008, 80, 3666-3676.(35) Reimhult, E.; Larsson, C.; Kasemo, B.; Hook, F. Anal. Chem. 2004, 76 (24),

7211-7220.(36) Reimhult, E.; Z€ach, M.; H€o€ok, F.; Kasemo, B. Langmuir 2006, 22,

3313-3319.(37) Salamon, Z.; Tollin, G. Biophys. J. 2001, 80 (3), 1557-1567.

(38) Lopez, A.; Dupou, L.; Altibelli, A.; Trotard, J.; Tocanne, J. F. Biophys. J.1988, 53 (6), 963-970.

(39) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W.Biophys. J. 1976, 16 (9), 1055-1069.

(40) Pasche, S.; Voros, J.; Griesser, H. J.; Spencer, N. D.; Textor, M. J. Phys.Chem. B 2005, 109 (37), 17545-17552.

(41) Pasche, S.; De Paul, S. M.; Voros, J.; Spencer, N. D.; Textor, M. Langmuir2003, 19 (22), 9216-9225.

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rinsing is given inTable 1.No significant desorptionwas observedafter rinsing. As observed in Table 1, -ΔD/Δf is stronglydecreasing with an increase in QAC density, while the massuptake (Δf) increases much less. The same trend was observedalso for other lipid mixtures, which varied in size, charge, andpresumably membrane stiffness. The -ΔD/Δf ratio is a measureof the softness of the adsorbed film and can hence be used as ameasure of the deformation for adsorbed liposomes. A higherdeformation would lead to more flattened and sterically con-strained liposomes which couple less water.31 The lower amountof coupled water as the liposomes are deformed and lose volumewill also reduce the amount of coupledmass per liposome and forthe entire film. At the ultimate deformation into a planar lipidbilayer the dissipation permass unit adsorbed goes toward zero.42

Thus, the strongly decreasing trend of-ΔD/ΔfwithQACdensitydemonstrates that a higher density of tethers effectively producesa more attractive surface potential for the surface and forces theliposomes to deformmore by overcoming the loss of entropy of amore deformed and rigid membrane. A similar trend of increas-ingly deformed liposomes as the liposome-substrate interactionis increased has previously been observed by dual polarizationinterferometry on fully packed liposome layers.43

The lack of desorption upon rinsing after saturated adsorptionfrom a high concentration of liposomes indicates that the maxi-mum surface loading was obtained for each polymer mixture.However, a comparison of the maximum loading reveals that10% PLL-g-PEG-QAC is not enough to adsorb a liposome layerto full coverage, since the obtained -Δf value is substantiallylower than for the higher QAC densities. This possibly also leadsto a more nonhomogenous coverage evidenced by the higherrelative error of the liposome loading. On the other hand, thedecrease in -Δf from 50% to 100% QAC density indicates thatnear full surface coverage has been obtained already at 50%QACdensity, since the total adsorbed mass decreases at 100% QACdensity. This results from the increased deformation of adsorbedliposomes and expulsion of water from the film.3.3. Influence of Quaternary Ammonium, Liposome

Charge, and Size. A comparison of adsorption of liposomeswith varied lipid composition to surfaces functionalized with

100% PLL-g-PEG-QAC is shown in Figure 2. Although thereare variations in liposome size between different lipid mixtureswhich will affect the mass uptake measured byQCM-D, it is clearthat all lipid compositions show similar uptake of highly de-formed liposomes (low -ΔD/Δf) except DOPC:DOPE:SM:CH(35:30:15:20 mol/mol), which is a lipid mixture developed byLentz et al. for optimized PEG-induced fusion between liposomesin the bulk.44 If the positive charge of the quaternary ammoniumgroups plays a significant role in the adsorption of liposomes, thiseffect would be expected to be pronounced for adsorption ofanionic, net negatively charged, liposomes, but not for zwitter-ionic, net neutral, liposomes. However, the lack of significantdifference in liposome uptake for the first four types of liposomesof increasing (0, 2, 20, and 30 mol %) anionic charge and similarsize displayed in Figure 2a and deformation led to the conclusionthat it is the total density of hydrophobic moieties at the interfaceand not the density of positively charged quaternary ammoniumwhich dominates the strength of the liposome-QAC interaction.This conclusionwas supported by very similar results at the lowerQACdensities. The observeddifferences in deformation andmassuptake, on the other hand, seem to correlate well with liposomesize as shown inFigure 2b. In fact, taking-ΔD/Δf as ameasure ofdeformation, this quantity seems to be linearly related (linearleast-squares fit withR=0.997) to the average liposome diametermeasured in the bulk solution. This is in spite of the largedifference in liposome charge and possibly also significant differ-ences in membrane elasticity especially between the two netneutral liposomes and the different charged liposomes.

While these results clearly show that the attractive interactionwith the polymer brush and the energy state of the adsorbedliposomes can be tuned by changing the QAC density (areadensity of hydrophobic moieties), in no case was spontaneousformation of an SLB on top of the PLL-g-PEG indicated byQCM-D or demonstrated by FRAP control measurements. It isinteresting to compare the deformation of the liposomes on top ofPLL-g-PEG-QAC(100%) to known cases of adsorbed liposomesthat are close to rupture. In Figure 2b is also data fromReimhultet al. for egg-PC liposomes adsorbed to TiO2 and SiO2, respec-tively, replotted as a function of liposome size.31 Liposomesadsorbed to TiO2 do not rupture even under additional, e.g.,

Figure 2. QCM-D results for saturated adsorption of bath sonicated liposomes on PLL-g-PEG-QAC(100%). (a) Frequency shifts withstandard errors of the mean (-Δf; solid squares) roughly corresponding to adsorbed mass or layer thickness and -ΔD/Δf (open circles), ameasure of liposome deformation, are plotted for the different lipid compositions ordered bymol% anionic lipids from left to right (0, 2, 20,30, and 0mol%, respectively). (b) The same data plotted as a function of liposome diameter, showing a linear scaling (linear least-squares fitwithR=0.997) of liposomedeformation,-ΔD/Δf, with liposomebulk diameter. In (b) is data also added for-ΔD/Δf for egg-PC liposomesadsorbed to TiO2 (solid stars) and to SiO2 at the verge of rupture (crosses) as a function of size obtained from Reimhult et al.31

(42) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397-1402.(43) Khan, T. R.; Grandin, H. M.; Mashaghi, A.; Textor, M.; Reimhult, E.;

Reviakine, I. Biointerphases 2008, 3 (2), FA90-FA95.(44) Haque, M. E.; McIntosh, T. J.; Lentz, B. R. Biochemistry 2001, 40 (14),

4340-4348.

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osmotic, stress and are very similarly deformed to those adsorbedto the PLL-g-PEG-QAC(100%). The deformation expressed as alower -ΔD/Δf is, on the other hand, significantly higher forliposomes adsorbing to SiO2 right before they rupture and forman SLB than they are for any of the corresponding sizes ofliposomes on PLL-g-PEG-QAC(100%).

The scaling of-ΔD/Δfwith liposome size almost regardless ofmembrane composition but not with surface attractive potentialfor the systems under study here also demonstrates that thismeasure of deformation can be applied to liposomes of the samesize to study surface-induced deformation. But in order tocompare the effect on liposomes of different size, normalizationwith respect to size should be performed, and the presented datacompared to Reimhult et al.31 suggest that this normalization isapproximately linear with liposome diameter in solution in therelevant size range.3.4. Induced Formation of Tethered Supported Lipid

Bilayer. An additional trigger step to induce liposome rupturewas added since even 100% QAC density was not sufficient toinduce the high deformation of the liposomes necessary to burstthem to form a laterally connected planar lipid bilayer. In bulksolutions addition of PEG of low molecular weight is known tocause fusion of vesicles,44 and the addition of low molecularweight PEG has also recently been applied to cause liposomerupture and formation of planar SLBs on solid substrates.27,45

Thus, PEG(8 kDa) was added at concentrations up to 30% w/v.In contrast to what has been reported by Bourdillon and co-workers, we did not observe rupture of liposomes for most of thedifferent lipid compositions in either QCM-D, OWLS, or FRAPexperiments. The same was true for these liposomes tethereddirectly to BSA-streptavidin-coated surfaces by incorporation ofbiotinylated lipids in control experiments following the protocolof Bourdillon using e.g. the egg-PC:DOPE mixture (experimentsnot shown)27 and also used by Taylor et al. in microfluidicchannels.45 The exception was a lipid composition of DOPC:DOPE:SM:CH (35:30:15:20mol/mol/mol/mol) chosen because ithas been claimed to be an optimized mixture for PEG-inducedfusion.44 These liposomes, despite not revealing a much higherthan expected deformation for its size (Figure 2b), showed apronounced mass loss upon addition of PEG(8 kDa) in OWLSmeasurements (Figure 3). While the initial adsorbed mass iscommensurate with a liposome layer and too high for an SLB,the lipid mass observed after the PEG adsorption is stable at avalue within 15% of that expected of an SLB, as compared tovalues from previously published measurements if the sameassumptions for lipid dn/dc is used.34,35,46 No change in adsorbedmass was observed for any other lipid composition when sub-jected to PEG injection. They all remained at a constant high lipidmass corresponding to a liposome monolayer.

The formation of a laterally continuous lipid bilayer in case ofthe DOPC:DOPE:SM:CH liposomes exposed to PEG(8 kDa)was verified by FRAP (Figure 4) with a recovered fraction afterphotobleaching of 98 ( 3%. The FRAP experiment verified theformation of a PEG brush supported lipid bilayer with anextraordinarily high diffusion coefficient of 10 ( 2 μm2/s. Thediffusion coefficient is ∼5 times higher than in control experi-ments for supported lipid bilayers of the same lipid compositionformed onUV/ozone-cleaned glass by liposome fusion (2 μm2/s),which were in agreement with typical values for supported lipidbilayers formed from PC lipid mixtures.47 Diffusion coefficients

as high as 19 μm2/s of PC lipids freely diffusing in liposomes havebeen recorded,48 which demonstrates that the proximity of the

Figure 3. OWLS measurement of adsorption and PEG-inducedfusion of fusogenic tethered liposome. OWLS measurement ofadsorption of DOPC:DOPE:SM:CH (35/30/15/20 mol/mol/mol/mol) liposomes onto PLL-g-PEG-QAC(100%), injected at t =0min. An additional injection at t=37min followed by rinsing ofthe cell at t = 83 min ensure a complete coverage and no freeliposomes in solution. PEG(8 kDa) 30% w/v in buffer is injectedinto the chamber at t = 100 min and rinsed at t = 110 min. Themass loss whichwas only observed for this lipidmixture resulted inamass similar towhat is observed for a supported lipid bilayer andindicates that rupture and formation of an SLB took place.

Figure 4. FRAPmeasurements on fusogenic liposome layers beforeand after PEG-induced fusion. FRAP experiments of fusion/rupturetriggered by PEG(8 kDa) 30%w/v for adsorbedDOPC:DOPE:SM:CH:NBD-PC(34:30:15:20:1 mol/mol/mol/mol/mol) lipid vesicles onPLL-g-PEG-QAC(100%). (a) Before PEG injection (the same resultwas observed for all other lipid compositions both before and afterPEG injection) and (b) after PEG injection. (c) The recovery curvefor the data shown in (b). The recovery after the PEG injection iscomplete and with a high diffusion coefficient ofD=10( 2 μm2/s.

(45) Taylor, J. D.; Phillips, K. S.; Cheng, Q. Lab Chip 2007, 7 (7), 927-930.(46) Richter, R. P.; Berat, R.; Brisson, A. R. Langmuir 2006, 22 (8), 3497-3505.(47) Lee, G. M.; Jacobson, K. In Cell Lipids; Academic Press: San Diego, 1994;

Vol. 40, pp 111-142. (48) Gaede, H. C.; Gawrisch, K. Biophys. J. 2003, 85 (3), 1734-1740.

DOI: 10.1021/la902039g 13539Langmuir 2009, 25(23), 13534–13539

Ye et al. Article

substrate severely reduces the diffusion of lipids within themembrane. The diffusion has however not been much higherfor tethered lipid bilayers aimed to decouple the membrane fromthe substrate where the density of tethers is high and the separa-tion from the substrate is small. While often not measured sincetethered lipid bilayers are typically created on fluorophorequenching gold substrates, lipid monolayers on hydrophobicSAMs have a diffusion coefficient an order of magnitude lower(∼0.2 μm2/s) than solid supported lipid bilayers on glass (∼2 μm2/s).4,49-51 However, a substantial decoupling distance of the SLBfrom the substrate without covalent tethers have been shown toresult in significantly higher diffusion coefficients and also toallow for insertion of large transmembrane proteins with retainedlateral fluidity.4,12,51-54 It should be mentioned that in a fewinstances also very large aggregates could be observed as high-intensity fluorescence at a few spots on the microscopy slidesurface. These could be the result of many liposomes fusing to alarge liposome instead of into the planar bilayer and either restingon themembrane surface due to difficulty with rinsing or tetheredto the PLL-g-PEG-QAC.

That the lipid composition with the highest -ΔD/Δf ratio foradsorbed liposome layers results in the only reliably producedPEGbrush supported lipid bilayers byPEG-induced fusionmightat first look like a contradiction given the reasonable hypothesisthat the higher the deformation of adsorbed liposomes, the morelikely they are to rupture and fuse.31,43,55 However, as shown inFigure 2b, the high absolute values for ΔD and Δf for theadsorbed DOPC:DOPE:SM:CH liposomes is a reflection of thatthese liposomes are on average substantially larger than the otherliposomes that were tested. It has previously been shown thatvesicle size for small and large unilamellar liposomes does notsignificantly affect rupture kinetics on for example SiO2, and thusthis size difference is not likely to be responsible for the increasedpropensity for rupture and fusion.31,56,57 An alternative explana-tion could be that the high -ΔD/Δf reflects that these liposomesare so fusogenic that they partially start fusing into larger

liposomes when deformed on the surface, a process previouslyobserved for liposomes adsorbed for example on mica.58 Suchfusion could relax deformations, increasewater coupling and leadto higher absolute ΔD, Δf, and ΔD/Δf. A further indication thatsuch fusionmight occur is that despite that a similar lipidmass perarea is expected regardless of size for OWLS measurements, theadsorbedmass is∼30% higher for these liposomes than for othercompositions (on average 730 ng/cm2 compared to 560 ng/cm2).However, the empirical trend in Figure 2b seems to fit well to theadsorption of a liposomal layer of the nominal bulk liposome size,and surface-induced fusion of the adsorbed liposomes is thusunlikely. In summary, the likely reason for the observed increasedpropensity of these liposomes to fusewhen exposed to free PEG insolution is the special lipid headgroup composition, since themechanical properties, as determined by the membrane deforma-tion exemplified in Figure 2b, are not significantly different to theother compositions.

In summary, we have shown that planar lipid bilayers withsubstantially improved lateral fluidity can be formed by a self-assembly process in three steps: (i) “dip-and-rinse” self-assemblyof a graft copolymer monolayer brushes applicable to anynegatively charged substrate; (ii) liposome adsorption to PEG-tethered quaternary ammonium compound functional groups;and (iii) fusion induced by free PEG(8 kDa) in solution. Thefluidity of the so-formed SLB was a factor of 5 higher than forsolid supported lipid bilayers and approaching that of freelydiffusing lipids in liposomes. The system was also shown tosensitively detect increased liposome deformation as a functionof tether density byQCM-D,which indicates the possibility to usethe PLL-g-PEG-functional linker coatingswithQCM-D to probethe nature and strength of membrane interacting functionalgroups by their effect on the adsorption kinetics and state ofadsorbed liposomes. The layer-by-layer “dip and rinse” buildupof the platform requiring no complicated preparative steps canpossibly be generalized as a facile platform for surface functio-nalization with biologically mimicking membranes which caninclude transmembrane proteins requiring substantial distanceand decoupling from the surface afforded by the water-rich PEGbrush spacer.

Acknowledgment. Swiss Competence Center for MaterialsResearch (CCMX) and BASF SA, Erich Nyfeler, and AndreasM€uhlebach are acknowledged for financial and material support.We especially thank Prof. Dr. Janos V€or€os for the kind access tothe Laboratory of Biosensors and Bioelectronics at ETH Z€urichandMarta Bally andDoroth�ee Grieshaber for providing trainingand advice for the FRAP and QCM-D measurements.

(49) Shen, W. W.; Boxer, S. G.; Knoll, W.; Frank, C. W. Biomacromolecules2001, 2 (1), 70-79.(50) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103 (2),

307-316.(51) Kaufmann, S.; Papastavrou, G.; Kumar, K.; Textor,M.; Reimhult, E. Soft

Matter 2009, 5 (14), 2804-2814.(52) Kunding, A.; Stamou, D. J. Am. Chem. Soc. 2006, 128 (35), 11328-

11329.(53) Albertorio, F.; Diaz, A. J.; Yang, T. L.; Chapa, V. A.; Kataoka, S.;

Castellana, E. T.; Cremer, P. S. Langmuir 2005, 21 (16), 7476-7482.(54) Diaz, A. J.; Albertorio, F.; Daniel, S.; Cremer, P. S. Langmuir 2008, 24 (13),

6820-6826.(55) Seifert, U. Adv. Phys. 1997, 46 (1), 13-137.(56) Reimhult, E.; Hook, F.; Kasemo, B. Langmuir 2003, 19 (5), 1681-1691.(57) Johnson, J. M.; Ha, T.; Chu, S.; Boxer, S. G. Biophys. J. 2002, 83 (6),

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