Liposomes Tethered to Omega-Functional PEG Brushes and Induced

(4) Tanaka and co-workers made use of thicker polymer cushions predeposited on the substrate of interest onto which membranes could be spread.(10) The...
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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 and Technology, Wolfgang-Pauli-Strasse 10, 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 to solid supported lipid bilayers and covalently tethered lipid bilayers for applications like sensing on transmembrane proteins which require a large aqueous volume between membrane and substrate. We present a simple dip-and-rinse method to produce poly(ethylene glycol) (PEG) brushes with sparse positively charged hydrophobic tethers, using poly(L-lysine)-graft-poly(ethylene glycol)-quaternary ammonium compound copolymers. The interaction of such polymer coatings with liposomes of different compositions and the conditions for formation of planar lipid bilayers of extraordinarily high fluidity on top of the >10 nm thick reservoir by liposome self-assembly and sequentially triggered rupture are investigated.

1. Introduction The cell membranes contain a variety of components which are associated with life-sustaining functions and medical disorders. Given the complexity of real cell membranes and the organism they surround there is an ongoing search for simpler model systems where their properties and the properties of their constituents can be investigated under controlled conditions with quantitative biosensor techniques. Two important examples of biomembrane model systems are unilamellar phospholipid vesicles (liposomes) and supported lipid bilayers (SLB).1 In the former, a bilayer of amphiphilic phospholipid molecules forms a spherical shell, separating an “intracellular” liquid volume from the “extra-cellular” space, while SLBs are planar, two-dimensional, extended bilayers adsorbed on a substrate. SLBs are preferably prepared by a method pioneered by McConnell et al.2 in which liposomes adsorb on a suitable surface. The surface interaction induces rupture and fusion of the vesicles to a continuous planar bilayer. The method produces; when successful;solvent-free fluid lipid bilayers spanning even macroscopic surface areas with few defects. For solid supported membranes the water reservoir between the membrane and the underlying solid substrate is typically only ∼1 nm thick.3 The small resulting volume does not prevent inserted transmembrane proteins from coming into contact with the underlying substrate, and this results in pinning and loss of function.4 A few SLB platforms have been suggested to remedy this problem. This has so far entailed assembling so-called tethered lipid bilayers using a mono- or submonolayer of covalently bound tethered lipids with a short hydrophilic spacer of typically a few *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.

13534 DOI: 10.1021/la902039g

ethylene glycol units between the lipid and the support5-9 or thick hydrophilic polymer cushions onto which membranes can be spread.10 Of these platforms the former requires rigorous surface preparation protocols for successful application11 and does not provide enough space for large membrane proteins like e.g. integrins which can have hydrophilic domains extending further than a couple of nanometers.4 Tanaka and co-workers made use of thicker polymer cushions predeposited on the substrate of interest onto which membranes could be spread.10 The technique has mainly been successful using cellulose polymer cushions, and on this platform a higher fraction of mobile large transmembrane proteins has been demonstrated.4,12 Despite the promise of this system, it has not been implemented by other groups, which might testify to a delicate process which is also difficult to implement in a nonexpert 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-gPEG can be self-assembled as monolayer by simple “dip-andrinse” processing on any substrate with negative surface potential.13 With a molecular weight of 2-3.4 kDa per PEG chain and optimized grafting density, there is approximately >10 nm of hydrophilic space provided between membrane and substrate, as estimated by Spencer and co-workers from force-volume spectroscopy of similar PLL-g-PEG films,14,15 mainly depending (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.; RuizTaylor, 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.

Published on Web 09/09/2009

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functional group (Figure 1a). QACs are thought to be membraneactive antimicrobial compounds17-25 and could thus be used to strongly interact with negatively charged liposome membranes at low surface densities. Instead of direct rupture and formation of the SLB on a rather low surface energy interface with a high density of hydrophobic moieties, we choose to investigate a strategy of sequential capture of liposomes (Figure 1b) and controlled deformation of liposomes into a high-energy state by tuning of the number of QAC groups (Figure 1c), followed by triggered fusion into a planar lipid bilayer (Figure 1d). A combination of techniques is explored to use this platform as a way to probe the interaction strength of ligands with different liposomal membranes on a pure nonfouling background. The platform is suitable for combination with a large range of biosensing techniques due to the well-defined proximity to the substrate. Finally, we demonstrate that planar lipid bilayers can be formed by tuning the lipid composition and number of presented QACs and using PEG induced fusion, as suggested by Lentz et al.26 and Berquand et al.27 Thanks to the larger hydrophilic space and lower tether density compared to previously reported tethered membranes the formed membranes are shown to possess much higher fluidity.

2. Materials and Methods

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

on how much osmotic pressure is experienced by the lipid membrane at the slightly attractive interface.14,16 To avoid costly synthesis of lipomimetic tethers, we investigated the effect of using a simple quaternary ammonium compound (QAC) consisting of a positively charged quaternary ammonium salt with a C12H25chain end-functionalized to the PEG as the membrane interacting (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.

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All lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1glycerol)] (POPG), L-R-phosphocholine (egg, chicken) (eggPC), 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 were suspended in 10 mM HEPES (Fluka, Switzerland) and 150 mM NaCl (Fluka, Switzerland) buffer prepared from ultrapure MilliQ water (Millipore) and adjusted to pH 7.4 using concentrated NaOH (Merck, Switzerland). Unilamellar liposomes were prepared by bath sonication (Branson Ultrasonics Corp.) at 5 mg/mL following a protocol described previously.28 In brief, the desired lipid composition was mixed in a round-bottom flask from stock solutions in chloroform. The chloroform was completely removed by drying the lipids to a thin film under nitrogen flow and the lipids then resuspended in buffer for sonication at 5 mg/mL. For the study of liposomes adsorbed to different densities of QACs, bath sonication was replaced by extrusion using a hand-held extruder and extruding the rehydrated lipid solution 31 times through double-stacked 100 nm pore size polycarbonate membranes (Avestin, Canada). Average vesicle size was determined by dynamic light scattering (Malvern 3000 Has, Malvern Instruments, UK) with the following average intensity-weighted hydrodynamic diameters recorded for bath sonicated liposomes: POPC:POPA (98/2 mol/mol) 52 nm, POPC:POPA (70/30 mol/mol) 45 nm, POPC:POPG (80/20 mol/ mol) 33 nm, egg-PC:DOPE (65:35) 74 nm, DOPC:DOPE:SM:CH (35/30/15/20 mol/mol/mol/mol) 170 nm, and extruded POPC: POPA (98/2 mol/mol) 114 nm. Liposome solutions were used at a diluted concentration of 0.1 mg/mL.

(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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Article The synthesis of poly(L-lysine)(20 kDa)-graft[7.4]-poly(ethylene glycol)(3.4 kDa)-Nþ(CH3)2(C12H25) (PLL-g-PEGQAC) and the interaction of formed monolayers with serum proteins 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 at 30% w/v. All sensor substrates were coated by magnetron sputtering with 12 and 50 nm of TiO2 on OWLS waveguides and cover glass and QCM-D crystals, respectively. Before adsorption of PLL-g-PEG/PLL-g-PEG-QAC to a substrate the magnetron sputtered TiO2-coated QCM crystal (Q-Sense, Sweden), OWLS waveguide (MicroVacuum, Hungary) or cover glass was cleaned by soaking in SDS, rinsing with Milli-Q water and 30 min exposure to UV/ozone (Boekel UV Clean model 135500, Boekel Industries). Quartz crystal microbalance with dissipation monitoring (QCM-D)30 experiments were conducted on a Q-Sense E4 instrument (Q-Sense, Sweden). Measurements were carried out at 50 μL/min buffer flow and 24 °C. After stabilization of the baseline in buffer the adsorption of PLL-g-PEG-QAC would be monitored until saturation as described below. After repeated rinsing of the flow cell with buffer liposome solution was added and the adsorption monitored for changes in resonant frequency Δf and energy dissipation ΔD using overtones 3-13 (15-65 MHz). The mass of the adlayer (including trapped water) is roughly proportional to the change in Δf according to the Sauerbrey relation, Δm=-kΔf (k=17.7/n ng/(cm2 Hz), where n=overtone number 1, 3, ...), but increasingly underestimating the total coupled mass for increasing liposome size.31 PLL-g-PEG-QAC followed by liposomes were injected using syringes into a liquid cell for Optical Waveguide Lightmode Spectroscopy (OWLS, MicroVacuum, Hungary)32 following the protocol described for QCM-D. OWLS determines the adsorbed lipid mass at the interface by probing changes in the refractive index within the evanescent field at the interface of the waveguide. The change in refractive index is related to the mass of adsorbed molecules by de Feijter’s formula, m=dA(nA - nC)/(dn/dc), where m is the adsorbed mass, dA the layer thickness, nA the refractive index of the adsorbed layer, nC the refractive index of the medium, and dn/dc the refractive index increment.33 The value of dn/dc is not well established for lipids, and calculation of the adsorbed mass is further complicated by the large optical anisotropy of the lipid membrane and changes in average distance of the lipids from the waveguide surface.28,34-37 On the basis of a survey of values for lipid refractive index and density used in the literature, dn/dc= 0.135 cm3/g was assumed for lipid the adsorbed lipid layers and used for relative comparisons.34 While relative comparisons are accurate, the calculated masses should not be taken as exact estimates of the adsorbed lipid mass. Fluorescence recovery after photobleaching (FRAP) was conducted using a confocal laser scanning microscope (Zeiss LSM 510, Zeiss, Germany) to verify formation of planar lipid membranes or liposome films. 1% NBD-PC was used as fluorescent lipids with all lipid mixtures. After vesicle adsorption on a coated cover glass in an open cell the vesicle solution was exchanged for (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€oo€k, F.; Kasemo, B. Langmuir 2006, 22, 3313-3319. (37) Salamon, Z.; Tollin, G. Biophys. J. 2001, 80 (3), 1557-1567.

13536 DOI: 10.1021/la902039g

Ye et al. Table 1. QCM-D Response with Standard Errors of the Mean for POPC:POPA (98:2 w/w) Liposome Adsorption on 10% (Three Independent Measurements), 50% (Three Independent Measurements), and 100% (Two Independent Measurements) Mixtures of PLL-g-PEG-QAC/PLL-g-PEGa 10% QAC

50% QAC

100% QAC

-Δf (Hz) 91 ( 15 190 ( 19 163 ( 19 13 ( 1 20 ( 6 11 ( 2 ΔD (10-6) -ΔD/Δf (1/GHz) 183 ( 31 102 ( 19 66 ( 9 a Liposome average diameter is 114 ( 30 nm. The same trend of decreasing -ΔD/Δf ratio with increased QAC density was observed for other lipid mixtures and liposome diameters.

pure buffer. A focused circular laser pulse (488 nm, 8.9 μm in diameter, 100% intensity) was used to bleach a spot in the membrane. The fluidity of the membrane was measured by the rate and percentage recovery of fluorescence intensity of the bleached spot. The calculation of the diffusion coefficient was done based on the evaluation of Lopez et al.38 and Axelrod et al.39

3. Results and Discussion 3.1. Formation of the PLL-g-PEG-QAC Coating. The PEG coatings with different QAC densities were obtained by mixing functionalized PLL-g-PEG-QAC with nonfunctionalized PLL-g-PEG at the molar ratios 10, 50, and 100%29 and exposing the required substrate cleaned according to above to the polymer mix dissolved in HEPES buffer. The incubation was monitored in situ for QCM-D and OWLS measurements and a saturated monolayer was observed to form within 10 min guided by the electrostatic interaction of the positively charged PLL backbone and the negatively charged TiO2 coated substrate,13 using a polymer concentration of 0.1 mg/mL. The substrate was thoroughly rinsed with buffer after 30 min incubation and a new baseline established for QCM-D and OWLS measurements. No significant mass loss was recorded during rinsing and so-prepared PLL-g-PEG-QAC-coated substrates were completely stable for measurements up to 10 h, which was the longest time recorded. Typical frequency and dissipation shifts obtained by QCM-D for the polymer adlayers were Δf ≈ 35 Hz and ΔD ≈ 3  10-6 for mixed PLL-g-PEG-QAC/PLL-g-PEG films, and Δf ≈ 40 Hz and ΔD ≈ 4  10-6 for 100% PLL-g-PEG-QAC films. For the same conditions the adsorbed mass was recorded as ∼145 ng/cm2 for pure PLL-g-PEG and 170 ng/cm2, using dn/dc = 0.139,40 for 100% PLL-g-PEG-QAC, in agreement with the higher molecular weight of the latter. 3.2. Influence of QAC Density. Liposomes were first exposed to the PLL-g-PEG-QAC with different densities of QAC ligands in order to probe the capture efficiency and the effect on liposome deformation, i.e., interaction strength. Table 1 shows the results for extruded POPC:POPA (98/2 w/w) liposomes when the QAC density was varied between 10, 50, and 100% of the grafted PEG, corresponding to an approximate surface density of 0.04, 0.2, and 0.4 QACs per nm2, respectively.15,41 The surface QAC density was assumed to correspond to the molar ratio of PLL-g-PEG-QAC:PLL-g-PEG in solution due to their similar molecular weights. The ratio of change in dissipation to change in frequency (-ΔD/Δf) obtained after saturated adsorption and (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. Langmuir 2003, 19 (22), 9216-9225.

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

rinsing is given in Table 1. No significant desorption was observed after rinsing. As observed in Table 1, -ΔD/Δf is strongly decreasing with an increase in QAC density, while the mass uptake (Δf) increases much less. The same trend was observed also for other lipid mixtures, which varied in size, charge, and presumably membrane stiffness. The -ΔD/Δf ratio is a measure of the softness of the adsorbed film and can hence be used as a measure of the deformation for adsorbed liposomes. A higher deformation would lead to more flattened and sterically constrained liposomes which couple less water.31 The lower amount of coupled water as the liposomes are deformed and lose volume will also reduce the amount of coupled mass per liposome and for the entire film. At the ultimate deformation into a planar lipid bilayer the dissipation per mass unit adsorbed goes toward zero.42 Thus, the strongly decreasing trend of -ΔD/Δf with QAC density demonstrates that a higher density of tethers effectively produces a more attractive surface potential for the surface and forces the liposomes to deform more by overcoming the loss of entropy of a more deformed and rigid membrane. A similar trend of increasingly deformed liposomes as the liposome-substrate interaction is increased has previously been observed by dual polarization interferometry on fully packed liposome layers.43 The lack of desorption upon rinsing after saturated adsorption from a high concentration of liposomes indicates that the maximum surface loading was obtained for each polymer mixture. However, a comparison of the maximum loading reveals that 10% PLL-g-PEG-QAC is not enough to adsorb a liposome layer to full coverage, since the obtained -Δf value is substantially lower than for the higher QAC densities. This possibly also leads to a more nonhomogenous coverage evidenced by the higher relative error of the liposome loading. On the other hand, the decrease in -Δf from 50% to 100% QAC density indicates that near full surface coverage has been obtained already at 50% QAC density, since the total adsorbed mass decreases at 100% QAC density. This results from the increased deformation of adsorbed liposomes and expulsion of water from the film. 3.3. Influence of Quaternary Ammonium, Liposome Charge, and Size. A comparison of adsorption of liposomes with varied lipid composition to surfaces functionalized with (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.

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100% PLL-g-PEG-QAC is shown in Figure 2. Although there are variations in liposome size between different lipid mixtures which will affect the mass uptake measured by QCM-D, it is clear that all lipid compositions show similar uptake of highly deformed liposomes (low -ΔD/Δf) except DOPC:DOPE:SM:CH (35:30:15:20 mol/mol), which is a lipid mixture developed by Lentz et al. for optimized PEG-induced fusion between liposomes in the bulk.44 If the positive charge of the quaternary ammonium groups plays a significant role in the adsorption of liposomes, this effect would be expected to be pronounced for adsorption of anionic, net negatively charged, liposomes, but not for zwitterionic, net neutral, liposomes. However, the lack of significant difference in liposome uptake for the first four types of liposomes of increasing (0, 2, 20, and 30 mol %) anionic charge and similar size displayed in Figure 2a and deformation led to the conclusion that it is the total density of hydrophobic moieties at the interface and not the density of positively charged quaternary ammonium which dominates the strength of the liposome-QAC interaction. This conclusion was supported by very similar results at the lower QAC densities. The observed differences in deformation and mass uptake, on the other hand, seem to correlate well with liposome size as shown in Figure 2b. In fact, taking -ΔD/Δf as a measure of deformation, this quantity seems to be linearly related (linear least-squares fit with R=0.997) to the average liposome diameter measured in the bulk solution. This is in spite of the large difference in liposome charge and possibly also significant differences in membrane elasticity especially between the two net neutral liposomes and the different charged liposomes. While these results clearly show that the attractive interaction with the polymer brush and the energy state of the adsorbed liposomes can be tuned by changing the QAC density (area density of hydrophobic moieties), in no case was spontaneous formation of an SLB on top of the PLL-g-PEG indicated by QCM-D or demonstrated by FRAP control measurements. It is interesting to compare the deformation of the liposomes on top of PLL-g-PEG-QAC(100%) to known cases of adsorbed liposomes that are close to rupture. In Figure 2b is also data from Reimhult et al. for egg-PC liposomes adsorbed to TiO2 and SiO2, respectively, replotted as a function of liposome size.31 Liposomes adsorbed to TiO2 do not rupture even under additional, e.g., (44) Haque, M. E.; McIntosh, T. J.; Lentz, B. R. Biochemistry 2001, 40 (14), 4340-4348.

DOI: 10.1021/la902039g

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osmotic, stress and are very similarly deformed to those adsorbed to the PLL-g-PEG-QAC(100%). The deformation expressed as a lower -ΔD/Δf is, on the other hand, significantly higher for liposomes adsorbing to SiO2 right before they rupture and form an SLB than they are for any of the corresponding sizes of liposomes on PLL-g-PEG-QAC(100%). The scaling of -ΔD/Δf with liposome size almost regardless of membrane composition but not with surface attractive potential for the systems under study here also demonstrates that this measure of deformation can be applied to liposomes of the same size to study surface-induced deformation. But in order to compare the effect on liposomes of different size, normalization with respect to size should be performed, and the presented data compared to Reimhult et al.31 suggest that this normalization is approximately linear with liposome diameter in solution in the relevant size range. 3.4. Induced Formation of Tethered Supported Lipid Bilayer. An additional trigger step to induce liposome rupture was added since even 100% QAC density was not sufficient to induce the high deformation of the liposomes necessary to burst them to form a laterally connected planar lipid bilayer. In bulk solutions addition of PEG of low molecular weight is known to cause fusion of vesicles,44 and the addition of low molecular weight PEG has also recently been applied to cause liposome rupture 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 coworkers, we did not observe rupture of liposomes for most of the different lipid compositions in either QCM-D, OWLS, or FRAP experiments. The same was true for these liposomes tethered directly to BSA-streptavidin-coated surfaces by incorporation of biotinylated lipids in control experiments following the protocol of Bourdillon using e.g. the egg-PC:DOPE mixture (experiments not shown)27 and also used by Taylor et al. in microfluidic channels.45 The exception was a lipid composition of DOPC: DOPE:SM:CH (35:30:15:20 mol/mol/mol/mol) chosen because it has been claimed to be an optimized mixture for PEG-induced fusion.44 These liposomes, despite not revealing a much higher than expected deformation for its size (Figure 2b), showed a pronounced mass loss upon addition of PEG(8 kDa) in OWLS measurements (Figure 3). While the initial adsorbed mass is commensurate with a liposome layer and too high for an SLB, the lipid mass observed after the PEG adsorption is stable at a value within 15% of that expected of an SLB, as compared to values from previously published measurements if the same assumptions for lipid dn/dc is used.34,35,46 No change in adsorbed mass was observed for any other lipid composition when subjected to PEG injection. They all remained at a constant high lipid mass corresponding to a liposome monolayer. The formation of a laterally continuous lipid bilayer in case of the DOPC:DOPE:SM:CH liposomes exposed to PEG(8 kDa) was verified by FRAP (Figure 4) with a recovered fraction after photobleaching of 98 ( 3%. The FRAP experiment verified the formation of a PEG brush supported lipid bilayer with an extraordinarily high diffusion coefficient of 10 ( 2 μm2/s. The diffusion coefficient is ∼5 times higher than in control experiments for supported lipid bilayers of the same lipid composition formed on UV/ozone-cleaned glass by liposome fusion (2 μm2/s), which were in agreement with typical values for supported lipid bilayers formed from PC lipid mixtures.47 Diffusion coefficients (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.

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Figure 3. OWLS measurement of adsorption and PEG-induced fusion of fusogenic tethered liposome. OWLS measurement of adsorption of DOPC:DOPE:SM:CH (35/30/15/20 mol/mol/mol/ mol) liposomes onto PLL-g-PEG-QAC(100%), injected at t = 0 min. An additional injection at t = 37 min followed by rinsing of the cell at t = 83 min ensure a complete coverage and no free liposomes in solution. PEG(8 kDa) 30% w/v in buffer is injected into the chamber at t = 100 min and rinsed at t = 110 min. The mass loss which was only observed for this lipid mixture resulted in a mass similar to what is observed for a supported lipid bilayer and indicates that rupture and formation of an SLB took place.

Figure 4. FRAP measurements on fusogenic liposome layers before and after PEG-induced fusion. FRAP experiments of fusion/rupture triggered by PEG(8 kDa) 30% w/v for adsorbed DOPC:DOPE:SM: CH:NBD-PC(34:30:15:20:1 mol/mol/mol/mol/mol) lipid vesicles on PLL-g-PEG-QAC(100%). (a) Before PEG injection (the same result was observed for all other lipid compositions both before and after PEG injection) and (b) after PEG injection. (c) The recovery curve for the data shown in (b). The recovery after the PEG injection is complete and with a high diffusion coefficient of D = 10 ( 2 μm2/s.

as high as 19 μm2/s of PC lipids freely diffusing in liposomes have been recorded,48 which demonstrates that the proximity of the (48) Gaede, H. C.; Gawrisch, K. Biophys. J. 2003, 85 (3), 1734-1740.

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substrate severely reduces the diffusion of lipids within the membrane. The diffusion has however not been much higher for tethered lipid bilayers aimed to decouple the membrane from the substrate where the density of tethers is high and the separation from the substrate is small. While often not measured since tethered lipid bilayers are typically created on fluorophore quenching gold substrates, lipid monolayers on hydrophobic SAMs 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 SLB from the substrate without covalent tethers have been shown to result in significantly higher diffusion coefficients and also to allow for insertion of large transmembrane proteins with retained lateral fluidity.4,12,51-54 It should be mentioned that in a few instances also very large aggregates could be observed as highintensity fluorescence at a few spots on the microscopy slide surface. These could be the result of many liposomes fusing to a large liposome instead of into the planar bilayer and either resting on the membrane surface due to difficulty with rinsing or tethered to the PLL-g-PEG-QAC. That the lipid composition with the highest -ΔD/Δf ratio for adsorbed liposome layers results in the only reliably produced PEG brush supported lipid bilayers by PEG-induced fusion might at first look like a contradiction given the reasonable hypothesis that the higher the deformation of adsorbed liposomes, the more likely they are to rupture and fuse.31,43,55 However, as shown in Figure 2b, the high absolute values for ΔD and Δf for the adsorbed DOPC:DOPE:SM:CH liposomes is a reflection of that these liposomes are on average substantially larger than the other liposomes that were tested. It has previously been shown that vesicle size for small and large unilamellar liposomes does not significantly affect rupture kinetics on for example SiO2, and thus this size difference is not likely to be responsible for the increased propensity for rupture and fusion.31,56,57 An alternative explanation could be that the high -ΔD/Δf reflects that these liposomes are so fusogenic that they partially start fusing into larger (49) Shen, W. W.; Boxer, S. G.; Knoll, W.; Frank, C. W. Biomacromolecules 2001, 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), 1132811329. (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), 3371-3379.

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Article

liposomes when deformed on the surface, a process previously observed for liposomes adsorbed for example on mica.58 Such fusion could relax deformations, increase water coupling and lead to higher absolute ΔD, Δf, and ΔD/Δf. A further indication that such fusion might occur is that despite that a similar lipid mass per area is expected regardless of size for OWLS measurements, the adsorbed mass is ∼30% higher for these liposomes than for other compositions (on average 730 ng/cm2 compared to 560 ng/cm2). However, the empirical trend in Figure 2b seems to fit well to the adsorption of a liposomal layer of the nominal bulk liposome size, and surface-induced fusion of the adsorbed liposomes is thus unlikely. In summary, the likely reason for the observed increased propensity of these liposomes to fuse when exposed to free PEG in solution is the special lipid headgroup composition, since the mechanical properties, as determined by the membrane deformation exemplified in Figure 2b, are not significantly different to the other compositions. In summary, we have shown that planar lipid bilayers with substantially improved lateral fluidity can be formed by a selfassembly process in three steps: (i) “dip-and-rinse” self-assembly of a graft copolymer monolayer brushes applicable to any negatively charged substrate; (ii) liposome adsorption to PEGtethered quaternary ammonium compound functional groups; and (iii) fusion induced by free PEG(8 kDa) in solution. The fluidity of the so-formed SLB was a factor of 5 higher than for solid supported lipid bilayers and approaching that of freely diffusing lipids in liposomes. The system was also shown to sensitively detect increased liposome deformation as a function of tether density by QCM-D, which indicates the possibility to use the PLL-g-PEG-functional linker coatings with QCM-D to probe the nature and strength of membrane interacting functional groups by their effect on the adsorption kinetics and state of adsorbed liposomes. The layer-by-layer “dip and rinse” buildup of the platform requiring no complicated preparative steps can possibly be generalized as a facile platform for surface functionalization with biologically mimicking membranes which can include transmembrane proteins requiring substantial distance and decoupling from the surface afforded by the water-rich PEG brush spacer. Acknowledgment. Swiss Competence Center for Materials Research (CCMX) and BASF SA, Erich Nyfeler, and Andreas M€uhlebach are acknowledged for financial and material support. We especially thank Prof. Dr. Janos V€or€ os for the kind access to the Laboratory of Biosensors and Bioelectronics at ETH Z€ urich and Marta Bally and Dorothee Grieshaber for providing training and advice for the FRAP and QCM-D measurements. (58) Reviakine, I.; Brisson, A. Langmuir 2000, 16 (4), 1806-1815.

DOI: 10.1021/la902039g

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