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May 27, 2010 - Zirconium Ion Mediated Formation of Liposome Multilayers. Sebastian C. Bürgel,† Orane Guillaume-Gentil,† Limin Zheng,‡ Janos Vö...
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Zirconium Ion Mediated Formation of Liposome Multilayers Sebastian C. B€urgel,† Orane Guillaume-Gentil,† Limin Zheng,‡ Janos V€or€os,† and Marta Bally*,† †

Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University Zurich, 8092 Zurich, Switzerland, and ‡School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Received December 17, 2009. Revised Manuscript Received April 15, 2010

Phospholipid vesicles have attracted considerable interest as a platform for a variety of biomolecular binding assays, especially in the area of membrane protein sensing. The development of liposome-based biosensors widely relies on the availability of simple and efficient protocols for their surface immobilization. We present a novel approach toward the creation of three-dimensional phospholipid vesicle constructs using multivalent zirconium ions as linkers between the liposomes. Such three-dimensional sensing platforms are likely to play a key role in the development of biosensing devices with increased loading capacity and sensitivity. After demonstrating the affinity of Zr4þ toward the phospholipids, we formed vesicle multilayers by sequential injections of solutions containing either liposomes or ZrOCl2. In situ adlayer characterization was carried out by optical waveguide lightmode spectroscopy (OWLS) and quartz crystal microbalance with dissipation (QCM-D) measurements while imaging was performed by atomic force microscopy (AFM) and fluorescence microscopy. Multilayers were successfully constructed, and as demonstrated in a model fluorescence-based biomolecular binding assay, the sensor’s loading capacity was increased. Furthermore, we observed that lipid exchange between the vesicles is promoted in the presence of Zr4þ and that addition of a phosphate-containing buffer leads to adlayer loosening and creation of lipidic tubular structures. The approach presented here could be applied to the study of membrane proteins in a highly sensitive manner due to the increased surface area or to produce functional coatings for controlled drug release and host response.

1. Introduction Liposomes are self-assembled colloidal hollow shells of amphiphilic molecules, most commonly phospholipids. They consist of an aqueous inner cavity delimited by a cell-membrane-like phospholipid bilayer presenting, on each side, the polar heads of the amphiphile. The hydrophobic tails are shielded in the interior of the lamella.1 Because of their unique chemical and physical properties as well as their three-dimensional structure, they have attracted considerable interest in a variety of bioanalytical and biomedical applications including drug delivery, therapeutics, and imaging.1-6 Phospholipid vesicles have also greatly contributed to the development of a variety of biosensors, *Corresponding author: Tel þ41 632 45 85; Fax þ41 632 11 93; e-mail [email protected]. (1) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. (2) Edwards, K. A.; Baeumner, A. J. Liposomes in analyses. Talanta 2006, 68 (5), 1421-1431. (3) Rongen, H. A. H.; Bult, A.; vanBennekom, W. P. Liposomes and immunoassays. J. Immunol. Methods 1997, 204 (2), 105-133. (4) Lian, T.; Ho, R. J. Y. Trends and developments in liposome drug delivery systems. J. Pharm. Sci. 2001, 90 (6), 667-680. (5) Forssen, E. Ligand-targeted liposomes. Adv. Drug Delivery Rev. 1998, 29 (3), 249-271. (6) Deissler, V.; Ruger, R.; Frank, W.; Fahr, A.; Kaiser, W. A.; Hilger, I. Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice. Small 2008, 4 (8), 1240-1246. (7) Bally, M.; V€or€os, J. Nanoscale labels: nanoparticles and liposomes in the development of high performance bionsensors. Nanomedicine 2009, 4 (4), 447-467. (8) Christensen, S. M.; Stamou, D. Surface-based lipid vesicle reactor systems: fabrication and applications. Soft Matter 2007, 3 (7), 828-836. (9) Boukobza, E.; Sonnenfeld, A.; Haran, G. Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J. Phys. Chem. B 2001, 105 (48), 12165-12170. (10) Svedhem, S.; Pfeiffer, I.; Larsson, C.; Wingren, C.; Borrebaek, C.; H€oo€k, F. Patterns of DNA-Labeled and scFv-Antibody-Carrying Lipid Vesicles Directed by Material-Specific Immobilization of DNA and Supported Lipid Bilayer Formation on an Au/SiO2Template. ChemBioChem 2003, 4 (4), 339-343.

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where they have been used as labels,7 as nanocontainers,8,9 or as a platform for bioligand surface immobilization.10,11 Especially, the potential of such lipidic structures in the development and implementation of membrane protein sensors is now widely recognized: liposomes provide the lipidic environment required to ensure a gentle and natural immobilization of membrane proteins, thus preventing denaturation.12-16 Furthermore, implementation as biosensing platforms is promoted by the fact that phosphocholine bilayers are inherently resistant against nonspecific protein adsorption and that a variety of protocols for biomolecule conjugation are readily available.1,17 A great number of bioanalytical tools require the surface immobilization of the biological species of interest for read-out. Along this line, a variety of strategies for the formation of tethered (proteo)liposome layers have been proposed over the past decades. These include direct adsorption onto a surface or attachment via chemical or biomolecular linkers (for a review see ref 8). (11) Stadler, B.; Bally, M.; Grieshaber, D.; Brisson, A.; Voros, J.; Grandin, H. M. Creation of a functional heterogeneous vesicle array via DNA controlled surface sorting onto a spotted microarray. Biointerphases 2006, 1 (4), 142-145. (12) Seddon, A.; Curnow, P.; Booth, P. Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophys. Acta 2004, 1666 (1-2), 105-117 (13) Bailey, K.; Bally, M.; Leifert, W.; Voros, J.; McMurchie, T. G-protein coupled receptor array technologies: Site directed immobilisation of liposomes containing the H-1-histamine or M-2-muscarinic receptors. Proteomics 2009, 9 (8), 2052-2063. (14) Pick, H.; Schmid, E. L.; Tairi, A. P.; Ilegems, E.; Hovius, R.; Vogel, H. Investigating cellular signaling reactions in single attoliter vesicles. J. Am. Chem. Soc. 2005, 127 (9), 2908-2912. (15) Bauer, B.; Davidson, M.; Orwar, O. Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks. Langmuir 2006, 22 (22), 9329-9332. (16) Davidson, M.; Karlsson, M.; Sinclair, J.; Sott, K.; Orwar, O. Nanotubevesicle networks with functionalized membranes and interiors. J. Am. Chem. Soc. 2003, 125 (2), 374-378. (17) Glasmastar, K.; Larsson, C.; Hook, F.; Kasemo, B. Protein adsorption on supported phospholipid bilayers. J. Colloid Interface Sci. 2002, 246 (1), 40-47.

Published on Web 05/27/2010

DOI: 10.1021/la9047566

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Additionally, Graneli et al. have demonstrated that surface bound three-dimensional liposome matrices can be obtained using short oligonucleotides as linkers.18,54 This strategy offers the possibility to increase the sensor’s loading capacity by building up in the third dimension. Furthermore, the membrane proteins embedded in the liposome are lifted off the sensor’s surface so that the harmful interactions with the solid substrate are limited. Tetravalent metal ions such as Zr4þ have long been known to interact strongly with phosphates and phosphonates. Especially in the 1990s, a large number of publications have reported the production of multilayered thin films consisting of organic biphosphonate sheets coordinating an inorganic network of Zr4þ ions. These films were shown to be well-organized and extremely stable.19,20 Furthermore, different functional groups could be incorporated conferring the adlayer a variety of optical, electrical, and physical properties.21 More recently, the affinity between the metal ion and phosphates/phosphonates has also been exploited in the bioanalytical and biomedical field: capillary columns22,23 or porous wafers24 coated with zirconium phosphonate have been used for phosphopeptide enrichment for mass spectroscopy. Zr4þ can also be the linker between the phosphates of surface bound nucleic acids and a particle label25,26 or a dye27 in a DNA sensor or for the creation of DNA thin films for gene delivery.28-30 Others (18) Graneli, A.; Benkoski, J. J.; Hook, F. Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes. Anal. Biochem. 2007, 367 (1), 87-94. (19) Thompson, M. E. Use of Layered Metal Phosphonates for the Design and Construction of Molecular Materials. Chem. Mater. 1994, 6 (8), 1168-1175. (20) Lee, H.; Kepley, L. J.; Hong, H. G.; Mallouk, T. E. Inorganic Analogs of Langmuir-Blodgett Films - Adsorption of Ordered Zirconium 1,10-Decanebisphosphonate Multilayers on Silicon Surfaces. J. Am. Chem. Soc. 1988, 110 (2), 618-620. (21) Katz, H. E. Multilayer Deposition of Novel Organophosphonates with Zirconium(Iv). Chem. Mater. 1994, 6 (12), 2227-2232. (22) Feng, S.; Ye, M.; Zhou, H.; Jiang, X.; Jiang, X.; Zou, H.; Gong, B. Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis. Mol. Cell. Proteomics 2007, 6 (9), 1656-1665. (23) Xue, Y. F.; Wei, J. Y.; Han, H. H.; Zhao, L. Y.; Cao, D.; Wang, J. L.; Yang, X. M.; Zhang, Y. J.; Qian, X. H. Application of open tubular capillary columns coated with zirconium phosphonate for enrichment of phosphopeptides. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2009, 877 (8-9), 757-764. (24) Zhou, H.; Xu, S.; Ye, M.; Feng, S.; Pan, C.; Jiang, X.; Li, X.; Han, G.; Fu, Y.; Zou, H. Zirconium Phosphonate-Modified Porous Silicon for Highly Specific Capture of Phosphopeptides and MALDI-TOF MS Analysis. J. Proteome Res. 2006, 5 (9), 2431-2437. (25) Fan, Y.; Chen, X. T.; Kong, J. M.; Tung, C. H.; Gao, Z. Q. Direct detection of nucleic acids by tagging phosphates on their backbones with conductive nanoparticles. Angew. Chem., Int. Ed. 2007, 46 (12), 2051-2054. (26) Fang, C.; Fan, Y.; Kong, J. M.; Gao, Z. Q.; Balasubramanian, N. Electrical Detection of Oligonucleotide Using an Aggregate of Gold Nanoparticles as a Conductive Tag. Anal. Chem. 2008, 80 (24), 9387-9394. (27) Fang, C.; Agarwal, A.; Buddharaju, K. D.; Khalid, N. M.; Salim, S. M.; Widjaja, E.; Garland, M. V.; Balasubramanian, N.; Kwong, D. L. DNA detection using nanostructured SERS substrates with Rhodamine B as Raman label. Biosens. Bioelectron. 2008, 24 (2), 216-221. (28) Wang, F.; Li, D.; Li, G. P.; Liu, X. Q.; Dong, S. J. Electrodissolution of Inorganic Ions/DNA Multilayer Film for Tunable DNA Release. Biomacromolecules 2008, 9 (10), 2645-2652. (29) Wang, F.; Liu, X. Q.; Li, G. P.; Li, D.; Dong, S. J. Selective electrodissolution of inorganic ions/DNA multilayer film for tunable DNA release. J. Mater. Chem. 2009, 19 (2), 286-291. (30) Wang, H. L.; Wang, F.; Xu, Z.; Wang, Y. Z.; Dong, S. J. Surface plasmon resonance and electrochemistry characterization of layer-by-layer self-assembled DNA and Zr4þ thin films, and their interaction with cytochrome c. Talanta 2007, 74 (1), 104-109. (31) Nonglaton, G.; Benitez, I. O.; Guisle, I.; Pipelier, M.; Leger, J.; Dubreuil, D.; Tellier, C.; Talham, D. R.; Bujoli, B. New approach to oligonucleotide microarrays using zirconium phosphonate-modified surfaces. J. Am. Chem. Soc. 2004, 126 (5), 1497-1502. (32) Monot, J.; Petit, M.; Lane, S. M.; Guisle, I.; Leger, J.; Tellier, C.; Talham, D. R.; Bujoli, B. Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: Critical influence of the location of the probe anchoring groups. J. Am. Chem. Soc. 2008, 130 (19), 6243-6251. (33) Bujoli, B.; Lane, S. M.; Nonglaton, G.; Pipelier, M.; Leger, J.; Talham, D. R.; Tellier, C. Metal phosphonates applied to biotechnologies: A novel approach to oligonucleotide microarrays. Chem.;Eur. J. 2005, 11 (7), 1981-1988.

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have reported the production of DNA microarrays on glass slides derivatized with zirconium phosphonate.31-33 In recent reports, phospholipid vesicles were shown to fuse and form monolayers34 or bilayers,35,36 depending on the lipid composition, when in contact with a surface modified with zirconium phosphonate. In our approach, we take advantage of this high-affinity interaction to create layer-by-layer multilayers of liposomes. In this paper, we study the interactions between this multivalent cation and phospholipids and demonstrate that a Zr4þ-based coordination chemistry provides novel means to obtain, with a simple protocol, three-dimensional vesicle constructs. With a model binding assay for the detection of biotin with fluorescently labeled streptavidin, we illustrate the potential of such threedimensional structures in the development of sensors with increased loading capacity and sensitivity.

2. Materials and Methods Liposome Preparation. All lipids were purchased from

Avanti Polar Lipids Inc. and stored in chloroform at -20 C. 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes were obtained by forming a lipid film on a glass flask by solvent evaporation (30 min under N2 flow), followed by hydration in 0.3 M NaCl solution to a lipid concentration of 10 mg/mL. Unilamellar vesicles were obtained by extrusion (31 times) through two polycarbonate filter membranes of 200 nm pore size (Avestin, Canada). The liposome suspension were stored at 4 C and used within 2 weeks. Fluorescent or biotinylated liposomes were obtained in a similar way by adding 2% (w/w) of either 2-(12(7-nitrobenz-2-oxa-1,3-diyzol-4-yl)amino)dodecanoyl-1-hexa-decanoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt), or 2% (w/w) 1,2-dioleoyl-sn-glycero-3-phophoethanolamineN-biotinyl (sodium salt) to the lipid solution before solvent evaporation. Liposomes loaded with BSA-AlexaFluor647 (Invitrogen, Switzerland) were obtained by hydrating the vesicles with a solution containing the fluorescent molecules (10 mg/mL). Aggregation Experiments. To study the effect of ZrOCl2 on a liposome suspension, equal volumes of solutions containing rhodamine-labeled liposomes or ZrOCl2 in 0.3 M NaCl at appropriate concentration were mixed together and inspected visually after 1 h. Surface Modifications. All experiments were performed in a 0.3 M NaCl solution obtained by dissolving NaCl (Fluka, Switzerland) in deionized water purified with a Milli-Q gradient A10 system (resistivity >18 MΩ/cm) (Millipore Corp., Switzerland). Zirconyl chloride octahydrate (ZrOCl2 3 8H2O) was purchased from Fluka (Switzerland) and dissolved in 0.3 M NaCl solution to a final concentration of 0.1 mM. All solutions were stored at 4 C and filtered prior use (200 nm filter, Minisart, Sartorius, Germany). The vesicles were immobilized on the sensor surface by incubation with a liposome suspension (lipid concentration: 0.5 mg/mL) for at least 30 min. After rinsing the adlayer, ZrOCl2 solution was added, followed by rinsing. The solution was injected twice at an interval of 5 min for in situ measurements (OWLS, QCM-D) or once for 15 min (AFM, microscopy experiments). These steps were repeated as many times as required to obtain the appropriate number of vesicle layers. Multilayer buildup was always terminated by liposome injection and rinsing. Streptavidin binding assays were performed by adding streptavidin-AlexaFluor488 (Invitrogen, Switzerland; 200 μg/mL in 10 mg/mL bovine serum (34) Oberts, B. P.; Blanchard, G. J. Formation of Air-Stable Supported Lipid Monolayers and Bilayers. Langmuir 2009, 25 (5), 2962-2970. (35) Brzozowska, M.; Oberts, B. P.; Blanchard, G. J.; Majewski, J.; Krysinski, P. Design and Characterization of Novel Tether Layer for Coupling of a Bilayer Lipid Membrane to the Surface of Gold. Langmuir 2009, 25 (16), 9337-9345. (36) Fabre, R.; Talham, D. R. Stable Supported Lipid Bilayers on Zirconium Phosphonate Surfaces. Langmuir 2009, ASAP article.

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B€ urgel et al. albumin, 30 min) to multilayers of biotinylated vesicles constructed as described above. Prior to injection, the layers were blocked for 15 min in 10 mg/mL bovine serum albumin. For multilayer dissolution, double concentrated phosphate buffer saline (PBS) (tablets obtained from Sigma-Aldrich, Switzerland; final composition: 0.02 M phosphate buffer, 0.0054 M potassium chloride, and 0.274 M sodium chloride) was added to the liposome construct. Optical Waveguide Lightmode Spectroscopy. Adlayer formation was first investigated in situ by optical waveguide lightmode spectroscopy (OWLS). The OWLS detection principle is based on coupling of a laser beam into a planar waveguide via a diffraction grating. Shifts in the incoupling angles of the TE and TM modes resulting from changes in the optical properties in vicinity of the surface are monitored and related to adlayer thickness and refractive index.37,38 The measured changes in refractive indexes can be converted into adsorbed (dry) mass using de Feijter’s formula.39 OWLS measurements were performed on an OWLS 120 (MicroVacuum, Hungary). The OWLS waveguides were purchased from MicroVacuum (Hungary) and sputter-coated with a 6 nm TiO2 layer at Paul Scherrer Institute (Switzerland). Prior to use, the substrates were cleaned by 10 min ultrasonication in isopropanol, followed by 10 min ultrasonication in cleaned water and finally 3 min oxygen-plasma cleaning. The thickness (dA), refractive index (nA), and optical mass (QA) of the adsorbed layer were estimated from raw data (incoupling angles for TE0 and TM0 modes) in the following way: a three-layer mode equation40 was used to determine the refractive index (nF) and the thickness (dF) of the waveguiding film from the baseline in solution. The refractive index of the cover media (nc) was 1.334 331, as measured with a J357 refractometer (Rudolph Research Analytical). These values were then used to estimate nA, dA, and QA = (nA - nC)dA of the adlayer using the extended model of the fourlayer mode equations (involving the adlayer) described by Picart et al.41,42 In order to obtain stable noncomplex solutions, 1 nm of the previously estimated dF value was subtracted before this step (and hence further treated as part of the adlayer). The final adlayer mass (ΔmOWLS) was calculated as described in the following equation

Article mass and thickness on the surface where the mass estimated from QCM-D includes the surrounding liquid associated with the oscillation. Additionally, QCM-D measures the damping of the oscillation which can be related to changes in viscoelastic properties of the adlayer.44,45 Thus, QCM-D as a complementary technique to OWLS can be used to detect morphological changes such as layer stiffening or water release from the adlayer.46 Experiments were carried out on a QSX 301 (Q-Sense AB, Sweden) using gold-coated AT-cut quartz crystals obtained from Q-sense (Sweden) exhibiting a fundamental resonance frequency of 4.95 MHz ( 50 kHz. The changes of overtones n = 3, 5, 7 of the fundamental frequency (indicated in normalized form (Δfn/n)) as well as the shifts in energy dissipation ΔDn were analyzed. Prior to use, all crystals were cleaned in 2% (w/w) sodium dodecyl sulfate solution for 30 min followed by rinsing with Milli-Q water, drying with a nitrogen stream and 30 min in a UV/ozone cleaner. A one-layer Voigt model47 was chosen to model the thickness and the viscosity of the adsorbed material. The following parameters were fixed: bulk liquid density: 103 kg m-3; bulk liquid viscosity: 10-3 kg m-1 s-1; adlayer density: 1.01  103 kg m-3 ; the adlayer shear modulus was first modeled and then fixed to an average value of 103 Pa. In order to evaluate the accuracy of the model the viscosity and thickness values obtained by fitting the third and fifth overtones were taken as fixed to estimate the expected values for ΔF7/7 and ΔD7. This resulted in an average error of less than 3% compared to the measured values. Fluorescence Microscopy. Fluorescence microscopy experiments were performed on a Zeiss LSM 510 confocal laser scanning microscope using an argon laser or a He-Ne laser and a 40X (LD, NA 0.7) objective. Glass slides sputter-coated with a 15 nm TiO2 layer at Paul Scherrer Institute (Switzerland) were used as substrates. To analyze diffusion of lipids within a liposome layer before and after injection of the ZrOCl2 solution, fluorescence recovery after photobleaching (FRAP) experiments were performed using a circular bleaching area (radius r = 25 μm). The intensity within the bleached area was measured at irregular time intervals (fluorescence recovery curve FK(t), bleaching ends at t = 0) and more conveniently displayed as the fractional fluorescence curve:48,49

ΔmOWLS ¼ ðQA - 0:001ðnF - nc ÞÞ=ðdn=dcÞ where QA is in μm. Since no value for the dn/dc of liposomes is reported in the literature, a value of 0.182 cm3/g43 was used for qualitative data comparison (Figure 3c).

Quartz Crystal Microbalance with Dissipation Monitoring. Quartz crystal microbalance with dissipation monitoring (QCM-D) was also carried out to monitor adlayer buildup in real time. QCM-D relies on the change of the fundamental resonance frequency and its overtones in thickness-shear modes upon mass adsorption on the surface of an oscillating quartz crystal. A decrease in frequency corresponds to an increase in (37) Ramsden, J. J. Optical biosensors. J. Mol. Recognit. 1997, 10 (3), 109-120. (38) Voros, J.; Ramsden, J. J.; Csucs, G.; Szendro, I.; De Paul, S. M.; Textor, M.; Spencer, N. D. Optical grating coupler biosensors. Biomaterials 2002, 23 (17), 3699-3710. (39) Defeijter, J. A.; Benjamins, J.; Veer, F. A. Ellipsometry as a Tool to Study Adsorption Behavior of Synthetic and Biopolymers at Air-Water-Interface. Biopolymers 1978, 17 (7), 1759-1772. (40) Tiefenthaler, K.; Lukosz, W. Sensitivity of Grating Couplers as IntegratedOptical Chemical Sensors. J. Opt. Soc. Am. B 1989, 6 (2), 209-220. (41) Picart, C.; Gergely, C.; Arntz, Y.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G.; Senger, B. Measurement of film thickness up to several hundreds of nanometers using optical waveguide lightmode spectroscopy. Biosens. Bioelectron. 2004, 20 (3), 553-561. (42) Picart, C.; Ladam, G.; Senger, B.; Voegel, J. C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. Determination of structural parameters characterizing thin films by optical methods: A comparison between scanning angle reflectometry and optical waveguide lightmode spectroscopy. J. Chem. Phys. 2001, 115 (2), 1086-1094. (43) Stadler, B.; Falconnet, D.; Pfeiffer, I.; Hook, F.; Voros, J. Micropatterning of DNA-tagged vesicles. Langmuir 2004, 20 (26), 11348-11354.

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f ðtÞ ¼

FK ðtÞ - FK ð0Þ FK ð¥Þ - FK ð0Þ

Each experiment was repeated at least three times and summarized by estimating the mean value and standard error of mean of five consecutive f values. Since the observed recovery was slow, the determination of exact values for FK(¥) was impossible to obtain without further analysis. We have used the model by Yguerabide et al.50 to estimate FK(0) and FK(¥) as well as the characteristic

(44) Marx, K. A. Quartz crystal microbalance: A useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface. Biomacromolecules 2003, 4 (5), 1099-1120. (45) Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. QuartzCrystal Microbalance Setup for Frequency and Q-Factor Measurements in Gaseous and Liquid Environments. Rev. Sci. Instrum. 1995, 66 (7), 3924-3930. (46) Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/ dissipation. Colloids Surf., B 2002, 24 (2), 155-170. (47) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic acoustic response of layered polymer films at fluid-solid interfaces: Continuum mechanics approach. Phys. Scr. 1999, 59 (5), 391-396. (48) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E. L.; Webb, W. W. Mobility Measurements by Analysis of Fluorescence Photobleaching Recovery Kinetics. Biophys. J. 1976, 16 (2), A217-A217. (49) Soumpasis, D. M. Theoretical-Analysis of Fluorescence Photobleaching Recovery Experiments. Biophys. J. 1983, 41 (1), 95-97. (50) Yguerabide, J.; Schmidt, J. A.; Yguerabide, E. E. Lateral Mobility in Membranes as Detected by Fluorescence Recovery after Photobleaching. Biophys. J. 1982, 40 (1), 69-75.

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Figure 2. Multiple layers of vesicles are stacked after sequential injection of vesicle suspensions and ZrOCl2 containing solutions.

Figure 1. Liposome aggregation behavior for various phospholipid and ZrOCl2 concentrations. (a) Behavior for different phospholipid/ ZrOCl2 molar ratios. Dark blue (þ) corresponds to clearly separated aggregates. White (-) corresponds to a homogeneous solution without visible aggregates. Light blue (() represents an intermediate state. (b) Aggregate images corresponding to the situations described in (a). diffusion time τD which can then be related to the diffusion coefficient: D = r2/4τD. The streptavidin-AlexaFluor488 binding assays were evaluated by measuring the fluorescence signal with identical instrument settings for different numbers of liposome layers. The average intensity was measured in an area of 50 μm  50 μm at 8 bit color depth. Atomic Force Microscopy. Atomic force microscopy (AFM) images were acquired in intermittent-contact fluid mode, using a Nanowizard I BioAFM (JPKInstruments, Berlin, Germany) and Mikromasch CSC38/noAl cantilevers. Height mode images were scanned at a fixed scan rate (0.3 Hz) with a resolution of 256 pixels  256 pixels. Several scans over a given surface area were performed in order to check for image reproducibility. The substrate was a silicon wafer sample sputter-coated at Paul Scherrer Institute (Switzerland) with a 21 nm TiO2 layer. Scanning over a scratched area allowed for the determination of the adlayer thickness (Figure 4d).

3. Results Liposome Aggregation Experiments. Solution-based experiments were performed in order to verify qualitatively the general linking mechanism between liposomes and Zr4þ. Here, a Zr4þ-containing solution was added to rhodamine-labeled liposomes. Different phospholipid/ZrOCl2 ratios were investigated. Observation after 1 h revealed three different domains, as depicted in Figure 1: (1) In a central domain corresponding to phospholipid/ZrOCl2 ratios ranging from 6 to 25 liposome aggregation is clearly visible. (2) At lower ratios, i.e., higher Zr4þ relative content (bottom left of the table in Figure 1), aggregation is inhibited, probably due to saturation of the vesicle surface with Zr4þ ions. (3) At higher ratios, aggregate formation was less pronounced since fewer liposome-Zr4þ-liposome contacts are available to achieve stable large scale aggregates, before being completely inhibited. Liposome Multilayer Buildup. A protocol for the creation of three-dimensional phospholipid constructs based on the 10998 DOI: 10.1021/la9047566

phosphate-zirconium chemistry and using Zr4þ as a linker between the liposomes was developed (Figure 2). Briefly, liposome layers were deposited by sequential injection of liposome and ZrOCl2 containing solutions. Vesicle surface immobilization was monitored in situ using OWLS and QCM-D measurements. OWLS Measurements. The successful formation of liposome multilayers was first confirmed by OWLS. A typical curve is shown in Figure 3a. As a result form the exponential decay of the evanescent field (which typically extends ∼200 nm above the waveguides41), OWLS measurements were affected by a decay in sensitivity with increasing layer thickness, characteristic for thick adlayers (>20 nm). The thickness estimated with the linear model proposed by Tiefenthaler et al.40 underestimates the mass adsorbed for layers thicker than a few tens of nanometers. Therefore, the extended model reported in ref 41 was used to calculate the refractive index and thickness of the adsorbed layer. The results summarized in Figure 3c show an increase in adsorbed mass upon injection of liposomes and ZrOCl2 and suggest a successful buildup of liposome multilayers. However, the mass values reported here can only be taken for qualitative comparison between layers of the same type since no appropriate dn/dc values are reported for lipids or Zr4þ layers. QCM-D. In addition to OWLS measurements, multilayer buildup was investigated by QCM-D to gain further insight into layer thickness, viscoelastic properties, and structural changes that might occur within the construct during the buildup process. Figure 3b shows a typical QCM-D curve for normalized frequency (ΔF3/3) and dissipation (ΔD3) changes of the third overtone. Liposome immobilization led to a significant decrease in frequency and increase in dissipation typical for the adsorption of soft layers with high water content,51-53 indicating that the liposomes were immobilized intact. Addition of ZrOCl2 resulted in a further decrease of the frequency together with a decrease in energy dissipation, at least for the first three layers. As described both theoretically and experimentally, thick viscoelastic layers exhibit a film resonance behavior which leads to a loss of sensitivity and eventually in positive frequency shifts.54,55 In our case signs of film resonance translated in positive frequency shifts for the seventh overtone at frequency shifts above 1050 Hz (see Supporting Information). Thus, because the adsorbed mass cannot be assumed to be proportional to the decrease in frequency (as in the case of thin layers), a one-layer Voigt model was chosen to model thickness and viscosity of the adlayer. As summarized in (51) Keller, C. Surface Specific Kinetics of Lipid Vesicle Adsorption Measured with a Quartz Crystal Microbalance. Biophys. J. 1998, 75 (3), 1397-1402. (52) Reimhult, E.; Hook, F.; Kasemo, B. Vesicle adsorption on SiO2 and TiO2: Dependence on vesicle size. J. Chem. Phys. 2002, 117 (16), 7401-7404 (53) Reviakine, I.; Rossetti, F. F.; Morozov, A. N.; Textor, M. Investigating the properties of supported vesicular layers on titanium dioxide by quartz crystal microbalance with dissipation measurements. J. Chem. Phys. 2005, 122 (20). (54) Graneli, A.; Edvardsson, M.; Hook, F. DNA-based formation of a supported, three-dimensional lipid vesicle matrix probed by QCM-D and SPR. ChemPhysChem 2004, 5 (5), 729-733. (55) Lucklum, R.; Behling, C.; Hauptmann, P. Role of mass accumulation and viscoelastic film properties for the response of acoustic-wave based chemical sensors. Anal. Chem. 1999, 71 (13), 2488-2496.

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Figure 4. AFM imaging of vesicle layers: (a, b) AFM height mode images of one vesicle layer after addition of Zr4þ. (c) AFM height mode images of two vesicle layers. (d) Typical height profiles obtained from images of one (left) or two (right) vesicle layers.

Figure 3. Liposome multilayers formation and dissolution. Multilayers were obtained by sequential injection of ZrOCl2 (small black arrows) and 200 nm liposomes (big green arrows). The flow cell was rinsed with NaCl solution between each injection. The measurement was terminated by injecting PBS (horizontal orange arrow) followed by rinsing with NaCl solution. (a) OWLS experiment displaying the optical (dry) mass for five liposome layers. (b) QCM-D displaying the normalized frequency (ΔF3/3) and dissipation changes of the third overtone for six liposome layers. (c) Comparison of optical mass determined from OWLS using the extended model described in ref 41 and layer thickness calculated from QCM-D experiments using the 1-layer Voigt model. Error bars indicate the standard error of mean.

Figure 3c, the adsorbed mass per layer increases upon liposome adsorption and decreases after subsequent ZrOCl2 injection. The mass of the first layer was 15.4 ( 0.6 μg cm-2, which corresponds to 152.1 ( 6 nm. The subsequent layers were of slightly smaller mass and thickness: 10.6 ( 1.1 μg cm-2 and 105.4 ( 1 nm, respectively. Characterization by AFM and Fluorescence Microscopy. As a further characterization tool, we have used atomic force Langmuir 2010, 26(13), 10995–11002

microscopy to image vesicle multilayer formation. AFM images confirmed that the vesicle layer stayed intact after addition of Zr4þ, with homogeneously distributed nonaggregated vesicles (Figure 4a). The imaged vesicles showed heights ranging typically from 100 to 150 nm, and a close-up view revealed areas of tightly packed vesicles (Figure 4b). We were also able to image the vesicles after addition of a second layer as shown in Figure 4c. Figure 4d shows typical height profiles for 1 and 2 vesicle layers. The profile of the second vesicle layer is shifted upward (ca. 50 nm), indicating that the vesicles are sitting between the vesicles of the first layer. Visualization of a multilayer of vesicles loaded with a fluorescent protein showed that the vesicles retain their content upon multilayer formation. An overnight bleaching experiment also indicated that the vesicles do not fuse into larger structures as no diffusion of vesicle content into the photobleached area was observed (Figure 5). Streptavidin Binding Assays. Three-dimensional constructs such as the one presented here can contribute to the development of sensing platforms with increased loading capacity and thus increased assay sensitivity. As a proof-of-concept, a model assay was performed using biotinylated liposomes. The binding of fluorescent streptavidin to constructs consisting of different numbers of layers was investigated by fluorescence microscopy. As depicted in Figure 6, streptavidin binding to biotin immobilized on the vesicle surface increased with the number of layers, indicating that molecules as large as streptavidin are able to diffuse into the 3D constructs, at least for up to 3 layers. Binding specificity was confirmed with control experiments performed with non-biotinylated liposomes: the signal was indistinguishable from the background noise (Figure 6, empty red circles). Multilayer Dissolution. The stability of the vesicle multilayers in phosphate containing buffer (PBS) was also investigated. PBS contains a high amount of phosphates which might interfere with the phosphocholine-Zr4þ bond and compete for it. As observed with OWLS and QCM-D measurements (Figure 3a,b) addition of PBS leads to a significant decrease of adsorbed mass (residual mass 1.8 ( 0.5 μg/cm2 in OWLS, 47 ( 2 μg/cm2 in QCM-D) coupled to an increase in dissipation (ΔD3 = 379  10-6 ( 71  10-6), indicating structural changes within the layer. DOI: 10.1021/la9047566

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Figure 7. Liposome multilayer dissolution investigated by fluorescence microscopy. A layer of fluorescent liposomes (a) before and (b) 20 min after addition of PBS. The multilayers consisted of 5 vesicle layers containing one layer of fluorescent NBD-labeled vesicles.

Figure 5. Bleaching experiment with vesicles loaded with BSAAlexaFluor 647. (a) Fluorescence Images of 3 layers of vesicles (i) right after multilayer formation and bleaching (ii) after 13 h. (b) Corresponding intensity profiles (middle of the image). Note that the slight increase in intensity after 13 h can be associated with bleaching of the partially quenched BSA-AlexaFluor647.

Figure 6. Fluorescent binding assays. Fluorescence resulting from the binding of streptavidin-AlexaFluor488 to multilayers of biotinylated (blue triangle) and non-biotinylated liposomes (empty red circles). The intensity was normalized against the intensity of a bleached area. Error bars are standard error of mean.

Fluorescence microscopy gave further insight into the dissolution process. As can be seen from Figure 7, addition of PBS led to the formation of tubular structures within 20 min. The mostly straight, up to a few tens of micrometers long tubes were pinned to the surface on one end, even during rinsing. Lipid Diffusion. We have used FRAP to analyze the diffusion properties of lipids in a single layer of fluorescent liposomes before and after injection of ZrOCl2. Figure 8 shows the recovery of intensity within the bleached area. Mobile fraction and diffusion coefficient were evaluated with the model proposed by Yguerabide et al.,50 yielding a mobile fraction of 15.9% ( 10.8% and 71.2% ( 5.5% before and after addition of ZrOCl2, respectively. Fitting of the FRAP data revealed an increase of the lipid diffusion coefficients of almost 2 orders of magnitude, with values of 3.3  10-11 ( 2.4  10-11 cm2/s in the absence of Zr4þ and 1.1  10-9 ( 0.3  10-9 cm2/s in the presence of the cation. 11000 DOI: 10.1021/la9047566

Figure 8. Diffusion of fluorescent species analyzed by fluorescence recovery after photobleaching. (a) NBD-lipid diffusion within a liposome layer before injections of ZrOCl2 (blue, filled circles) and after injection of ZrOCl2 (red, empty triangles). Error bars are the standard error of means. (b) Fluorescence images of a layer of vesicles stained with NBD before injection of ZrOCl2 (i) immediately after bleaching and (ii) after 5 h, (iii) in the presence of ZrOCl2 immediately after bleaching, and (iv) in presence of ZrOCl2 after 2 h. Scale bars are 50 μm.

4. Discussion Multilayer Buildup and Adhesion between the Liposomes. After demonstrating the interaction between phospholipids and Zr4þ ions in solution, we have taken advantage of the linkage formed between phosphocholine lipids and Zr4þ ions to build up, layer-by-layer, three-dimensional lipidic constructs. Successful buildup was confirmed by in situ QCM-D and OWLS measurements as well as by fluorescence microscopy. QCM-D experiments were performed on gold substrates while all other measurements were performed on TiO2-coated waveguides and TiO2-coated glass slides. The behavior of phospholipid vesicles in contact with metal and metal oxide surfaces widely depends on the experimental conditions (surface properties of the substrate, vesicle charge, vesicle size, buffer molarity and composition, etc.). Liposomes are known to either adsorb intact or to fuse into supported phospholipid bilayers or monolayers.56,57 In the case of TiO2 and (56) Chan, Y. H. M.; Boxer, S. G. Model membrane systems and their applications. Curr. Opin. Chem. Biol. 2007, 11 (6), 581-587. (57) Sackmann, E. Supported membranes: Scientific and practical applications. Science 1996, 271 (5245), 43-48.

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gold, a large number of studies performed with zwitterionic phosphocholine lipids have confirmed that liposomes adsorb intact on both substrates, forming a homogeneous densely packed vesicle layer51-53,58-60 so that the multilayer experiments are directly comparable. This was also confirmed by control experiments performed on TiO2-coated QCM-D crystals (see Supporting Information). OWLS and QCM-D measurements gave clear evidence that a thick viscoelastic film is formed on the sensor surface upon subsequent addition of liposomes: for OWLS, the sensitivity of the measurement decreased with an increase in number of layers while film resonance effects due to the establishment of a standing acoustic wave within the film were observed in QCM-D experiments. The latter phenomenon has already been described in detail for a similar system by Graneli et al.54 In our case, however, signs of film resonance translated into positive frequency shifts already at ΔF7/7 = -1050 Hz (see Supporting Information). This value is nevertheless in agreement with the theoretical model of 47 predicting film resonance effects (depending on the material properties) in bulk water at ∼2 kHz. Thus, for QCM-D as well as OWLS, data modeling was performed in order to correct for deviation from the linear behavior usually assumed for thin rigid films (Sauerbrey equation in the case of QCM-D,61 de Feijter’s formula in the case of OWLS39) and to get insight into the total mass adsorbed. For QCM-D the adsorbed mass was derived using a 1-layer Voigt model. This is in contradiction with the 2-layer model used by Graneli et al.54 However, one should note that those authors linked liposomes together via short oligonucleotide strands and argue that the first layer, in direct contact with the surface, is likely to be more deformed than the subsequent layer. This is further supported by the ΔD vs Δf plot which clearly show that the first layer is behaving differently that the following layers. In our case, the response was essentially linear for all the layers (see Supporting Information). Furthermore, a similar deformation behavior is expected if a large number of attachment points (the surface is saturated with Zr4þ) is available for all the layers, including the first one. The QCM-D modeled thickness provides a realistic representation of the system. The obtained thickness for the first layer is only slightly smaller than the liposome diameter (152 ( 6 nm), in good agreement with the data obtained by others on similar systems54 and with our AFM measurements. The upper layers are thinner, as expected considering that liposomes will not stack on top of each other but occupy the partially free spaces between liposomes of the previous layer as also observed by AFM (Figure 4). Addition of ZrOCl2 to the vesicle layers did not result in significant content release from the vesicles as suggested from the QCM-D data where no increase in frequency shift, characteristic for the release of content from the vesicles,58 is observed. However, a clear dissipation decrease is observed upon addition of Zr4þ ions (Figure 3b). As shown by the data modeled from QCMD, this correlates with a decrease in adlayer thickness but with no mass loss as indicated by the OWLS measurements and can thus be associated with a film stiffening and compacting resulting from (58) Reimhult, E.; Hook, F.; Kasemo, B. Intact vesicle adsorption and supported biomembrane formation from vesicles in solution: Influence of surface chemistry, vesicle size, temperature, and osmotic pressure. Langmuir 2003, 19 (5), 1681-1691. (59) Rossetti, F. F.; Bally, M.; Michel, R.; Textor, M.; Reviakine, I. Interactions between titanium dioxide and phosphatidyl serine-containing liposomes: Formation and patterning of supported phospholipid bilayers on the surface of a medically relevant material. Langmuir 2005, 21 (14), 6443-6450. (60) Groves, J. T.; Ulman, N.; Cremer, P. S.; Boxer, S. G. Substrate-membrane interactions: Mechanisms for imposing patterns on a fluid bilayer membrane. Langmuir 1998, 14 (12), 3347-3350. (61) Sauerbrey, G. Verwendung Von Schwingquarzen Zur Wagung Dunner Schichten Und Zur Mikrowagung. Z. Phys. 1959, 155 (2), 206-222.

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Zr4þ-mediated attraction between the walls of adjacent liposomes. This hypothesis is further confirmed by the lipid diffusion data which shows a clear increase in lipid transfer in the presence of ZrOCl2. It is also interesting to note that AFM imaging of a single vesicle layer without addition of zirconium was difficult because of the low adhesion between vesicles and substrates; the vesicles were easily displaced by the cantilever upon scanning. This can also be taken as an indirect indication that the vesicle layer is more compact and tightly bound after addition of the cations. Liposome multilayer buildup was confirmed indirectly by a binding assay performed with fluorescently labeled streptavidin. The amount of protein bound to biotin immobilized on the liposome surface increased linearly with the number of layers demonstrating the applicability of such three-dimensional constructs for the creation of sensors with increased loading capacity. PBS Addition and Lipid Tubes Formation. The stability of the liposome multilayers in PBS was also investigated. PBS contains a high amount of phosphates which interfere with the phosphocholine-Zr4þ bond and compete for it. Phosphate is expected to exhibit a stronger affinity since interaction between the phospholipid and Zr4þ is likely to be weakened by the presence of the choline group on the lipid head. Addition of PBS resulted in a substantial mass loss as observed both with QCM-D and OWLS and in the formation of tubular structures as observed by fluorescence microscopy. The latter observation translated into an oscillation damping of the QCM-D experiment. Here, two main scenarios are possible: (1) Addition of phosphates induces a structural change within the lipid bilayer structure, resulting in the formation of bilayer or monolayer tubes. The formation of such tubular structures has been reported e.g. by Rossetti et al.,62 but the underlying mechanism is poorly understood. (2) There is also a possibility for an induced organization of the liposomes into “vesicle chains” as observed by others with gold nanoparticles and a tuned attractive (Zr4þ-mediated) and repulsive (electrostatic) force.26,63 Analysis of lipid diffusion within the tubes together with a determination of the tube diameter (using e.g. transmission electron microscopy) and an in-depth analysis of the parameters influencing tube formation would give further insight into this process but are beyond the scope of this work. Lipid Diffusion. A comparison of lipid diffusion coefficients within a single liposome layer in the absence and presence of ZrOCl2 revealed a significant increase in lipid exchange in the presence of Zr4þ (Figure 8). Although a few vesicles are indeed desorbing and resorbing, leading to the appearance of single bright dots in the bleached area as can be seen from Figure 8b(ii), lipid exchange between the vesicles is most likely to be the major mechanism here. Lipid transfer between phospholipid membranes has been studied by several groups, and the following mechanisms have been proposed (summarized e.g. in ref 64): (1) a phospholipid is transferred from one membrane to the other after diffusion through the aqueous phase; (2) lipid transfer occurs when the membranes are in close contact either after collision65,66 or after (62) Rossetti, F. F.; Reviakine, I.; Csucs, G.; Assi, F.; Voros, J.; Textor, M. Interaction of poly(L-lysine)-g-poly(ethylene glycol) with supported phospholipid bilayers. Biophys. J. 2004, 87 (3), 1711-1721. (63) Fang, C.; Fan, Y.; Kong, J. M.; Gao, Z. Q.; Balasubramanian, N. Preparation of nanochain and nanosphere by self-assembly of gold nanoparticles. Appl. Phys. Lett. 2008, 92 (26), -. (64) Kunze, A.; Svedhem, S.; Kasemo, B. Lipid Transfer between Charged Supported Lipid Bilayers and Oppositely Charged Vesicles. Langmuir 2009, 25 (9), 5146-5158. (65) Jones, J. D.; Thompson, T. E. Spontaneous Phosphatidylcholine Transfer by Collision between Vesicles at High Lipid-Concentration. Biochemistry 1989, 28 (1), 129-134. (66) Jones, J. D.; Thompson, T. E. Mechanism of Spontaneous, ConcentrationDependent Phospholipid Transfer between Bilayers. Biochemistry 1990, 29 (6), 1593-1600.

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extended contact e.g. through electrostatic interactions;64,67-69 (3) contact between the liposomes leads to hemifusion, i.e., fusion of the outer leaflets of both membranes into a single connected membrane.70 While mechanism 1 might be governing, in the absence of zirconium (a slow overnight recovery is observed in this case), processes based on the direct exchange of lipids in close vicinity are likely to play a key role for the case where Zr4þ is connecting the vesicles through a larger boundary. This is also widely supported by the observation that in the presence of zirconium, diffusion occurs from the edges into bleached area and not homogeneously through the entire spot, as it is the case in the absence of the cation. Although vesicle fusion events cannot completely be excluded, we postulate that no extended hemifusion (case 3) or liposome fusion is taking place, since this would translate in fast diffusion with a diffusion coefficient in the same order of magnitude as for a supported phospholipid bilayer (usually in the order of 10-8 cm2/s; see for example ref 59). AFM images (Figure 4) confirm that no complete liposome fusion takes place since no larger liposome aggregates are observed (the diameters were in the order of 100-200 nm) as expected. Additionally, experiments performed with vesicles loaded with a fluorescent protein confirm that the loading is retained within the multilayer and does not diffuse within the structure (Figure 5).

5. Conclusions We presented a simple approach for the formation of threedimensional liposome networks based on high affinity between phosphocholine and tetravalent zirconium ions. QCM-D and OWLS measurements revealed the successful formation of a few hundred nanometer thick viscoelastic adlayer. Addition of zirco(67) Sapuri, A. R.; Baksh, M. M.; Groves, J. T. Electrostatically targeted intermembrane lipid exchange with micropatterned supported membranes. Langmuir 2003, 19 (5), 1606-1610. (68) Wikstrom, A.; Svedhem, S.; Sivignon, M.; Kasemo, B. Real-Time QCM-D Monitoring of Electrostatically Driven Lipid Transfer between Two Lipid Bilayer Membranes. J. Phys. Chem. B 2008, 112 (44), 14069-14074. (69) Reinl, H. M.; Bayerl, T. M. Lipid Transfer between Small Unilamellar Vesicles and Single Bilayers on a Solid Support - Self-Assembly of Supported Bilayers with Asymmetric Lipid Distribution. Biochemistry 1994, 33 (47), 14091-14099. (70) Jahn, R.; Grubmuller, H. Membrane fusion. Curr. Opin. Cell Biol. 2002, 14 (4), 488-495.

11002 DOI: 10.1021/la9047566

nium was shown to increase the contact boundary between the liposomes, a morphological change translating in layer stiffening and in an increase in lipid exchange between the liposomes. The role of liposomes in biosensing, especially for the study of membrane proteins, has been widely recognized over the past years. As demonstrated by a simple biomolecular binding assay, such three-dimensional structures have a potential as sensing interfaces with increased loading capacity and thus sensitivity. Preliminary experiments have also demonstrated that our approach is directly applicable to the construction of liposome multilayers obtained from GPCR containing cell membrane extracts (results not shown). Beyond biosensing, a deeper understanding of the interactions between biomolecules and Zr4þ is likely to be of more basic scientific interest. Despite zirconium’s low toxicity and presence in a variety of animal species in ppb concentrations, its biological role remains still unclear. This is despite the fact that zirconium has been reported, several decades ago, to interact strongly with a variety of functional groups found in biomolecules. Moreover, phosphates, phosphonates, and related chemical groups are not only widely represented among the phospholipids of the cell membrane but do also play a primordial role in a variety of cellular processes. Here, we have tackled several fundamental aspects by describing zirconium-mediated lipid diffusion and tube formation processes. Acknowledgment. The Swiss Commission for Technology and Innovation CTI (Project No. 7241.1 NMPP-NM), the Competence Centre for Materials Science and Technology CCMX, and the ETH Zurich are acknowledged for financial support. The software used for the modeling of OWLS data was kindly provided by C. Picart. Supporting Information Available: QCM-D curve displaying the normalized frequency (ΔFn/n) and dissipation changes (ΔDn) for all overtones, ΔF vs ΔD plot for the formation of vesicle multilayers, and QCM-D curve displaying the normalized frequency ((ΔF3/3) and the dissipation change (ΔD3) for the formation of vesicle multilayers on TiO2. This material is available free of charge via the Internet at http://pubs.acs.org.

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