Surface-Bound Cytomimetic Assembly Based on Chemoselective and

Oct 12, 2010 - sively for biosensors and functional and biocompatible bioma- terial surface engineering applications (5, 6). However, in most cases, g...
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Bioconjugate Chem. 2010, 21, 1994–1999

Surface-Bound Cytomimetic Assembly Based on Chemoselective and Biocompatible Immobilization and Further Modification of Intact Liposome Yong Ma, Hailong Zhang, and Xue-Long Sun* Department of Chemistry, Cleveland State University 2121 Euclid Avenue, Cleveland Ohio 44115, United States. Received May 5, 2010; Revised Manuscript Received September 20, 2010

A surface-bound cytomimetic assembly based on chemically selective and biocompatible immobilization and further modification of intact liposome is described. Liposomes carrying PEG-triphenylphosphine were chemoselectively immobilized onto azide-modified glass slides through Staudinger ligation, followed by modification with azide-modified lactose as a model biomolecule through Staudinger ligation to afford the surface-bound cytomimetic assembly. The intact liposome immobilized and modified and its protein binding activity were confirmed by fluorescence imaging, fluorescent dye releasing kinetics, and AFM techniques. The resultant surfacebound cytomimetic assembly showed sustained stability and fluorescent dye releasing kinetics and specific protein binding activity. The reported method provides a robust platform for preparation of a complex immobilized liposome system with multifunctional components, which mimics the cell surface in both geographical and content features and thus will find important biomedical applications.

INTRODUCTION Cell surface biomolecules, especially glycans, have become attractive biomimetic targets for potential biomedical applications, since they act as receptors for a variety of protein ligands and are involved in a wide range of biological processes, including immune recognition events (1) and virus and bacterial invasion (2), as well as tissue growth and repair (3). On the other hand, cell surface glycosylated molecules play a significant role in maintaining the nonadhesive/nonthrombogenic property of the native intravascular luminal wall (4). Therefore, mimicking cell surface glycan structure by immobilizing glycan onto solid surface, namely, glycosurface has been explored extensively for biosensors and functional and biocompatible biomaterial surface engineering applications (5, 6). However, in most cases, glycans are directly conjugated onto the solid surfaces, which lack cell surface glycoconjugates’ three-dimensional (3D) structure properties and cell lipid membrane’s supplement effect and thus showed lower activity in both affinity and selectivity points and had limited success. For cell surface mimetic engineering, surface chemistry characterized by the type of cell-binding ligand (carbohydrates, proteins, etc.) and their surface density (7), spatial distribution (8), and conformation (9) have been demonstrated to be important surface cues. Immobilization of liposome onto solid surface has shown great potential in biological and biomedical research and applications (10). This discipline has been inspired by the fact that liposome structurally retains the properties inherent in natural lipid membranes and functionally can serve as a biomembrane model and can encapsulate both hydrophobic and hydrophilic compounds such as drug and gene for delivery applications (11). For example, immobilized liposomes have been investigated as model systems presenting lipid membranes for bioseparation (12), biosensor (13), and nanobioreactor (14) applications. Recently, immobilized liposomes have been explored for medical device surface functionalization for sustained drug elution at the site of interest (15). Crucial to the * Address correspondence to Xue-Long Sun, Ph.D., 2121 Euclid Avenue SI 313, Cleveland, OH 44115. Phone: (216) 687-3919. Fax: (216) 687-9298. E-mail: [email protected].

further development of immobilized liposomes for practical application is their stability and the immobilization methods that allow chemoselective and biocompatible attachment of the intact liposomes with multifunctional components. Early studies using physisorptive entrapment (12, 15) for liposome immobilization suffered from their low reproducibility and stability due to their nonspecific interaction features. In response to these problems, biotin-streptavidin/avidin (13, 14) and oligonucleotide-based specific binding (16), histidine-nickel (17), and covalent immobilization strategies such as using glutaraldehyde (18), disulfide linkage (19), carbamate (20), and carbodiimide (21) chemistry have been explored. The major limitations to all these approaches lie in the fact that the immobilized liposomes tend to unravel, rupture, and fuse with other surrounding liposomes during and after the immobilization process, as well as nonselective and harsh reaction conditions. In addition, to the best of our knowledge, there has been no report on further covalent modification of the immobilized liposome. Herein, a chemically selective and biocompatible immobilization and further modification of intact liposome was investigated for preparation of a surface-bound cytomimetic assembly. The immobilized and functionalized liposome-based system mimics the cell surface in both geographical and content features and thus is expected to have high biological activity. Specifically, liposomes carrying PEG-triphenylphosphine were chemoselectively immobilized onto azide-PEG-modified glass slides through Staudinger ligation and were further modified with azidemodified glycans through Staudinger ligation with triphenyphosphine localized on the immobilized liposome exterior surface (Figure 1). This strategy fulfills the following important design criteria: (1) PEG spacers on both glass side and liposome surface will stabilize the immobilized liposome (22); (2) the azide group tolerates a diverse array of other functionalities in the complex and undergoes chemoselective Staudinger ligation with triphenylphosphine under mild conditions. Several research groups have demonstrated the superior chemoselectivity and biocompatibility of Staudinger ligation, for example, in the recently highlighted metabolic engineering of cell surface (23), microarray (24), and liposome surface functionalization (25) applications. In addition, Staudinger ligation is a catalyst-free

10.1021/bc100220j  2010 American Chemical Society Published on Web 10/12/2010

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Figure 1. Illustration of chemoselective and biocompatible immobilization and further glyco-modification of intact liposome based on Staudinger ligation and PEG chemistry.

coupling reaction and thus does not bring in any residue to the immobilized liposome during the reaction. Therefore, the reported method will provide a robust platform for preparation of a complex immobilized liposome system with multifunctional components for a variety of biological and biomedical research and practices.

cence imaging study, 1 mol % of DSPE-Rodamine was embedded in the lipid membrane as the same liposome preparation above. For liposome size determination, 20 µL of extruded liposome sample was diluted to 2 mL with PBS (pH 7.4) buffer and then measured with a 90plus particle size analyzer (Brookhaven Co., USA).

MATERIALS AND METHODS

Staudinger Immobilization of Liposome. The liposome prepared above was diluted to desired lipid concentration (2.0 mg/mL, total lipid concentration) by PBS (pH 7.4) buffer and then was incubated on an azide-PEG6-functionalized glass slide for 2.0 h at room temperature. Next, MeO-PEG2000-triphenylphosphine was added (20 µg/mL) to quench the azide group left on the glass slide for 2 h at room temperature, followed by removing the glass slide from the reaction solution. Finally, the liposome-immobilized glass slide was washed by rinsing with PBS (pH 7.4) buffer for 2.0 h, repeated three times.

Materials. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (DSPE-Rodamine) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol, 5,6-carboxyfluorescein, and lectin (Arachis hypogae, FITC-labeled) were purchased from Sigma (USA). All other solvents and reagents were purchased from commercial sources and were used as received, unless otherwise noted. Deionized water was used as a solvent in all experiments. DSPEPEG2000-triphenylphosphine, MeO-PEG2000-triphenylphosphine, azide-PEG6-Glass slide, and 2-azideethyle-lactoside were synthesized as described in the Supporting Information. General Measurement. The fluorescence imaging experiments of the immobilized liposomes on a glass slide were performed by using Typhoon 9410 variable mode imager (Amersham Biosciences, USA). A two-channel stage-scanning fluorescent image was used. Channel 1 used 521 nm output (526 nm) from laser blue 2 to selectively excite 5,6-CF encapsulated inside the liposome, and Channel 2 used 571 nm output (580 nm) from laser green to selectively excite Liss Rhod-DSPE embedded in the lipid membrane. AFM imaging experiments of the immobilized liposomes on a glass slide were performed by using PicoPlus 3000 (Molecular Imaging, USA). Liposome Preparation. DSPC (15 mg, 20.4 µmol), cholesterol (4 mg, 10.2 µmol), and DSPE-PEG2000-Triphenylphosphine (0.9 mg, 0.3 µmol) (2:1:1% molar ratio) were dissolved in 3.0 mL of chloroform. The solvent was gently removed on an evaporator under reduced pressure to form a thin lipid film on the flask wall, which was kept in a vacuum chamber overnight. The lipid film was swelled with 2.5 mL of PBS (pH 7.4) buffer containing 5,6-carboxyfluorescein (50 mM for fluorescence imaging or 85 mM for releasing study) to form the multilamellar vesicle suspension, followed by 10 freeze-thaw cycles involving quenching in liquid N2 and then immersion in a 65 °C water bath. The lipid suspension thus formed was extruded through polycarbonate membranes (pore size 600, 200, and 100 nm, gradually) to afford small unilamellar vesicles (SUVs). Nonentrapped CF was separated from the 5,6-CF-encapsulated vesicles with Sephadex G-50 gel chromatography (1.5 × 20 cm column) using PBS (pH 7.4) buffer as eluent. For fluores-

Staudinger Glyco-Functionalization of Immobilized Liposome. The immobilized liposome carrying triphenylphosphine was incubated with 2-azidoethyllactoside in PBS buffer (pH 7.4, 40 mg/mL) at room temperature for 2.0 h, followed by removing the glass slide from the reaction solution. The glass slide was washed by rinsing with PBS (pH 7.4) buffer for 2.0 h, repeated three times. Specific Lectin Binding onto Lactosylated Immobilized Liposome. The lactosylated liposomes immobilized onto a glass slide were incubated with lectin (Arachis hypogae, FITC-labeled, Sigma) in PBS (pH 7.4) buffer solution (50 µg/mL) at room temperature for 2.0 h, followed by removing the glass slide from the reaction solution. The glass slide was washed by rinsing with PBS (pH 7.4) buffer for 2.0 h, repeated three times. The control experiment was performed with lectin-FITC presaturated with free lactose (0.1 mM in PBS (pH 7.4) buffer). Measurement of Release of 5,6-CF from Liposome. 5,6CF released from free liposomes, immobilized liposomes, and glycosylated immobilized liposomes in PBS (pH 7.4) buffer at room temperature was measured over time. The excitation and emission wavelengths of 5,6-CF were 497 and 520 nm, respectively. The variation of the fluorescent intensity with release time was calculated according to the equation below fraction of CF remaining in liposomes ) 1 - F/F0 where F is the fluorescent intensity measured at any time during the experiment and F0 is the total fluorescent intensity measured

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Figure 2. Fluorescent-labeled immobilized liposome (A); fluorescence images of immobilized liposomes obtained for selectively exciting PERhodamine embedded in the liposome membrane (B) and for selectively exciting 5,6-CF encapsulated in the liposome (C). Bar size: 200 µm.

after disrupting liposomes completely with 0.5% Triton X-100 in PBS (pH 7.4) buffer.

RESULTS AND DISCUSSION The synthesis of surface-immobilized liposomes starts with the synthesis of anchor group modified liposomes followed by attachment onto a solid surface carrying a counterpart group of the anchor group on the liposome surface. The anchor lipid DSPE-PEG2000-Triphenylphosphine was synthesized by amidation of commercially available DSPE-PEG2000-NH2 (Avanti Polar Lipid, Inc.) with 3-diphenylphosphino-4-methoxycarbonylbenzoic acid NHS active ester (see the Supporting Information). Small unilamellar vesicles composed of DSPC and cholesterol (2:1 mol ratio) and 1.0 mol % of the anchor lipid in PBS (pH 7.4) buffer were prepared by rapid extrusion through polycarbonate membrane with pore size of 600, 200, and 100 nm diameter, sequentially at 65 °C. In the present study, DSPC was used as a model lipid; different lipids such as unsaturated lipids will be used to mimic the real cellular surface in continued study. This produced small unilamellar vesicles show an average mean diameter of 120 ( 5 nm as determined by dynamic light scattering (see Supporting Information). On the other hand, the azid-PEG6-glass slide was used as model surface and was prepared by amidation of commercially available amine glass slide (Xenopore, Co) with azido-PEG6-COO-NHS (see the Supporting Information). Next, immobilization of the preformed liposomes was performed by incubating azide-PEG-glass slides with preformed liposomes in PBS (pH 7.4) buffer (2 mg/mL of total lipid concentration) for 2.0 h at room temperature, followed by addition of MeO-PEG2000-triphenylphosphine (20 µg/mL) to quench the azide group left on the glass slide for 2 h at room temperature. Finally, removing the glass slide from the reaction solution followed by washing with PBS (pH 7.4) buffer three times to remove the unreacted liposomes afforded immobilized liposomes. To confirm the intact liposome immobilized on the glass slide surface, a fluorescence imaging study was conducted first. DSPE-Rodamine (1 mol %, Avanti Polar Lipid, Inc.) was doped in the liposomes so as to label the lipid membrane, and 5,6carboxyfluorescein (5,6-CF, Sigma) (50 mM) was encapsulated into the liposome so as to image the inner compartment of the liposome (Figure 2A). As a result, detecting either Rhodamine (Figure 2B) or 5,6-CF (Figure 2C) yielded a fluorescence image of the immobilized liposome for an azide-PEG glass slide treated with liposomes carrying anchor group triphenylphosphine, while there was no apparent fluorescence image observed for an azidePEG glass slide treated with liposomes without anchor group triphenylphosphine (data not shown). These results indicated that the immobilization of intact liposomes was achieved through Staudinger ligation. Both fluorescence images were not so uniform. This might be due to non-uniform functionalization of the glass slide surface or the original glass slide surface being bumpy.

To examine whether the immobilization reaction condition could provoke liposome disruption, fluorescent dye releasing kinetics from the liposome during immobilization reaction was investigated. Briefly, we have exposed our standard conditions to the same type of liposomes but with encapsulated selfquenching concentration (85 mM) of 5,6-CF. On the basis of the fluorescence quenching determinations (Figure 3A), we could demonstrate that less than 7.2% leakage of the total loading was triggered during the immobilization reaction (2.0 h) compared with 5.2% leakage for the same liposome during storage without reaction (2.0 h). The leakage percentiles were calculated by destroying liposomes with surfactant 0.5% Triton X-100 in PBS (pH 7.4) buffer to release all 5,6-CF encapsulated after 2.0 h reaction. These results indicated that the immobilization reaction condition is harmless for liposome integrity. Continually, the subsequent fluorescent dye releasing kinetics over time for the non-destroyed immobilized liposomes was examined to verify the stability of the intact immobilized liposome in PBS (pH 7.4) buffer at room temperature. It was found that immobilized liposome showed a constant leakage of approximately 25%/day for three days (Figure 3B), whereas free liposome in PBS (pH 7.4) solution showed a leakage rate of approximately 20%/day (Figure 3C), which is slightly less than that of immobilized liposome. The extended fluorescent dye releasing results further demonstrated that intact liposomes had been successfully immobilized onto the glass slide and showed sustained stability as well. Furthermore, atomic force microscopy (AFM) was used to confirm the intact liposomes immobilized on the glass slide surface, since it is a powerful tool for investigating surfacerelated physical and chemical properties in the nanoscale. Typical AFM images of immobilized liposomes were observed on the azide-PEG glass slide treated with liposomes carrying anchor group triphenylphosphine (Figure 4B), while there were no immobilized liposomes observed for the azide-PEG glass slide treated with liposomes without anchor group triphenylphosphine (Figure 4A). These results further indicated the successful immobilization of intact liposomes via Staudinger ligation. Lactose was chosen as a model glycan for chemoselective modification of the immobilized liposome since it serves as ligand for lectin (26) and cells such as hepatocytes that express galactose-specific binding receptor asialoglycoproteins on the surface (27). Briefly, glyco-modification was performed by incubation of the immobilized liposomes carrying triphenylphosphine on the exterior surface with 2-azidoethyllactoside (28) in PBS buffer (pH 7.4) at room temperature under an argon atmosphere for 2 h. Similarly, fluorescent dye releasing kinetics from the glycosylated immobilized liposome was examined by using liposome encapsulating self-quenching concentrations (85 mM) of 5,6-CF (Figure 3D). It was found that the 5,6-CF releasing kinetics from the glycosylated immobilized liposome is slower than that of its precursor, but slightly faster than that

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Figure 3. 5,6-CF releasing from liposome during the immobilization reaction: before reaction, after reaction, and after adding 20 µL of 0.5% Triton X-100 (A); the 5,6-CF releasing kinetics from immobilized liposome (B); the 5,6-CF releasing kinetics from free liposome (C); and the 5,6-CF releasing kinetics from glycosylated immobilized liposome (D) during storage in PBS (pH 7.4) buffer at rt during 0-72 h, and after adding 20 µL of 0.5% Triton X-100 at the final point.

of free liposome (Figure 5A), which indicated that glycosylated immobilized liposome showed sustained stability. It can be explained by the fact that liposome surface glycans prevent 5,6CF release and stabilize the immobilized liposome. Furthermore, the glyco-stabilizing function was defined by a detergent rupturing experiment (29), in which the immobilized liposomes were ruptured by adding surfactant 0.5% Triton X-100 immediately after immobilization and glycosylation reaction, respectively. As shown in Figure 5B, 90% of encapsulated 5,6CF was released from the non-glycosylated immobilized liposomes in 30 min, while it took 120 min for the glycosylated immobilized liposomes. In other words, the glycosylated immobilized liposomes released encapsulated 5,6-CF much more slowly than its precursor did. This result demonstrated the capacity of the surface glycans to protect liposomes against the action of surfactant, which is consistent with the literature report (29). Next, to determine whether the grafted lactose residues are easily accessible at the surface of the immobilized liposomes, specific lectin binding assay was conducted by incubating lactosylated immobilized liposomes in the presence of β-galactose binding lectin (Arachis hypogae, 120 kDa, FITCLabeled, Sigma) in PBS (pH 7.4) buffer at room temperature for 2.0 h. As shown in Figure 6, the specific binding of fluorescent-labeled lectin was observed on the lactosylated immobilized liposome (Figure 6A), while no fluorescent image was observed on non-glycosylated immobilized liposomes treated with the same lectin (Figure 6B). Furthermore, lectin binding was not observed when lactose-saturated lectin was used (Figure 6C). These results indicated specific binding of lectin

onto the lactosylated immobilized liposomes. Finally, AFM images were investigated to confirm lactosylated immobilized liposomes as well as its lectin binding. As result, there is no apparent size change after lactosylation of the immobilized liposomes (Figure 4C); however, morphological change was observed after lectin binding to the lactosylated immobilized liposomes (Figure 4D). This phenomenon can be explained as the glycosylated immobilized liposome might be ruptured due to specific multivalent binding interaction between grafted lactoses on the immobilized liposome surface and lectin. These results further demonstrated the successful glyco-functionalization of the immobilized liposome and its lectin binding capacity.

CONCLUSION A surface-bound cytomimetic assembly was demonstrated by chemically selective and biocompatible immobilization and further glyco-modification of intact liposome through Staudinger ligation and PEG chemistry. The integrity and stability of the intact glycosylated immobilized liposomes and its specific lectin binding were confirmed by fluorescence imaging, fluorescent dye releasing kinetics, and AFM techniques. The high specificity and efficiency and biocompatible reaction conditions make the present approach an attractive alternative to all currently used protocols for liposome immobilization and modification. Continued study on maximum and optimized density of immobilized liposomes and the glycans on the surface of the immobilized liposomes correlating to its stability and biological activity are under investigation and will be reported accordingly. The reported method will provide a robust platform for preparation of immobilized liposome systems with multifunctional compo-

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Figure 4. AFM image of immobilized intact liposomes onto glass slide: azide-PEG-glass slide incubated with liposomes without DSPE-PEG2000TP (A), azide-PEG-glass slide incubated with liposomes carring PEG2000-TP (B), glycosylated immobilized liposome (C), glycosylated immobilized liposome after lectin binding (D).

Figure 5. 5,6-CF releasing kinetics from liposomes during storage (A): free liposome (green square), immobilized lipsomes (blue dot), glycosylated immobilized liposomes (pink triangle). 5,6-CF releasing kinetics from liposome after adding 20 µL of 0.5% Triton X-100 in PBS (pH 7) buffer (B): immobilized liposomes (blue dot), glycosylated immobilized liposomes (pink triangle).

Figure 6. Fluorescent image of lectin (FITC-labeled Arachis hypogaea) binding onto the immobilized liposome surface: lactosylated immobilized liposomes (A); bare glass slide (B), lactosylated immobilized liposomes incubated with free lactose presaturated lectin (C). Bar size: 200 µm.

nents that can be used in cell surface models and biosensors and biomedical device surface functionalization and will find important use in biological and biomedical research and applications.

ACKNOWLEDGMENT This work was financially supported by the grant from NIH (1R01HL102604-01), American Health Assistance Foundation

(AHAF-H2007027), and the Startup Fund from Cleveland State University. Thanks to Dr.s Su-Tung Yau and Mekki Bayachou at CSU for AFM study. Supporting Information Available: Syntheses of DSPEPEG2000-triphenylphosphine, azide-PEG6-Glass slide, and 2-azidoethyllactoside. This material is available free of charge via the Internet at http://pubs.acs.org.

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LITERATURE CITED (1) Sodetz, J. M., Paulson, J. C., and McKee, P. A. (1979) Carbohydrate composition and identification of blood group A, B, and H oligosaccharide structures on human Factor VIII/von Willebrand factor. J. Biol. Chem. 254, 10754–10760. (2) Karlsson, K. A. (1995) Microbial recognition of target-cell glycoconjugates. Curr. Opin. Struct. Biol. 5, 622–635. (3) Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130. (4) Evans, W. H., and Graham, J. M. (1991) Membrane structure and function, pp 1-86, Oxford University Press, New York. (5) Zhang, Y., Luo, S., Tang, Y., Yu, L., Hou, K., Cheng, J., Zhang, Y., Telyatnikov, V., Sathe, M., Zeng, X. Q., and Wang, P. G. (2003) Studying the interaction of R-gal carbohydrate antigen and proteins by quartz-crystal microbalance. J. Am. Chem. Soc. 125, 9292–92933. (6) Gupta, A. S., Wang, S., Link, E., Anderson, E. H., Hofmann, C., Lewandowski, J., Kottke-Marchant, K., and Marchant, R. E. (2006) Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface. Biomaterials 27, 3084–3095. (7) Michael, K. E., Vernekar, V. N., Keselowsky, B. G., Meredith, J. C., Latour, R. A., and Garcia, A. J. (2003) Adsorption-induced conformational changes in fibronectin due to interactions with well-defined surface chemistries. Langmuir 19, 8033–8040. (8) Rajagopalan, P., Marganski, W. A., Brown, X. Q., and Wong, J. Y. (2004) Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectinand RGD-modified substrata. Biophys. J. 87, 2818–2827. (9) Luk, Y. Y., Kato, M., and Mrksich, M. (2000) Self-assembled monolayers of alkanethiolates presenting mannitol groups are inert to protein adsorption and cell attachment. Langmuir 16, 9604–9608. (10) Christensen, S. M., and Stamou, D. (2007) Surface-based lipid vesicle reactor systems: fabrication and applications. Soft Matter 3, 828–836. (11) Torchilin, P. V. (2005) Recent advances with liposomes as pharmaceutical carriers. Nat. ReV. Drug DiscoVery 4, 145–160. (12) Zhang, Y. X., Aimoto, S., Lu, L., Yang, Q., and Lundahl, P. (1995) Immobilized liposome chromatography for analysis of interactions between lipid bilayers and peptides. Anal. Biochem. 229, 291–298. (13) Ngo, A. T., Karam, P., Fuller, E., Burger, M., and Cosa, G. (2008) Liposome encapsulation of conjugated polyelectrolytes: Toward a liposome beacon. J. Am. Chem. Soc. 130, 457–459. (14) Jung, S. L., Shumaker-Parry, S. J., Campbell, T. C., Yee, S. S., and Gelb, H. M. (2000) Quantification of tight binding to surfaceimmobilized phospholipid vesicles using surface plasmon resonance: Binding constant of phospholipase A2. J. Am. Chem. Soc. 122, 4177–4184. (15) Kallinteri, P., Antimisiaris, S. G., Tsota, I., Kalogeropoulou, C., Karnabatidis, D., and Siablis, D. (2002) Dexamethasone

Bioconjugate Chem., Vol. 21, No. 11, 2010 1999 incorporating liposomes: An in vitro study of their applicability as a slow release delivery system of dexamethasone from covered metallic stents. Biomaterials 23, 4819–4826. (16) Yoshina-Ishii, C., and Boxer, S. G. (2003) Arrays of mobile tethered vesicles on supported lipid bilayers. J. Am. Chem. Soc. 125, 3696–3697. (17) Stora, T., Dienes, Z., Vogel, H., and Duschl, C. (2000) Histidine-tagged amphiphiles for the reversible formation of lipid bilayer aggregates on chelator-functionalized gold surfaces. Langmuir 16, 5471–5478. (18) Yoshimoto, M., Wang, S., Fukunaga, K., Fournier, D., Walde, P., Kuboi, R., and Nakao, K. (2005) Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase. Biotechnol. Bioeng. 90, 231–238. (19) Khaleque, M. A., Okamura, Y., Yabushita, S., and Mitani, M. (2003) Liposome immobilization on polymer gel particles by in situ formation of covalent linkages. Chem. Lett. 32, 416– 417. (20) Lunelli, L., Pasquardini, L., Pederzolli, C., Vanzetti, L., and Anderle, M. (2005) Covalently anchored lipid structures on amine-enriched polystyrene. Langmuir 21, 8338–8343. (21) Morita, S., Nukui, M., and Kuboi, R. (2006) Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins. J. Colloid Interface Sci. 298, 672–678. (22) Allen, T. (1996) Nanocompartments enclosing vesicles, colloids, and macromolecules via interdigitated lipid bilayers. Curr. Opin. Colloid Interface Sci. 1, 645–651. (23) Prescher, A. J., Dube, H. D., and Bertozzi, R. C. (2004) Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877. (24) Stamou, D., Duschl, C., Delamarche, E., and Vogel, H. (2003) Staudinger ligation: a new immobilization strategy for the preparation of small-molecule arrays. Angew. Chem., Int. Ed. 42, 5830–5834. (25) Zhang, H., Ma, Y., and Sun, X.-L. (2009) Chemically-selective surface glyco-functionalization of liposomes through staudinger ligation. Chem. Commun. 3032–3034. (26) Sun, X.-L., Cui, W., Haller, C., and Chaikof, E. L. (2004) Site-specific multivalent carbohydrate labeling of quantum dots and magnetic beads. ChemBioChem 5, 1593–1596. (27) Wu, J., Nantz, M. H., and Zern, M. A. (2002) Targeting hepatocytes for drug and gene delivery: emerging novel approaches and applications. Front. Biosci. 7, d717-d725. (28) Sun, X.-L., Grande, D., Baskaran, S., and Chaikof, E. L. (2002) Glycosaminoglycan mimetic biomaterials. 4. Synthesis of sulfated lactose-based glycopolymers that exhibit anticoagulant activity. Biomacromolecules 3, 1065–1070. (29) Maza, A., Lo´pez, O., Co´cera, M., and Parra, J. L. (2000) Protection of liposomes against triton X-100 by means of the new exopolymer excreted by Pseudoalteromonas antarctica NF3. J. Colloid Surf. A 166, 91–99. BC100220J