Characterization of Surface-Immobilized Layers of Intact Liposomes

Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095 ... Received January 26, 2004; Revised Manuscript Received March 8, ...
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Biomacromolecules 2004, 5, 1496-1502

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Characterization of Surface-Immobilized Layers of Intact Liposomes Patrick Vermette,*,†,‡ Hans J. Griesser,†,§ Peter Kambouris,† and Laurence Meagher†,‡ CSIRO Molecular Science, Bag 10, Clayton South, VIC 3169 Australia, Cooperative Research Centre for Eye Research and Technology, The University of New South Wales, Sydney, NSW 2052 Australia, and Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095 Australia Received January 26, 2004; Revised Manuscript Received March 8, 2004

Surface-immobilized liposome layers are of interest for various potential applications such as localized drug delivery, but their characterization is challenging. We have employed an AFM method and fluorescent dye release to analyze anchored liposomes. In addition, we studied whether the liposomes are surfacebound solely via specific interaction (NeutrAvidin/biotin) or whether physisorptive binding also plays a role. Liposomes containing PEG-biotin lipids were affinity bound to NeutrAvidin molecules which had been immobilized onto solid supports via three different hydrogel interlayers. After liposome docking, approaching the surface with a colloid probe mounted onto an AFM cantilever showed considerable compression behavior, consistent with expectation based on intact, deformable liposomes but not lipid bilayers, thus showing that disruption of liposomes did not occur upon immobilization onto these support surfaces. Plastic deformation suggestive of liposome disruption on compression was not observed. The kinetics of fluorescent dye release also demonstrated that intact liposomes had been successfully immobilized onto all three supports. Blocking surface-immobilized NeutrAvidin molecules with excess biotin in solution before exposure to liposomes showed that the docking of liposomes was dependent largely but not exclusively on biotin-NeutrAvidin affinity binding, with evidence for some nonspecific physisorption, as the extent of liposome binding onto blocked NeutrAvidin surfaces was appreciably lower than for unblocked surfaces but not zero. Finally, consecutive addition of further NeutrAvidin and liposome layers enabled fabrication of multilayers, and this was clearly seen in AFM compressibility and fluorescent dye release measurements. Introduction Typically, lipid vesicles rupture upon contact with solid surfaces, thus forming a surface-adsorbed bilayer. Under certain conditions and with selected surfaces. however, it has been reported that intact liposomes can be immobilized onto solid substrates.1-9 It is, however, a considerable challenge to verify the presence of intact surface-bound liposomes, and early reports1,2 did not provide sufficient characterization to demonstrate integrity of bound liposomes. The related question also arises whether intact liposomes are bound to support surfaces via specific interfacial interactions or whether they simply physisorb via nonspecific interfacial forces; in fact, it may be postulated that specific binding may be necessary as physisorption above a certain interfacial interaction force may inevitably lead to rupture upon surface contact. The study of these questions requires appropriate methods for the detailed characterization of putative surface* To whom correspondence should be addressed. Prof. Patrick Vermette: Laboratoire de Bioinge´nierie et de Biophysique de l’Universite´ de Sherbrooke, Department of Chemical Engineering and Research Centre on Aging, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada J1K 2R1. Phone: 819-821-8000 ext. 2826. Fax: 819-821-7955. E-mail: Patrick. [email protected]. † CSIRO Molecular Science. ‡ The University of New South Wales. § University of South Australia.

bound liposome layers and their surface/liposome interactions, which was the motivation for several reported studies3-8 as well as the present work. Quartz crystal microbalance (QCM) measurements showed that the binding of immunoliposomes to a hapten-coated quartz crystal was decreased but not completely inhibited by the preincubation of immunoliposomes with “free” hapten,3 suggesting the presence of nonnegligible nonspecific attractive interactions between the hapten-coated liposomes and the solid surface. This accorded with another study of specific and nonspecific liposome attachment, which found that the latter could not be eliminated altogether.4 Evidence for specific binding was also observed when monitoring the adhesion of 100-nm unilamellar biotin-doped vesicles to avidin-coated gold surfaces by QCM, which showed an increased shift in resonance frequency with increasing biotin concentration.5 Another study found that the avidity of a targeted biotinylated liposome for streptavidin-coated ELISA plates was influenced by liposomal lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface.6 It was shown that the formation of domains of tight adhesion between giant biotinylated liposomes and streptavidin-coated surfaces was a function of time.7 More recently, a method based on intact lipid vesicles immobilized on a surface plasmon resonance (SPR) sensor was developed to obtain dissociation equilib-

10.1021/bm049941k CCC: $27.50 © 2004 American Chemical Society Published on Web 04/17/2004

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Figure 1. Schematic drawing (not to scale) of the multilayer construct used for immobilizing intact PEG-biotinylated liposomes onto solid carrier materials via NeutrAvidin binding.

rium constants, Kd, between phosphatidylcholine vesicles and cobra venom phospholipase A2.8 Although these reports provide good evidence for the docking of intact lipid vesicles onto solid substrates, it is nevertheless desirable to develop and apply a surface characterization method that reveals directly the presence of surface-bound liposomes. Such characterization, and study of how they dock, will benefit both fundamental understanding as well as applications-oriented research. This need prompted us to investigate additional and more direct methods for the characterization of surface-bound liposomes, which for this study we immobilized onto polymeric supports via multilayer schemes and NeutrAvidin /biotin binding, as shown in Figure 1. Poly(ethylene glycol) (PEG)-stabilized liposomes were selected because they are usually preferred for therapeutic applications on the basis of their low recognition by opsonizing proteins.9 The fabrication of the multilayer structures has been reported earlier.10-16 A main advantage is their generic nature; the first layer in all cases being a plasma-deposited polymeric thin coating makes these schemes readily transferable to a variety of solid supports, without the need for specific reactive groups on the solid support surface nor extensive re-design and optimization of attachment strategies. This is pertinent to our interest in developing intact layers of surface-bound liposomes loaded with therapeutic agents for local drug delivery applications from biomedical devices; many of the most useful polymeric materials used for biomedical devices and implants, such as polyurethanes, poly(tetrafluoroethylene), and poly(dimethylsiloxane), possess chemically inert or relatively nonreactive surfaces. To verify the intact nature of liposomes immobilized by our routes, we have in this work employed a method based on atomic force microscopy (AFM) but using a colloid-modified tip with which we compress the putative surface-bound liposomes; the compression behavior of liposome layers will differ markedly from that of lipid bilayers (from liposome rupture), thus providing direct evidence. In

addition, we have observed extended release of a fluorescent dye as expected from intact liposomes. Experimental Section Materials. Silicon wafers and 24-well TCPS plates were used as substrates for liposome immobilization. 24-well TCPS plates were used as received. Acetaldehyde (99.5% purity), n-heptylamine (99% purity), poly(ethylenimine) (PEI, 70 KDa), poly(acrylic acid) (PAAC, 250 kDa), sodium cyanoborohydride (NaCNBH3), N-hydroxysuccinimide (NHS), HEPES (99.5%), t-octylphenoxypolyethoxyethanol (Triton X-100), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) (> 99%), cholesterol (CHOL) (>99%), and N-[w-(Biotinoylamino) poly(ethylene glycol) 2000]-1,2distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)-Biotin) (>96%) were purchased from Northern Lipids Inc. (Vancouver, BC, Canada). Biotin-PEG-CO2-NHS (NHSPEG-Biotin) was purchased from Shearwater Polymers, Inc. (Huntsville, AL). 5-(and-6)-Carboxyfluorescein (CF, mixed isomers, 99%) was obtained from Molecular Probes (Eugene, OR) and used as received. Dextran (70 kDa, Sigma-Aldrich, Castle Hill, NSW, Australia) was carboxymethylated using a published procedure.10 NeutrAvidin (ImmunoPure NeutrAvidin Biotin-Binding Protein) and D-Biotin (ImmunoPure D-Biotin) were purchased from Pierce (Rockford, IL). NeutrAvidin is a modified avidin with low nonspecific binding properties (information supplied by manufacturer). Methods Preparation of Buffer. The buffer used to prepare the liposome suspensions contained 10 mM HEPES pH 7.4 and a NaCl concentration that was adjusted to an osmolarity of 290 mOsm, using The Advanced Osmometer, model 3D3

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(Advanced Instruments Inc., Norwood, MA). Milli-Q gradient water (Millipore Australia Pty limited, North Ryde, NSW, Australia) with a resistivity of not less than 18.2 MΩcm was used to prepare the buffer solutions. Immobilization of Liposomes. Procedures for obtaining extruded liposomes have been previously reported.11 The substrate surfaces were activated using plasma (also known as radio frequency glow discharge) polymerization to deposit a polymeric thin film that served as an adhesive interlayer with reactive surface groups. Deposition of thin plasma polymer films was carried out from the vapor of n-heptylamine or acetaldehyde in a custom-built reactor as described elsewhere.10,12-14 The root-mean-squared (RMS) roughness of these plasma polymer films was measured by AFM imaging and found to be approximately 0.3 nm over regions of several square micrometers.15 The PAAC layer was attached directly to the n-heptylamine plasma polymer (HApp) layer using a method previously reported.16 The preparation of the carboxymethyl-dextran (CMD), NHSPEG-Biotin, and NeutrAvidin layers has also been previously reported.10,14 For convenience, the immobilization of liposomes onto these three different hydrogel interlayers is shown in Figure 1. Immobilization of liposomes was performed by incubating the NeutrAvidin surfaces in a 1 mg/cm3 (total lipid concentration) biotinylated-liposome suspension made of DSPC: CHOL:DSPE-PEG(2000)-Biotin (2:1:5 mol %) for 4 h. In the case of the 24-well TCPS plates, 1 cm3 of the biotinylated-liposome suspension was added per well. To remove loosely adsorbed liposomes, the samples were rinsed overnight at room temperature in the HEPES buffer described above. The buffer solution was changed four times. It is important to note that liposomes should be exposed only to iso-osmolar solutions.17 Multilayers of liposomes were fabricated by repeated application of layers of NeutrAvidin and biotinylated liposomes. The term layers is used loosely here as we do not envisage a regular layer structure when adding NeutrAvidin solution followed by additional biotinylated liposomes to the first liposome layer. A control experiment was carried out in order to determine if liposome immobilization was specific or nonspecific. A 1 mg/cm3 (total lipid concentration) liposome suspension containing biotin (DSPC:CHOL:DSPE-PEG(2000)-Biotin; 2:1:5mol % mole ratio) was added to pre-blocked NeutrAvidin surfaces. The binding of DSPE-PEG(2000)-Biotin to NeutrAvidin molecules can be blocked by preexposing NeutrAvidin to excess biotin (10mM D-biotin made in 10mM HEPES buffer). The nonspecific adsorption was then determined by monitoring the total release of a fluorescent dye (carboxyfluorescein), as described below. Atomic Force MicroscopysColloid Probe Force Measurements. AFM was performed with a commercial instrument (Nanoscope Multimode, Digital Instruments, Santa Barbara, CA). The interaction forces between a silica particle and immobilized liposomes layers were measured using the colloid probe method developed by Ducker et al.18,19 In this method, a spherical colloidal particle is attached to the microfabricated AFM cantilever spring via an epoxy adhesive (Epon 1004, Resolution Performance Products, Houston, TX)

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and an XYZ translation stage, providing a surface of known geometry. In our case, the spherical particles were pure silica (diameter 4-5 µm), prepared by a modified Stober method20,21 and obtained from Bangs Laboratories, Inc. (Fishers, IN). To scale the force measurements correctly, the spring constant of the AFM cantilever must be known accurately. This was achieved using the resonance method proposed by Cleveland et al.22 The cantilevers used were gold coated, triangular Si3N4 cantilevers obtained from Digital Instruments Inc. (model NP) with spring constants of 0.076 and 0.155 Nm-1. Conversion of the cantilever deflection curves to plots of the force/radius as a function of separation distance was carried out using a custom designed computer program. In the analysis and scaling of the force profiles, the compliance, or linear region of the deflection curve was used to calibrate both the photodetector and to define zero separation distance. Cleaning protocols used for AFM experiments have been described in detail elsewhere.16 For AFM interaction force measurements, freshly prepared surfaces containing immobilized liposomes were mounted into the AFM apparatus, brought to within a separation distance of 30 µm, injected with a solution of HEPES buffer, and allowed to equilibrate for approximately 1 h. Force curves were then obtained. At least 10 force curves were obtained for each sample at several locations. The diameters of the spherical surfaces were measured using a video camera and screen attached to a high power optical microscope. Stability of Immobilized Liposomes. Measurements of the stability of immobilized liposomes were performed using a technique adapted from the fluorescence self-quenching (FSQ) method.23,24 For these stability experiments, liposomes were produced using the same technique,11 except that the lipids were hydrated in the dark using a solution containing 85 mM CF and 10 mM HEPES at pH 7.4. As before, the osmolarity of this CF solution was determined to be approximately 290 mOsm. Unilamellar vesicles were then produced as described above. Separation of the dye-containing vesicles from nonentrapped CF was achieved by gel chromatography, which involved passage through a 2.5 × 25 cm column of Sephadex G-50 Fine (Amersham Pharmacia Biotech, Castle Hill, NSW, Australia). The column was eluted at room temperature with HEPES buffer at a flow rate of 20 mL/min, which was adjusted using a peristaltic pump connected to the column inlet. The total lipid concentration was determined using a phosphorus assay (Australian Government Analytical Laboratories, South Melbourne, Australia). Briefly, the samples were digested with nitric acid using microwave digestion, and the solutions were analyzed by inductively coupled plasma atomic emission spectroscopy. The total lipid concentration of the liposome suspension collected at the column outlet was adjusted to the desired concentration (i.e., 1 mg/cm3) using HEPES buffer. Liposomes containing CF were immobilized onto NeutrAvidin surfaces (in 24-well TCPS plates) as described above, and the release of CF from liposomes was monitored over time (at 37 °C) using a Perkin-Elmer LS50-B spectrofluorometer (Perkin-Elmer Co., Wellesley, MA). Experiments were performed in 24-well TCPS plates typically as follows: 1 cm3 of the appropriate medium (either

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0.5% (wt./v.) Triton X-100 solution made with Milli-Q water or HEPES buffer) was added to each well containing immobilized liposomes. Immobilized liposomes were incubated at 37 °C under mild shaking. At low concentration and neutral pH, Triton X-100 has been shown to instantaneously disrupt the vesicles and liberate their contents without significant interference with the intrinsic fluorescence of the CF dye (though lower pH or the presence of serum components can affect CF fluorescence intensity).23,24 Pure CF has its absorption maximum (at pH 7.5) at 487 nm, which is not significantly changed by the addition of 0.5% (wt./v.) Triton X-100.23,24 At given intervals, the 1-cm3 solution contained in each well was withdrawn from the wells and added to a 2-cm3 Erlenmeyer flask. The wells were then immediately rinsed with 1 cm3 of HEPES buffer, added with the 1 cm3 incubating solution previously withdrawn, and mixed thoroughly. This step facilitates the removal of CF molecules loosely adsorbed on the walls of the TCPS plates. Finally, 1 cm3 of the resulting solution, which contains 1 cm3 of the incubating solution + 1 cm3 of the rinsing HEPES buffer solution, was added to a 1 × 1 cm quartz fluorometer cuvette (1-cm path length). The fluorescence signal was monitored at 520 nm. Each experiment was done in triplicate. The variation of the CF release with time was calculated by applying eq 1 fraction of CF remaining in vesicles ) 1 - F/FT

(1)

where F is the fluorescence (at 520 nm) measured at any time during the experiment, and FT is the total CF fluorescence determined after disruption of the vesicles with Triton X-100. Results and Discussion In our earlier study,11 the presence of a signal from P in XPS analysis had clearly shown that phospholipids had been immobilized onto AApp:PEI:PEG-Biotin:NeutrAvidin supports, and the samples were very hydrophilic upon removal from the liposome suspension. In the vacuum environment of the XPS instrument, however, liposomes, if present on the surface, would be disrupted and flattened; thus, XPS analysis cannot distinguish whether the intended immobilization had indeed led to a layer of intact liposomes or a lipid bilayer consequent upon liposome disruption. AFM images, on the other hand, showed structures assignable to liposomes but also suggested that the surface was not completely covered by liposomes,11 i.e., a liposome surface concentration well below dense packing, whereas XPS suggested fairly dense packing. This apparent contradiction is likely due to disruption and flattening of liposomes in the vacuum of the XPS unit. The fluorescence kinetics of release of CF clearly verified that the liposomes were immobilized as intact structures as the release behavior after surface binding accords with the release behavior of free liposomes in solution. The data also allow quantification of surface density; on the basis of the concentration of the CF dye loaded into the liposomes (85mM) and the surface area of a well in the 24-well TCPS plates (4.9 cm2), this corresponds to approximately 5

Figure 2. (A) Approaching force vs distance curves and (B) force vs indentation curves of PEG-biotinylated liposome layers docked onto a NeutrAvidin layer (AApp:PEI:PEG-Biotin:Neut) in HEPES buffer.

immobilized liposomes per µm2 or 5 × 108 immobilized liposomes per cm2. Given a liposome diameter of ∼120 nm,25 it is clear that this coverage is submonolayer. This surface density of immobilized liposomes is lower than that reported by Jung et al.,8 which was 1.8 × 1010 liposomes per cm2. However, the value calculated from the fluorescence measurements appears to be significantly lower than the estimate that can be derived from the AFM images,11 which was close to the value of Jung et al.8 It is difficult to determine the surface density of immobilized liposomes because there is no established method known to accurately probe the surface density of intact lipid vesicles immobilized on solid supports. The compression of liposome layers by an approaching silica colloid sphere, mounted on an AFM cantilever, can be quantified via force vs distance curves and theoretical models. For the present purposes, we will simply compare the measured force vs distance curves, as the applicability of models such as the Kelvin-Voigt viscoelastic description is still the subject of ongoing study. Comparing the force vs distance curves measured with a spherical silica particle approaching NeutrAvidin (on hydrogel interlayers) surfaces and putative immobilized liposome surfaces revealed directly the existence of substantial deformation of a “soft” layer, assignable to immobilized liposomes, for the liposomeexposed surfaces but not for the NeutrAvidin surfaces (Figure 2A). The compression of the latter surfaces was restricted to a range of ∼10 nm, as expected for such thin hydrogel coatings (for clarity, only one system is shown in Figure 2A), whereas a single layer of liposomes could be compressed by ∼70-80 nm. Clearly, a lipid bilayer resulting from liposome disruption upon surface contact would not show such compressibility; we would expect to see a force curve very similar to that of the NeutrAvidin surface if a

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lipid bilayer had formed on this surface from approaching liposomes. Hence, such measurements are capable of directly revealing intact or disrupted liposomes on support surfaces. In such compression measurements inherently the problem arises as to where the “zero separation” (D ) 0) is. With the noncompressible NeutrAvidin surface, the point of hard contact is clearly definable, but with the liposome layers we do not know when they are fully compressed. Accordingly, we have plotted all of the force curves obtained with the lipid vesicles with arbitrary x-axis shifts for ease of visual comparison; the distance scale is correct but the origins for the different curves are different and undefined. In Figure 2A, instead of setting zero distance, we set zero force. This protocol has been used in the literature for deformable liquid surfaces.26 It shows clearly that the range of the forces measured was very different in each case. NeutrAvidin on AApp:PEI:PEG-Biotin showed a steep slope in the contact regime, which indicated a high compliance slope and little deformation. The short range of the electrical double layer force observed for NeutrAvidin was due to the high salt concentration used in this experiment. In the force vs distance curve for surfaces with the immobilized liposomes, the less steep slope indicated that the surface layer was deformed. We did not observe any changes in the force vs distance curves with repeated cycles of approach and separation; such changes would indicate plastic deformation associated with disruption of the liposomes. Therefore, we postulate that the immobilized liposomes remained intact upon contact with and compression by the silica colloid surface but were quite compressible. The range of the interaction forces (approximately 70 nm) also confirms that the liposomes were not disrupted upon immobilization onto the solid supports. To better illustrate the compression of the immobilized liposomes, the force vs distance curves were transformed into force vs indentation curves (Figure 2B) in the same way as in previous studies.27,28 It is clearly shown that the addition of additional liposome layers produced a substantially thicker and even “softer” coating, as indicated by the less steep slope with more liposome layers and an increase in the range of the interaction forces (up to approximately 220 nm for 5 “layers”). It is thus clear that immobilized liposomes can be substantially compressed without rupturing. The stiffness of the lipid vesicles could be a function of the curvature of the membrane.29 Do (some of) the liposomes rupture upon surface immobilization, or if not, is the subsequent release rate affected by the surface confinement? When immobilizing CF loaded liposomes, the immediate release of CF was less than 5% of the total loading, indicating immobilization of mostly intact liposomes. The kinetics of CF release is shown in Figure 3. At 37 °C, we found that “free” liposomes showed a leakage of approximately 0.2%/h, whereas for an immobilized liposome layer this leakage rate was approximately 0.5%/h (Figure 3B). This increase is marginal given experimental uncertainties. However, this differs from observations by Rongen et al.2 who found that “free” liposomes showed a leakage of approximately 2%/h; however, for surface-bound liposomes (using biotin-streptavidin docking), the leakage was doubled. The lower leakage rate observed

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Figure 3. (A) Kinetics of carboxyfluorescein (CF) release from liposomes immobilized on the different NeutrAvidin surfaces. (B) Fraction of CF remaining in liposomes immobilized on the different NeutrAvidin surfaces.

in our study might be due to the presence of PEGylated lipids in the liposome formulation. PEGs have been shown to stabilize lipid vesicles and also to affect the release of solute from liposomes. The addition of up to 8 mol % of DSPE-PEG(2000) into liposome made of DSPC:CHOL was found to reduce the permeation of CF into buffer solution.30 On the molecular scale, the course of the release of CF may be complex, but the form of the empirical rate law shown in Figure 3A suggests that the particular path via which the release of CF takes place is a first-order pathway. This suggests that CF was released by diffusion through the bilayer membrane rather than by disruption of the liposomes. As shown in Figure 4, different amounts of liposomes could be immobilized using the three different types of interlayers (PAAC, CMD, and PEG-Biotin) we used to attach NeutrAvidin molecules. In addition, Figure 4 also shows the results obtained when docking liposomes onto NeutrAvidin coatings that had been blocked/deactivated by prior exposure to biotin. Theoretically, this should inhibit liposome docking via specific interaction (if assuming that all NeutrAvidin binding sites can be blocked with solution biotin) and only allow liposome immobilization via nonspecific, physisorptive interfacial forces. Thus, differentiating specific interactions from nonspecific liposome attachment, it becomes clear that the latter could not be fully inhibited on our support surfaces.

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Figure 4. Total encapsulation capacity of surface-bound liposomes carrying carboxyfluorescein. The total encapsulation capacity of the liposomes immobilized on the different NeutrAvidin surfaces was determined after disrupting the surface-bound liposomes with the detergent Triton X-100 and measuring the total fluorescence. The control experiments were carried out by de-activating (pre-blocking) the surface-bound NeutrAvidin proteins with excess D-Biotin.

It is important to appreciate that nonspecific interactions between lipid vesicles and solid surfaces often result in rupture of lipid vesicles upon contact with the surface.31-34 Accordingly, to immobilize intact lipid vesicles onto solid substrates, several studies have suggested that the use of specific interactions (ligand-receptor binding) rather than nonspecific, physicochemical interactions (hydrophobic and electrostatic forces, etc.) is preferable. The present study shows that biotinylated liposomes were immobilized intact (i.e., do not rupture upon contact) on NeutrAvidin coatings prepared in different ways, regardless of the specific chemical composition and properties (such as electrostatic charge) of the hydrogel interlayer. We used hydrogel layers because we speculated that they may give rise to substantially less liposome disruption than solid polymer surfaces; our data and comparison with literature data bear this out. By monitoring the fluorescence, we observed that the liposomes released less than 5% of their content (CF solution) upon adhesion with the NeutrAvidin surfaces. The use of the NeutrAvidin-Biotin specific interaction was also demonstrated to be a suitable strategy for the aggregation of liposomes, which likewise remained intact upon aggregation,25,35-37 and this aggregation can be turned into a scheme for the formation of liposome multilayers by the alternating addition of NeutrAvidin molecules and liposomes. Nonspecific interactions generally contribute to some extent to the affinity between the liposomes and the solid surfaces; even though specific ligand-receptor interactions are the main driving force for the surface immobilization in our cases. Presumably, nonspecific attractive interactions would depend on the mechanical properties of the lipid vesicles and on the surface properties of both the liposomes and the host surfaces. It is clear from our data that nonspecific interaction forces were involved in the adhesion process between the liposomes made of DSPC:CHOL:DSPE-PEG(2000)-Biotin and the different NeutrAvidin surfaces, as shown by monitoring the release of the fluorescent dye, CF, from biotinylated liposome layers immobilized on deactivated NeutrAvidin coatings. Figure 4 shows that even when NeutrAvidin was blocked before immersion of the sample in liposome solution some liposomes were able to

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attach onto the NeutrAvidin coatings. Therefore, specific interactions could be differentiated from nonspecific liposome attachment (which is less efficient), but the results show that liposomes have inherent affinity for protein-coated surfaces. This observation is in agreement with other studies.3,4,38 In the case of the avidin-biotin mediated binding process, it is important to test whether biotinylated liposomes can nonspecifically adsorb onto (strept)avidin coatings, a question which can readily be studied by pre-blocking with a solution of excess “free” biotin. Interestingly, only specific interactions were observed in the aggregation of liposomes using aqueous-phase NeutrAvidin.25 Liposome preparations (not aggregated and aggregated) were observed for two months (at 4 °C) and showed no appreciable increase in the mean liposome diameter, indicating that nonspecific aggregation forces did not interfere, in solution, with the aggregation modulated by NeutrAvidin. This observation indicates that the nonspecific interactions reported in the present study are related to the surface properties of the various hydrogel coatings. Thus, the interactions between the coatings and the liposomes must be interpreted in terms of not only interactions between the liposomes and the NeutrAvidin molecules but also in terms of interactions involving other constituents of the multilayer coating structure. These interactions involve biologically nonspecific, physicochemical forces which at present are not understood owing to the complexity of the multilayer structures we used. One possibility is that there is a finite attractive force between the hydrogel layers and liposomes. It is also important to elucidate in more detail the structure of “multilayers” of intact liposomes, by repeated application of layers of NeutrAvidin and biotinylated liposomes. We previously demonstrated, by ELISA and XPS, that at the NeutrAvidin concentration used during the attachment of the NeutrAvidin onto the different gel interlayers (i.e., PAAC, CMD, and PEG-Biotin) saturation was reached.14 Although it is clear that the repeated application of layers of NeutrAvidin and biotinylated liposomes produced more compliant surfaces (Figure 2) and also allowed the encapsulation of a larger amount of the fluorescent dye (Figure 4), the structure and organization of “multilayered” liposome coatings are not well understood as yet and should not be viewed as clearly defined, discrete layers, particularly as we start from submonolayer coverage of liposomes in the first layer. NeutrAvidin proteins added to the first liposome layer bind to remaining, accessible biotin molecules on the immobilized biotinylated liposomes. Conclusions AFM compression and fluorescent dye release experiments verified the intact nature of liposomes immobilized onto solid supports via three different hydrogel interlayer schemes and biotin/avidin binding. The liposomes had biotinylated PEGlipids incorporated into the vesicle membrane; the resultant biotin moieties on the outer surface of the liposomes were capable of docking onto NeutrAvidin molecules immobilized onto the hydrogel interlayers, which were designed to provide a platform for binding NeutrAvidin molecules while mini-

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mizing nonspecific adsorption events and minimizing the disruption of liposomes upon contact with the surface. Using an AFM cantilever modified by attachment of a silica colloid, compression curves indicative of soft, compressible layers of a size expected of intact liposomes were observed, as opposed to the abrupt force curves one would see with lipid bilayers formed upon liposome disruption. Comparison of the release characteristics of fluorescent dye from liposomes in solution and liposomes bound to the three different hydrogel interlayers showed that the release characteristics were little affected, indicating that there was minimal disruption of the liposomes upon surface binding. The release rate of the surface-bound liposomes is ∼2.5 times that of liposomes in solution but again independent of the mode of attachment. By blocking surface-immobilized NeutrAvidin molecules with excess biotin in solution before exposure to liposomes, we investigated whether the docking of liposomes was dependent exclusively on biotin-NeutrAvidin affinity binding. Results show that this is not completely so, although the extent of binding onto blocked NeutrAvidin surfaces was appreciably lower. Thus, there exists a physicochemical, attractive force of nonnegligible magnitude between PEGylated liposomes and hydrogel surfaces decorated with NeutrAvidin, and this physicochemical binding mechanism can surface-attach liposomes when the biologically specific biotin-avidin mechanism is absent. Acknowledgment. This work was partially supported by the Australian Commonwealth Government under the Cooperative Research Centres Scheme (CRC for Eye Research and Technology) and the FCAR-Que´bec (P.V.). References and Notes (1) Yang, Q.; Liu, X.-Y.; Ajiki, S.-I.; Hara, M.; Lundahl, P.; Miyake, J. J. Chromatogr. B 1998, 707, 131-141. (2) Rongen, H.; van Nierop, T.; van der Horst, H.; Rombouts, R.; van der Meide, P.; Bult, A.; van Bennekom, W. Anal. Chim. Acta 1995, 306, 333-341. (3) Yun, K.; Kobatake, E.; Haruyama, T.; Laukkanen, M.-L.; Keinanen, K.; Aizawa, M. Anal. Chem. 1998, 70, 260-264. (4) Liebau, M.; Bendas, G.; Rothe, U.; Neubert, R. Sens. Actuators B 1998, 47, 239-245. (5) Pignataro, B.; Steinem, C.; Galla, H.; Fuchs, H.; Janshoff, A. Biophys. J. 2000, 78, 487-498. (6) Redelmeier, T.; Guillet, J.-G.; Bally, M. Drug DeliVery 1995, 2, 98109. (7) Albersdorfer, A.; Feder, T.; Sackmann, E. Biophys. J. 1997, 73, 245257. (8) Jung, L.; Shumaker-Parry, J.; Campbell, C.; Yee, S.; Gelb, M. J. Am. Chem. Soc. 2000, 122, 4177-4184.

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