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* Department of Pharmaceutical Sciences, Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada
Correspondence: Address reprint requests to Peter S. Pennefather, E-mail: p.pennefather{at}utoronto.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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; Lipowsky and Seifert, 1991; Nissen et al., 1999; Keller et al., 2000). However, unless some input of energy is applied, such as by homogenization or Langmuir-Blodgett type processes, the lipid membrane formed on substrate surfaces may be heterogeneous, and their properties hard to predict. For instance, Gao and Huang (1987) showed that membrane formation depends critically on the lipid composition and the size and shape of polymer cores. Major et al. (1997), Kiser et al. (1998, 2000), and De Miguel et al. (2000) needed to cohomogenize phospholipids and hydrogel beads to form a stable encapsulating membrane. Reviakine and Brisson (2000) demonstrated that the ability of liposomes to fuse on mica surfaces depends on vesicle size, surface potential, lipid concentration, and the presence or absence of calcium ions.
In a previous article (Ng et al., 2001), we reported the synthesis of pre-Lipobeads, hydrogel beads with covalently attached phospholipid anchors on the surface. We showed that upon incubation with liposome those hydrophobic anchors drove the formation of a phospholipid membrane on the surface of the pre-Lipobeads (Fig. 1) to form Lipobeads. The manner in which anchors attract liposomes to the hydrogel may be similar to the way that fatty anchors on solid substrates, such as silica, drive the self-assembly of a supported phospholipid membrane by the hydrophobic effect (Plant, 1999).
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; Wagner and Tamm, 2000, 2001; Theato and Zentel, 1999, 2000), the mechanically stable hydrophilic support platform for the lipid membrane in Lipobeads is provided by the crosslinked hydrogel core. Thus, the volume of the hydrophilic compartment accessible to the "inner" leaflet of the membrane is greatly increased.
In this article, we examine the mobility and barrier properties of the Lipobead membrane. The transport of different sized solutes through the membrane is measured to probe the barrier properties of the membrane. Cobalt quenching of fluorescent phospholipids (Lakowicz, 1999) is used to determine the homogeneity of the bilayer. Lateral mobility of lipids within the membrane is studied by fluorescence recovery after photobleaching (FRAP) and compared to other membrane systems. In addition, since the membrane is tethered to the surface of the hydrogel, we also explore how the nature of the support can influence membrane properties. Our long-term goal is to demonstrate that the hydrogel-supported and lipid-anchored Lipobeads can be useful in the study of membrane biology and transmembrane protein properties.
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MATERIALS AND METHODS |
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. Spectroscopic grade chloroform was obtained from Fisher Scientific Canada (Nepean, ON). All other reagents used were analytical grade.
Unilamellar liposome preparation
The selected phospholipids and cholesterol were dissolved in chloroform at known concentrations in the range of 1–5 mg/mL. To evaporate chloroform, air was blown over glass vials containing the solution, which then results in a thin lipid film of the desired composition at the bottom of the vial. HEN buffer (20 mM HEPES, 1 mM EDTA, 160 mM sodium chloride, pH 7.4) was added to the dried film to make the final concentration of lipid 1 mg/mL. The suspension was subjected to three freeze/thaw cycles to promote the formation of larger lipid aggregates. The lipid aggregates were then extruded at 40°C through a polycarbonate membrane, with an etched pore size of 100 nm, using the Avanti Mini-Extruder apparatus (Avanti Polar Lipids). Extruded liposomes, checked by dynamic light scattering (90Plus Particle Size Analyzer, Brookhaven Instruments, Holtsville, NY) before use, had a unimodal distribution with an average particle size of 120 nm and a polydispersity (standard deviation over mean of the distribution) of <0.05.
Lipobead synthesis
The pre-Lipobead was synthesized according to a previously described one-step method in which the phospholipid anchors are covalently attached to the surface of the hydrogel at the same time as the polymerization and crosslinking of the hydrogel (Ng et al., 2001). An aqueous solution of the monomer dimethylacrylamide (DMAA) (1.9 mL 
1.9 g), the crosslinker n,n'-ethylene-bis(acrylamide) (E-BIS) (150 mg), and ammonium persulfate (50 mg) was placed in a round bottom reaction flask along with a hexane:carbon tetrachloride (50 mL:29 mL) organic phase mixture. A 90-mg mixture of surfactant composed of sorbitan monostearate and the specially synthesized anchor (in various ratios ranging from 0 to 100 wt % of the anchors) was then added to the flask and stirred to form a water-in-oil emulsion. N,n,n',n'-tetramethylethylene-diamine was then added to react with ammonium persulfate to generate free radicals and to initiate polymerization. The phospholipid anchors localize at the oil/water interface and become covalently attached to the hydrogel surface upon polymerization. The ratio of the anchor mass to the total mass of the surfactant mixture provided an estimate for the expected surface anchor density on the pre-Lipobead. Assuming that the crosslinker is completely consumed by the polymerization process, the molar ratio of E-BIS to the sum of DMAA and E-BIS was taken as the hydrogel crosslinking density.
Approximately 7 mg of dried pre-Lipobeads were prehydrated with 50 µL of HEN buffer for 15 min, then incubated with 300 µL of a 1 mg/mL suspension of unilamellar liposomes in HEN buffer for 2 h at room temperature. Unbound liposomes were removed by rinsing with HEN buffer and by decanting the supernatant four times. Fusion experiments with fluorescently labeled liposomes showed that the liposomes fuse, upon mixing, with the hydrogel beads and form a uniform phospholipid membrane on the bead surface (Ng et al., 2001).
Fluorescent probe encapsulation and imaging by laser scanning confocal microscopy
Pre-Lipobeads were first immersed in buffer then incubated in a 0.5 mg/mL dextran-tetramethylrhodamine conjugate solution until hydration equilibrium was achieved. At that point, a 1 mg/mL egg phosphatidylcholine (ePC) liposome suspension containing 3.2 wt % of PC-NBD was added and equilibrated with the loaded beads for 2 h at room temperature. Before imaging, the beads were thoroughly washed with buffer to remove unbound liposomes and fluorescent dextrans that were not encapsulated. Crosslinked poly(dimethylacrylamide) beads with no surface anchors were hydrated in the same dextran solution as controls.
Laser scanning confocal microscopy images were obtained using a Model 5.10 Carl Zeiss Axiovert 100-M laser scanning confocal microscope equipped with a C-Apochromat 63x/1.2 NA water immersion lens; a 10x/0.5 NA Fluar lens; an argon laser (488-nm line); a helium/neon laser (543-nm line); a beam splitter, NFT 488/543; and two emission filters, BP505-530 and LP 560. A 120-µm pinhole was used along with the 10x and 63x lens, which generated optical sections of 10 and 1 µm, respectively.
Fluorescence quenching by cobalt ions
A 0.5 M cobalt chloride hexahydrate stock solution in HEN buffer was prepared. A 1-mg/mL liposome suspension containing PC-NBD was allowed to fuse with pre-Lipobeads for 2 h at room temperature. Free liposomes were removed by washing with buffer. The fluorescent intensity of the liposome and Lipobead samples were determined by a Delta Scan spectrofluorometer (Photon Technology International, South Brunswick, NJ), with the excitation monochrometer set at 460 nm. The cobalt solution was added incrementally while stirring, and the average emission from 535 to 545 nm was measured after each cobalt addition at 90° angle of the incident excitation beam. The background fluorescence was determined using a sample containing pre-Lipobeads in buffer and subtracted from the fluorescence intensity of the Lipobead sample. The collected data were fitted to a modified Stern-Volmer model which assumes that a fraction of membrane phospholipids is not accessible to quenching by the externally added cobalt ions (Lakowicz, 1999),
 | (1) |
1); and K is the quenching constant. The parameters K and f were determined by obtaining the best fit of Eq. 1 by linear regression through the data points.
Diffusion coefficients and mobile fractions determined by fluorescence recovery after photobleaching (FRAP)
Lipobeads were prepared from the fusion of ePC/PS/cholesterol/PC-NBD (11:12:1:1) liposomes, and the diffusion coefficient of the fluorescently labeled phospholipids in the Lipobead membrane at 20°C was determined by FRAP. The same laser scanning confocal microscope described above was used to measure the fluorescence intensity of the samples. A 15-mW argon laser was used to bleach the membrane. The bleached membrane width varied among experiments, but was between 3 and 10 µm in all cases. Fluorescence recovery in the bleached area was measured using the same laser at 10% of the maximum transmission level, and there was <10% photobleaching in other parts of the membrane during recovery. A beamsplitter, HFT 488, and a long-pass filter, LP505, were used to isolate the emission.
The diffusion coefficient of the fluorescently labeled phospholipid was determined by finding the values that gave the best fit between the experimental fluorescence intensity time profile and the solution of the transient one-dimensional diffusion model (Baker and Lonsdale, 1974),
 | (2) |
Since the total intensity recovered was the sum of fluorescence contributed by the mobile and immobile fractions and assuming only the fluorescence of the mobile fraction recovered (see Fig. 5),
 | (3) |
 | (4) |
is the fraction unbleached and is equal to 
; Iim(i) is the average fluorescence intensity contributed by the immobile fraction in the bleached area before photobleaching.
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 | (5) |
is the fluorescence intensity recovered at infinite time after photobleaching. |
RESULTS |
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with a different Lipobead formulation made of a polyvinyl alcohol hydrogel core and phosphatidic acid anchors. In that case, the Lipobead membrane restricted flux of both calcium and the indicator dye Fluo-3.
Cobalt ion quenching
Fluorescence quenching experiments by cobalt ions were conducted to reveal the nature of the phospholipid membrane on the Lipobeads. The access of cobalt ions to fluorescently labeled lipids is through hydrophilic pathways in bilayer membranes. Since cobalt ions do not penetrate an intact lipid membrane readily and are known to quench the fluorescence of NBD molecules (Lakowicz, 1999), the addition of excess cobalt to an NBD-labeled, wholly formed liposome will quench fluorescence in the outer membrane leaflet, thereby reducing the fluorescence intensity to 
50% of the initial value.
Fig. 4 A shows fluorescence intensity, normalized as the fraction of initial fluorescence, plotted against the concentration of added cobalt ions for Lipobeads and two control liposome preparations. The same data are replotted according to a modified Stern-Volmer model (Fig. 4 B) which accounts for quencher-accessible and quencher-inaccessible populations of fluorescent probes. The inverse of the y-intercept in Fig. 4 B is indicative of the quenchable fraction of fluorophores. Liposomes made from ePC prepared by extrusion are known to have a bilayer membrane (Macdonald et al., 1991). Fig. 4 A shows that the fluorescence of ePC liposomes asymptotes toward 50% in the presence of excess cobalt ions, whereas the y-intercept of the corresponding line in Fig. 4 B is 1.96 ± 0.15, indicating that two distinct membrane leaflets are present and that, as expected, 50% of all fluorescently labeled phospholipid can be quenched by CO2+. Quenchable fluorophores are located exclusively on the outer membrane leaflet and the quencher-inaccessible fluorophores face the inner liposome core.
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). PS liposomes, consisting of ePC/PS/cholesterol/PC-NBD (11:12:1:1), shows quenching curves that are consistent with 100% of the fluorophores being accessible to CO2+. The curve in Fig. 4 A asymptotes toward F/F0 = 0, whereas the line in Fig. 4 B gives a y-intercept of 0.97 ± 0.02. Lipobeads made by incubating pre-Lipobeads with ePC/PC-NBD (30:1) liposomes show a quenching curve that asymptotes toward F/F0 = 0 (Fig. 4 A), and a Stern-Volmer plot with a y-intercept of 0.86 ± 0.08 (Fig. 4 B), indicating that nearly 100% of the phospholipid fluorophore are accessible to the quencher. Since Lipobead membranes allow transmembrane transport of calcium ions (see above), it is expected that the divalent cobalt ions can also transport through Lipobead membranes and quench the fluorophores in both leaflets of a phospholipid bilayer. The nearly complete quenching observed does not exclude the possibility of multiple bilayers; however, multiple bilayers are unlikely to form since steric repulsion would prevent the close approach of two bilayers, and without additional attractive forces, liposomes are unlikely to fuse with a bilayer-covered Liposome surface. The complete quenching also suggests that lipids in the membrane are not arranged in aggregated or globular constructs that may prevent CO2+ access.
Phospholipid mobility in the Lipobead membrane and the influence of substrate
The time course of fluorescence intensity for a typical FRAP experiment is shown in Fig. 5 A; the asymptote of the curve is then used to determine the fraction of mobile phospholipids. The fluorescence recovery of the mobile fraction is then replotted in Fig. 5 B as suggested by the form of Eq. 2, and the diffusion coefficient of the mobile fraction is extracted from the slope of the line. Fig. 6 A shows that as the anchor density on the pre-Lipobead surface increases, both the lipid diffusivity and the fraction of mobile phospholipid of the Lipobead membrane decrease slightly. In contrast, Fig. 6 B shows that increasing the hydrogel crosslinking density only has a marginal effect on diffusivity and mobile fraction. The diffusivity of PC-NBD in the Lipobead membrane at 20°C ranges from 0.30 to 5.0 x 10–10 cm2/s over the range of experimental variables examined.
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DISCUSSION |
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); therefore any defect in the membrane may be <6.7 nm2 in area, assuming a circular hydrodynamic cross section. The presence of hydrophobic anchors on the hydrogel seems to promote lipid fusion and a certain degree of bilayer healing that facilitates the formation of a coherent bilayer, whereas bilayers supported on unmodified hydrophilic polymers were reported to be patchy and defective (Kühner et al., 1994; Reviakine and Brisson, 2000). The ability of Lipobeads to encapsulate preloaded content and either prevent or modulate their release needs further development but points to potential drug delivery, antigen presentation, and diagnostic applications.
The quenching experiments showed that the fluorescent phospholipids in the Lipobead membrane are all accessible to cobalt quenching. The membrane is likely composed of two leaflets of phospholipids in a bilayer configuration that permit cobalt transport, as multiple bilayers stacked on top of one another would lead to protection of some of the fluorescent phospholipid from quenching by cobalt. Previous attempts to determine the number of phospholipid leaflets on polyvinyl alcohol bead surfaces by dipping experiment using Liposheets also suggested that the formed Lipobead membrane was a bilayer (Jin et al., 1996). Maltodextrin encapsulated liposomes (SMBV) prepared by the homogenization of crosslinked maltodextrin cores with liposomes, characterized by cryoelectron microscopy, demonstrated a 5-nm-thick membrane around the hydrogel and suggested the membrane was also a monobilayer (De Miguel et al., 2000). In contrast to the SMBVs, however, the membrane of Lipobead self-assembles without any homogenization, probably by using the anchors as pinning centers.
The extent of polymerization (yield) achieved during pre-Lipobead synthesis was >90% as determined by performing a mass balance calculation of the reagent added and product produced. This indicates that nearly all reactants, including the anchors, were incorporated into the pre-Lipobeads. Assuming that the reactivity of anchors does not change significantly with changing anchor concentration in the reaction mixture, then the surface density of anchors on pre-Lipobead surfaces can be controlled by varying the fraction of anchors relative to surfactants during bead synthesis. By maintaining the same level of hydrogel crosslinking, while using a larger fraction of anchors in the anchor-surfactant mixture during polymerization, the anchor density on the hydrogel surface increases. As shown in Fig. 6 A, the lateral diffusivity of membrane phospholipid decreases slightly as the fraction of anchors in the surfactant mixture of the reaction medium increases, presumably because a larger fraction of covalently coupled anchor on the Lipobead surface retards phospholipid mobility. The mobile membrane fraction also decreases as the surface anchor density increases.
At a constant anchor/surfactant ratio 1:1 (i.e., 50% anchor in the surfactant mixture) during pre-Lipobead synthesis we expect that 50% of the inner leaflet phospholipid of the Lipobead membrane could be covalently attached to the hydrogel core. With such Lipobead membranes, phospholipid diffusivity decreases as hydrogel crosslinking increases from 2% to 5%, but showed no further decrease as hydrogel crosslinking density increased to up to 15% (Fig. 6 B). The mobile fraction of phospholipid also showed a similar trend, dropping from 80% to 40% as crosslink density increased from 2% to 5%, remained approximately constant with higher crosslink density. One reason for the higher phospholipid mobility at lower crosslink densities may be that the hydrogel core is less rigid and more hydrated when crosslink density is low. Perhaps there are also fewer interactions between the bilayer and more widely spaced polymer chains. Qualitatively, a similar trend was observed by Naumann et al. (2001) who found that lateral diffusivity varied inversely with the amount of lipid-polymer conjugate used in creating a polymer cushion with anchor lipids for supporting a lipid bilayer. However, they showed that at lower concentrations of surface lipid-polymer conjugate anchor (5–30%; the absolute value cannot be compared with our experiment due to different setup), the diffusivities measured were independent of conjugate concentration. They argued that diffusion slows significantly as the polymer density in the cushion layer increases above a certain level, presumably reflecting the possibility of direct interactions between adjacent polymer chains, forming a more rigid network. Our results may indicate that at crosslinking densities above a critical value, further increase in network rigidity has little effect on the already low diffusivity values. It is clear that future development of the Lipobead platform will require more experimentation with anchor structures. In particular, introducing hydrophilic spacers between the polymer core matrix and the hydrophobic anchor molecule may reduce coupling between the bilayer and the core. In addition, use of more hydrophilic polymer cores may increase hydrogel swelling and reduce interactions with the lipid bilayer.
Comparisons of the diffusion coefficients of PC-NBD in the Lipobead membrane determined in this study with values measured by FRAP in other similar supported systems are summarized in Table 1. Differences in diffusivity value may be due to 1), the nature of the underlying support substrate; 2), the fluorescent probe used; 3), the lipid composition of the membrane under study; and 4), the gel-fluid phase transition of the membrane.
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reported that the measured diffusivity of a phospholipid in a polyethylene oxide (PEO) supported membrane was reduced by the presence of lipid-PEO conjugate deposited on the silica surface, despite claims that PEO is a polymer which has near ideal behavior in water and negligible interaction with proteins. It is likely that substantial interaction arises between the headgroups of the phospholipid and the free ends of the polymer. For many of the studies of polymer-supported membrane discussed above, the fluorescent probe used was NBD-PE in which the probe NBD is conjugated to the headgroup of the phospholipid. The probe we used was PC-NBD in which NBD is attached to one of the acyl chains. We attempted to measure the diffusivity of NBD-PE in a 3 mol % ePC membrane and found the fluorescence emission of NBD in water to be weak and insufficient in intensity for FRAP measurement using our instrumentation.
Lipid composition affects the fluid-to-gel transition of a membrane, and thus at temperatures below the transition, the diffusivity of a probe is typically 100-fold lower than at temperatures above the transition. Our FRAP experiments were conducted at 20°C, and the low diffusivity of the PC-NBD probe may indicate a gel phase ordering of the Lipobead bilayer. Differential scanning calorimetry experiments did not show a clear phase transition in the Lipobeads; however, this is not surprising given the presence of cholesterol in the membrane. It is notable that in studying a poly(ethyloxazaline)-supported phospholipid membrane in which the polymer was photocrosslinked to the substrate, Naumann et al. (2002) reported that the membrane remained in the gel phase even when heated to 52°C; their membrane seems to have been stiffened by the polymer support. When the polymer support was not conjugated to the substrate surface, the membrane gel-to-fluid transition was between 27°C and 30°C, and the measured phospholipid diffusivity in the sol state varied between 1 and 18 x 10–8 cm2/s depending on the amount of lipid-polymer conjugate used in the support.
The addition of an ionic phospholipid also affects mobility. The PEO-supported membrane described by Wagner and Tamm (2001) had a measured NBD-PE diffusivity of 6 x 10–9 cm2/s. However, the value dropped to 4 x 10–9 cm2/s when 15% of dioleoylphosphoserine, a common phospholipid found in the cytoplasmic leaflet of cell membrane, was added to the system. Our Lipobead membrane lipid composition was 48% ePC, 48% PS, and 4% cholesterol. This composition was selected in anticipation of experiments with transmembrane receptor constituted proteoliposomes (see Park et al., 2004). Thus, the high fraction of phosphatidylserine used here may have further restricted probe diffusivity.
Another interesting observation from the FRAP measurements was that fluorescence intensity of the bleached area did not recover completely to the prebleached value. The apparent diffusivity measured by FRAP is likely dominated by the fastest diffusing species in the membrane. The fraction of fluorescence not recovered is considered to reflect the immobile or slower diffusing phospholipids in the membrane. In artificial membranes supported at air/water interfaces, only a limited amount of immobile phospholipid is present. In a real cell membrane, on the other hand, membrane phospholipids and proteins show anomalous diffusion, which generally is attributed to the interaction with the underlying cytoskeleton elements (Feder et al., 1996; Schutz et al., 1997; Cherry et al., 1998). Single-particle tracking (Elliott et al., 2003) or image correlation spectroscopy (Rocheleau et al., 2003) may be a better technique to measure diffusion coefficients in such complex, multicomponent, and multicompartment systems.
A three-step mechanism to liposome fusion on a substrate surface has been proposed and partly validated (Lipowsky and Seifert, 1991; Keller and Kasemo, 1998; Nissen et al., 1999). To form a membrane on a substrate surface via liposome fusion, it was shown that liposomes first have to adhere to the substrate surface. By using hydrophobic anchors, the hydrogel surface likely attracts a large amount of liposomes to minimize anchor exposure to water. As the concentration of liposome rises on the hydrogel surface, the liposomes may fuse and rupture. Because the underlying substrate is in proximity, yet yielding and flexible, defects may be healed by a combination of bilayer rolling and spreading. Since the hydrophobic surface is the main driving force for liposome fusion, the self-assembly of a membrane on Lipobead is likely to be relatively unaffected by the size of the beads and the composition of the lipid mixture. The minimum anchor density to effect a complete, self-assembled bilayer needs still to be determined.
A brush-like, noncrosslinked polymer without any hydrophobic anchors on the surface may actually prevent vesicle adhesion and thus lower the effective vesicle concentration on that substrate surface. The flexible polymer chain may also trap intact vesicles and prevent the vesicles from fusing to form a complete bilayer. This may explain why membranes could only form with specific polymer or specific lipid composition on unmodified hydrophilic polymer surfaces (Kiser et al., 1998). Because of the presence of anchors on the surface of pre-Lipobeads, the adhesion wall potential of the anchor-rich hydrogel surface is so strong that the shape, size, and surface charges of the hydrogel surface do not seem to be critical in promoting formation of the supported membrane. In addition, using pre-Lipobeads to form a supported membrane seems to be a more robust strategy, as lipid composition, ionic strength, and the size of liposomes are no longer critical.
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CONCLUSIONS |
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, 2000; Kiser et al., 1998; Wong et al., 1999) which require homogenization to form a membrane, our self-assembly system allows easy and robust incorporation of a phospholipid membrane. The nature of the hydrogel as well as the amount and identity of anchors can be varied because of the unique synthesis scheme. As a result, the ability to make a variety of Lipobead with distinct functionality allows one to examine different membrane-protein properties. Furthermore, the micron size of pre-Lipobeads facilitates the ease of handling and possibly its use as a new platform in high throughput drug screening. We are currently investigating the use of Lipobeads to support and study transmembrane protein binding properties (Park et al., 2004). Submitted on July 2, 2003; accepted for publication November 21, 2003.
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, was composed of ePC/PC-NBD (30:1); PS liposome, 
, was composed of ePC/PS/cholesterol/PC-NBD (11:12:1:1). (A) A plot of the fraction of fluorescence remaining versus cobalt ion concentration. (B) The same set of data fitted with the modified Stern-Volmer model. The inset is an expanded view of the plot near the y-intercept region.