Controlled Self‐Assembly of Vesicles by Electrospray Deposition

The self‐assembly of amphiphilic molecules produces structures of diverse dimensions, encompassing micelles, tubules, lamellae, and vesicles. This study focuses on elucidating the controllability of amphiphilic lipid molecule self‐assembly in size and uniformity to facilitate our understanding of the molecular characteristics that correlate with the functions and attributes of the assembled structures. Electrospray deposition allows micropatterning of lipid molecules in conjunction with a conductive–nonconductive patterned substrate. The solvent in the sprayed mist undergoes evaporation during flight, leading to the deposition of dry lipids exclusively on the conductive regions of the substrate. This process enables homogeneous lipid micropatterning, effectively circumventing the coffee‐ring effect. Subsequent hydration of the lipid pattern triggers the spontaneous formation of a size‐controlled, unilamellar vesicle array on the substrate, spanning an area of a few square millimeters. The vesicles exhibits monodispersity, with a coefficient of variation below 8% for sizes ranging from 5 to 20 μm. The size‐controlled self‐assembly process is adaptable to various lipid compositions, thereby demonstrating that the molecular characteristics manifest in the morphological features appear as phase separation, budding, and curvature of vesicle membranes. The approach further validates its suitability for conducting time‐resolved analyses of molecular transport and ligand binding on the monodispersed vesicle array.

The self-assembly of amphiphilic molecules produces structures of diverse dimensions, encompassing micelles, tubules, lamellae, and vesicles.This study focuses on elucidating the controllability of amphiphilic lipid molecule self-assembly in size and uniformity to facilitate our understanding of the molecular characteristics that correlate with the functions and attributes of the assembled structures.Electrospray deposition allows micropatterning of lipid molecules in conjunction with a conductive-nonconductive patterned substrate.The solvent in the sprayed mist undergoes evaporation during flight, leading to the deposition of dry lipids exclusively on the conductive regions of the substrate.This process enables homogeneous lipid micropatterning, effectively circumventing the coffee-ring effect.Subsequent hydration of the lipid pattern triggers the spontaneous formation of a size-controlled, unilamellar vesicle array on the substrate, spanning an area of a few square millimeters.The vesicles exhibits monodispersity, with a coefficient of variation below 8% for sizes ranging from 5 to 20 μm.The size-controlled selfassembly process is adaptable to various lipid compositions, thereby demonstrating that the molecular characteristics manifest in the morphological features appear as phase separation, budding, and curvature of vesicle membranes.The approach further validates its suitability for conducting time-resolved analyses of molecular transport and ligand binding on the monodispersed vesicle array.
generate monodispersed self-assembled structures (Figure 1).We demonstrate a technique for controlled deposition of dry solutes for micropatterning, departing from conventional liquid-based methods.[22] The enlarged surface area of the sprayed mist facilitates solvent evaporation during flight, ensuring thorough desiccation of the solutes prior to deposition on a substrate, [23,24] thus avoiding the coffee-ring effect.Additionally, a conductivenonconductive micropattern is designed on the substrate to regulate the ESD area, exploiting the inherent characteristic of electrospray to selectively accumulate solutes in conductive regions (Figure 1a).The combination of ESD and the substrate enables homogeneous patterning of amphiphilic molecules.We apply this patterning method to amphiphilic phospholipid molecules and explore the homogeneity of micropatterning as well as the controllability of size and shape in assembled lipid vesicles.Furthermore, we demonstrate the potential for spatiotemporal monitoring of ligand binding and molecular transport through the incorporation of membrane proteins into a set of monodispersed lipid vesicles.

Controlled Self-Assembly of Amphiphilic Lipid Molecules Using ESD
First, we present the homogeneous micropatterning of an amphiphilic molecule through the utilization of ESD for lipid molecules on a conductive-nonconductive patterned substrate (Figure 1, refer to Experimental Section in details).The conductivenonconductive pattern was achieved by fabricating an array of polymer microwells, exposing a substrate consisting of indium tin oxide (ITO)-deposited glass at its base (Figure 1b).Lipid molecules, dissolved in a solvent (a mixture of chloroform/acetonitrile or chloroform/methanol), were sprayed by the application of an electric field of 0.5-2.0kV between the tip of a glass capillary and the ITO surface (Figure 1c).Both bright-field (BF) and fluorescence micrographs illustrate the deposition of the sprayed lipids exclusively onto the conductive ITO within an area of 5 Â 5 mm 2 , but not on the nonconductive polymer region (Figure 1d).It is noteworthy that no deposition was observed at the periphery of the bottom of the wells, plausibly attributed to the charged state of the insulated polymer barrier, which repelled the sprayed lipids. [25,26]In theory, lipids are desiccated upon deposition onto the substrate surface.According to the principles of droplet evaporation, the sprayed mist, composed of submicrometer droplets, undergoes rapid evaporation within a time span of 1 ms.The maximum distance traveled by the mist prior to evaporation is estimated to be less than 500 μm, which is notably shorter than the experimental setup of 20 mm (Text S1, Supporting Information for the theoretical estimation).A representative atomic force microscopy (AFM) image of the lipid pattern is presented in Figure 1e.The trapezoidal shape in the cross-sectional profile provides compelling evidence that the electrospray technique successfully averted the formation of coffee-ring patterns, thus affirming the deposition of desiccated lipids.At each designated location, the apparent lipid volume was evaluated by calculating the product of the area and mean height derived from AFM images (Figure S2, Supporting Information).The distribution of volumes exhibited uniformity across the pattern, with a CV of 6.0% (n = 88; Figure 1e).In contrast, our attempts to uniformly deposit the lipid using a liquidbased method proved unsuccessful, as evidenced by the presence of ring stains (Figure S1, Supporting Information).
Subsequently, we demonstrate the monodispersed selfassembly of lipid vesicles arising from the aforementioned patterns (Figure 2).The introduction of an aqueous solution onto the homogeneous patterns initiated the self-assembly of desiccated lipids, resulting in the formation of uniformly sized vesicles on the substrate (refer to Experimental Section in details).The morphology of these resulting vesicles underwent a transition from dome-like structures (Figure 2a) to spherical entities (Figure 2b), depending on the quantity of deposited lipids; spray durations extended from 120 to 150 s exhibited a tendency for generating spherical vesicles.A vast array exceeding 1000 lipid vesicles was successfully acquired from a pattern as shown in Figure 2c and characterized by an average diameter of 20.2 AE 1.2 μm (CV 5.9%, n > 450, derived from a region featuring unilamellar lipid vesicles).We postulate that the homogeneous patterning ensured the monodispersity of the resulting vesicles.The hydration process started instantaneously upon the introduction of the aqueous solution and was completed within 5 min (see Movie S1, Supporting Information).Initially, numerous small vesicles of a few micrometers or less in diameter emerged on each lipid pattern, which rapidly coalesced to form larger vesicles, eventually resulting in the formation of a single lipid vesicle on each pattern.We consider that a large surface area of deposited lipid particles on the pattern facilitated the faster vesicle formation process compared to the common gentle hydration method.As previously discussed, the lipid pattern was formed through the accumulation of desiccated lipid particles by ESD.

Characterization of Self-Assembled Vesicles
Vesicle size could be precisely regulated by modulating the quantity of deposited lipids through the design of the microwell diameter: an expanded area of the exposed ITO pattern increased the quantity of lipid deposition within each microwell.As shown in Figure 2d-h, the vesicle size increased with increasing the diameter of microwells.The CV for the resulting dome-shaped vesicles remained below 8% (n = 250) across four different diameters ranging from 5 to 20 μm.We found that attaining vesicles larger than 30 μm proved challenging due to the vesicle formation process.Although fusion is a crucial process in the maturation of larger vesicles, as vesicle size increases, their enhanced physical stability poses an obstacle to the fusion process, making it increasingly difficult to achieve successful fusion events.Nevertheless, our approach demonstrated improved size controllability compared to previous studies. [12,14]As an alternative to modulating the microwell diameter, the quantity of deposited lipid was regulated by varying the ESD duration; longer spray durations led to an increase in vesicle size as well as a change in shape, as shown in Figure 2a-b, despite both patterns exhibiting a diameter of 10 μm.
Next, the unilamellarity of the vesicle membrane was confirmed using a previously established methodology based on a discrete distribution of fluorescence intensities. [27,28]Five sets of confocal micrographs capturing dome-shaped vesicles and vesicle-in-vesicles, both containing fluorescently labeled lipids, were acquired for lamellarity analysis.Vesicle-in-vesicles consist of a smaller vesicle enclosed within an outer vesicle.The fluorescence intensities of these two vesicle types were projected onto the x-axis, as depicted in Figure 2i, and histograms were generated from the projected intensities for the vesicle (blue) and vesicle-in-vesicle (red), respectively.Based on the histograms, the first intensity level of the vesicle membrane (blue) overlapped with that of the outer membrane of the vesicle-in-vesicles (red).The second intensity level, representing the combined intensity of the inner and outer membranes of the vesicle-in-vesicles (red), exhibited a discretized distribution with the same increment observed between the first intensity level and the background level.This observation suggests that the single line observed in the confocal micrographs of the vesicles corresponds to a unilamellar bilayer membrane.
To examine the robustness of our approach, we investigated the self-assembly of lipids with various compositions using the ESD on the substrate.The standard conditions involved employing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid and a 1:1 molar ratio mixture of DOPC/1,2-dioleoyl-sn-glycero-3phospho-L-serine (DOPS).These lipids are prominent constituents of both animal and plant cell membranes, [29,30] and the net negative charge of DOPS facilitates the hydration of desiccated lipids. [27]By adjusting the spray voltage between 0.5 and 2.0 kV cm À1 , we achieved a homogeneous lipid pattern for different lipid mixtures.We successfully generated uniform, dome-shaped vesicle arrays using a 9:1 DOPC/DOPS ratio and a 1:1:1 DOPC/DOPS/cholesterol mixture (Figure 3a,b).The CVs for these vesicles were also estimated to be below 8%.Furthermore, we demonstrated the generation of specific patterns using DOPC, DOPS, N-hexadecanoyl-D-erythrosphingosylphosphorylcholine from chicken egg (SM) and cholesterol (Figure 3c-f ).Following vesicle formation, phase separation occurred within these uniform vesicle arrays, leading to the budding of small vesicles. [31,32]Fluorescent lipids were distributed among the liquid-disordered phases of the lipid membrane.The size distribution of these vesicles was not estimated because the liquid-ordered phase was not fluorescently visible and the budding process altered their shape and size over time.The morphology of assembled lipid vesicles is influenced by both the substrate pattern and the properties of the lipid molecules.We introduced elliptical microwells with varying axis lengths to replace the circular ones and used the electrospray technique to deposit three different lipid compositions.The ellipticity of the resulting vesicles upon hydration was examined through confocal micrographs (Figure 4).While a spherical shape was preferred for all lipid compositions, elongated dome-like vesicles were more prominently observed with L-α-phosphatidylcholine from soybean (soy PC) and DOPC/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) compared to DOPC/DOPS.The presence of polyunsaturated acyl chains in soy PC suppressed membrane bending rigidity, which allowed a small curvature radius, that is, the formation of elongated vesicles adhered on the elliptical patterns. [33,34]A previous study theoretically derived that the bending rigidity and curvature radius have a proportional relationship at constant adhesion energy. [35]Ellipsoidal vesicles with DOPC/ DOPE were similarly attributed to low bending rigidity arising from their monounsaturated acyl chains and the conical molecular shape of DOPE, distinct from the cylindrical DOPC, although vesicle formation was less reproducible.Conversely, the net negative charge of DOPS may have caused membrane stretching, thereby impeding the formation of an ellipsoidal shape. [34]

Statistical and Temporal Analyses Using a Monodispersed Vesicle Array
We conducted time-resolved monitoring of molecular transport across a vesicle membrane to explore the advantages of employing monodispersed lipid vesicles for statistical analyses (refer to Experimental Section in details).A monodispersed array of spherical lipid vesicles, akin to the array depicted in Figure 2b, was formed through lipid pattern hydration.The vesicle volume was estimated to be 3.3 AE 0.8 pL (n = 32) based on diameter measurements.The diffusion kinetics of the fluorescent molecule calcein into vesicles via the nanopore-forming protein α-hemolysin (αHL) incorporated into the vesicle membrane are presented in Figure 5a-e.Due to disturbances caused by convection flow during calcein infusion, we selectively analyzed the fluorescence intensity of lipid vesicles that remained on the substrate and did not exhibit calcein permeation prior to αHL introduction (Figure 5a,b).Upon the addition of αHL to the external solution, the fluorescence intensity within the vesicles gradually increased (Figure 5c,d), indicating the successful incorporation of a large number of αHL nanopores into the membrane, enabling calcein diffusion into the vesicles. [36,37]onsidering that nanopore formation occurs exclusively at the outermost membrane due to αHL addition to the surrounding solution of the vesicles, the influx of calcein into the vesicles serves as further evidence of the unilamellarity of the vesicle membrane.Moreover, we observed temporal changes in the variation of fluorescence intensity among vesicles, reflecting temporal differences in calcein influx (Figure 5e).Specifically, for a subset of vesicles, calcein influx commenced immediately after αHL addition, while less influx was observed in others.Evaluation of the CV of calcein influx over time revealed an initial increase followed by a subsequent decrease, eventually reaching a plateau (refer to Figure S3, Supporting Information).This finding indicates that the number of incorporated αHL nanopores varies among vesicles during the initial stages, although the variability diminishes over time.
To evaluate the efficacy of the vesicles in incorporating membrane proteins, we conducted a ligand-binding assay employing the adrenergic receptor β2 (ADRB2) on a dome-shaped vesicle array, akin to the one depicted in Figure 2a (refer to Experimental Section in details).ADRB2-reconstituted vesicles were prepared using the budded virus (BV)-vesicle fusion method. [38,39]ADRB2, expressed in the BV envelope, was incorporated into the vesicle membrane through the process of membrane fusion.Initially, we verified the occurrence of membrane fusion between the BV envelope and the lipid vesicle under an acidic pH condition.Following the fusion process incubated at pH 4.5, the red fluorescence emitted by labeled lipids on the BV envelope was observed on the vesicle membrane (Figure S4a, Supporting Information).Subsequently, the reconstitution of ADRB2 was confirmed by evaluating the binding of a fluorescently labeled agonist to ADRB2 (Figure 5f ).Upon the addition of the labeled agonist, a substantial increase in membrane fluorescence was observed in the ADRB2-reconstituted vesicles compared to the control vesicles lacking ADRB2 (p < 0.01, n = 100; Figure 5g).This result indicates that the agonist specifically bound to ADRB2 on the membrane, and the tertiary structure of the protein around the binding site was preserved following its incorporation into the vesicle membrane.Notably, there was significant variability in fluorescence intensity among the ADRB2-reconstituted vesicles (Figure S4b, Supporting Information), suggesting that the number of fused BV envelopes was not consistent across the vesicles.We also investigated the displacement of the labeled agonist bound to ADRB2 by nonlabeled agonists.As depicted in Figure 5h, the fluorescence intensity decreased over time as the labeled agonist was substituted with a nonlabeled agonist.Distinct temporal profiles were observed for epinephrine and norepinephrine, likely attributable to the higher binding affinity of epinephrine for ADRB2. [40]

Discussion
ESD enabled the deposition of molecules on a surface in a solvent-free state, effectively circumventing the coffee-ring effect (Figure 1e).For liquid-based patterning, the evaporation of a  droplet on a substrate is influenced by the interfacial tension between the solvent and the substrate surface.Typically, desiccation initiates at the periphery of the droplet, resulting in the formation of a ring-shaped residue (Figure S1, Supporting Information). [18]By virtue of the negligible interfacial tension during the ESD process, a wide range of solvents, including aqueous buffers, can be employed for patterning, as in the case of electrospray or electrospinning methods. [41]This adaptability to diverse solvents expands the repertoire of solutes in the ESD method, thereby broadening the spectrum of amphiphilic molecules (including biomolecules) amenable to design.In previous studies, the coffee ring effect was prevented by avoiding liquidbased patterning.For example, a damp lipid film was prepared by adsorbing a nanosized vesicle suspension in liquid, [42] or a lipid pattern was obtained by removing a dried lipid film except for the pattern region using the microcontact stripping technique. [43]n this study, we provided compelling evidence that the inherent characteristics of amphiphilic molecules are reflected in the characteristics of the assembled structures.The presence of specific molecules within a vesicle membrane system induced phase separation and budding phenomena (Figure 3), [31,32] while the molecular attributes dictated the curvature of the assembled vesicles (Figure 4).These findings underscore the potential of monodispersed self-assembly as a means to elucidate the intricate relationship between assembled structures and molecular attributes, encompassing factors such as configuration, charge/dipole distribution, and composition. [44,45]One potential application of this method lies in investigating the localization and phase separation of specific lipids on a vesicle, such as cardiolipin, a diphosphatidylglycerol lipid with two negative charges and four alkyl chains.Cardiolipin is considered to be closely related to the structure and functions of mitochondria and to be localized to membrane regions of large curvature. [46,47]olecular self-assembly achieved through the developed methodology exemplified the advantages of precise control over both size and position.The size of the vesicles was regulated by both the pattern size and the deposition amount of amphiphilic lipid molecules, while the vesicles were assembled directly above the substrate's pattern.The controlled size distribution of the vesicles was exploited for comprehensive analyses, harnessing their monodispersity.For instance, statistical comparisons were performed on vesicle morphologies dependent on the molecular compositions, as discussed previously (Figure 4).The elliptical vesicles were assembled on the lipid patterns with elliptical shapes, where the uniformity of the vesicles played a crucial role in elucidating variations in membrane curvature attributed to the lipid properties.Such statistical comparisons would have been infeasible without size controllability, as the ellipticity was contingent on the vesicle size.Another application capitalizing on precise positioning was the time-course observation of vesicles incorporating membrane proteins (Figure 5).The monodispersed vesicles on the substrate provided a confocal micrograph of a vesicle array at a specific z-plane, enabling simultaneous and time-resolved monitoring of multiple vesicles for quantitative and statistical analyses.The disturbance of vesicles in xy-positions caused by pipetting can be improved by the use of a flow chamber and a syringe pump.Figure 5e shows the influx of a fluorescent molecule into a vesicle array, where the variation in influx across the vesicles exhibited significant temporal changes (Figure S3, Supporting Information).As discussed in the Results, the variability in the process of nanopore reconstitution within the vesicle membranes might account for the observed increase in variation during the early stages.Thus, the analysis utilizing monodispersed vesicles at controlled positions would not only uncover averaged time traces but also shed light on the variations attributed to the molecular characteristics involved in the process.

Conclusion
We investigated the methodology for achieving precise control over the self-assembly of lipid vesicles through the hydration of micropatterned lipid molecules and validated the suitability of the proposed approach through the characterization of individual vesicles generated.The ESD technique in conjunction with a conductive-nonconductive patterned substrate was employed for the homogeneous micropatterning of amphiphilic molecules.The deposition of lipids exhibited a narrow distribution across the attern, facilitating the controlled assembly of lipid vesicles with diameters ranging from 5 to 20 μm.The vesicles derived from individual spots exhibited a high degree of monodispersity, with a CV below 8%.The self-assembly procedure yielded numerous monodispersed vesicles arranged in an area spanning a few square millimeters, thus establishing a lipid vesicle array amenable to comprehensive statistical and timeresolved analyses.We believe that the ability to precisely manipulate the size and position of vesicles holds significant potential for diverse applications in research areas focused on studying the relationships and interactions among vesicles and/or membrane proteins.For instance, the vesicle array could serve as a viable platform for constructing a 2D assembly that mimics a tissue-like structure, [48] or for investigating substance transport between vesicles via lipid tubule connections, [49,50] or for studying distribution and the function of membrane proteins on different membrane curvatures. [46]atterning of a Polymer Thin Film: A conductive-nonconductive patterned substrate was fabricated by processing a polymer thin film on an ITO-coated glass slide (Figure 1b).First, a layer of poly(chloro-pxylylene) with a thickness of 1 μm was deposited on the ITO-glass slide using chemical vapor deposition (PDS-2010, Specialty Coating Systems, Indianapolis, IL, USA).Subsequently, a thin aluminum layer was vapor deposited, followed by the spin coating of positive photoresist S1818G (Shipley Corp, Whitehall, PA, USA) onto the aluminum layer.The polymer film on the ITO-glass slide was patterned by standard photolithography, employing oxygen plasma dry etching to obtain microwells with diameters between 5 and 30 μm. [51] ESD: ESD of lipids onto the aforementioned substrate is schematically illustrated in Figure 1c (see also Figure S5a, Supporting Information).A thin glass capillary, serving as the spray nozzle (15/20 μm inner/outer diameter, Prime Tech Ltd., Ibaraki, Japan), was connected to a gas-tight syringe via a lure-lock needle and affixed to a syringe pump (Pico Plus, Harvard Apparatus, Holliston, MA, USA).This setup facilitated the application of a spray solution containing a total lipid concentration of 0.5 mg mL À1 , dissolved in a mixture of chloroform/acetonitrile (95/5, v/v) or chloroform/methanol (2/1, v/v). 1 wt% of fluorescently labeled lipids were mixed for fluorescence microscopy.The metallic portion of the needle was connected to a high-voltage power source (HJPQ-20P0.25,Matsusada Precision Inc., Kusatsu, Japan), while the ITO electrode was grounded.The distance between the target substrate and the capillary tip was set to 20 mm.Under these conditions, a uniform electrospray was achieved within a voltage range of 0.5-2.0kV cm À1 and a flow rate of 0.5 μL/min.The optimized spray durations were 120 s for domeshaped lipid vesicles and 150 s for spherical lipid vesicles in target areas of 5 Â 5 mm 2 .We varied the spray duration to regulate the deposition amount since time proportionally affects the quantity.The amount of spray was also influenced by the concentration of the spray solution and the flux of the spray.However, these two parameters had a significant impact on the spray mode, necessitating the adjustment of electrospray conditions to achieve a fine mist.Following deposition, the sample was stored under vacuum until further use.It is important to note that appropriate safety precautions must be taken to mitigate electrical shock hazards during this process.

Experimental Section
Lipid Vesicle Formation and Membrane Protein Assays: An array of vesicles was formed from the dried lipid pattern through hydration with a 20 mM sucrose solution or deionized water (Figure S5b, Supporting Information).Note that the infusion of aqueous solution was carefully performed to avoid deformation of the vesicles by flow.Microscopic and topographic images were acquired using a confocal laser scanning microscope system (TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) and AFM (NanoWizard, JPK Instruments AG, Berlin, Germany).The obtained images were processed and analyzed using ImageJ (National Institute of Health, Bethesda, MD, USA) and VHX Viewer5 (Keyence Corp., Osaka, Japan) software.
The transport kinetics of calcein molecules were observed after hydration of the dried lipid (DOPC/DOPS at a 1:1 molar ratio) in a 20 mM sucrose solution.Following the formation of lipid vesicles, a calcein solution was added to the exterior of the vesicles at a final concentration of 2.5 μM.An αHL solution (1 or 2 μL of a 50Â concentrate) was subsequently introduced to obtain a final concentration of 500 nM.A control experiment was conducted without αHL.Timelapse images were acquired using the confocal laser scanning microscope TCS SP5.
The agonist-binding assay with ADRB2-reconstituted lipid vesicles was performed as follows.First, dome-shaped vesicles were hydrated in 20 μL of deionized water.Then, 30 μL of 20 mM sodium acetate buffer was added to adjust the pH to 4.5.Recombinant ADRB2 protein was reconstituted into the vesicles using the BV-envelope fusion method. [35,36]pproximately 2 μg of ADRB2 BVs were infused onto a vesicle array (DOPC/DOPS at a 1:1 molar ratio) at pH 4.5 and incubated for 30 min at 25 °C, leading to BV fusion and ADRB2 reconstitution into the vesicle membrane.The acetate buffer was then replaced to phosphate buffered saline (PBS, pH 7.4).A BODIPY630/650-conjugated ADRB2 agonist was subsequently mixed onto the ADRB2-reconstituted vesicle array at a final concentration of 100 nM and incubated at 25 °C for 30 min.Finally, the agonist was rinsed with PBS.Note that the osmotic balance across the vesicle membrane was considered in the assay to avoid deformation of the vesicles.Agonist binding on the vesicles was observed using the confocal laser scanning microscope TCS SP5 (excitation/emission at 633/640-660 nm).The competitive agonist-binding assay was performed by subsequently adding a nonfluorescent ADRB2 agonist (epinephrine or norepinephrine) at a final concentration of 100 nM.The time course of the reduction in fluorescence intensity was monitored to evaluate agonist replacement on ADRB2-reconstituted lipid vesicles.A control experiment was conducted in the absence of competitive agonists.The fluorescence intensity was quantified from the acquired images.The preparation of the ADRB2-expressed BV-envelope is described in Text S2, Supporting Information.

Figure 1 .
Figure 1.Homogeneous micropatterning of amphiphilic phospholipid molecules by ESD.a) Controlled self-assembly of lipid vesicles in an array format.Micropatterning of lipids on a polymer/ITO-glass substrate is achieved by ESD; hydration of the lipid pattern leads to a vesicle array with a uniform diameter determined by the substrate pattern.b) Patterning of a polymer thin film by standard photolithography.c) Schematic illustration of ESD on the polymer/ITO-glass substrate.The sprayed mist is deposited onto the conductive area.d) BF micrographs before/after lipid deposition onto the substrate (left), and a fluorescence micrograph of a lipid-deposited substrate (right).e) Topographic analysis of a lipid pattern by AFM.The cross-sectional profile (top right), acquired at the dashed line in the left image.The variation in lipid deposition over the pattern (bottom right).The apparent volume was estimated from the AFM images.The solid line shows Gaussian fitting.The polymer film was peeled off before AFM observation.A mixture of DOPC/ DOPS (1:1 molar ratio, 1 wt% Rhod-DOPE) was sprayed d,e).

Figure 2 .
Figure 2. Fluorescence confocal micrographs of lipid vesicles self-assembled from homogeneous micropatterns of phospholipids.a) Images of domeshaped lipid vesicles and b) spherical lipid vesicles on substrates, reconstructed from a series of confocal 2D slices.c) A large array of lipid vesicles of defined size.The polymer pattern was 22.7 μm in diameter.The entire image was pieced together from 4 Â 5 images.Variation in fluorescence intensity across the array is due to photobleaching.d-g) Arrays of vesicles with various sizes obtained by varying the pattern size along with h) histograms showing the vesicle size distribution for each pattern size.Average diameters: 4.2 AE 0.3, 9.7 AE 0.7, 20.4 AE 0.8, and 34.4 AE 2.6 μm, respectively (n = 250; mean AE standard deviation).Blue arrowheads indicate the diameter of the polymer pattern (5.0, 11.2, 22.2, and 33.0 μm, respectively).i) Lamellarity of the vesicle membrane evaluated based on a discrete distribution of fluorescence intensities.The histograms were obtained from the projected intensities of five vesicles (blue) and five vesicle-in-vesicles (red), respectively.The projected intensity is an x-axial projection of fluorescence intensity extracted from a confocal micrograph of a vesicle or a vesicle-in-vesicle (right).The z-plane for the confocal images was set at 6.5 μm above the substrate surface and the other scanning conditions were adjusted equally.The peak at the lowest intensity was attributed to the background level.Lipid patterns of DOPC were hydrated with deionized water c,i), while the patterns of DOPC and DOPS mixture (1:1 molar ratio) were hydrated with 20 mM sucrose solution a,b,d-h).

Figure 5 .
Figure 5. Statistical and temporal analyses using vesicle arrays.a-e) Transport kinetics of calcein through αHL nanopores reconstituted in patterned lipid vesicles.Timelapse micrographs after a) calcein addition, b) αHL addition, and c,d) calcein transport through nanopores (enlarged view of the dashed box in b).Each image is a merged view of the labeled lipid (red) and calcein (green).e) Fluorescence intensity inside lipid vesicles (n = 32, 18.4 AE 1.4 μm diameter).The red line represents the average profile.f-h) Kinetics of agonist binding to ADRB2 reconstituted in dome-shaped lipid vesicles.f ) Typical confocal micrographs of fluorescently labeled agonist (magenta) bound to ADRB2 vesicles (green).BV envelopes with ADRB2 (þADRB2, at pH 4.5) or without ADRB2 (ÀADRB2, at pH 7.0) were added to the vesicles.g) Fluorescence intensity of labeled agonist bound to vesicles (þADRB2) is significantly different (p < 0.01; Welch's test) from the unbound control (ÀADRB2) (n = 100; error bars represent standard deviation).h) Competitive agonist binding assay with ADRB2-reconstituted lipid vesicles (þADRB2).Time course of displacement of labeled agonist by epinephrine (♦), norepinephrine (▪), or negative control (▴) (n = 20; error bars represent standard deviation).