Layer-by-Layer Deposition of Vesicles Mediated by Supramolecular

Article pubs.acs.org/Langmuir

Layer-by-Layer Deposition of Vesicles Mediated by Supramolecular Interactions Oliver Roling,†,§ Christian Wendeln,† Ulrike Kauscher,† Patrick Seelheim,§,‡ Hans-Joachim Galla,§,‡ and Bart Jan Ravoo*,†,§ †

Organic Chemistry Institute, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, 48149 Münster, Germany Institute of Biochemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 2, 48149 Münster, Germany § NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Strasse 10, 48149 Münster, Germany ‡

S Supporting Information *

ABSTRACT: Vesicles are dynamic supramolecular structures with a bilayer membrane consisting of lipids or synthetic amphiphiles enclosing an aqueous compartment. Lipid vesicles have often been considered as mimics for biological cells. In this paper, we present a novel strategy for the preparation of three-dimensional multilayered structures in which vesicles containing amphiphilic β-cyclodextrin are interconnected by proteins using cyclodextrin guests as bifunctional linker molecules. We compared two pairs of adhesion molecules for the immobilization of vesicles: mannose−concanavalin A and biotin−streptavidin. Microcontact printing and thiol−ene click chemistry were used to prepare suitable substrates for the vesicles. Successful immobilization of intact vesicles through the mannose−concanavalin A and biotin−streptavidin motifs was verified by fluorescence microscopy imaging and dynamic light scattering, while the vesicle adlayer was characterized by quartz crystal microbalance with dissipation monitoring. In the case of the biotin−streptavidin motif, up to six layers of intact vesicles could be immobilized in a layer-by-layer fashion using supramolecular interactions. The construction of vesicle multilayers guided by noncovalent vesicle−vesicle junctions can be taken as a minimal model for artificial biological tissue.

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INTRODUCTION Due to their property to separate an aqueous inner compartment from a bulk external aqueous medium with a bilayer membrane, vesicles have been proposed as cell precursors involved in the origin of life.1 Since living tissue is a complex and hierarchical assembly of many different cells, it is conceivable to employ vesicles for the constructions of biomimetic soft materials and tissue engineering.2−6 Vesicles are also promising systems for encapsulation and delivery of biologically active substances because they can easily be loaded with a broad spectrum of compounds. Although there are many reports on the immobilization of a single layer of intact vesicles,7−15 only a few examples for three-dimensional systems containing vesicles have been described to date. Liposomes were for instance embedded in polyelectrolyte multilayers which were obtained by layer-by-layer deposition.16,17 Bürgel et al. built up vesicle multilayers by sequential deposition of liposomes and tetravalent Zr4+ ions as linkers. Delivery systems or solid-supported sensors relying on the release of substances from vesicles are often afflicted with weak loading capacities. Bürgel et al. were able to show that their three-dimensional constructs featured increased loading capacities with a fluorescence-based binding assay.18 A different approach is © 2013 American Chemical Society

the multilayer structure introduced by Granéli et al. in which small unilamellar vesicles carrying single-stranded DNA tags on their surface were applied to a gold sensor functionalized with the complementary single-stranded DNA. By alternately adding vesicle dispersions carrying complementary DNA strands, a well-defined multilayered vesicle structure was obtained on the surface.19,20 The key challenge in the preparation of vesicle multilayers is the identification of a noncovalent binding motif that provides robust yet specific interconnections of the vesicles, without affecting the integrity and stability of the vesicles immobilized in the layer. In this paper we present a noncovalent strategy for the preparation of vesicle multilayers that relies on orthogonal interactions of cyclodextrins (CDs) and proteins (see Figure 1). Bifunctional linker molecules 1 and 2 equipped with suitable guest units for CDs and ligands for proteins are used to prepare vesicle multilayers in a layer-by-layer fashion, in which supramolecular complexes function as noncovalent adhesion points for the vesicles. CDs are cyclic oligosaccharides with 6−8 Received: March 24, 2013 Revised: May 28, 2013 Published: July 12, 2013 10174

dx.doi.org/10.1021/la4011218 | Langmuir 2013, 29, 10174−10182

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Figure 1. Schematic representation of the concept of layer-by-layer deposition of vesicle multilayers mediated by supramolecular interactions. Cyclodextrin vesicles decorated with guest molecules, either 1 or 2, display a high density of mannose or biotin, respectively, on their surface. Addition of concanavalin A or streptavidin, respectively, leads to the formation of noncovalent intervesicular links. If applied in succession on a suitable surface, vesicle multilayers can be obtained by layer-by-layer deposition of vesicles and protein.

protein interactions for host−guest-assisted vesicle immobilization and layer-by-layer deposition of vesicles and protein on surfaces. We employed reactive microcontact printing (μCP), which is a straightforward method for the patterning and functionalization of surfaces,35−37 to immobilize mannose thiol or biotin thiol derivatives on glass-supported alkene-terminated self-assembled monolayers (SAMs) by photoinduced thiol−ene addition. These bioadhesive glass slides were used for the sitespecific binding of ConA or streptavidin, respectively, and for the verification of host−guest-assisted tethering of intact vesicles on the protein layers by fluorescence microscopy imaging. In addition, mannose and biotin were immobilized on gold-coated quartz resonators, and the buildup of vesicle multilayers was monitored in real time by quartz crystal microbalance with dissipation monitoring (QCM-D).

glucose units that are capable of forming inclusion complexes with hydrophobic guest molecules (e.g., adamantane, azobenzene, ferrocene, or tert-butylbenzene derivatives).21−24 Ravoo and Darcy reported the first example of amphiphilic CDs, 3, that combine the capability to form vesicles and to act as artificial receptors.25 Guest molecules bind to the surface of the cyclodextrin vesicles (CDVs) and thus change the surface properties of CDVs. We have described CDVs functionalized with DNA, carbohydrates, or peptides in the literature.26−28 In a recent paper, we have also demonstrated that amphiphilic CD 3 can be mixed with phospholipids to provide liposomes with embedded synthetic receptors.29 In the current study, CDVs are decorated with adamantane derivatives which are functionalized with either mannose (1) or biotin (2). CDVs can be decorated with 1 to provide a dense coverage of mannose on the vesicle surface. Concanavalin A (ConA) is a protein belonging to the group of lectins and is capable of selectively binding α-D-mannosides with an association constant Ka on the order of ∼104 M−1 for a 1:1 carbohydrate−lectin complex.30 We have previously shown that CDVs decorated with 1 agglutinate in the presence of ConA due to the formation of multiple (weak) intervesicular links.31Alternatively, CDVs can be decorated with 2 to display a dense coverage of biotin on the vesicle surface. The addition of streptavidin, which can bind up to 4 equiv of biotin, should result in the formation of multiple strong intervesicular links. With an association constant Ka on the order of ∼1014 M−1, the biotin−streptavidin binding is the strongest noncovalent biological interaction known, which is why it has found widespread application for solid-supported sensors.32−34 In this paper, we exploit the noncovalent interactions of CDVs decorated with either 1 or 2 for the formation of vesicle multilayers on an adhesive substrate. To this end, we prepared solid-supported sensors presenting mannose or biotin to compare the suitability of the two ligand−

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EXPERIMENTAL SECTION

General Procedures. Chemicals were purchased from SigmaAldrich, Acros Organics, or Fluka and used without further purification. Silicon wafers were kindly donated by Siltronic AG (Burghausen, Germany). Glass substrates were prepared from IDL microscope slides (Interessengemeinschaft der Laborfachhändler). QCM-D resonators were acquired from LOT-QuantumDesign (Darmstadt, Germany). The adsorbates 16-mercaptohexadecanol and 16-mercapto(8-biotinamido-3,6-dioxaoctyl)hexadecanamide for the preparation of biotinylated QCM-D sensors were purchased from ProSpec. Thiol−ene reactions on alkene-terminated substrates were initiated photochemically using a high-power UV-LED (P8D236, Seoul Semiconductor, 365 nm peak wavelength, 18 nm spectrum halfwidth, 90 mW optical power output) which was supplied by Conrad Electronics. Detailed information concerning the synthesis of biotin derivatives can be found in the Supporting Information. The syntheses of amphiphilic CD,38 mannose thiol,39 tetraethylene glycol thiol,39 and the adamantane−mannose conjugate31 are described in the literature. Vesicle Preparation. For the preparation of unilamellar CDVs, a 1 mM stock solution of amphiphilic β-CD 3 or fluorescently labeled 10175


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amphiphilic β-CD S1 (see the Supporting Information) in CHCl3 was added to a 1 mM mixture of lipid mix (DOPC/DOPE/cholesterol, 2:1:1) in CHCl3 to yield a total volume of 500 μL. By varying the ratio of these stock solutions, vesicles with the desired CD content (CDV10’s with 10% CD and CDV50’s with 50% CD) were obtained. The CHCl3 was evaporated, and the resulting lipid film was suspended in 2.5 mL of HEPES buffer (20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MnCl2, pH 7.5), stirred overnight, sonicated at 40 °C for 15 min, and extruded 11 times through a 100 nm polycarbonate membrane to yield a vesicle dispersion with a size distribution around 100 nm. For the preparation of CDV10’s encapsulating sulforhodamine B, a lipid film was prepared as described before. This lipid film was suspended in 1 mL of a 20 mM solution of sulforhodamine B in HEPES buffer. The vesicle suspension was stirred vigorously overnight and sonicated for 15 min. Sulforhodamine B-encapsulating vesicles were separated from excess fluorophore by size exclusion chromatography (Sephadex G-50 Superfine, Aldrich) with HEPES buffer as the eluent. The vesicle fraction was diluted to a volume of 5 mL to yield a 0.1 mM CDV dispersion. Dynamic Light Scattering (DLS). DLS data were obtained using a Nano ZS Zetasizer (Malvern Instruments Ltd.). All measurements were performed in HEPES buffer using low-volume disposable PMMA cuvettes with a volume of 1 mL. Data were processed by using Zetasizer Software 6.32. Functionalization of QCM-D Resonators. QCM-D resonators were immersed into a freshly prepared mixture of ammonia (25%)/ H2O2 (30%)/deionized water (1:1:5) for 15 min at 70 °C. They were rinsed with deionized water and ethanol and dried in a stream of nitrogen. Then the sensors were exposed to an argon plasma for 10 min. QCM-D sensors for the investigation of the streptavidin−biotin interaction were incubated overnight with a 50:1 mixture of 16mercaptohexadecanol (26 μL, 7.65 mM in CHCl3) and 16mercapto(8-biotinamido-3,6-dioxaoctyl)hexadecanamide (39 μL, 0.1 mM in CHCl3) in CHCl3 (1940 μL). QCM-D-sensors for the investigation of the ConA−mannose interaction were incubated overnight with 2 mL of a 1 mM solution of 11-undecenethiol in ethanol. The alkene-terminated QCM-D-sensors were reacted with a mixture of mannose thiol 5 (40 mM) and α,α-dimethoxy-αphenylacetophenone (DMPA) (20 mM) in diethylene glycol, by placing 20 μL of this reaction mixture on the QCM-D-sensors and capping it with microscopy cover slides. The surfaces were irradiated at room temperature with a 365 nm high-power UV-LED for 90 min while the light source was placed approximately 2 cm above the sensors. After the reaction was stopped, the QCM-D resonators were thoroughly washed with deionized water and ethanol and dried in a stream of argon. The unreacted alkene moieties were then blocked by reacting them with tetraethylene glycol thiol 6. Therefore, DMPA (3 wt %) was dissolved in 6, and a drop of this solution was applied to the substrate, which was then capped with a microscopy cover slide. The substrates were irradiated at room temperature with a 365 nm highpower UV-LED, which was positioned approximately 2 cm above the substrates. After 10 min, the reaction was stopped and the substrates were thoroughly washed with deionized water and ethanol. QCM-D. QCM-D data were obtained by using a Q-Sense E4 with an ISMATEC IPC model ISM 935C pump system. Gold-coated QCM-D resonators QSX 301 with a resonance frequency of 4.95 ± 0.05 MHz were purchased from LOT-QuantumDesign and prepared as described above. All measurements were performed at 20 °C and with flow rates of 100 μL/min, and all solutions were prepared in HEPES buffer. Prior to the binding of ConA (50 μg/mL) or streptavidin (10 μg/mL), the surfaces were blocked with bovine serum albumine (BSA; 3 wt %) in HEPES buffer. Every incubation step was followed by a rinsing step with HEPES buffer. Vesicles (CDV10’s with 10% CD and CDV50’s with 50% CD content; 0.1 mM total amphiphile concentration) were decorated with the adamantane conjugate 1 or 2 (5.0 μM for CDV10’s and 25 μM for CDV50’s to provide 100% external cavity saturation; 5.0 μM for CDV50’s to provide 20% external cavity saturation). SAM Preparation on Glass and Silicon. Glass or silicon slides were cut into pieces of approximately 2.6 × 1.4 cm2 and cleaned by

sonication in pentane, acetone, and deionized water. The substrates were dried in a stream of argon and immersed into a freshly prepared piranha solution (concentrated H2SO4/H2O2 (30%), 3:1). After 30 min the substrates were extensively washed with deionized water and ethanol, dried in a stream of argon, and put into a freshly prepared solution of 11-undecenyltrichlorosilane in toluene (0.1 vol %). After 40 min the substrates were taken out of the solution, washed with deionized water and ethanol, and dried in a stream of argon. PDMS Stamps. PDMS stamps were prepared by casting a 10:1 mixture of poly(dimethylsioxane) and curing agent (Sylgards 184, Dow Corning) on a patterned silicon master. The PDMS mixture was cured at 80 °C overnight. Patterned stamps were cut out with a knife and put into a UV ozonizer (PSD-UV, Novascan Technologies Inc.) for 55 min prior to use. Preparation of a flat stamp was analogous to the described procedure apart from the use of a nonpatterned master. Microcontact Printing of Biotin and Mannose. Oxidized stamps were incubated with 20 μL of a mixture of 4 (40 mM) and DMPA (20 mM) in methanol or 5 (40 mM) and DMPA (20 mM) in ethanol, respectively. After incubation for 30 s, excess reaction mixture was removed in a stream of argon and the stamps were carefully placed on the alkene-modified glass or silicon substrate, respectively. The substrates were irradiated with a 365 nm high-power UV-LED, which was placed approximately 2 cm above the contact area of the stamps and substrates. After 5 min, the reaction was stopped and the substrates were thoroughly washed with deionized water and ethanol. Finally, the surfaces were dried in a stream of argon. The unreacted alkene moieties were saturated with 6. To this end, DMPA (3 wt %) was dissolved in 6 and a drop of this solution was applied to the substrate, which was then capped with a microscopy cover slide. The substrates were irradiated with a 365 nm high-power UV-LED, which was positioned approximately 2 cm above the substrates. After 10 min, the reaction was stopped and the substrates were thoroughly washed with deionized water and ethanol. Finally, the surfaces were dried in a stream of argon to obtain mannose- or biotin-functionalized substrates. Fluorescence Microscopy. Fluorescence microscopy images were obtained by using an inverted fluorescence microscope, CKX41 (Olympus), combined with a Kappa DX L-FW camera (Kappa Optoelectronics GmBH) and a U-RFL-T mercury burner as light source. Kappa Imagebase Control v2.7.2 was used as the data processing software. Preparation of biotin- and mannose-modified substrates for fluorescence microscopy investigation was done with solutions in HEPES buffer. The substrates were blocked with a 3 wt % solution of BSA in HEPES buffer (500 μL, 30 min) and washed with HEPES buffer afterward (2 × 5 min). In the following step the surfaces were incubated with streptavidin (200 μL, 50 μg/mL, HEPES buffer) or ConA (200 μL, 50 μg/mL, HEPES buffer), respectively, for 30 min each. Subsequently, the surfaces were washed with HEPES buffer and incubated with the fluorescently labeled biotin- or mannose-decorated CDVs for 5 min. Afterward the substrates were rinsed with HEPES buffer.

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RESULTS AND DISCUSSION A first proof for the specific host−guest-assisted tethering of intact CDVs via ConA−mannose or biotin−streptavidin interaction was obtained from fluorescence microscopy. To this end, reactive microcontact printing was employed, reacting biotin thiol 4 and mannose thiol 5 (Figure 2) with a glasssupported alkene-terminated SAM to obtain patterned substrates with 10 μm dots that were spaced by 5 μm. X-ray photoelectron spectra, atomic force microscopy images, and contact angles verifying the successful ligation of the bioactive molecules to the substrates can be found in the Supporting Information (Figures S1−S3). To avoid unspecific binding of the proteins, the unreacted alkene groups were blocked with tetraethylene glycol thiol 6 (Figure 2). To further minimize unspecific protein adsorption of ConA and streptavidin, the substrates were incubated with BSA (3 wt % in HEPES buffer) prior to protein application. BSA blocks the surface and can 10176


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Figure 2. Molecular structures of biotin thiol 4, mannose thiol 5, and tetraethylene glycol thiol 6 used for the functionallization of alkeneterminated SAMs via thiol−ene click reaction.

only be replaced by proteins that interact specifically with a ligand on the surface. For fluorescence microscopy analysis vesicles with a CD fraction of 10% (CDV10’s) were prepared. These CDV10’s carried either rhodamine B-labeled β-CD S1 or encapsulated sulforhodamine B (see the Supporting Information, Figure S4). The functionalized glass slides were incubated with CDV10’s (0.1 mM total amphiphile) in the presence of the adamantane mannose linker 1 (5.0 μM) or the adamantane biotin linker 2 (5.0 μM). Fluorescence microscopy analysis clearly shows the specific immobilization of a first layer of CDV10’s with intact bilayer membranes since vesicles labeled with rhodamine CD S1 as well as CDV10’s encapsulating sulforhodamine reproduce the printed patterns (Figures 3 and 4). Both the ConA−mannose and the streptavidin−biotin interactions result in images with fine pattern resolutions. The orthogonal interaction of linker 1 (and comparable carbohydrates) with CDVs is documented in a number of preceding papers.27−29,31 The orthogonal interaction of linker 2 with CDVs was verified using isothermal titration calorimetry and agglutination measurements (see Figures S5 and S6 in the Supporting Information). QCM-D Investigation of Vesicle Multilayers Based on ConA−Mannose Interaction. For a more detailed investigation of the CDV immobilization and to monitor the layerby-layer deposition of vesicles and protein, QCM-D was employed. Sensors displaying mannose were obtained by reacting an alkene-terminated QCM-D sensor with a mixture of mannose thiol 5 and DMPA in diethylene glycol. The mounted mannose-functionalized QCM-D sensors were blocked with BSA prior to the immobilization of ConA (50 μg/mL in HEPES). Figure 5a shows the QCM-D data of two representative sensors to which ConA and mannose-decorated CDV10’s were applied. Incubation with ConA (50 μg/mL) leads to a frequency decrease, whereas the dissipation does not display a significant change, which is indicative of the tethering of a rigid protein layer (Figure 5a, green arrow). Upon application of mannose-decorated CDV10’s (0.1 mM total amphiphile and 5.0 μM linker 1), both the frequency and the dissipation show a drastic decrease or increase, respectively (Figure 5a, red arrow). The dissipation values provide a strong indication of the immobilization of intact CDVs.40 Sauerbrey introduced a relationship between the normalized resonance frequencies and the adsorbed mass (Δm = CQCMΔf n/n), in which Δm represents the adsorbed mass, CQCM is the mass sensitivity constant, and Δf n is the frequency

Figure 3. Fluorescence microscopy images of vesicles containing 10% CD (CDV10’s) immobilized by linker 1 and ConA on glass substrates patterned with mannose via μCP: (a) CDV10’s labeled with rhodamine CD S1; (b) CDV10’s encapsulating sulforhodamine B.

shift of the nth overtone.41 This equation is only valid for thin rigid adlayers that cause no dissipation shift. For the adsorption of sufficiently soft layers with nonzero dissipation shifts, the Sauerbrey equation is no longer valid, and the adsorbed mass cannot be calculated. During the application of ConA to the sensor surface, the Sauerbrey equation is valid, since no change in dissipation is observed. Due to the immobilization of CDVs, an energy-dissipative layer is generated, which results in the invalidity of the Sauerbrey equation, and hence, the normalized resonance frequencies drift apart (Figure 5b). However, it can be deducted from the QCM-D data that the tethering of CDVs to the sensor surface via ConA−mannose is not stable since the rinsing step with HEPES buffer leads to a large mass loss of between 69% and 92% estimated from the increase in frequency upon rinsing with HEPES (Figure 5a). We suggest that the ineffective CDV binding results from the rather weak association constant of the ConA−mannose interaction, which is comparatively low. Figure 5c clearly shows that ConA is released from the sensor surface during rinsing with HEPES buffer (blue arrow). CDVs without added adamantane mannose linker 1 did not bind to the sensor since no significant decrease in frequency was detected (Figure 5c, red arrow). This observation clearly demonstrates that the interaction of CDVs and the sensor surface is exclusively due to the mannose−ConA interaction. The variation of the amount of CDVs that dissociate from the sensor (Figure 5a) is probably related to variations in the homogeneity of the ConA monolayer since a defect-rich protein layer could be more vulnerable for mass loss initiated by rinsing with buffer. By applying a 20 mM mannose solution as a competitor, all 10177


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remaining CDVs were released. The outflow from the QCM-D was subjected to DLS analysis, showing vesicles in the size range of 100 nm, i.e., similar in size to the CDVs that were immobilized on the sensor. CDVs which were not exposed to the QCM-D were taken as a reference. Both dispersions were measured again after two days. While the reference still contained CDVs with a diameter of around 100 nm, the sample from the QCM-D showed bigger aggregates. These clusters can be explained by ConA that was released from the sensor, leading to agglutination of the mannose-decorated CDVs (Figure 5d).31 Since the interaction of mannose-decorated CDV10’s with ConA proved to be insufficient for the immobilization of a second protein layer, the β-CD fraction in the CDVs was increased to 50% (CDV50’s) to enhance the density of recognition units on the vesicle surface. CDV50’s (0.1 mM total amphiphile) were decorated with linker 1 (5.0 μM) so that 20% of the available (external) β-CD cavities would be occupied with mannose. These mannose-decorated CDV50’s were applied to the sensor surfaces, resulting in a frequency decrease and a dissipation increase owing to the adhesion of intact CDV50’s on the sensor surface (Figure 6a, black curve, 1). However, as observed for CDV10’s the stability of the immobilization of CDV50’s via ConA−mannose interaction was insufficient since rinsing with HEPES buffer led to a massive frequency and dissipation change, indicating a substantial detachment of CDVs from the surface. As a negative control experiment vesicles lacking linker 1 were applied, showing no adsorption. Next a mixture of 1 and ConA was flown through the QCM-D, causing a frequency decrease and thus signaling the immobilization of ConA on top of the

Figure 4. Fluorescence microscopy images of vesicles containing 10% CD (CDV10’s) immobilized by linker 2 and streptavidin on glass substrates patterned with biotin via μCP: (a) CDV10’s labeled with rhodamine CD S2; (b) CDV10’s encapsulating sulforhodamine B.

Figure 5. QCM-D data for the immobilization of CDV10’s via ConA−mannose interaction. (a) Fifth resonance frequency overtone of two vesicle samples with adamantane mannose linker 1: top, dissipation data; bottom, frequency data. (b) Third to eleventh resonance frequency overtones of a vesicle sample with linker: top, dissipation data; bottom, frequency data. (c) Frequency data of a negative control (CDV10’s and ConA without linker 1, green arrows), HEPES buffer (blue arrows), CDV10’s with linker 1 (red arrows), and 20 mM mannose solution in HEPES buffer (black arrows). (d) DLS data of CDV10’s which were not immobilized (black, blue) and CDV10’s which were released from the QCM-D sensor surface (red, green). 10178


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could occur. Therefore, CDV50’s carrying 1 were used after the sensor was incubated with a mixture of ConA and 1 again. Once more a frequency decrease and a dissipation increase were observed. However, the data suggest that no vesicle double layer was obtained since the measured frequency values are similar to the ones measured for the first layer (Figure 6a, black curve, 3). In the fourth and last incubation steps with CDV50’s the linker 1 was also added to CDVs in the reference channel, resulting in a frequency decrease and a dissipation increase as expected (Figure 6a, blue curve, 4). The comparison between the data of the CDV50 monolayer in the reference cell with those of the attempted triple-layer construction once more suggests that the ConA−mannose interaction is insufficient for a vesicle multilayer construct since the observed dissipation and frequency values are very similar. Also the systems’ deviation from the Sauerbrey equation should drastically increase with every adlayer, which was not observed (Figure 6b). The measurement was terminated by a rinsing step with a 20 mM mannose solution, which resulted in the release of vesicles (Figure 6a, blue curve). As before the outflow was subjected to DLS analysis. The suspension from the QCM-D showed large aggregates that originate from the repetitive incubation of ConA, 1, and CDV50’s, which led to a cross-linking of vesicles within the monolayer. In summary, we conclude that although the interaction of ConA and mannose is highly specific and although it results in agglutination of CDVs in solution,31 it is not suitable for the layer-by-layer deposition of vesicles due to the unavoidable loss of CDVs and/or ConA upon rinsing with buffer. We note this conclusion is in contrast to a recent paper by Barboiu et al.,42 who prepared multilayers of mannose-functionalized gold nanoparticles by layer-by-layer deposition with ConA. However, the density of mannose and ConA binding sites in the multilayerand therefore the effective multivalency of the systemis much higher in the case of nanoparticles (diameter 15 nm) than in the case of CDVs (diameter 100 nm). QCM-D Investigation of Vesicle Multilayers Based on Streptavidin−Biotin Interaction. For the functionalization of QCM-D gold sensors with biotin, clean sensors were immersed in a solution of 16-mercaptohexadecanol and 16mercapto(8-biotinamido-3,6-dioxaoctyl)hexadecanamide (molar ratio 50:1) to yield a hydrophilic SAM displaying biotin. The mounted biotin-functionalized QCM-D sensors were blocked with BSA prior to the immobilization of streptavidin. Upon application of streptavidin (10 μg/mL in HEPES), the sensors showed a frequency decrease, indicating a mass adsorption to the surface. Since no increase in dissipation was observed, the adsorption of a rigid protein layer took place (Figure 7a, green arrow). The protein adsorption to the surface was stable during rinsing with HEPES buffer, which is proven by the constant frequency. This was followed by flowing CDV10’s decorated with biotin linker 2 (Figure 7a, black curves, red arrow) and vesicle dispersions lacking the biotin linker over the sensors (Figure 7a, blue curve, red arrow). On the basis of the observed plots, no more than about 2% of the CDVs were immobilized by unspecific adsorption. These findings demonstrate that in this case the interaction of CDVs with the sensor is driven exclusively by the highly specific interaction of biotin and streptavidin. For biotin-decorated CDVs a drastic frequency decrease and a dissipation increase were observed, indicating the immobilization of soft matter which was stably tethered to the sensor surface. The obtained dissipation values indicate the immobi-

Figure 6. QCM-D data for the immobilization of CDV50’s via ConA− mannose interaction. (a) Fifth resonance frequency overtone of a vesicle sample decorated with 1 (black curve) and negative control measurement (blue curve): top, dissipation data; bottom, frequency data. (b) Third to eleventh resonance frequency overtones of a vesicle sample with 1: top, dissipation data; bottom, frequency data. Application of vesicle suspensions is marked with red bars, and incubation with a 20 mM mannose solution is marked with blue bars. (c) DLS data of CDV50’s that were not immobilized (blue curve) and CDV50’s which were released from the QCM-D sensor surface (red curve).

vesicle layer. The simultaneous decrease in the dissipation can be explained by cross-linking of the CDVs. Since the second protein layer should be saturated with 1, the following CDV50 solution was applied without the mannose linker. The anticipation that CDV50’s without linker 1 should bind to the protein layer carrying the adamantane mannose linker was proven wrong since no frequency decrease was observed (Figure 6a, black curve, 2). As before, this observation can be explained by taking the comparatively low association constant of the ConA−mannose interaction into account. It is likely that the adamantane mannose linker 1 exposed to the aqueous medium was detached upon rinsing, and hence, no binding 10179


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Figure 7. QCM-D data for the immobilization of CDV10’s via streptavidin−biotin interaction. (a) Fifth resonance frequency overtone of vesicle suspensions with adamantane biotin linker 2 (black curve) and a negative control without linker 2 (blue curve): top, dissipation data; bottom, frequency data. (b) Third to eleventh resonance frequency overtones of a vesicle sample with linker: top, dissipation data; bottom, frequency data. The arrows indicate injection of streptavidin (green), HEPES buffer (blue), vesicles (red), and deionized water (black).

Figure 8. QCM-D data for the immobilization of CDV50’s via biotin− streptavidin interaction. (a) Fifth resonance frequency overtone of two vesicle samples decorated with 2 (black curve) and negative control measurement (blue curve): top, dissipation data; bottom, frequency data. (b) Third to eleventh resonance frequency overtones of a vesicle sample with 2: top, dissipation data; bottom, frequency data. Application of vesicle suspensions is marked with red bars, and incubation with Triton X is marked with black arrows.

shifts of up to 189 × 10−6 were observed. The increasing diversion of the signals for different overtones with every incubation step with CDV50’s also indicates the formation of soft vesicle multilayers (Figure 8b). The CDV50’s for the second and subsequent layers did not require additional linker 2 since instead of the CDVs in this case the adamantane− biotin−streptavidin complex provided linker 2. As Figure 8a shows an “x − 1”-vesicle layer buildup took place in the reference cell. An explanation for this is that the adamantane− biotin−streptavidin complex solution contained excess adamantane−biotin linker. Thereby the first streptavidin layer in the reference cell was functionalized with adamantane upon application with adamantane−biotin−streptavidin complex, and hence, CDV50’s were capable of tethering to the sensor surface. The measurement was terminated by the application of the surfactant Triton X, which dissolved the entire multilayer construct so that the original frequency and dissipation of the sensor were restored (Figure 8, black arrows). In summary, we conclude that multilayers of intact vesicles can be readily obtained by alternating layer-by-layer deposition of biotindecorated CDVs and streptavidin.

lization of intact CDVs. The deviation from the Sauerbrey equation is consistent with this interpretation (Figure 7b). In contrast to the ConA−mannose interaction, no signal change was observed upon rinsing with buffer, which indicates a stable immobilization of CDVs. In cases in which the immobilized CDVs were rinsed with deionized water, the CDVs were exposed to significant osmotic stress, which led to a partial rupture of vesicle membranes and hence a mass loss on the sensor indicated by an increase in the frequency (Figure 5a,b). Taken together, the QCM-D experiments provide consistent evidence that intact CDV10’s can be immobilized on the sensor using the biotin−streptavidin interaction. Finally, the potential of the streptavidin−biotin interaction for the layer-by-layer deposition of vesicles was investigated. To this end, CDV50’s (0.1 mM total amphiphile) were decorated with linker 2 (5.0 μM) so that 20% of the available (external) βCD cavities would be occupied with biotin. These biotindecorated CDV50’s were applied to the streptavidin-functionalized sensors (Figure 8a, black curves) to form a vesicle monolayer. In the reference channel, CDV50’s without linker 2 were flowed over the sensor (Figure 8a, blue curve). This was followed by a rinsing step with HEPES buffer, the application of a premixed adamantane−biotin−streptavidin complex, an iterated rinsing with HEPES buffer, and incubation with CDV50’s without linker. By executing these steps repeatedly, a stable three-dimensional supramolecular construct consisting of x = 6 vesicle layers was generated, which can be derived from the fact that frequency shifts of up to −700 Hz and dissipation

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CONCLUSION In this study we investigated the host−guest-assisted immobilization of vesicles in multilayers using the mannose− ConA and biotin−streptavidin recognition motifs. While both interactions proved to be appropriate for the construction of a monolayer of intact vesicles in a static aqueous environment, as 10180


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verified by fluorescence microscopy analyses, the mannose− ConA interaction did not qualify for the immobilization of vesicles under flow. As a consequence, the construction of vesicle multilayers by layer-by-layer deposition of mannosedecorated vesicles and ConA was not successful. However, QCM-D data obtained for the analogous system based on the biotin−streptavidin interaction clearly indicated that a threedimensional vesicle construct of up to six vesicle layers was achieved. In summary, we could demonstrate that the combination of CD host−guest chemistry and biotin− streptavidin interaction can be developed to a powerful tool for the preparation of vesicle multilayers on solid substrates. These results indicated that it is feasible to prepare tailor-made soft nanomaterials on target substrates using layer-by-layer deposition guided by molecular recognition. Such materials may be considered versatile mimics of artificial tissues.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis of biotin derivatives, surface analysis of substrates prepared via microcontact printing, isothermal titration calorimetry, optical density measurements, and encapsulation experiments with CDVs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the NRW Graduate School of Chemistry for fellowships to O.R. and P.S. Silicon wafers were kindly donated by Siltronic AG. We thank Michael Kurlemann and Benjamin Vonhören for contributing isothermal titration calorimetry and X-ray photoelectron spectroscopy measurements.

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REFERENCES

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