Multivalency at Interfaces: Supramolecular Carbohydrate

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Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection zhenhui qi, Priya Bharate, Chian-hui Lai, Benjamin Ziem, Christoph Böttcher, Andrea Schulz, Fabian Beckert, Benjamin Hatting, Rolf Mülhaupt, Peter H Seeberger, and Rainer Haag Nano Lett., Just Accepted Manuscript • Publication Date (Web): 03 Aug 2015 Downloaded from http://pubs.acs.org on August 3, 2015

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Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection Zhenhui Qi,†,# Priya Bharate,ǁ,‡, # Chian-Hui Lai,ǁ,‡ Benjamin Ziem,† Christoph Böttcher,§ Andrea ⊥ ⊥ Schulz,§ Fabian Beckert, Benjamin Hatting,△ Rolf Mülhaupt, Peter H. Seeberger,*,ǁ,‡ and

Rainer Haag*, † †

Institut für Chemie und Biochemie, Freie Universität Berlin, Takustrasse 3, 14195, Berlin,

Germany ǁ

Biomolecular Systems Department, Max Planck Institute of Colloids and Interfaces, Am

Mühlenberg 1, 14476 Potsdam, Germany ‡

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195 Berlin,

Germany §

Research Center for Electron Microscopy and Core Facility BioSupraMol, Institut für Chemie

und Biochemie, Freie Universität Berlin, Fabeckstr. 36a, 14195, Berlin, Germany ⊥

Freiburg Materials Research Center (FMF) and Institute for Macromolecular Chemistry of the

University of Freiburg, Stefan-Meier-Strasse 31, D-79104 Freiburg, Germany

ACS Paragon Plus Environment

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△

Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany

ABSTRACT: A supramolecular carbohydrate-functionalized two-dimensional (2D) surface was designed and synthesized by decorating thermally reduced graphene sheets with multivalent sugar ligands. The formation of host-guest inclusions on the carbon surface provides a versatile strategy, not only to increase the intrinsic water solubility of graphene-based materials, but more importantly to let the desired biofunctional binding groups bind to the surface. Combining the vital recognition role of carbohydrates and the unique 2D large flexible surface area of the graphene sheets, the addition of multivalent sugar ligands makes the resulting carbon material an excellent platform for selectively wrapping and agglutinating E. coli. By taking advantage of the responsive property of supramolecular interactions, the captured bacteria can then be partially released by adding a competitive guest. Compared to previously reported scaffolds, the unique thermal IR-absorption properties of graphene derivatives provide a facile method to kill the captured bacteria by IR-laser irradiation of the captured graphene-sugar-E. coli complex.

KEYWORDS: Multivalency, host-guest chemistry, thermally reduced graphene oxide, carbohydrates, self-assembly

Multivalency plays a pivotal role in biological processes, such as the adhesion of bacteria or viruses to the cell surfaces.1 In contrast to weak monovalent binding, multivalent interactions result in high specificity and in thermodynamic and kinetic stability. The attachment of multiple


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weakly binding ligands on the surface results in significantly stronger adhesion at interfaces than those that have been produced from monovalent interactions.2 Carbohydrates, as a class of weakly binding ligands for cell surface recognition, require many individual binding events through multivalent carbohydrate–protein interactions.3 These interactions occur during the initial phase of an infection, when the host and bacteria come in contact,4 and during the ensuing immune response.5 Considerable effort has therefore been devoted to constructing synthetic multivalent glycoconjugates with diverse spatial arrangements of ligands that can be used to interfere with the pathogen adhesion process and serve as antibacterial or antiviral agents.6

Scaffolds, such as dendrimers,7 nanoparticles,8 calixarenes,9 and fullerenes,10 offer a zerodimensional (0D) carrier platform for functionalizing carbohydrates. One-dimensional (1D) scaffolds based on peptides,11 carbon nanotubes,12 and self-assembled fibrous nanostructures13 can be used to investigate cell targeting and anti-adhesive properties, whereby glycoconjugate valency and the size of the scaffold-pathogen interface play a key role for effective inhibition of large microorganism particles and for controlling their agglutination.14 As examples, Myongsoo et al. reported that both valency and size increase the density of presented sugars and that the nanofiber length leads to enhanced bacterial cell agglutination.15 Recently, Huang and coworkers discovered that when the length of nanotubes was several micrometers long, the galactosyl nanotubes that had been assembled from pillar[5]arene amphiphiles exhibited a higher affinity to the pathogenic bacteria.16 However, soluble multivalent glycolconjugate two-dimensional (2D) scaffolds that have adequate dimensions for biological pathogens can act as potent but elusive inhibitors.17


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The discovery of graphene has fundamentally changed the field of materials science.18 This micrometer-scaled material provides an unprecedentedly large 2D area of flexible film and has exceptional mechanical, electronic, thermal, and optical properties.19 Graphene is generally described as a single-atom-thick planar sheet of sp2-bonded carbon atoms that are arranged in a 2D honeycomb lattice. The thermally reduced graphene oxide (TRGO), where some carbon atoms of the graphene lattice bear hydroxyl groups (on the basal planes of the lattice), retains a similar structure but also has arrays of identical reactive groups that facilitate surface functionalization.20 Despite the abundant demonstration of TRGO for electronic, optical, or sensor applications, only a few reports have utilized these unique size and chemical characteristics of TRGO to conjugate multivalent carbohydrates and to implement the regulation of cell mobility21 – in spite of their great practical value.22 Here, we describe a versatile platform for constructing multivalent carbohydrate-functionalized 2D scaffolds by placing cyclodextrin-based sugar ligands on adamantyl-functionalized TRGO sheets (AG4) (Scheme 1). Owing to the adamantyl groups on the surface of AG4, the inclusion complex of β-cyclodextrin (β-CD) and adamantyl units reversibly connects the reduced graphene sheet and heptamannosylated β-CD (ManCD) in aqueous medium. The resulting supramolecular carbohydrate-functionalized TRGO derivative can agglutinate Escherichia coli, and the agglutination ability of the 2D sheets is much higher than the parent water-soluble macrocyclic host (ManCD) or the TRGO itself. Taking advantage of the responsive property of the supramolecular interaction, the captured bacteria can be partially released upon addition of a competitive guest. Moreover, because of their unusual infrared absorption property, these TRGO derivatives exhibit excellent bacteriostatic properties (99% elimination) following near-infrared (NIR) laser irradiation of the graphene-sugar-E. coli complexes.


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SCHEME 1. (a) Chemical structure of adamantyl-functionalized graphene derivatives AG4 and heptamannosylated ß-cyclodextrin (ManCD). (b) Schematic representation of supramolecular carbohydrate-functionalized graphene complexes. Binding of bacteria to the complex resulted in a reversible multivalent inhibition of the bacteria. The adamantyl-functionalized TRGO sheets AG4 were synthesized stepwise (see Supporting Information). The (ethylenedioxy)bis(ethylamine)-based linker produced sufficient flexibility and solvation. The degree of adamantyl moieties covalently attached on the TRGO could be readily tuned by using different TRGOs (Table S1, Supporting Information). The TRGO reduced at 400 °C (TRGO400) afforded a higher density of adamantyl units - one adamantyl group for each of the 27 aromatic rings according to elemental analysis (see Supporting Information). AG4 formed a stable suspension in organic solvents such as chloroform and dimethyl formamide


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(DMF). The adamantyl units significantly weakened the strong aggregation force caused by the large area π-π interaction. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) disclosed that the size of AG4 sheets was several micrometers (Figure S9 and S10, Supporting Information). The corresponding area was similar in size to the surface area of bacteria (E. coli has approximately 0.5 µm in width and 2 µm in length). Mannosylatedcyclodextrin (ManCD) equipped with heptamannosyl groups at the upper rim of the cavity was supposed to provide sufficient sugar density for attachment on the adamantylated TRGO surface. The ManCD was prepared according to established methods (Scheme S4, Supporting Information).23 Supramolecular functionalization is widely recognized as a convenient strategy for constructing sophisticated architectures.24 The supramolecular carbohydrate-functionalized graphene derivative ManCD@AG4 was prepared by directly mixing the guest molecule AG4 and the host ManCD in DMF as cosolvent and was subsequently transferred into aqueous solution. After centrifugation at 12,000 rpm (twice), the sediment could be readily dispersed in water with considerable stability (0.83 mg/mL AG4, see Figure S12a, Supporting Information). Under identical conditions, AG4 formed a precipitation during the redispersion in water. The improved solubility implied the formation of host–guest complexes that had been retained at the interface due to the high binding constant (Ka) between adamantyl and the ß-CD moiety (Ka ~ 105 M-1).25 Notably, the heptamannosyl groups on the cyclodextrin were important for increased solubility. The complexation with neat ß-CD led to only a limited water solubility (Figure S12b, Supporting Information). Besides the enhanced solubility, we also used AFM to obtain a second proof for the successful functionalization with mannosyl-functionalized cyclodextrin. The height of AG4, bearing the adamantyl moieties (Figures S9 and S11), was approximately 2-3 nm. After


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installing the mannosylated cyclodextrin ManCD, the height increased to 4-5 nm for ManCD@AG4 (Figure S13, Supporting Information). Computer modeling revealed that the height of ManCD was approximately 1.2 nm. Therefore, the increased height (1-2 nm) was in agreement with the expected increase for these systems. The decisive evidence came from the addition of a competitive guest to the ManCD@AG4 solution. Since the host-guest inclusion complexation between ManCD and AG4 enabled the binary system to dissolve in water, it was reasonable to expect that once the cyclodextrin cavity was occupied by a competitive guest, the hydrophobic AG4 would be excluded from the hydrid system and consequently precipitate. Indeed, when the water-soluble sodium adamantine carboxylate powder (AdCNa, 100 equiv. to ManCD unites) was added to the ManCD@AG4 hybrid system in an aqueous media, a precipitate formed (Figure S15, Supporting Information). These results demonstrated that the host-guest interaction between cyclodextrin and adamantyl units provided enough driving force to transfer the reduced graphene sheets into the water phase. A similar cyclodextrin-based desolvation effect was observed in the case of supramolecular amphiphiles.26 For each batch of ManCD@AG4 system, the carbohydrate loading efficiency was approx. 16% and the final concentration of ManCD in the system was determined to be 98.1 µM. A TEM measurement of the sugar-coated nanostructures revealed the formation of highly dispersed 10 µm2 large 2D sheets that were suitable for optimal interactions with bacteria (Figure 1a). The new multivalent 2D ManCD@AG4 scaffold was studied for its ability to bind mannosespecific E. coli adhesin FimH of type 1 pili. Type 1 pili are filamentous proteinaceous appendages that extend from the surface of many gram-negative organisms.27 Multivalent binding of mannose residues in nanostructures with FimH in E. coli ORN178 leads to agglutination and/or motility inhibition of the bacteria.28 In contrast to previous 0D and 1D


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scaffolds that induced agglutination, the E. coli were entirely wrapped by Man@AG4 hybrid. The TEM images clearly show that ManCD@AG4 binds the E. coli ORN178 strain (Figure 1c and Figure S17, Supporting Information). Several rod-shaped bacteria were tightly wrapped by these sheet-like scaffolds, which indicated a strong multivalent binding. The visible shape of the pili at the external shell of the aggregates (Figure S17e, Supporting Information) provided evidence that the entrapped objects were bacteria. This finding is consistent with the surface morphology observed in a single E. coli (Figure 1b) as well as with the localization pattern of FimH protein along type 1 pili.29


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FIGURE 1. TEM images of the supramolecular carbohydrate-functionalized graphene derivative for bacterial capture: (a) ManCD-AG4 hybrid, (b) ORN178 E. coli, and (c) E. coli agglutination incubated with ManCD@AG4. The dashed yellow circles outline the captured bacteria. The inserted figure shows the different responses of the ManCD@AG4s after respectively adding E. coli strain ORN208 and E. coli strain ORN178. The photographs were taken an hour after incubation at room temperature. No staining agent was used in Figure 1a. Figures 1b and 1c used 1% uranyl acetate solution as staining agent.


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FIGURE 2. Confocal laser scanning microscopy images of ManCD@AG4 incubated with (a) E. coli bacteria strain ORN178, (b) ORN208 and incubation of CD@AG4 with (c) ORN178 and (d) ORN208, respectively. The insert in Figure 2a is a software-processed merged image combining Figure 2a and its transmitted light form. The location of ManCD@AG4 in the agglutination of


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bacteria can be easily visualized in the insert. The white arrows in Figure 2a show the graphene sheets’ position according to merged image. (e) The agglutination index was obtained from confocal microscopy images. Due to the insolubility of AG4 in aqueous medium, it was not included in the comparison. The specificity of the interaction of ManCD@AG4 with the mannose binding FimH receptors on the bacterial surface was evaluated by examining binding to two E. coli strains, ORN178 and ORN208. In contrast to ORN178, the ORN208 strain expressed abnormal type 1 pili that do not bind D-mannose. Both strains were stained by fluorescein isothiocyanate (FITC) to monitor cell agglutination by fluorescence confocal microscopy (CLSM). Mixtures of ManCD@AG4 and ORN178 produced a strongly fluorescent cluster where the bacteria were bound (Figure 2a), whereas no apparent binding of ManCD@AG4 to ORN208 was detected (Figure 2b). Due to the large interference contrast between graphene sheets and bacteria, the location of ManCD@AG4 in the cluster could be easily distinguished in the merged form of CLSM images. The merged form combines the fluorescent image and transmitted light image by software processing. As shown in the insert of Figure 2a, the black species (assigned to ManCD@AG4) existed randomly within the agglutinated area of bacteria, which indicates the agglutination of bacterial cells was due to the bilateral interaction. The macroscopic observation further supported the CLSM data that the precipitation of graphene sheets only occurred in the case of ManCD@AG4 and ORN178 (Figure 1c, insert), which was absent with ORN208. Agglutination experiments on a ßCD-functionalized TRGO sheet (CD@TRGO) served as a control to assign the contribution of mannose to the hybrid materials. Neither ORN178 (Figure 2c) nor ORN208 (Figure 2d) strains aggregated in this case. Based on the TEM and fluorescence confocal microscopy results, we concluded that ManCD@AG4 selectively bound to the bacterial surface via mannose-FimH


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receptor interactions and that the heptamannosylated moiety played a determining role for binding and selectivity. Following the established protocol,15,16 we defined the average number of E. coli cells contacting each other based on CLSM images as the agglutination index (A.I.) Thereby, the capture efficiency was quantified and the ability of ManCD@AG4, CD@AG4 and the macrocyclic host ManCD to agglutinate bacterial cells was evaluated (Figure 2e). The A.I. value of ManCD towards ORN178 at 117 was much higher than for ORN208 without mannose binding sites (14), as well as for CD@AG4 with ORN178 (18) and ORN208 (16). Without the assistance of the graphene scaffold, the mannosyl-functionalized macrocycle ManCD only slightly induced agglutination (17), which clearly reflected the benefit of the 2D large flexible carbon surface for capturing pathogenic cells.

FIGURE 3. Release effect of the captured E. coli strain ORN178 upon addition of a competitive guest. The top dashed-line is the original fluorescence intensity of E. coli before adding the competitive guest.


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Multivalent binding and the responsive release potential of ManCD@AG4 towards E. coli were determined using a competition experiment: Co-incubation of the FITC-stained ORN178 E. coli with varying concentrations of the monovalent binder: methyl α-D-mannopyranoside (Me-Man) and ManCD@AG4. The competitive assay was further evaluated by a fluorescence read-out after reaching the equilibrium (see Supporting Information). We found that free Me-Man at concentrations up to 2000 times of ManCD@AG4 concentration had little effect on the agglutination of ManCD@AG4 to bacteria: ~20% of captured bacteria were released. However, the competitive release of captured E. coli from ManCD@AG4 was induced by addition of sodium adamantine carboxylate (AdCNa) (Figure 3). Since there was only a monovalent binding between cyclodextrin and the adamantyl unit, the addition of excess AdCNa to the system was more likely to result in ~50% release of the captured E. coli. The completive assay clearly showed that multivalent ManCD@AG4 bound FimH at least 2000-fold better than monovalent mannose.


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FIGURE 4. (a) Schematic illustration of ManCD@AG4 under NIR irradiation causing the death of captured E. coli. (b) Thermal image of an E. coli sample incubated with ManCD@AG4 under NIR laser irradiation (0.5 W/cm2). After 10 min, the temperature of irradiated spot reached 70.9 ± 1.4°C. (c) Temperature evolution profiles of the E. coli samples with and without ManCD@AG4, respectively. Images of E. coli bacterial colonies (captured by ManCD@AG4) (d) without and (e) with treatment of NIR irradiation. In the next step, a strong infrared-absorption of TRGO30 was exploited to utilize this photothermal effect to selectively destroy captured bacteria. We irradiated the captured E. coli in


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ManCD@AG4 (AG4 in 0.83 mg mL-1, incubated for 1 h) at a NIR (785 nm) and 0.5 W cm-2 for 10 min (Figure 4a). During irradiation, the sample temperature gradually increased to 70.9± 1.4°C (Figures 4b and 4c). After irradiation, the bacteria were spread on the agar plates to characterize cell viability: The abundance of E. coli colonies on the agar plate indicated the amount of live cells. After irradiation, the agar plate contained almost no live bacteria (Figure 4e). In a parallel experiment without IR treatment, live bacteria could be seen in large bacterial colonies (Figure 4d). In the official profile of the World Health Organization (WHO),31 it is stated that E. coli bacteria can be destroyed by thoroughly cooking foods until all parts reach a temperature of 70 °C or higher. Neither ManCD nor E. coli can expose enough IR absorption to elevate the surrounding temperature (both aqueous solution of ManCD and E. coli have no absorption at 785 nm). Therefore, the bacterial killing effect here can be ascribed to the unusual IR-absorption mechanism of TRGO, which leads to a sufficiently high temperature to disinfect the E. coli. In a diluted sample, the bacteriostatic efficiency was determined at > 99% (Figure S25c, Supporting Information). In summary, we designed a novel, multivalent sugar-functionalized graphene sheet that selectively binds E. coli. A host-guest inclusion complex between heptamannosylated βcyclodextrin and adamantyl fixed to the graphene sheet made the resulting carbon sheets more soluble in water. The addition of carbohydrate via a supramolecular binding afforded the carbon materials to agglutinate E. coli bacteria. The multivalent interactions were responsible for cell agglutination. The agglutination ability of graphene-based 2D sheets greatly exceeded those of pure ManCD due to a large interphase with the E. coli bacteria. The supramolecular linkage at the interface of graphene allowed a controlled release of the captured bacteria through the addition of a competitive guest, such as AdCNa. Considering a number of sugar-modified


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cyclodextrins are available,32 our system can be further extended to endow the graphene surface with more different functionalities. The unique thermal IR-absorption properties of TRGO enable targeted bacterial killing upon IR-laser irradiation. The integration of multivalent, supramolecular binding on graphene also provides a new strategy for the design of functional materials for filtration in healthcare and environmental protection. ASSOCIATED CONTENT Supporting Information. Detailed materials and methods and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Present Addresses (C.-H.L.) Institute of Chemistry, Academia Sinica, Taipei, Taiwan Author Contributions #These

authors contributed equally

Notes Any additional relevant notes should be placed here. ACKNOWLEDGMENT


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R.H and P.H.S. thank the Deutsche Forschungsgemeinschaft (SFB 765) and Focus Area Nanoscale for financial support. P.H.S. and C-H.L thank the Max-Planck Society and the European Union FP7 (CARMUSYS) for financial support. B.L. thanks the German Federal Ministry of Education and Research (BMBF, Fkz. 0315446) for financial support. The authors acknowledge: Dr. Pamela Winchester for editing the manuscript; Dr. Chakkumkal Anish and Bopanna Ponnappa Monnanda for guiding biological experiments.

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Table of Contents Graphic


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(a) Chemical structure of adamantyl-functionalized graphene derivatives AG4 and heptamannosylated ßcyclodextrin (ManCD). (b) Schematic representation of supramolecular carbohydrate-functionalized graphene complexes. Binding of bacteria to the complex resulted in a reversible multivalent inhibition of the bacteria. 103x118mm (300 x 300 DPI)


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TEM images of the supramolecular carbohydrate-functionalized graphene derivative for bacterial capture: (a) ManCD-AG4 hybrid, (b) ORN178 E. coli, and (c) E. coli agglutination incubated with ManCD@AG4. The dashed yellow circles outline the captured bacteria. The inserted figure shows the different responses of the ManCD@AG4s after respectively adding E. coli strain ORN208 and E. coli strain ORN178. The photographs were taken an hour after incubation at room temperature. No staining agent was used in Figure 1a. Figures 1b and 1c used 1% uranyl acetate solution as staining agent. 99x142mm (300 x 300 DPI)


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Confocal laser scanning microscopy images of ManCD@AG4 incubated with (a) E. coli bacteria strain ORN178, (b) ORN208 and incubation of CD@AG4 with (c) ORN178 and (d) ORN208, respectively. The insert in Figure 2a is a software-processed merged image combining Figure 2a and its transmitted light form. The location of ManCD@AG4 in the agglutination of bacteria can be easily visualized in the insert. The white arrows in Figure 2a show the graphene sheets’ position according to merged image. (e) The agglutination index was obtained from confocal microscopy images. Due to the insolubility of AG4 in aqueous medium, it was not included in the comparison. 139x239mm (300 x 300 DPI)


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Release effect of the captured E. coli strain ORN178 upon addition of a competitive guest. The top dashedline is the original fluorescence intensity of E. coli before adding the competitive guest. 92x73mm (300 x 300 DPI)


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(a) Schematic illustration of ManCD@AG4 under NIR irradiation causing the death of captured E. coli. (b) Thermal image of an E. coli sample incubated with ManCD@AG4 under NIR laser irradiation (0.5 W/cm2). After 10 min, the temperature of irradiated spot reached 70.9±1.4°C. (c) Temperature evolution profiles of the E. coli samples with and without ManCD@AG4, respectively. Images of E. coli bacterial colonies (captured by ManCD@AG4) (d) without and (e) with treatment of NIR irradiation. 177x185mm (300 x 300 DPI)


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Multivalency at Interfaces: Supramolecular Carbohydrate

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