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Glycan functionalized microgels for scavenging and specific binding of lectins Alexander Jans, Ruben R. Rosencrantz, Ana D. Mandic, Naveed Anwar, Sarah Boesveld, Christian Trautwein, Martin Moeller, Gernot Sellge, Lothar Elling, and Alexander J. C. Kuehne Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01754 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017
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Glycan functionalized microgels for scavenging and specific binding of lectins Alexander Jans†a, Ruben R. Rosencrantz†b, Ana D. Mandićc, Naveed Anwara, Sarah Boesveldc, Christian Trautweinc, Martin Moellera, Gernot Sellgec, Lothar Ellingb and Alexander J.C. Kuehnea* a
DWI – Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50,
52076 Aachen, (Germany). b
Laboratory for Biomaterials Institute for Biotechnology and Helmholtz-Institute for Biomedical
Engineering, RWTH Aachen University, Pauwelsstr. 20, 52074 Aachen (Germany). c
Department of Internal Medicine III, University Hospital, RWTH Aachen University, Pauwelsstr. 30,
52074 Aachen (Germany).
KEYWORDS lectin • glycopolymer • microgel • ECL • LacNAc
ABSTRACT: Lectins are proteins with a well-defined carbohydrate recognition domain. Many microbial proteins such as bacterial toxins possess lectin or lectin-like binding domains to interact with cell membranes that are decorated with glycans recognition motifs. Here, we report a straightforward way to prepare monodisperse and biocompatible polyethylene glycol microgels, which carry glycan motifs for specific binding to lectins. The sugar-functionalized colloids exhibit a wide mesh size and a highly accessible volume. The microgels are prepared via drop-based microfluidics combined with radical polymerization. GSII and ECL are used as model lectins that bind specifically to the corresponding carbohydrates namely GlcNAc and LacNAc. LacNAc microgels bind ECL with a high ACS Paragon Plus Environment
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capacity and high affinity (Kd ~ 0.5 – 1 µM) suggesting multivalent binding of the lectin to the LacNAc-decorated flexible microgel network. Glycan functionalized microgels present a useful tool for lectin scavenging in biomedical applications.
INTRODUCTION Lectins are omnipresent proteins with a well-defined carbohydrate recognition domain (CRD) that exists in fauna[1] and flora.[2] Lectins exhibit glycan-binding domains with the ability to bind specifically and reversibly to precise carbohydrate recognition motifs.[3,4] For the interaction with lectins, the size, shape, charge and arrangement of the saccharide are paramount for the specificity of the binding event, also known as the “cluster glycoside effect”.[5][6] Many pathogenic viruses, bacteria and parasites express lectins that bind to glycans on host cell surfaces.[7] These lectins motifs are incorporated in hemagglutinis, adhesins and toxins and are often key virulence factors mediating cellular adhesion, uptake, biofilm formation and host cell death.[8][9][10] Several bacterial toxins make use of lectin binding domains for attachment to cells and intracytoplamic delivery of a cytotoxic domain. Examples are the cholera toxin produced by Vibrio cholerae[11–13], Shiga and Shiga-like toxins of Shigella dysenteriae[14] and enterohemorrhagic Escherichia coli (EHEC)
[15][16][17]
and toxin A and B of Clostridium
difficile.[18,19] Interruption of pathogen lectin binding to host glycans offers great potential for the development of new anti-microbial drugs. A common feature of lectin-glycan interactions is the relatively weak binding affinity in single glycan binding events. However, lectins often exhibit multiple bindings sites, and simultaneous binding to several glycan moieties enhances the binding affinity exponentially across several orders of magnitude.[20] The design of high affinity molecular entities, which bind multivalently to lectin structures, is a formidable task.[21][22][23] We have previously shown, that we can selectively bind lectins to surface bound glycan functionalized ethylene glycol polymer (PEG)brushes[24][25] and glycan-functionalized PEG hydrogels.[26,27] Water soluble polymers such as polyethylene glycol (PEG) are the materials of choice due to their non-fouling and biocompatible properties.[28,29] ACS Paragon Plus Environment
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In this study, we developed a material system based on porous micron-sized PEG based polymer gel entities, which carry glycan moieties for specific lectin binding. The microgels are produced using microfluidics, which offers the possibility for parallelized and up-scaled synthesis of porous polymer colloids with diameters of several microns.[30] These microgels are swollen polymer networks and differ significantly from established colloidal and polymeric systems. The mesh size of a microgel can be designed to be wide enough to take up guest molecules. Furthermore, it is possible to purposefully introduce chemical functionality to different compartments of the microgel.[31,32] Their soft and water swollen molecular structure entails excellent biocompatibility and makes them ideal candidates to serve as a porter for glycans in order to construct micro-carriers capable of multivalent lectin binding for medical application. These could in the future be used to scavenge the above described toxins and transport them out of the body, evading antibiotic treatments and potential danger of relapse. We here apply legume lectins as a model system for our binding study. We show that the microgels can take up large guest molecules and that lectin binding is specific depending on the type of glycan used. EXPERIMENTAL SECTION Materials Paraffin oil, n-hexane, propan-2-ol, 2-Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone (HEMP), N-Acetylglucosamine and SPAN 80 were purchased from Sigma-Aldrich (Germany). Sylgard 184 elastomer kit for the PDMS devices was obtained from Dow Corning (USA). PE tubing was purchased from Smiths medical (USA). Hydroxy terminated starPEG was obtained from CHT R. Beitlich GmbH (Germany). All chemicals for preparation of buffers were purchased from Carl Roth (Karlsruhe, Germany) and used without further purification. Nucleotide sugars were from Carbosynth (Berkshire, UK). FITC-labeled ECL and GSII were from VectorLabs (Burlingame, CA, US). All other origins of materials are stated within the text.
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Production of glycosyltransferases The glycosyltransferases were expressed and purified as described elsewhere[25].Briefly, β4GalT was expressed in E. coli SHuffle T7 Express (New England Biolabs, Frankfurt am Main, Germany) to enhance the activity. Purification was performed by using HisTrap HP columns and Aekta Systems (GE Healthcare, Solingen, Germany). β4GalT was used directly after purification. The enzyme was stored at 4°C. Synthesis of 1-allyl-N-acetylglucosamine (Allyl-2-acetamido-2-deoxy- β-D-glucopyranoside) The synthesis of 1-allyl-N-acetylglucosamine was carried out according to the procedure previously published[33]. During the modified Königs-Knorr reactions allyl-alcohol was used and attached to the chlorinated compound via its hydroxyl groups. (See Fig. S2 in the SI). Glycosyltransfer reaction The reaction was generally performed as described elsewhere for β4GalT[25]. Briefly, up to 500 mg of GlcNAc-allyl was dissolved at a concentration of 5 mM in a solution containing final concentrations of 7.5 mM UDP-α-D-galactose, 25 mM HEPES-NaOH, pH=7.6, 2 mM MnCl2, 5 U alkaline phosphatase (life technologies, Darmstadt, Germany) and 5 U β4GalT. The reaction was incubated at 30°C for 24 h 48h. As no residual substrate was detected anymore by RP-HPLC-MS the reaction was terminated by heating and the precipitated, denatured enzyme was filtered off. The product (LacNAc-allyl) was obtained after preparative HPLC and freeze-drying as white powder with a yield of 85 %. Analysis was carried out by ESI-MS and NMR. (See Fig. S3 in the SI). RESULTS AND DISCUSSION Generation of glycan functionalized microgels. To facilitate incorporation of the glycan motifs during the radical polymerization, we functionalize GlcNAc with an allyl functionality. The allyl moiety is less reactive in radical polymerization compared to the acrylate moiety. This prevents spontaneous polymerization during the enzymatic glycan synthesis. Furthermore, the allyl group allows for
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incorporation of the glycan compound preferentially towards the end of the growing polymer chains during photo-crosslinking into microgels. We extend the allyl functionalized GlcNAc to N-acetyl-Dlactosamine (LacNAc-allyl) using UDP-Gal in an enzymatic galactosyltransferase conversion with β4GalT, an enzyme that tolerates allyl functionalities (Fig. 1b). We now have two binding motifs available, which bind specifically to legume lectins. GlcNAc binds to GSII and LacNAc to ECL. To produce microgels, we rely on microfluidic synthesis (Fig. 2a), guaranteeing highly monodisperse microgels with good control over the shape and size (Fig. 2b). For this, the prepolymer is polymerized in aqueous solution in a photo-initiated radical polymerization. The prepolymer strategy allows the production of microgels with a homogeneous network and an invariant mesh-size distribution across the entire microgel volume.[34] To produce microgels with a diameter between 10 and 20 µm we apply microfluidic channel intersections with a channel height of 20 µm, produced via PDMS soft-lithography.[35] To passivate the PDMS and avoid swelling of the microfluidic chips when using organic non-polar solvents we coat the entire chip with Parylene-C using chemical vapor deposition. This leads to a Parylene-C coating of a thickness of around 250 nm on the inside of the channels.[36] We apply a 50/50 mixture of paraffin oil together with hexane to obtain a hydrophobic
Figure 1. Reaction scheme of (a) an acrylation reaction of the six armed star-shaped polyethylene glycol prepolymer based on a sorbitol core. (b) Glycolsyltransfer reaction of N-acetylglucosamine-allyl via β4GalT to N-acetyl-D-glucoseamine.
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Preparation of glycan funtionalized microgels by (a) dispersion of an aqueous phase
containing the polymerprecursor and an allyl-funtionalized LacNAc, in a continuous oil phase on a PDMS-based microfludic drop-maker with a channel diameter of 20 µm. (b) Photocrosslinked swollen monodisperse glycan containing microgels in an aqueous media with an average diameter of 20 µm. (c) Reaction scheme of the synthesis of porous starPEG-glycan modified monodisperse micorgels. The scale bars represent 100 µm.
continuous phase of suitable viscosity. We add Span 80 as a surfactant. The dispersed phase consists of an aqueous solution with 10 wt% of acrylate terminated starPEG prepolymer (3 kDa, 6 kDa or 18 kDa; see Fig. S1 for 1H-NMR), 0.5 wt% of 2-Hydroxy-4’-(2-hydroxyethoxy)-2-methyl-propiophenone (HEMP), a radical photo-initiator and varying amounts of allyl functionalized N-acetylglucosamine (GlcNAc) or N-acetyl-D-lactosamine (LacNAc) glycans (see Fig. S2 and S3 in the SI for 1H-NMR spectra). We produce droplets by guiding the two phases into the microfluidic channels at constant pressures (see Supplementary Movie 1). The droplets are produced at the intersection of the microfluidic channels and then exposed to UV-light (λmax = 380 nm,
power: 200 mW) to initiate
crosslinking. This exposure is performed while the droplets exit the device and are transported through UVtransparent polyethylene tubing to a collection container. For a channel width of 20 µm the resulting microgels are monodisperse with a diameter of 20 µm (Fig. 1c). Smaller microgels can be produced by varying the microfluidic channel width. ACS Paragon Plus Environment
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Figure 3. Diffusion study with in (a) Graphical plot of the radial intensity of TG for the determination of the diffusion constant over 60 min. (b) Optical images of the starPEG microgels over 60 minutes with increasing fluorescence due to the diffusion of the Atto665 labeled thyroglobulin into the porous network. The white ring is a guide for the eye (c) Non-functionalized, GlcNAc-, and LacNAc-decorated microgels with the corresponding fluorescently labeled biomolecules to show the accessibility of the microgel network. The scale bars represent 20 µm.
Accessibility of the microgel network and binding. We prepare microgels using microfluidics with 6 kDa, 12kDa and 18 kDa starPEG prepolymer. Only the 18 kDa starPEG prepolymer produces microgels with an accessible network structure. We estimate the smallest microgel mesh size by considering the smallest possible loop for two acrylate functionalized arms reacting with each other. ACS Paragon Plus Environment
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This minimal loop would have a circumference of around 18 nm. To test the accessibility of the microgel network mesh we apply a thyroglobulin (TG) (M = 660 kDa), labeled with Atto665, a red fluorescent dye, to be able to follow diffusion of the high molecular weight molecule. We incubate the microgels with the fluorescently labeled TG for 60 min. During this time we take aliquots and subject the samples to a single centrifugation step to separate the microgels from the fluorescent supernatant. We record the fluorescence of the microgels using confocal microscopy. We examine the distribution of fluorescence and therefore the biomolecular access to the microgel (Fig. 3a). Given sufficient time, the large molecule diffuses deep into the microgel, verifying a uniform network and large enough mesh size. By fitting the obtained data with a simplified diffusion equation ignoring all interactions (see SI, Equation 1) we obtain the diffusion constant D = 2.50 ± 0.39 µm2/s, which is on the order of diffusion of a globular object through a macromolecular network (Fig. 3b).[37] To test the accessibility of the network also in glycan functionalized microgels, we incubate them with high concentrations of fluorescently labeled lectins. GSII and ECL specifically bind to the multivalently present glycan motifs in the microgels, as demonstrated previously,[25] whereas eGFP does not interact. We use FITC labeled GSII (Griffonia Simplicifolia Lectin II; M = 113 kDa) for binding to GlcNAc, and FITC labeled ECL (Erythrina Cristagalli Lectin; M = 54 kDa) for binding to LacNAc functionalized microgels. GSII and ECL contain two binding sites for GlcNAc and LacNAc, respectively. As an unspecific non-binding control molecule, we apply eGFP (M = 32.7 kDa) with non-functionalized (Nf) microgels (Fig. 3c). After incubation for 60 min the fluorescent supernatant is removed and the microgels are redispersed in fresh lectin binding buffer and immediately transferred to the confocal microscope for inspection. All microgels exhibit fluorescence throughout the entire spherical volume substantiating complete access also for interacting molecules. However, GlcNAc and LacNAc functionalized microgels show stronger fluorescence at the borders, revealing local accumulation of the fluorescently labeled biomolecules. As a result of the different acrylate and allyl reactivities during microgel generation, the sugar moieties are expected to concentrate at chain ends and therefore in the corona of the microgel. The stronger
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fluorescence signal at the microgel borders might originate from a higher number of binding events of fluorescent biomolecules to the glycan moieties. Having proven sufficient accessibility- of the microgel network we carry on to investigating the specificity of our glycan functionalized microgels towards respective lectins. We compared microgels with 1 equivalent of GlcNAc or LacNAc per starPEG molecule and we use non-functionalized microgels as a negative control. We again apply FITC-labeled GSII and ESL as specific binding lectins (to GlcNAc and LacNAc respectively). We analyze the samples using flow cytometry, which is usually
Figure 4. Specific binding of lectin to glycan functionalized microgels. (a) Flow cytometry analysis demonstrate binding of FITC-labeled GSII and ECL (1 µM) to microgels. (b) Specific binding of ECL to LacNAc microgels ([LacNAc + ECL] – [Nf + ECL]) (Nf = non-functionalized) analysed by flow cytometry. Data are expressed as fold increase over the background mean florescence intensity (MFI) of LacNAc microgels. (c) Binding of FITC-ECL and FITC-GSII to LacNAc- and Nf-microgels analysed by fluorimetric measurement of microgels. ACS Paragon Plus Environment
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applied to investigate populations of cells. Flow cytometry allows us to investigate the scattering behavior and fluorescence intensity of multiple microgel samples in a short period of time. Forward scatter (FSC) is a measure for particle size and sideward scatter (SSC) a measure for granularity (see Fig. S4 in the SI). The non-functionalized and LacNAc microgels show relatively homogeneous FSC/SSC behavior indicating a homogenous size and structure (Fig. S4). Gating within the FSC/SSC dot blot allows excluding small debris and larger microgel aggregates from the subsequent analysis (Fig. S4). We find very low amounts of non-specific binding (ECL and GSII to non-functionalized microgels; ECL to GlcNAc microgels and GSII to LacNAc microgels). GSII binds to GlcNAc microgels with slightly higher affinity; however, very strong binding of ECL to LacNAc microgels is detectable (Fig. 4a). To verify that binding is indeed mediated through the LacNAc moieties in the microgel and not some other attractive effect, we perform a competition experiment using lactose as a free compound, which interacts and binds to ECL. For increasing lactose concentrations ECL is removed from the microgels as it binds to the over presented free lactose (see Fig. S5). The determined Kd for ECL binding to LacNAc microgels measured by the flow cytometry method is 0.51 ± 0.02 µM (Fig. 4b). For absolute quantification of ECL binding to LacNAc microgels, we incubate microgels at a defined concentration (1000 microgels/µl) with different concentrations of lectins. Bound FITC-labeled lectins are measured by a fluorimeter within the microgel fraction after washing and centrifugating twice. The calculated binding affinity by this method is Kd = 1.06 ± 0.32 µM, which is on the order of Kd measured via flow cytometry. Overall, the calculated binding affinities are in the range that has been reported for bivalent binding of ECL to LacNAc in other assays, proving that we have multivalent binding of lectins in our microgels.[38] The maximum binding capacity (Bmax) is 1,5 fmol / microgel corresponding to approximately 9 x 108 ECL molecules per microgel. Considering that one ECL molecule binds to two LacNAc, we estimate that 1.8 x 109 LacNAc molecules are accessible for binding by ECL. This correspond to approximate 13 % of total LacNAc molecules within the microgels, since we estimate that one microgel consist of 1.4 x ACS Paragon Plus Environment
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1010 starPEG and LacNAc molecules, respectively (with a ratio of ca. 1:1 as confirmed by magic angle spinning solid state 1H-NMR). CONCLUSION In this work, we demonstrate facile functionalization of PEG-based microgels with glycan moieties. The microgels exhibit an accessible interior due to the designed large mesh size. The functionalized microgels specifically bind complementary lectins with high affinity suggesting polyvalent binding. These materials pave the way for lectin scavenger systems with the potential for biomedical applications. In the future, systems could be developed that are able to specifically bind bacterial toxins with lectin binding domains at a high capacity and selectivity. This will afford high avidity and affinity in the nanomolar range of sugar modified microgels towards the respective targets. These microgels may be used as alternative non-antibiotic treatments for intestinal pathogens such as Clostridium difficile or enterohemorrhagic Escherichia coli (EHEC). ASSOCIATED CONTENT Supporting Information. Experimental details and movie on microgel generation, starPEG and glycan modification, lactose competition test and flow cytometry are supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors e-mails:
[email protected],
[email protected],
[email protected] Author Contributions †
These authors contributed equally to this work.
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ACKNOWLEDGMENTS The authors wish to acknowledge support via the Deutsche Forschungsgemeinschaft DFG through the collaborative research center SFB 985 “Functional microgels and microgel systems” (project C3). This work was performed in part at the Center for Chemical Polymer Technology CPT, which was supported by the EU and the federal state of North Rhine-Westphalia (grant no. EFRE 30 00 883 02). REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
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