Glycopolymer-Functionalized Cryogels as Catch and Release Devices

Letter pubs.acs.org/macroletters

Glycopolymer-Functionalized Cryogels as Catch and Release Devices for the Pre-Enrichment of Pathogens Christian von der Ehe,†,‡,⊥ Tanja Buś,†,‡ Christine Weber,†,‡ Steffi Stumpf,†,‡ Peter Bellstedt,† Matthias Hartlieb,†,‡,§ Ulrich S. Schubert,*,†,‡,⊥ and Michael Gottschaldt*,†,‡ †

Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstraße 10, 07743 Jena, Germany ‡ Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena, Germany ⊥ Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB Eindhoven, The Netherlands S Supporting Information *

ABSTRACT: A highly porous cryogel is prepared and subsequently functionalized with an atom transfer radical polymerization (ATRP) initiator at the surface. Two new glycomonomers are introduced, which possess deprotected mannose as well as glucose moieties. These are copolymerized with N-isopropylacrylamide (NiPAm) from the cryogel surface, providing a highly hydrophilic porous material, which is characterized by SEM, FTIR spectroscopy, and NMR spectroscopy. This functionalized support can be applied for affinity chromatography of whole cells owing to the high pore space and diameter. Such an application is exemplified by investigating the ability to capture Escherichia coli bacteria, revealing selective binding interactions of the bacteria with the mannose glycopolymer-functionalized cryogel surface. Thus, the presented glycopolymer-cryogel represents a promising material for affinity chromatography or enrichment of cells. ffinity chromatography represents a powerful technique to selectively separate different analytes, even complete cells from each other, which is of major importance in applications such as wastewater treatment or analysis of biological contaminants.1,2 In particular, the application of catching bacteria is very auspicious.3−6 For this purpose a solid support matrix for attachment of affinity ligands is required. Cryogels represent a promising material in this respect owing to the large cryogel pore size, which ranges from 10 to 100 μm so that complete cells can elute through the support. If equipped with a suitable affinity ligand, they can be designed as stationary phase material for affinity chromatography of whole cells.7,8 Examples of used affinity ligands include metal ions,5,9 protein A,10 lectins,4,11 cationic polymers,12 or dyes13 for DNA purification. Surprisingly, no cryogel surfaceimmobilized glycopolymers have been reported up to now, even though sugars represent a very selective type of ligand found throughout nature in specific recognition and binding contexts.14,15 The interaction with lectins16 is even enhanced due to the cluster glycoside effect in the case of polymeric sugars (glycopolymers).17 Glycopolymers are also able to selectively bind to certain bacteria,18 such as mannose binding Escherichia coli (E. coli) bacteria.19−22 Cryogel surfaces have been functionalized with polymers, mainly applying free radical polymerization initiated by potassium diperiodatocuprate.12,23−25 However, a cryogel surface-initiated atom transfer radical polymerization (SI-

A

© XXXX American Chemical Society

ATRP)26 represents a more versatile strategy for the surface functionalization with bioactive polymers.27 The present communication describes a similar SI-ATRP approach, resulting in the first cryogel-supported glycopolymer and its application for selective binding of E. coli bacteria. The synthesis of cryogels results in a monolith which possesses the shape of the container it was prepared in. Thus, it can be engineered to match the desired application.28 Therefore, the cryogel synthesis was performed inside stainless steel HPLC columns to yield a cryogel support (CG−OH) from N,N-dimethylacrylamide as the main constituent (Scheme 1A). Methylenebis(acrylamide) was added as a cross-linker, while hydroxyl functionalities required for further functionalization were introduced via the comonomer N-(2-hydroxyethyl)acrylamide (ratio 94:4:2). The common redox initiating system ammoniumperoxodisulfate/tetramethylethylene diamine proved suitable. To obtain a large pore size, the polymerization mixtures were frozen at −13 °C followed by incubation at −20 °C for 40 h to ensure quantitative conversion. A monomer concentration of 3.5% (w/ V) was found to provide an optimum compromise between mechanical stability and sufficient flow rate. Subsequent to Received: November 26, 2015 Accepted: February 8, 2016

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DOI: 10.1021/acsmacrolett.5b00856 ACS Macro Lett. 2016, 5, 326−331

Letter

ACS Macro Letters

Scheme 1. Schematic Representation of the Cryogel Synthesis (A), the Glycomonomer Synthesis (B), as well as the SI-ATRP from the Cryogel Surface (C)

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drying, the resulting hydroxyl-functionalized gel CG−OH could be reswollen in water as well as in DMF to reach its original volume within a few seconds. In order to polymerize glycopolymers from the surface of the synthesized gels, an ATRP initiator moiety was attached to the pendant hydroxyl groups (Scheme 1A). To avoid hydrolysis of the acid bromide, the cryogels were dried by extensive washing with anhydrous DMF directly inside the stainless steel columns. Subsequently, a large excess of bromoisobutyryl bromide was eluted through the column to maximize the conversion of the hydroxyl groups. After extensive washing and drying, the degree of initiator functionalization was determined by elemental analysis, revealing a bromine content of 0.16 mmol g−1, which corresponds to 84% conversion of the functional comonomer. In order to polymerize glycomonomers from the surface, the synthetic approach requires deprotected glycomonomers for two reasons: Since the cryogel needs to be swollen during the polymerization process, a hydrophilic solvent has to be used. As a matter of fact, water-soluble (glyco)monomers are required. This strategy has the further advantage of omitting the additional deprotection step of the protected sugar moieties, which would be necessary to obtain the biologically active polymer. Since full conversion of this deprotection is difficult to achieve as soon as the saccharides are attached to a solid support,29 this is especially advantageous. More importantly, a postpolymerization deprotection of the acetyl groups would result in simultaneous cleavage of the ester bonds that connect the glycopolymer to the cryogel support. For this purpose, acetyl-protected glycomonomers15,30 of mannose and glucose were deprotected using a catalytic amount of sodium methoxide in anhydrous methanol, yielding the monomers ManMAmOH and GlcMAmOH (Scheme 1B). The 1H and

C NMR spectra show the expected signals, which could be assigned with the help of the HSQC NMR spectra (Figure S1 and Figure S2, Supporting Information). To enable selective binding interactions with E. coli bacteria, NiPAm was copolymerized with the mannose glycomonomer from the cryogel surface by SI-ATRP. NiPAm was chosen for this purpose since it represents the best investigated hydrophilic and water-soluble acrylamide monomer. Furthermore, poly(NiPAm) shows lower critical solution temperature (LCST) behavior, which might be exploited in future applications. In order to compare any biological experiments with a negative control (i.e., a glycopolymer of a different sugar type and a similar polymer without sugar), the analogue glucose copolymer was synthesized as well as the homopolymer of NiPAm (Scheme 1). When conducting a surface-initiated polymerization from a flat substrate, the active polymerizing species can be reached easily by the monomer since it is not sterically shielded. However, for cryogels this is not the case. Transport of a monomer by diffusion is required to reach the inner pores of a gel, which might result in a depletion of monomer concentration within the gel and, thus, contributes to low conversions, which are also reported in the literature.26 Hence, a different experimental setup was chosen to enhance the mixing and circulation of the monomer solution. The monomer mixture was circulated through the stainless steel column containing the gel using a peristaltic pump setup (6 mL min−1) instead of stirring, which can obviously not be applied in the present heterogeneous system (Figure S3, Supporting Information). The polymerization was conducted using the catalyst system CuBr/CuBr2/Me6TREN, which has been reported to yield 327


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ACS Macro Letters Table 1. Selected Characterization Data of the Glycopolymer-Functionalized Cryogels

a

sample

glycomonomer in feed [mol %]

mass increase

conversion [%]a

DPb

Mn cleaved [g/mol]c

Đcleavedc

CG-Br CG-PMan CG-PGlc CG-PNiPAm

10 10 0

220% 145% 180%

19 7 13

57 21.1 37.5

51 600 24 100 30 500

1.34 1.23 1.30

swelling degree [mgwater/mggel] 35.3 30.3 34.7 36.6

± ± ± ±

0.9 0.2 4.2 0.7

Calculated from mass increase. bCalculated from conversion. cDetermined by SEC (linear PS standard).

Figure 1. Overlay of the 13C NMR spectra obtained by solid-state NMR spectroscopy of the three functionalized supports CG-PGlc, CG-PMan, and CG-PNiPAm (left). Magnified area of the sugar signals showing the overlays with the 13C NMR spectra of the corresponding monomers (right, monomers measured in D2O solution).

good results for acrylamide monomers.29,31 ATRP in pure water often results in uncontrolled polymerizations due to destabilization of the deactivator, whereas addition of methanol (or organic cosolvents in general) decreases the polymerization rate but increases control.32,33 For this reason, a water/ methanol mixture (2:1) was used as polymerization medium. The copolymerizations of the glycomonomers with NiPAm (10% glycomonomer) from the cryogel surface as well as the NiPAm homopolymerization were conducted at 0 °C since the aqueous ATRP of NiPAm is reported to proceed with better control at low temperatures.34 Activators generated by electron transfer (AGET) ATRP conditions were applied (Scheme 1C) by in situ reduction of CuBr2 with ascorbic acid, applying 0.9 equiv. of reducing agent to retain 10% of CuBr2 as deactivator. Subsequent to polymerization, the gels were washed extensively with water and dried under reduced pressure. The dry mass of the gels revealed an increase of 220% for the mannose polymer CG-PMan, 145% for the glucose polymer CG-PGlc, and 180% in the case of the PNiPAm homopolymer CG-PNiPAm, as compared to the initial cryogel masses (Table 1). These weight increases correspond to monomer conversions of 19%, 7%, and 13%, respectively, which is comparable to the conversions achieved via cryogel SI-ATRP reported in the literature.26 The limitation of the conversion may be due to residual oxygen within the cryogels, which is difficult to remove and leads to termination of the ATRP or to recombination reactions occurring between two growing polymer chains.

The functionalized cryogels were characterized by methods suitable for solid-state structural analysis. In the ATR FT-IR spectra of all functionalized cryogels the peak corresponding to the N−H stretch vibration at 3500 cm−1 is strongly pronounced compared to the unfunctionalized CG−OH (Figure S4, Supporting Information), indicating successful initiator functionalization. Furthermore, CG-PMan and CGPGlc reveal a broad shoulder at 3250 cm−1 corresponding to the hydroxyl groups of the attached sugar moieties, suggesting successful functionalization by SI-ATRP. To gain further information about the chemical composition of the glycopolymer-functionalized cryogels, solid-state NMR spectroscopy was applied (Figure 1). In the 13C NMR spectra of all samples the peaks corresponding to the poly(acrylamide) backbone of the cryogel support as well as the PNiPAm backbone can be observed (22, 36, 38, 42, and 176 ppm). For CG-PMan, additional signals between 60 and 86 ppm appear, which can be assigned to the sugar ring carbon atoms (61, 67, 70, 72, 74, and 85 ppm). To allow the assignment of the broad peaks in the solid-state spectra, these are overlaid with the solution-phase NMR spectra of the respective monomers (Figure 1 (right)). As can be clearly seen, the according peaks appear in solid-state spectra at the same chemical shift, confirming the sugar functionalization of the cryogel CGPMan. Similar observations were made for the glucose functional cryogel CG-PGlc showing signals that correspond to the sugar ring carbon atoms as well (62, 70, 73, 78, 81, and 86 ppm). In contrast, the 13C NMR spectrum of CG-PNiPAm does not reveal signals in this region of the spectrum since no 328


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ACS Macro Letters glycomonomer was used in this case. These findings prove the successful attachment of the polymers to the cryogel surface. To characterize the grafted polymer chains separately in terms of molar mass (distribution), PMan, PGlc, and PNiPAm were detached from the cryogel via transesterification using 0.5 M sodium methoxide solution in anhydrous methanol. Subsequently, analysis by means of size exclusion chromatography (SEC) was performed (Figure 2), revealing monomodal molar mass distributions with dispersities Đ around 1.3 (Table 1).

bacteria) or other particle-containing solutions from biological samples. Compared to the initial cryogel CG-Br the wateruptake capacity is not increased (35 mgwater/mggel) since the main constituent of the cryogel matrix is not changed by the SIATRP. The porous character is further illustrated by SEM micrographs (Figure 3), revealing pores in the size range from 10 to 100 μm. To investigate if the developed sugar functional cryogels would indeed possess an affinity toward certain pathogens, they were incubated with E. coli bacteria. E. coli is a Gram-negative bacterium commonly found in the intestine part of organisms but also known as a widespread pathogen. It is a frequent source of infections from water and food and, thus, is widely accepted as an indicator bacterium for water contamination.35 That is why the European law demands its quantification in drinking water.36 E. coli is known to bind selectively to mannose moieties.19,37,38 The E. coli strain W3110, a derivative of E. coli K-12, was used as a model in this study, as it is known that this strain has mannose-specific binding lectins located at the bacterial fimbriae (type I fimbriae).39 To assess the lectin-based affinity of E. coli W3110 to the mannose-functionalized cryogels, scanning electron microscopy (SEM) measurements were performed (Figure 3). Following incubation with an E. coli suspension (OD600 = 1), the functionalized cryogel samples were washed with phosphate-buffered saline (PBS) to remove bacteria bound unspecifically. As can be concluded from the SEM images (Figure 3, left column) CG-PNiPAm and CGPGlc do not bind E. coli bacteria from solution. The opposite is observed for CG-PMan, the surface of which is densely covered with bacteria.

Figure 2. Size exclusion chromatograms of the polymers cleaved from the cryogel supports (eluent DMAc/LiCl, RI detection).

The glycopolymer-functionalized cryogels feature a high water-uptake capacity, ranging from 30 to 37 times of the own mass (Table 1). This illustrates the high pore space, elasticity, and hydrophilic character of the functionalized material, which, therefore, is ideally suited for elution of whole cells (e.g.,

Figure 3. SEM images of representative areas of the functionalized cryogels after incubation with E. coli bacteria. The incubation was followed by cumulative washing steps with PBS, α-methyl glucoside, and α-methyl mannoside solutions prior to recording of the SEM images (after dehydration with ethanol mixtures and fixation with glutaraldehyde). 329


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ACS Macro Letters These findings suggest the mannose-specific binding interaction. In order to verify this effect, cryogels treated identically were additionally washed with an excess of α-methyl glucoside, which acts as a competing ligand with analogous structure to PGlc. As is clearly evident from the SEM images in Figure 3 (second column), the mannose gel is still densely populated with bacteria subsequent to the washing step. A third set of gels was additionally washed with the analogue α-methyl mannoside solution as competing ligand for CG-PMan (Figure 3, third column). As can be concluded from the SEM images, the binding interaction of the E. coli to CG-PMan is significantly reduced, confirming the mannose-specific nature of the binding interaction between the E. coli bacteria and the functionalized cryogel. Furthermore, these results provide an easy method for the release of the captured bacteria after accumulation from suspension by the use of a sugar-containing eluent. In conclusion, the successful polymerization of cryogels and the surface functionalization with mannose as well as glucose glycopolymers could be shown. The accumulation of E. coli bacteria on the mannose-functionalized gel as well as the subsequent release were achieved, revealing a possible application of the presented functional material for E. coli capture. Thus, the synthesized solid support represents a promising material for affinity chromatography of cell-containing fluids. This could be, e.g., applied to improve the detection of E. coli in drinking water, which is regulated according to ISO 93081:2014.36 However, the statutory detection is to date severely limited by the presence of contaminating organisms, which might be selectively filtered by a device such as the monolithic affinity column presented in this communication.

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facilities of the Jena Center for Soft Matter (JCSM) were established with a grant from the German Research Council (DFG) and the European Fonds for Regional Development (EFRE). The authors also acknowledge the Elektronenmikroskopisches Zentrum (EMZ) of the Universitätsklinikum Jena for providing the critical point drying device. MH gratefully acknowledges the German Research Foundation (DFG, GZ: HA 7725/1-1) for funding. CW acknowledges the Carl-Zeiss foundation. TB acknowledges the German Federal Ministry of Education & Research (BMBF #031A518B Vectura).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00856. Experimental procedures, cryogel synthesis, ATRP, bacteria experiments, SEM study (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected]. Present Address

§ Department of Chemistry, University of Warwick, Library Rd, Coventry, CV4 7AL, GB.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Dutch Polymer Institute (DPI, project area bioinspired polymers, project #686). Furthermore, the German Research Council (DFG), the European Fonds for Regional Development (EFRE), as well as the Carl Zeiss foundation contributed to the funding of this work. The SEM 330


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Glycopolymer-Functionalized Cryogels as Catch and Release Devices

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