Temperature-Controlled Adhesion to Carbohydrate Functionalized Microgel Films: An E. coli and Lectin Binding Study.

The preparation of thermoresponsive mannose functionalized monolayers of poly(N-isopropylacrylamide) microgels and the analysis of the specific binding of concanavalin A (ConA) and E. coli above and below the lower critical solution temperature (LCST) are shown. Via inhibition and direct binding assays it is found that ConA binding is time-dependent, where at short incubation times binding is stronger above the LCST. Given larger incubation times, the interaction of ConA to the microgel network is increased below the LCST when compared to temperatures above the LCST, possibly due to increased ConA diffusion and multivalent binding in the more open microgel network below the LCST. For E. coli, which presents only monovalent lectins and is too large to diffuse into the network, binding is always enhanced above the LCST. This is due to the larger mannose density of the microgel layer above the LCST increasing the interaction to E. coli. Once bound to the microgel layer above the LCST, E. coli cannot be released by cooling down below the LCST. Overall, this suggests that the carbohydrate presenting microgel layers enable specific binding where the temperature-induced transition between swollen and collapsed microgels may increase or decrease binding depending on the receptor size.

Lectin and E. coli Binding to Carbohydrate-Functionalized Oligo(ethylene glycol)-Based Microgels: Effect of Elastic Modulus, Crosslinker and Carbohydrate Density.

The synthesis of carbohydrate-functionalized biocompatible poly(oligo(ethylene glycol) methacrylate microgels and the analysis of the specific binding to concanavalin A (ConA) and Escherichia coli (E. coli) is shown. By using different crosslinkers, the microgels' size, density and elastic modulus were varied. Given similar mannose (Man) functionalization degrees, the softer microgels show increased ConA uptake, possibly due to increased ConA diffusion in the less dense microgel network. Furthermore, although the microgels did not form clusters with E. coli in solution, surfaces coated with mannose-functionalized microgels are shown to bind the bacteria whereas galactose (Gal) and unfunctionalized microgels show no binding. While ConA binding depends on the overall microgels' density and Man functionalization degree, E. coli binding to microgels' surfaces appears to be largely unresponsive to changes of these parameters, indicating a rather promiscuous surface recognition and sufficiently strong anchoring to few surface-exposed Man units. Overall, these results indicate that carbohydrate-functionalized biocompatible oligo(ethylene glycol)-based microgels are able to immobilize carbohydrate binding pathogens specifically and that the binding of free lectins can be controlled by the network density.

Switchable Adhesion of E. coli to Thermosensitive Carbohydrate-Presenting Microgel Layers: A Single-Cell Force Spectroscopy Study.

Adhesion processes at the cellular scale are dominated by carbohydrate interactions, including the attachment and invasion of pathogens. Carbohydrate-presenting responsive polymers can bind pathogens and inhibit pathogen invasion by remote stimuli for the development of new antibiotic strategies. In this work, the adhesion forces of E. coli to monolayers composed of mannose-functionalized microgels with thermosensitive poly(N-isopropylacrylamide) (PNIPAM) and poly(oligo(ethylene glycol)) (PEG) networks are quantified using single-cell force spectroscopy (SCFS). When exceeding the microgels' lower critical solution temperature (LCST), the adhesion increases up to 2.5-fold depending on the polymer backbone and the mannose density. For similar mannose densities, the softer PNIPAM microgels show a significantly stronger adhesion increase when crossing the LCST as compared to the stiffer PEG microgels. This is explained by a stronger shift in swelling, mannose density, and surface roughness of the softer gels when crossing the LCST. When using nonbinding galactose instead of mannose, or when inhibiting bacterial receptors, a certain level of adhesion remains, indicating that also polymer-fimbria entanglements contribute to adhesion. The presented quantitative analysis provides insights into carbohydrate-mediated bacterial adhesion and the relation to material properties and shows the prospects and limitations of interactive polymer materials to control the attachment of bacteria.

Temperature-Switchable Glycopolymers and Their Conformation-Dependent Binding to Receptor Targets.

The temperature-dependent binding of copolymers from poly(N-isopropylacrylamide) (PNIPAM) and mannose ligands to Escherichia coli and concanavalin A (ConA) is determined. Through polymer analogous reactions using poly(N-acryloxysuccinimide) and amine-linked mannose residues with different linkers, glycopolymers are prepared with the variation of the mannose density. Quantitative adhesion inhibition assays show the inhibitory potential of the glycopolymers as a function of the mannose/NIPAM ratio and linker type above and below their lower critical solution temperature (LCST). Intriguingly, opposite temperature effects on the binding to E. coli and ConA are observed. While the E. coli inhibition is stronger above the LCST, the ConA inhibition is, in overall, weaker at elevated temperatures. When going beyond the LCST, the polymers undergo a coil-to-globule transition, forming microphases with surface-enriched hydrophilic sugar moieties exhibiting increased E. coli inhibition through steric shielding. However, the formation of such microphases above the LCST renders a fraction of carbohydrate ligands inaccessible,and the polymers remaining in the solution phase then have coil sizes below the minimum binding site spacing of the ConA receptor, explaining reduced ConA inhibition. Overall, these results suggest that the coil-to-globule transition of glycopolymers may induce lower or higher inhibitory potentials due to the adverse effects of steric shielding and carbohydrate ligand accessibility.

Thermosensitive Display of Carbohydrate Ligands on Microgels for Switchable Binding of Proteins and Bacteria.

The synthesis of carbohydrate-functionalized thermosensitive poly(N-isopropylacrylamide) microgels and their ability to bind carbohydrate-binding pathogens upon temperature switch are reported. It is found that the microgels' binding affinity is increased above their lower critical solution temperature (LCST), enabling thermo-triggerable capture of pathogens. Here, a series of microgels with comparatively low mannose functionalization degrees below 1 mol % is achieved by a single polymerization step. Upon increase in mannose density, the microgel size increases, and the LCST decreases to 26 °C. Clustering with concanavalin A indicated that binding affinity is enhanced by a higher mannose content and by raising the temperature above the LCST. Binding studies with Escherichia coli confirm stronger specific interactions above the LCST and formation of mechanically stable aggregates enabling efficient separation of E. coli by filtration. For small incubation times above the LCST, the microgels' potential to release pathogens again below the LCST is confirmed also. Compared to existing switchable scaffolds, microgels nearly entirely composed of a thermosensitive material undergo a large change in volume, which allows them to drastically vary the density of ligands to switch between capture and release. This straightforward yet novel approach is likely compatible with a broad range of bioactive ligands. Therefore, thermosensitive microgels represent a promising platform for the specific capture or release of cells or pathogens.