Sweet Janus Particles: Multifunctional Inhibitors of Carbohydrate-Based Bacterial Adhesion.

Escherichia coli and other bacteria use adhesion receptors, such as FimH, to attach to carbohydrates on the cell surface as the first step of colonization and infection. Efficient inhibitors that block these interactions for infection treatment are multivalent carbohydrate-functionalized scaffolds. However, these multivalent systems often lead to the formation of large clusters of bacteria, which may pose problems for clearing bacteria from the infected site. Here, we present Man-containing Janus particles (JPs) decorated on one side with glycomacromolecules to target Man-specific adhesion receptors of E. coli. On the other side, poly(N-isopropylacrylamide) is attached to the particle hemisphere, providing temperature-dependent sterical shielding against binding and cluster formation. While homogeneously functionalized particles cluster with multiple bacteria to form large aggregates, glycofunctionalized JPs are able to form aggregates only with individual bacteria. The formation of large aggregates from the JP-decorated single bacteria can still be induced in a second step by increasing the temperature and making use of the collapse of the PNIPAM hemisphere. This is the first time that carbohydrate-functionalized JPs have been derived and used as inhibitors of bacterial adhesion. Furthermore, the developed JPs offer well-controlled single bacterial inhibition in combination with cluster formation upon an external stimulus, which is not achievable with conventional carbohydrate-functionalized particles.

Specific Binding of Ligand-Functionalized Thermoresponsive Microgels: Effect of Architecture, Ligand Density, and Hydrophobicity.

The biomolecular interaction of ligand-presenting switchable microgels is studied with respect to the polymer type, composition, and structure of the microgels. Monodisperse microgels are prepared through precipitation polymerization of N-isopropylacrylamide (PNIPAM microgels) or oligo(ethylene glycol methacrylamide)s (POEGMA microgels) in the presence of crosslinkers or in their absence (self-crosslinked). Functionalization with mannose or biotin as model ligands and affinity measurements upon heating/cooling are conducted to obtain mechanistic insights into how the microgel phase transition affects the specific interactions. In particular, we are interested in adjusting the crosslinking, swelling degree, and ligand density of mannose-functionalized microgels to reversibly catch and release mannose binding Escherichia coli by setting the temperature below or above the microgels' volume phase transition temperature (VPTT). The increased mannose density for collapsed microgels above the VPTT results in stronger E. coli binding. Detachment of E. coli by reswelling the microgels below the VPTT is achieved only for self-crosslinked microgels showing a stronger decrease in ligand density compared to microgels with dedicated crosslinkers. Owing to a reduced mannose density in the shell of POEGMA microgels, their E. coli binding was lower compared to PNIPAM microgels, as supported by ultraresolution microscopy. Importantly, an inverse temperature-controlled binding of microgels decorated with hydrophilic mannose and hydrophobic biotin ligands is observed. This indicates that hydrophobic ligands are inaccessible in the collapsed hydrophobic network above the VPTT, whereas hydrophilic mannose units are then enriched at the microgel-water interface and thus are more accessible.

Elastic modulus distribution in poly(N-isopopylacrylamide) and oligo(ethylene glycol methacrylate)-based microgels studied by AFM.

The spatial elastic modulus distribution of microgel networks in presence and absence of bifunctional crosslinkers is studied by AFM. Thermoresponsive poly(N-isopopylacrylamide) (PNIPAM) and poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol)methacrylate) (P(MEO2MA-co-OEGMA)) microgels are synthesized via precipitation polymerization above their lower critical solution temperature (LCST). High-resolution elastic modulus profiles are acquired using AFM force-indentation mapping of surface-deposited microgels at 25 °C. For both microgel systems, the use of a bifunctional crosslinker leads to a strong elastic modulus gradient with stiff microgel cores and soft networks toward the edge. In absence of a dedicated crosslinker (self-crosslinking), PNIPAM microgels show a homogeneous elastic modulus distribution, whereas self-crosslinked P(MEO2MA-co-OEGMA) microgels still show decreasing elastic moduli from the centre to the edge of the microgels. However, POEGMA microgels without comonomer showed no elastic modulus gradient suggesting that different incorporation rates of MEO2MA and OEGMA result in a radial variation of the polymer segment density. In addition, when varying the molecular weight of OEGMA the overall elastic modulus was affected, possibly due to molecular weight-dependent phase behavior and different reactivity. This shows that quite different microgel architectures can be obtained by the simple "one-pot" precipitation reaction of microgels which may open to new avenues toward advanced applications.

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.

Effect of PEGylation on Receptor Anchoring and Steric Shielding at Interfaces: An Adhesion and Surface Plasmon Resonance Study with Precision Polymers.

This study aims at quantifying the steric shielding effect of multivalent glycoconjugates targeting pathogens by blocking their carbohydrate binding sites. Specifically, PEGylated and non-PEGylated glycoconjugates are studied as inhibitors of lectins and bacterial adhesins evaluating the steric repulsion effect of the nonbinding PEG chains. We use the soft colloidal probe (SCP) adhesion assay to monitor the change in the adhesion energy of mannose (Man)-decorated hydrogel particles on a layer of concanavalin A (ConA) in the presence of sequence-defined multivalent glycoconjugate inhibitors over time. The results show that PEGylated glycoconjugates achieve a stronger adhesion inhibition when compared to non-PEGylated glycoconjugates although the dissociation constants (KD) of the PEGgylated compounds to ConA were larger. These results appear in line with Escherichia coli adhesion inhibition assays showing a small increase of bacteria detachment by PEGgylated glycoconjugates compared to non-PEGylated compounds. This suggests that an increase of sterical shielding via PEGylation may help reduce the invasiveness of pathogens even after they have adhered. Adhesion studies based on electrostatic interactions using amine-linked PEG of varying molecular weight confirm that such sterical shielding effect is not limited to carbohydrate-mediated adhesion.

Biomimetic estrogen sensor based on soft colloidal probes.

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Selective Adhesion and Switchable Release of Breast Cancer Cells via Hyaluronic Acid Functionalized Dual Stimuli-Responsive Microgel Films.

The detection of tumor cells from liquid biopsy samples is of critical importance for early cancer diagnosis, malignancy assessment, and treatment. In this work, coatings of hyaluronic acid (HA)-functionalized dual-stimuli responsive poly(N-isopropylacrylamide) (PNIPAM) microgels are used to study the specificity of breast cancer cell binding and to assess cell friendly release mechanisms for further diagnostic procedures. The microgels are established by straightforward precipitation polymerization with amine bearing comonomers and postfunctionalization with a UV-labile linker that covalently binds HA to the microgel network. Well-defined microgel coatings for cell binding are established via simple physisorption and annealing. The HA-presenting PNIPAM microgel films are shown to specifically adhere CD44 expressing breast cancer cell lines (MDA-MB-231 and MCF-7), where an increase in adhesion correlates with higher CD44 expression and HA functionalization. Upon cooling below the lower critical solution temperature of PNIPAM microgels, the cells could be released; however, 10-30% of the cells still remained on the surface even after prolonged cooling and mild mechanical agitation. A complete cell release is achieved after applying the light stimulus by short UV treatment cleaving HA units from the microgels. Owing to the comparatively straightforward preparation procedures, such dual-responsive microgel films could be considered for the effective capture, release, and diagnostics of tumor cells.