Evaluation of boronate-containing polymer brushes and gels as substrates for carbohydrate-mediated adhesion and cultivation of animal cells

Boronate-containing thin polyacrylamide gels (B-Gel), polymer brushes (B-Brush) and chemisorbed organosilane layers (B-COSL) were prepared on the surface of glass slides and studied as substrates for carbohydrate-mediated cell adhesion. B-COSL- and B-Brush-modified glass samples exhibited multiple submicron structures densely and irregularly distributed on the glass surface, as found by scanning electron microscopy and atomic force microscopy. B-Gel was ca. 0.1 mm thick and contained pores with effective size of 1–2 μm in the middle and of 5–20 μm on the edges of the gel sample as found by confocal laser scanning microscopy. Evidence for the presence of phenylboronic acid in the samples was given by time-of-flight secondary ion mass-spectrometry (ToF SIMS), contact angle measurements performed in the presence of fructose, and staining with Alizarin Red S dye capable of formation specific, fluorescent complexes with boronic acids. A comparative study of adhesion and cultivation of animal cells on the above substrates was carried out using murine hybridoma M2139 cell line as a model. M2139 cells adhered to the substrates in the culture medium without glucose or sodium pyruvate at pH 8.0, and then were cultivated in the same medium at pH 7.2 for 4 days. It was found that the substrates of B-Brush type were superior both regarding cell adhesion and viability of the adhered cells, among the substrates studied. MTT assay confirmed proliferation of M2139 cells on B-Brush substrates. Some cell adhesion was also registered in the macropores of B-Gel substrate. The effects of surface microstructure of the boronate-containing polymers on cell adhesion are discussed. Transparent glass substrates grafted with boronate-containing copolymers offer good prospects for cell adhesion studies and development of cell-based assays.     

Control of endothelial cell adhesion to polymer surface by ion implantation

The bio‐compatibility of ion implanted polymers has been studied by means of in vitro attachment measurements of bovine aorta endothelial cells. The specimens used were polystyrene (PS), polyethylene (PE), polypropylene (PP) and expanded polytetrafluoroethylene (ePTFE). He⁺ and Ne⁺ ion implantation were performed at an energy of 150 keV with fluences between 1 × 10 ¹³ to 1 × 10 ¹⁷ ions/cm ² at room temperature. Wettability was estimated by means of a sessile drop method. The chemical and physical structures of ion implanted polymers were investigated by contact angle measurements, atomic force microscopy and X‐ray photoelectron spectroscopic analysis in relation to cell attachment behavior. The strength of cell attachment on ion implanted specimens at static and under flow conditions was also measured. Ion implanted PP and ePTFE were found to exhibit remarkably higher adhesion and spreading of endothelial cells than non‐implanted specimens. In contrast to these findings, ion implanted PS and PE only demonstrated a little improvement of cell adhesion in this assay. Copyright © 2001 John Wiley & Sons, Ltd.     

Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.     

Inhibitory effect of hydrophilic polymer brushes on surface-induced platelet activation and adhesion

Poly(N,N-dimethylacrylamide) (PDMA) brushes are successfully grown from unplasticized poly(vinyl chloride) (uPVC) by well-controlled surface-initiated atom transfer radical polymerization (SI-ATRP). Molecular weights of the grafted PDMA brushes vary from ≈ 35,000 to 2,170000 Da, while the graft density ranges from 0.08 to 1.13 chains · nm(-2). The polydispersity of the grafted PDMA brushes is controlled within 1.20 to 1.80. Platelet activation (expression of CD62) and adhesion studies reveal that the graft densities of the PDMA brushes play an important role in controlling interfacial properties. PDMA brushes with graft densities between 0.35 and 0.50 chains · nm(-2) induce a significantly reduced platelet activation compared to unmodified uPVC. Moreover, the surface adhesion of platelets on uPVC is significantly reduced by the densely grafted PDMA brushes. PDMA brushes that have high molecular weights lead to a relatively lower platelet activation compared to low-molecular-weight brushes. However, the graft density of the brush is more important than molecular weight in controlling platelet interactions with PVC. PDMA brushes do not produce any significant platelet consumption in platelet rich plasma. Up to a seven-fold decrease in the number of platelets adhered on high graft density brushes is observed compared to the bare PVC surface. Unlike the bare PVC, platelets do not form pseudopodes or change morphology on PDMA brush-coated surfaces.     

Combined QCM-D/GE as a tool to characterize stimuli-responsive swelling of and protein adsorption on polymer brushes grafted onto 3D-nanostructures

A combined setup of quartz crystal microbalance and generalized ellipsometry can be used to comprehensively investigate complex functional coatings comprising stimuli-responsive polymer brushes and 3D nanostructures in a dynamic, noninvasive in situ measurement. While the quartz crystal microbalance detects the overall change in areal mass, for instance, during a swelling or adsorption process, the generalized ellipsometry data can be evaluated in terms of a layered model to distinguish between processes occurring within the intercolumnar space or on top of the anisotropic nanocolumns. Silicon films with anisotropic nanocolumnar morphology were prepared by the glancing angle deposition technique and further functionalized by grafting of poly-(acrylic acid) or poly-(N- isopropylacrylamide) chains. Investigations of the thermoresponsive swelling of the poly-(N-isopropylacrylamide) brush on the Si nanocolumns proved the successful preparation of a stimuli-responsive coating. Furthermore, the potential of these novel coatings in the field of biotechnology was explored by investigation of the adsorption of the model protein bovine serum albumin. Adsorption, retention, and desorption triggered by a change in the pH value is observed using poly-(acrylic acid) functionalized nanostructures, although generalized ellipsometry data revealed that this process occurs only on top of the nanostructures. Poly-(N-isopropylacrylamide) is found to render the nanostructures non-fouling properties.     

Reversible conformational transitions of a polymer brush containing boronic acid and its interaction with mucin glycoprotein

Reversible changes of the height of a polymer brush containing phenylboronic acid were studied. The polymer brush thickness underwent reversible changes of 0.5–1 nm, in response to the changes in composition of the contacting aqueous phase from deionized water to bicarbonate buffer and vice versa, apparently due to the conformational transition of the weak polyelectrolyte to the more extended electrically charged state. Adsorption of mucin glycoprotein to the polymer brush took place due to boronate/sugar interactions between the glycoprotein and the graft copolymer and resulted in further increase of the brush height by ca. 1.5 nm, as observed by means of spectral correlation spectroscopy and ellipsometry.

     

Adsorption of ions onto polymer surfaces and its influence on zeta potential and adhesion phenomena

 The adhesion behavior that governs many technologically and biologically relevant polymer properties can be investigated by zeta potential measurements with varied electrolyte concentration or pH. In a previous work [1] it was found that the difference of the adsorption free energies of Cl^- and K⁺ ions correlates with the adhesion force caused by van der Waals interactions, and that the decrease of adhesion strength by adsorption layers can be elucidated by zeta potential measurements. In order to confirm these interrelations, zeta potential measurements were combined with atomic force microscopy (AFM) measurements. Force–distance curves between poly(ether ether ketone) and fluorpolymers, respectively, and the Si₃N₄ tip of the AFM device in different electrolyte solutions were measured and analysed. The adsorption free energy of anions calculated from the Stern model correlates with their ability to prevent the adhesion between the polymer surface and the Si₃N₄ tip of the AFM device. These results demonstrate the influence of adsorption phenomena on the adhesion behavior of solids. The results obtained by AFM confirm the thesis that the electrical double layer of solid polymers in electrolyte solutions is governed by ion adsorption probably due to van der Waals interactions and that therefore van der Waals forces can be detected by zeta potential measurements. Received: 18 November 1997 Accepted: 19 January 1998     

Design and characterization of endosomal-pH-responsive coiled coils for constructing an artificial membrane fusion system

A weakly acidic pH-responsive polypeptide is believed to have the potential for an endosome escape function in a polypeptide-triggered delivery system. For constructing a membrane fusion device with pH-responsiveness, we have designed novel polypeptides that are capable of forming an α2 coiled coil structure. Circular dichroism spectroscopy reveals that a polypeptide, AP-LZ(EH5), with a Glu and His salt-bridge pair at a staggered position in the hydrophobic core forms a stable coiled coil structure only at endosomal pH values (pH 5.0 to 5.5). On the basis of their endosomal-pH responsiveness, a boronic acid/polypeptide conjugate (BA-H5-St) was also designed as a pilot molecule to construct a pH-responsive, one-way membrane fusion system with a sugarlike compound (phosphatidylinositol: PI)-containing liposome as a target. Membrane fusion behavior was characterized by lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays. These studies reveal that membrane fusion is clearly observed when the pH of the experimental system is changed from 7.4 (physiological condition) to 5.0 (endosomal condition).     

Discrimination and classification of ginsenosides and ginsengs using bis-boronic acid receptors in dynamic multicomponent indicator displacement sensor arrays

Ginsenosides are complex natural products with a diverse array of biological activities, but their molecular recognition and sensing is challenging. A library of simple bis-boronic acid-based receptors with various spacers was synthesized for the sensing of ginsenosides. The incorporation of two boronic acids allowed the pairing of two indicators, which can simultaneously bind the receptors or two saccharides within the ginsenosides. A cross-reactive sensing array was therefore constructed using the receptors in conjunction with different pairs of indicators. LDA plots created from the colorimetric response of the hosts and indicator pairs reveal excellent classification of the ginsenosides, and the corresponding loading plots reveal the cross-reactivity of the receptors. In addition, several commercial ginseng extracts were unambiguously classified using the same sensing array. The assay reported here should be applicable to the analysis of other large saccharide-based natural products.     

Molecular Encryption and Reconfiguration for Remodeling of Dynamic Hydrogels

Dynamic materials have been widely studied for regulation of cell adhesion that is important to a variety of biological and biomedical applications. These materials can undergo changes mainly through one of the two mechanisms: ligand release in response to chemical, physical, or biological stimuli, and ligand burial in response to mechanical stretching or the change of electrical potential. This study demonstrates an encrypted ligand and a new hydrogel that are capable of inducing and inhibiting cell adhesion, which is controlled by molecular reconfiguration. The ligand initially exhibits an inert state; it can be reconfigured into active and inert states by using unblocking and recovering molecules in physiological conditions. Since molecular reconfiguration does not require the release of the ligand from the hydrogels, inhibiting and inducing cell adhesion on the hydrogels can be repeated for multiple cycles.     

Plasticization of a polymer layer harnessed to a silicon microcantilever as a highly sensitive and selective means to detect nitroaromatic derivatives

A new approach to the chemo‐mechanical detection of trace amounts of nitroaromatics, even in the presence of high concentrations of background materials, is presented. The detection scheme is based on the plasticization of an aminopropyl silane layer that is harnessed to a silicon beam following its reaction with nitroaromatic systems. The reaction‐induced plasticization attenuates the temperature induced bending of the polymer‐beam sandwich, offering a simple and very sensitive tool for the detection of nitroaromatic systems. Using this approach, it was possible to detect a sample of 100 pg TNT even in the presence of a ～10⁹ fold excess of a background material such as acetonitrile. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 2124–2130     

Preparation of polymer particles having ethyleneurea groups at their surfaces by emulsifier-free seeded emulsion polymerization and wet adhesion of its emulsion film

Polymer emulsion having ethyleneurea groups at particle surfaces was produced by emulsifier-free seeded emulsion copolymerization of n-butyl methacrylate (BMA) and methacrylamide ethylethyleneurea (EU) with poly(BMA) seed particles utilizing the starved-fed monomer addition method. This emulsion film, prepared by casting the polymer emulsion on an alkyd resin plate, had a superior adhesion in water, as well as on stainless steel. Such superior wet adhesions seem to be based on a large amount of EU predominantly localized at the particle surfaces.Part CCXLIX of the series "Studies on suspension and emulsion"     

RANKL stimulates proliferation, adhesion and IL-7 expression of thymic epithelial cells

In many clinical situations which cause thymic involution and thereby result in immune deficiency, T cells are the most often affected, leading to a prolonged deficiency of T cells. Since only the thymic-dependent T cell production pathway secures stable regeneration of fully mature T cells, seeking strategies to enhance thymic regeneration should be a key step in developing therapeutic methods for the treatment of these significant clinical problems. This study clearly shows that receptor activator of NF-kappaB ligand (RANKL) stimulates mouse thymic epithelial cell activities including cell proliferation, thymocyte adhesion to thymic epithelial cells, and the expression of cell death regulatory genes favoring cell survival, cell adhesion molecules such as ICAM-1 and VCAM-1, and thymopoietic factors including IL-7. Importantly, RANKL exhibited a significant capability to facilitate thymic regeneration in mice. In addition, this study demonstrates that RANKL acts directly on the thymus to activate thymus regeneration regardless of its potential influences on thymic regeneration through an indirect or systemic effect. In light of this, the present study provides a greater insight into the development of novel therapeutic strategies for effective thymus repopulation using RANKL in the design of therapies for many clinical conditions in which immune reconstitution is required.     

Use of polystyrene brushes to investigate the role of interface between substrates and thin homogeneous films

The viscoelastic properties of thin polystyrene (PS) films depend on confinement, as it can modify the molecular dynamics affecting the glass transition. In the recent past, the authors have investigated the region next to the free interface by means of an atomic force microscope suitably modified to monitor the indentation of a tip into a film during a given lapse of time while applying a constant load. Herein, to explore the interface with the substrate, the authors report on experiments in which PS brushes grafted to native silicon oxide were used. It was found that the film wettability on brushes and H‐terminated silicon can be highly improved when compared with native silicon oxide. In addition, the glass transition temperature of thin films increases up to the bulk value in the case of film/brush combinations with high molecular weight or films with high molecular weight on H‐terminated silicon. Data are discussed according to hypotheses such as residual solvent presence, interface free volume, and molecular mechanical coupling. These observations can be of great interest for nanotechnological applications, especially in those instances where one needs to tailor the temperature dependence of viscoelastic properties of thin films. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013, 51, 1149–1156     

Boronate‐Affinity Glycan‐Oriented Surface Imprinting: A New Strategy to Mimic Lectins for the Recognition of an Intact Glycoprotein and Its Characteristic Fragments

Lectins possess unique binding properties and are of particular value in molecular recognition. However, lectins suffer from several disadvantages, such as being hard to prepare and showing poor storage stability. Boronate‐affinity glycan‐oriented surface imprinting was developed as a new strategy for the preparation of lectin‐like molecularly imprinted polymers (MIPs). The prepared MIPs could specifically recognize an intact glycoprotein and its characteristic fragments, even within a complex sample matrix. Glycan‐imprinted MIPs could thus prove to be powerful tools for important applications such as proteomics, glycomics, and diagnostics.     

Diversity of the surface properties of Lactococci and consequences on adhesion to food components

Bacteria possess surface properties, related to their charge, hydrophobicity and Lewis acid/base characteristics, that are involved in the attachment processes of microorganisms to surfaces. Fermentation bulks and food matrixes are complex heterogeneous media containing various components with different physicochemical characteristics. The aim of the present study was to investigate whether (i) bacteria present in a food matrix, interacted physicochemically at their surface level with the other constituents and (ii) the diversity of bacterial surface properties could result in a diversity of microbial adhesion to components and thus in a diversity of tolerance to toxic compounds. The surface properties of 20 lactic acid bacteria were characterized by the MATS method showing their relatively hydrophilic and various basic characteristics. The results obtained from a set of representative strains showed that (i) the strains with higher affinity for apolar solvents adsorbed more to lipids and hydrophobic compounds, (ii) the more the strains adsorbed to a toxic solvent, the less they were tolerant to this solvent. A diversity of bacterial surface properties was observed for the strains in the same species showing the importance of choosing bacteria according to their surface properties in function of technological objectives.     

Diversity of the surface properties of Lactococci and consequences on adhesion to food components

Bacteria possess surface properties, related to their charge, hydrophobicity and Lewis acid/base characteristics, that are involved in the attachment processes of microorganisms to surfaces. Fermentation bulks and food matrixes are complex heterogeneous media containing various components with different physicochemical characteristics. The aim of the present study was to investigate whether (i) bacteria present in a food matrix, interacted physicochemically at their surface level with the other constituents and (ii) the diversity of bacterial surface properties could result in a diversity of microbial adhesion to components and thus in a diversity of tolerance to toxic compounds. The surface properties of 20 lactic acid bacteria were characterized by the MATS method showing their relatively hydrophilic and various basic characteristics. The results obtained from a set of representative strains showed that (i) the strains with higher affinity for apolar solvents adsorbed more to lipids and hydrophobic compounds, (ii) the more the strains adsorbed to a toxic solvent, the less they were tolerant to this solvent. A diversity of bacterial surface properties was observed for the strains in the same species showing the importance of choosing bacteria according to their surface properties in function of technological objectives.