Carbohydrate-specific, boronate-containing copolymers: from sugar-sensing to cell adhesion

Carbohydrates play a major role in many cell recognition events and metabolic pathways of living organisms. Synthetic carbohydrate receptors are of great interest for the chemical design of sugar sensors, materials for cell separation, cell immobilization, drug delivery and other biomedical/biotechnological applications. These receptors are more stable under harsh conditions and have lower cost as compared to natural carbohydrate-binding proteins, such as lectins, antibodies or enzymes. Phenylboronic acid (PBA) is a highly stable, well-studied synthetic ligand that exhibits specificity to carbohydrates and has been employed for the molecular recognition of sugars, nucleotides, nucleosides, glycoproteins, and other biological molecules containing diol or polyol moieties. Water-soluble boronate-containing copolymers (BCCs) are extremely attractive for preparing glucose-responsive materials that can be used for both sugarsensing and drug delivery. Another important property of BCCs is their reversible multivalent binding to the carbohydrates present on the surfaces of living cells, thus enabling the cell adhesion to artificial BCC-grafted surfaces. This thesis presents a study of several BCCs and their applications to sugar-sensing and the reversible adhesion of carbohydrate particles and cells. Novel BCC-containing polymer brushes were prepared on glass slides by graft copolymerization, and these BCC brushes were characterized by time-of-flight secondary ion mass-spectrometry, atomic force microscopy and contact angle measurements. The brushes were able to interact strongly with carbohydrates of living mammalian cells surfaces and cause cell adhesion. It was possible to detach the adhered living cells on demand by competitive elution with an aqueous fructose solution under mild conditions. Thus, a promising technique for manipulation of mammalian cells was discovered. A boronate-containing hydrogel was used for quantitative sugar assays based on the optical detection of PBA-sugar binding. The interaction of sugar molecules with the boronate groups of the gels provided changes in the gel turbidity, which were detected by a spectrophotometer. BCC gels gave linear and reversible optical response to sugars in a broad concentration range at physiological pH. Scanning electron microscopy revealed the existence of micrometer-sized pores in the gels, which explained the good gel permeability and accessibility of the PBA ligands to the analytes. Moreover, the gel was able to maintain its properties after autoclaving, which allows for sterilization and repeated use of the sensor for sugar monitoring during fermentation processes

Boronate-containing copolymers: Polyelectrolyte properties and sugar-specific interaction with agarose gel

Copolymers of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with acryamide (AA), N,N-dimethylacrylamide (DMAA), and N-isopropylacrylamide (NIPAM) were found to adsorb on cross-linked agarose gel (Sepharose CL-6B) in the pH range from 7.5-9.2, due to specific boronate-sugar interactions. The molar percentages of phenylboronic acid (PBA) groups in the boronate-containing copolymers (BCCs), as estimated by H-1 NMR spectroscopy, were 13, 10, and 16%, respectively, whereas the apparent ionization constants, the pK(a) values, of the copolymers were similar and equal to 9.0 +/- 0.2 at 20 degrees C. The copolymers adsorption capacities were in the range of 15-30 mg center dot ml(-1) gel (14-36 mu mol pendant PBA ml(-1) gel) at pH 9.2 and decreased with decreasing pH value. The interaction of monomeric NAAPBA with Sepharose CL-6B was characterized by an equilibrium association constant of 53 +/- 17 m(-1), the chromatographic capacity factor k ' = 1.8, and a total content of binding sites of 27 +/- 10 mu mol center dot ml(-1) gel at pH 9.2. The weak reversible binding of monomeric NAAPBA and almost irreversible binding of NAAPBA copolymers to the gel at pH 9.2 suggested a multivalent character of the copolymer adsorption. At pH 7.5, the maximal adsorption capacity was displayed by the AA-NAAPBA copolymer (45 mg center dot ml(-1) gel). All the BCCs could be completely desorbed from the gel by 0.1 m fructose in aqueous buffered media with pH values from 7.5-9.2. The strong adsorption of AA-NAAPBA on agarose gel probably relates to the conformation of the copolymer in aqueous solution and provides opportunities for biomedical applications of the copolymer under physiological conditions. Multivalent, weak-affinity adsorption of BCCs to the agarose gel seems to be a tentative model for the copolymers' binding to oligo- and polysaccharides of cell membranes and mucosal surfaces

Fluorescence of Single Conjugated Chains of MEH-PPV Dispersed in Polymer Matrix : What Do We See in the Microscope?†

It has been a mystery that the detected fluorescence intensity of single MEH-PPV molecules is much lower than expected based on their chain length. In this review, after re-evaluating of the literature data in the light of new specially designed experiments, we present a comprehensive explanation of this issue: The actual size of MEH-PPV molecules at single molecule level is much smaller than expected due to de-aggregation and chain scission, while static quenching ("dark matter") also exists for large molecules, further reducing their brightness. (Figure presented.

Affinity adhesion of carbohydrate particles and yeast cells to boronate-containing polymer brushes grafted onto siliceous supports

Cross-linked agarose particles (Sepharose CL-6B) and baker's yeast cells were found to adhere to siliceous supports end-grafted with boronate-containing copolymers (BCCs) of N,N-dimethylacrylamide at pH >= 7.5, due to boronate interactions with surface carbohydrates of the particles and the cells. These interactions were registered both on macroscopic and on molecular levels: the BCCs spontaneously adsorbed on the agarose gel at pH >= 7.5, with adsorption increasing with pH. Agarose particles and yeast cells stained with Procion Red HE-3B formed stable, monolayer-like structures at pH 8.0, whereas at pH 7.0-7.8 the structures on the copolymer-grafted supports were less stable and more random. At pH 9.0, 50% saturation of the surface with adhering cells was attained in 2 min. Stained cells formed denser and more stable layers on the copolymer-grafted supports than they did on supports modified with self-assembled organosilane layers derivatized with low-molecular-weight boronate, presumably due to a higher reactivity of the grafted BCCs. Quantitative detachment of adhered particles and cells could be achieved by addition of 20 mm fructose - a strong competitor for binding to boronates - at pH 7.0-9.0. Regeneration of the grafted supports allowed several sequential adhesion and detachment cycles with stained yeast cells. Affinity adhesion of micron-sized carbohydrate particles to boronate-containing polymer brushes fixed on solid supports is discussed as a possible model system suggesting a new approach to isolation and separation of living cells

Optical responses, permeability and diol-specific reactivity of thin polyacrylamide gels containing immobilized phenylboronic acid

Thin semitransparent gels were prepared by radical copolymerization of N-acryloyl-m-aminophenylboronic acid (NAAPBA) and acrylamide (AAm) taken in molar ratios from 8:92 to 16:84, respectively, in water. The gels were characterized by the content of immobilized NAAPBA and monomer conversion. Scanning electron microscopy revealed the micrometer size pores in the dried gels. The wet gels displayed a linear optical response to sugars with sensitivity decreasing in the series: D-fructose, D-galactose, D-glucose, D-mannose, N-acetyl-D-glucosamine in the sugar concentration range from 1 to 40-60 mM at pH 7.3. Cross-linking of the gels with N,N-methylene-bis-acrylamide decreased the strength of optical response. Specific binding capacity of a diol-containing dye Alizarin Red S in the gels at pH 7.0 coincided with the content of immobilized NAAPBA indicating the 1: 1 stoichiometry of the reaction and, therefore, good accessibility of the boronic acid ligands for water-soluble diols. Permeability of the gels was studied with a non-interacting dye Ethyl Orange exhibiting the pore diffusion coefficient of 1.4 x 10(-7) cm(2)/s. The rate of optical response of the gels to glucose was found to be determined by diffusion of sugar into the relatively thick gels (1 = 0.35-1 mm) with effective diffusion coefficients of 2 x 10(-7) cm(2)/S. In the thinner gels (l = 0.1 mm) the input of other kinetic processes, such as affinity binding or structural rearrangements of the gel, was noticeable. (c) 2008 Elsevier Ltd. All rights reserved

Thin semitransparent gels containing phenylboronic acid: porosity, optical response and permeability for sugars.

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes. Copyright (c) 2008 John Wiley & Sons, Ltd

Molecular Weight Determination by Counting Molecules

Molecular weight (MW) is one of the most important characteristics of macromolecules. Sometimes, MW cannot be measured correctly by conventional methods like gel permeation chromatography (GPC) due to, for example, aggregation. We propose using single-molecule spectroscopy to measure the average MW simply by counting individual fluorescent molecules embedded in a thin matrix film at known mass concentration. We tested the method on dye molecules, a labeled protein, and the conjugated polymer MEH-PPV. We showed that GPC with polystyrene calibration overestimates the MW of large MEH-PPV molecules by 40 times due to chain aggregation and stiffness. This is a crucial observation for understanding correlations between the conjugated polymer length, photophysics and performances of devices. The method can measure the MW of fluorescent molecules, biological objects, and nanoparticles at ultimately low concentrations and does not need any reference; it is conformation-independent and has no limitations regarding the detected MW range

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. 0.1mm thick and contained pores with effective size of 1-2mum in the middle and of 5-20mum 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

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

Boronate-containing polymer brushes: Characterization, interaction with saccharides and mammalian cancer cells.

Boronate-containing polymer brushes were synthesized by free radical copolymerization of N,N-dimethylacrylamide (DMAA) and N-acryloyl-m-phenylboronic acid (NAAPBA) (9:1) on the surface of 3-mercaptopropyl-silylated glass plates and capillaries. The brushes were characterized with time-of-flight secondary ion mass-spectrometry (ToF SIMS), atomic force microscopy and contact angle measurements. Fructose caused a well-expressed drop spreading on the surface of copolymer-grafted glass, due to the strong interaction with the boronate groups. Sedimentation of murine hybridoma cells M2139 or human myeloid leukemia cells KG1 onto the DMAA-NAAPBA copolymer-grafted glass plates from 10 mM phosphate buffer solution (pH 8.0) resulted in the cell adhesion. The adhered M2139 and KG1 cells could be quantitatively detached from the grafted plates with 0.1M fructose, which competed with cell surface carbohydrates for binding to the boronates. Evaluation of the binding strength between M2139 cells and the copolymer brush was performed by exposure of the adhered cells to a shear stress. Detachment of a fraction of 18% of the adhered M2139 cells was obtained at a shear force of 1400-2800 pN/cell generated by the running phosphate buffer (pH 8.0), whereas the remaining adhered cells (70%) could be detached with 0.1M fructose dissolved in the same buffer. Possible applications of the boronate-containing polymer brushes to affinity cell separation can be based upon the facile recovery of the attached cells. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 2008