Determination of oversulfated chondroitin sulfate and dermatan sulfate impurities in heparin by capillary electrophoresis

Recently, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. The United States Pharmacopeia (USP) and European Pharmacopeia (Eur.Ph.) announced an immediate revision of their monographs for heparin sodium by adding two US Food and Drugs Administration-recommended tests for OSCS based on nuclear magnetic resonance and capillary electrophoresis (CE). However, the proposed CE method provides only partial separation of the OSCS contaminant from heparin, thereby hindering appropriate impurity profiling. Here we present an improved CE method that is especially useful for the reliable quantification of OSCS in heparin samples, but also allows determination of the common impurity dermatan sulfate (DS). Parameters such as type and concentration of background electrolyte, capillary temperature, sample concentration and injection volume were investigated and optimized. Enhancement of the OSCS–heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. High currents and excessive Joule heating were prevented by employing fused-silica capillaries with an internal diameter of 25 μm. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits injection of relatively high heparin concentrations (up to 50 mg/ml) and large sample volumes (up to 5% of the capillary volume) allowing OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method is shown to be repeatable and linear (R² > 0.99) for OSCS, heparin and DS. CE analyses of OSCS-contaminated heparin samples and different heparin standards further demonstrate the utility of the method.     

硫酸皮肤素的共振瑞利散射法测定

研究了硫酸皮肤素对十六烷基三甲基溴化铵(CTMAB)发生缔合反应的条件、共振瑞利散射特征.结果表明,在pH 5.8的BR缓冲溶液中,硫酸皮肤素能与CTMAB形成离子缔合物,使共振瑞利散射RRS急剧增强并产生新的RRS光谱.硫酸皮肤素浓度在0.04～4.0μg/mL之间与散射强度呈线性关系,方法检出限(3σ)为13 ng/mL,并以CTMAB体系为例研究了共存物质的影响,表明方法选择性好.     

溴代十六烷基吡啶光谱探针RRS法测定硫酸皮肤素

研究了溴代十六烷基吡啶与硫酸皮肤素发生缔合反应的条件、共振瑞利散射特征.结果表明,在pH5.0的BR缓冲溶液中,硫酸皮肤素能与溴代十六烷基吡啶形成离子缔合物,使共振瑞利散射RRS急剧增强并产生新的RRS光谱.硫酸皮肤素浓度在0.015μg/mL～2.5μg/mL之间与散射强度呈线性关系,方法具有较高的灵敏度,其检出限(3σ)为4.5ng/mL,还研究了共存物质的影响,表明该方法选择性较好.该法用于血样和尿样中硫酸皮肤素含量的测定,结果令人满意.     

Nanomechanical properties of polymer brushes by colloidal AFM probes

Nanomechanical properties of end grafted polymer layers were studied by AFM based, colloidal probe compression measurements. Zwitterionic poly(sulfobetaine methacrylate) (PSBMA) brush was grafted from planar Si surface and poly(methyl methacrylate) (PMAA) brush was grown on colloidal probe by surface initiated atom transfer radical polymerization. PMAA brush was further modified with adhesion promoting arginyl-glycyl-aspartic acid (RGD) peptide sequences. Force–distance curves were obtained for systems where the polymer brushes were probed on unmodified surfaces or face to each other. For each systems the grafting density of the polymer brush was determined applying a ‘box’ like polymer brush model based on the theory by de Gennes. ‘Average’ grafting density was calculated in cases when two polymer brushes face each other: RGD functionalized PMAA or PMAA against PSBMA. For our systems the values for the grafting density was between 0.04 and 0.11 nm^?2. Furthermore the measured approach force–distance curves were fitted according to the Hertz model and the apparent Young’s modulus was determined for all measurements being in a range of around 250 kPa at physiological conditions.     

Online preconcentration and determination of chondroitin sulfate,dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis

Capillary electrophoresis with large‐volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and ‐16 kV. Exploiting large‐volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.     

Effect of charge asymmetry and charge screening on structure of superlattices formed by oppositely charged colloidal particles

Colloidal suspensions made up of oppositely charged particles have been shown to self-assemble into substitutionally ordered superlattices. For a given colloidal suspension, the structure of the superlattice formed from self-assembly depends on its composition, charges on the particles, and charge screening. In this study we have computed the pressure-composition phase diagrams of colloidal suspensions made up of binary mixtures of equal sized and oppositely charged particles interacting via hard core Yukawa potential for varying values of charge screening and charge asymmetry. The systems are studied under conditions where the thermal energy is equal or greater in magnitude to the contact energy of the particles and the Debye screening length is smaller than the size of the particles. Our studies show that charge asymmetry has a significant effect on the ability of colloidal suspensions to form substitutionally ordered superlattices. Slight deviations of the charges from the stoichiometric ratio are found to drastically reduce the thermodynamic stability of substitutionally ordered superlattices. These studies also show that for equal-sized particles, there is an optimum amount of charge screening that favors the formation of substitutionally ordered superlattices.     

Synthesis and anti-inflammatory activity of gold-nanoparticle bearing a dermatan sulfate disaccharide analog

A novel gold glyconanoparticle coating with DS disaccharide analog has been synthesized, and the potential anti-inflammatory activity was studied using carrageenan-induced paw edema model.     

Energy, charge, and spin transport in molecules and self-assembled nanostructures inspired by photosynthesis

Electron transfer in biological molecules provides both insight and inspiration for developing chemical systems having similar functionality. Photosynthesis is an example of an integrated system in which light harvesting, photoinduced charge separation, and catalysis combine to carry out two thermodynamically demanding processes, the oxidation of water and the reduction of carbon dioxide. The development of artificial photosynthetic systems for solar energy conversion requires a fundamental understanding of electron-transfer reactions between organic molecules. Since these reactions most often involve single-electron transfers, the spin dynamics of photogenerated radical ion pairs provide important information on how the rates and efficiencies of these reactions depend on molecular structure. Given this knowledge, the design and synthesis of large integrated structures to carry out artificial photosynthesis is moving forward. An important approach to achieving this goal is the development of small, functional building blocks, having a minimum number of covalent bonds, which also have the appropriate molecular recognition sites to facilitate self-assembly into a complete, functional artificial photosynthetic system.     

Experimental study of electrostatically stabilized colloidal particles: colloidal stability and charge reversal

We consider the interaction of colloidal spheres in the presence of mono-, di-, and trivalent ions. The colloids are stabilized by electrostatic repulsion due to surface charges. The repulsive part of the interaction potential Ψ(d) is deduced from precise measurements of the rate of slow coagulation. These "microsurface potential measurements" allow us to determine a weak repulsion in which Ψ(d) is of the order of a few k(B)T. These data are compared to ζ potential measured under similar conditions. At higher concentrations both di- and trivalent counterions accumulate at the very proximity of the particle surface leading to charge reversal. The salt concentration c(cr) at which charge reversal occurs is found to be always above the critical coagulation concentration c(ccc). The analysis of Ψ(d) and of the ζ potential demonstrates, however, that adsorption of multivalent counterions starts far below c(cr). Hence, colloid stability in the presence of di- and trivalent ions cannot be described in terms of a DLVO ansatz assuming a surface charge that is constant with regard to the ionic strength.     

Adhesion of single polyelectrolyte molecules on silica, mica, and bitumen surfaces

In a recent study (Energy Fuels 2005, 19, 936), a partially hydrolyzed polyacrylamide (HPAM) was used as a process aid to recover bitumen from oil sand ores. It was found that HPAM addition at the bitumen extraction step not only improved bitumen recovery but also enhanced fine solids settling in the tailings stream. To understand the role of HPAM, single-molecule force spectroscopy was employed for the first time to measure the desorption/adhesion forces of single HPAM molecules on silica, mica, and bitumen surfaces using an atomic force microscope (AFM). Silicon wafers with an oxidized surface layer and newly cleaved mica were used, respectively, to represent sand grains and clays in oil sands. The force measurements were carried out in deionized water and in commercial plant process water under equilibrium conditions. The desorption/adhesion forces of HPAM obtained on mica, silica, and bitumen surfaces were approximately 200, 40, and 80 pN in deionized water and approximately 100, 50, and 40 pN in the plant process water, respectively. The measured adhesion forces together with the zeta potential values of these surfaces indicate that the polymer would preferentially adsorb onto clay surfaces rather than onto bitumen surfaces. It is the selective adsorption of HPAM that benefits both bitumen recovery and tailings settling when the polymer was added directly to the bitumen extraction process at an appropriate dosage.     

Flocculation of colloidal clay by bacterial polysaccharides: effect of macromolecule charge and structure

The molecular mechanism of montmorillonite flocculation by bacterial polysaccharides was investigated, with special emphasis on the effect of carboxylic charges in the macromolecules on the mechanisms of interaction with the clay surface. An indirect way to quantify the energy of interaction was used, by comparing the flocculation ability of variously acidic polysaccharides. Data on tensile strength of aggregates in diluted suspension were collected by timed size measurements in the domain 0.1-600 microm, using laser diffraction. The flow behavior of settled aggregates was studied by rheology measurements. Flocculation of colloidal clay suspension by polysaccharides requires cancelling of the electrostatic repulsions by salts, which allows approach of clay surfaces close enough to be bridged by adsorbing macromolecules. The amount of acidic charges of the polysaccharides, and especially their location in the molecular structure, governs the bridging mechanism and the resulting tensile strength of the aggregates. The exposure of carboxylate groups located on side chains strongly promotes flocculation. In turn, charges located on the backbone of the polysaccharide are less accessible to interaction, and the flocculation ability of such polysaccharides is lowered. Measurements at different pH indicate that adsorption of acidic polysaccharides occurs via electrostatic interactions on the amphoteric edge surface of clay platelets, whereas neutral polysaccharides rather adsorb via weak interactions. Increased tensile strength in diluted aggregates due to strong surface interactions results in proportionally increased viscosity of the concentrated aggregates.     

Design and elaboration of colloidal molecules: an overview

The concept of colloidal molecules was first evoked by van Blaaderen in 2003 for describing small non-spherical colloids made of the aggregation of a small number of particles. He predicted original properties to the complex assemblies of such colloids, in particular in optics. This critical review deals with the different strategies reported for creating robust clusters of spherical particles which could mimic the space-filling models of simple conventional molecules. These routes concern either the controlled clustering of preformed colloids directed by coalescence, physical routes, chemical routes, or 2-D/3-D geometrical confinement, or strategies starting from a single colloid which is decorated by satellite colloids by taking advantage of controlled phase separation or nucleation and growth phenomena. These routes are compared from the viewpoint of the accessible shapes, their tunability and scalability (146 references).     

Influence of molecular size,polarity and charge on the retention of organic molecules by nanofiltration

Because of the growing interest in nanofiltration for industrial use, a better insight in the retention mechanisms in nanofiltration is needed, which will make it possible to understand membrane performances for specific applications. In this paper, the retention of a series of organic molecules by four nanofiltration membranes was studied. The membranes that were used are NF70 (Dow/FilmTec), NTR 7450 (Nitto-Denko), UTC-20 (Toray Ind.) and Zirfon^® (VITO). In order to correlate the retention with the size of the molecule, which is the main factor that determines the retention, use was made of different parameters for the molecular size: molecular weight, the Stokes diameter, the equivalent molar diameter, and a diameter obtained with energy minimisation calculations. For each size parameter, the correlation with retention in nanofiltration experiments was calculated. For the Zirfon^® membrane, retentions were too low to obtain a good correlation. For the three other membranes, a good correlation with retention was found for each of the size parameters. Two other factors were found to have an influence on retention of organic molecules: the polarity of the molecule, and the charge of the molecule. The importance of these factors depends on the molecules as well as on the type of membrane.     

Investigation of charge localization and charge delocalization in model molecules by multiphoton ionization photoelectron spectroscopy and DFT calculations

In this work we focus on the question to which degree a surplus charge is localized or delocalized in extended molecular systems. Molecules consisting of a flexible tail and the benzene chromophore, such as n-propylbenzene, 2-phenylethyl alcohol and 2-phenylethylamine, are used as model molecules. Their S0-S1 resonance enhanced multiphoton ionization (MPI) spectra containing origin transitions of different conformers appear at similar wavelengths. This shows, that in the neutral the electronic excitation is localized at the benzene chromophore. Geometry differences between the neutral and the cation can be qualitatively derived from intensities of vibrational transitions or the onset behavior in MPI high-resolution photoelectron (MPI-PE) spectra. We identify two possible reasons for structural changes: Charge-dipole interaction and charge delocalization. Whereas both effects can be active for the folded gauche conformers, the charge-dipole interaction is expected to be small for the extended anti conformers and geometry changes are attributed to charge delocalization. Density functional calculations of structures and energies qualitatively confirm the experimental results for all molecules and their conformers. They predict charge delocalization into the end group of below 20% for n-propylbenzene and 2-phenylethyl alcohol. In the case of 2-phenylethylamine the charge is equally shared by the near-isoenergetic charge sites of the benzene chromophore and the amine group.     

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by <Superscript>1</Superscript>H NMR spectroscopy and PLS regression

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of ¹H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed ¹H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the ¹H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of ¹H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.     

Computer simulations of stretching and collapse of polymer molecules in solution

Computer simulations are reported for system of linear polymer molecules, diblock copolymer and dendrimer in dilute solution without and with elongational flow. The effect of fluctuating hydrodynamics interactions (HI) on the coil-stretch transition of linear polymers and dendrimers in elongational flow is studied. The process of coiling of homo- and blockcopolymer from completely extended state is also simulated.     

Deformation and adhesion of elastomer poly(dimethylsiloxane) colloidal AFM probes

We report on the synthesis and characterization of elastomer colloidal AFM probes. Poly(dimethylsiloxane) microparticles, obtained by water emulsification and cross-linking of viscous prepolymers, are glued to AFM cantilevers and used for contact mechanics investigations on smooth substrates: in detail cyclic loading-unloading experiments are carried on ion-sputtered mica, the deformation rate and dwell time being separately controlled. We analyze load-penetration curves and pull-off forces with models due respectively to Zener; Maugis and Barquins; and Greenwood and Johnson and account for bulk creep, interfacial viscoelasticity, and structural rearrangements at the polymer-substrate interface. A good agreement is found between experiments and theory, with a straightforward estimation of colloidal probes' material parameters. We suggest the use of such probes for novel contact mechanics experiments involving fully reversible deformations at the submicrometer scale.     

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L-iduronic acid (L-IdoA) or D-glucuronic acid (D-GlcA) residues linked to N-acetyl-galactosamine. High-performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D-GlcA or L-IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2-aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser-induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized delta-saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for delta-disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L-IdoA- or D-GlcA-containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.     

Bond stretching force constants and compressibilities of nitride,oxide, and sulfide coordination polyhedra in molecules and crystals

Molecular quadratic stretching force constants are calculated for a variety of MX bonds (X = N, O, S) in coordinated polyhedra containing row 1 and 2 metal atoms, M, using SCF molecular orbital methods and 6-31G^* basis sets. The resulting data scatter along three distinct trends, depending on whether the bonds involve row 1 atoms, row 1 and row 2 atoms, or row 2 atoms. When compared with spectroscopically determined force constants, the calculated force constants are found to be 20% larger. A single trend seems to obtain when the calculated force constants are plotted as a function of the effective nuclear charges of the bonded atoms and their interatomic separations. Scaled force constants calculated for the SiO bond are in rough agreement with values provided by spectroscopic measurements for silicic acid molecules and silicate crystals. Polyhedral compressibilities for nitride-, oxide-, and sulfide-coordinated polyhedra are inversely related with force constants calculated for their MX bonds. The close similarity of these compressibilities and those recorded for chemically similar crystals suggests that force constant-compressibility relationships in chemically similar molecular and crystalline systems are not significantly different.