Binding of methyl orange and its homologs by polyion complexes consisting of a piperidinium cationic polymer and various polyanions in aqueous solution

A study was made of the formation of polyion complexes between a piperidinium cationic polymer and polyanions and of the binding of azo-dye anions (methyl, ethyl, propyl, and butyl orange) by these complexes. Sodium poly(acrylate), poly(styrenesulfonate), dextran sulfate, and carboxy-methylcellulose were used as polyanions. The resultant polyion complexes (insoluble in aqueous solutions) were compared for their ability to bind the small organic molecules in aqueous solutions, for example, of urea and an inorganic electrolyte (KCI), and exhibited a strong binding affinity toward these small anions. Polyion complexes that consisted of sodium poly(acrylate), dextran sulfate, and carboxymethylcellulose as polyanions cooperated in the binding, whereas the polyion complex of sodium poly(styrenesulfonate) did not. It was suggested that small organic anions interact with the polyion complexes primarily through electrostatic and hydrophobic forces.     

Time-dependent conformational change of thrombin molecules induced by sulfated polysaccharides

Dextran sulfate (DS) had a greater ability to elute thrombin adsorbed on a small Sepharose 6B column than heparin did, while chondroitin sulfate A had little ability. It is probable that the strength of the interaction of thrombin depends mostly on the charge-density of strongly acidic sulfate groups in the polysaccharides. The change in intrinsic fluorescence intensity of thrombin with time was closely correlated with the rate of inactivation of the enzyme in the presence of sulfated polysaccharides. Both rates were affected by the pH of the solution in the presence of the polyanions. The rates in the presence of DS were highest at pH 6.05 among the three pHs tested, while they were enhanced only at pH 6.05 by heparin, but not by chondroitin sulfate A. Therefore, extensive charge-neutralization of thrombin by the sulfated polysaccharides is able to induce time- and temperature-dependent intramolecular conformational change (irreversible denaturation) of the enzyme molecules.     

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 micro m in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5'-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length.An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.     

Synthesis and luminescent properties of water-soluble nanobiocomposite CdSe/polysaccharide quantum dots

Water-soluble nanobiocomposite CdSe quantum dots were obtained using the stabilizing potential of natural biologically active polysaccharides galactomannan and κ-carrageenan. A complex of physicochemical methods was used to investigate the biphasic amorphous-crystalline structure of the obtained nanocomposites, the size range (4.8—6.9 nm) of the formed CdSe particles was determined, and, additionally, it was shown that aqueous solutions of the nanocomposites in question were characterized by the presence of photoluminescence in two spectral regions, namely, 410—450 and 510—580 nm.     

Quantitative determination of the glycosaminoglycan Delta-disaccharide composition of serum, platelets and granulocytes by reversed-phase high-performance liquid chromatography

Seven Delta-disaccharide standards from heparan sulfate/heparin (HS/H) and nine Delta-disaccharide standards from chondroitin/dermatan sulfate (CS/DS) and hyaluronic acid (HA) were derivatized with the fluorophore 2-aminoacridone (AMAC) and separated in two runs each by reversed-phase HPLC with baseline separation and very short run times. This novel method facilitates the separation of the largest number of Delta-disaccharides from both CS/DS/HA and HS/H with one column and buffer system after fluorophore labeling in two runs at present. For the first time nine glycosaminoglycan (GAG) Delta-disaccharides from CS/DS/HA were separated after fluorophore labeling in one run. The limits of quantification (LOQs) were below 0.2 pmol for CS/DS/HA and HS/H Delta-disaccharides. We demonstrated applicability of our method for biological samples. Furthermore, normal ranges of the GAG Delta-disaccharide compositions from platelets and granulocytes were determined for the first time.     

pH-sensitive polyelectrolyte complex gel microspheres composed of chitosan/sodium tripolyphosphate/dextran sulfate: swelling kinetics and drug delivery properties

Porous chitosan (CS) polyelectrolyte complex (PEC) hydrogel microspheres were prepared via either wet phase-inversion or ionotropic crosslinking with sodium tripolyphosphate (Na+ - TPP) and dextran sulfate (DS). The resulting microspheres were characterized using scanning electron microscopy (SEM) and elemental analysis (EA). The controlled release behavior of ibuprofen (IBU) from these microspheres was investigated. The PEC microspheres were about 700-950 microm in diameter with large pores and open porous structure. The CS/TPP/DS microspheres resisted hydrolysis in strong acid and biodegradation in enzymatic surroundings. The swelling kinetics for CS microspheres was close to Fickian diffusion, whereas those for CS/TPP and CS/TPP/DS were non-Fickian. Furthermore, the equilibrium water content (EWC) and water diffusion coefficient (D) increased with the pH of the media. The release profiles of IBU from CS/TPP/DS microspheres were slow in simulated gastric fluid (SGF, pH 1.4) over 3 h, but nearly all of the initial drug content was released in simulated intestinal fluid (SIF, pH 6.8) within 6 h after changing media. Overall the results demonstrated that CS/TPP/DS microspheres could successfully deliver a hydrophobic drug to the intestine without losing the drug in the stomach, and hence could be potential candidates as an orally administered drug delivery system.     

Dependence of the rate of thrombin/antithrombin III reaction upon the turnover rate of a catalytic amount of heparin

Commercially available heparin preparations slightly enhanced the rate of thrombin/antithrombin (AT) III reaction at pH 6.05 in the absence of NaCl. However, this accelerative activity was significantly lower than that induced by heparin with high affinity for AT III (HA-heparin), probably due to the formation of the binary complexes of HA-heparin-AT III as well as that composed of thrombin and heparin with low affinity for AT III (LA-heparin). The HA-heparin-catalyzed thrombin/AT III reaction was faster in the presence of 0.1 M NaCl at pH 6.05 than that in the absence of the salt. LA-heparin and dextran sulfate (DS) were also found to accelerate the thrombin/AT III reaction rate, but neither substance catalyzed the formation of the complex in the presence of 0.1 M NaCl at pH 7.4. LA-heparin was also confirmed to compete with HA-heparin for enhancement of the thrombin/AT III reaction. Thus, it appears that AT III tends to form a ternary complex with the thrombin-DS or thrombin-LA-heparin complex, even in the presence of 0.1 M NaCl, whereas factor Xa reacts with the AT III-DS or AT III-LA-heparin complex. These results indicate that HA-heparin is the only substance having the ability to catalyze the thrombin/AT III reaction, and that its turnover rate is markedly elevated in the presence of strongly electropositive and electronegative ions because of the decreased affinity of the enzyme for heparin under such conditions.     

Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All

A sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1%). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited.     

Light scattering investigation of association phenomena in saline carrageenan solutions

Intermolecular association of ι- and κ-carrageenan in aqueous solution has been studied by wide-angle laser light scattering as a function of salt composition and temperature. For ι-carragenan, the effect of three different salts (LiCl, NaCl, NaI) on the associative properties was investigated. As to κ-carrageenan, the association-inducing effect of Cs⁺ ions was studied, with particular interest in the time dependence of the aggregation process.     

The SARS-CoV-2 Entry Inhibition Mechanisms of Serine Protease Inhibitors,OM-85, Heparin and Soluble HS Might Be Linked to HS Attachment Sites

This article discusses the importance of D-xylose for fighting viruses (especially SARS-CoV-2) that use core proteins as receptors at the cell surface, by providing additional supporting facts that these viruses probably bind at HS/CS attachment sites (i.e., the hydroxyl groups of Ser/Thr residues of the core proteins intended to receive the D-xylose molecules to initiate the HS/CS chains). Essentially, the additional supporting facts, are: some anterior studies on the binding sites of exogenous heparin and soluble HS on the core proteins, the inhibition of the viral entry by pre-incubation of cells with heparin, and additionally, corroborating studies about the mechanism leading to type 2 diabetes during viral infection. We then discuss the mechanism by which serine protease inhibitors inhibit SARS-CoV-2 entry. The biosynthesis of heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (Hep) is initiated not only by D-xylose derived from uridine diphosphate (UDP)-xylose, but also bioactive D-xylose molecules, even in situations where cells were previously treated with GAG inhibitors. This property of D-xylose shown by previous anterior studies helped in the explanation of the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This explanation is completed here by a preliminary estimation of xyloside GAGs (HS/CS/DS/Hep) in the body, and with other previous studies helping to corroborate the mechanism by which the D-xylose exhibits its antiglycaemic properties and the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This paper also discusses the confirmatory studies of regarding the correlation between D-xylose and COVID-19 severity.     

Adsorptive Voltammetry of Polyelectrolyte Complex Between 11‐Ferrocenyltrimethylundecylammonium and Polyanions at Carbon Paste Electrode

For polyelectrolyte complex between cationic surfactant and polyanion, the adsorptive voltammetry at carbon paste electrode using an electroactive cationic surfactant was examined. The adsorption state of the cationic surfactant in the complexes at CPE was estimated from the half‐height width of the oxidation waves. The half‐height width for poly(styrene sulfonate) was independent of the molecular weight, and was same as that for poly(vinyl sulfate). The half‐height width for heparin was broad and different from that of the vinyl polyanions. According to the analysis by Frumkin isotherm, the interaction between cationic surfactants was attractive in heparin complex at CPE, however, in the vinyl polyanion complexes at CPE the interaction was non‐cooperative as that predicted with the Langmuir isotherm. In spite of the same adsorption state, the concentration dependency of the peak current for poly(styrene sulfonate) was quite different from that for poly(vinyl sulfate). The concentration dependence indicated the reactive property of each polyanion on the association with the cationic surfactant in aqueous solution.     

The effect of polyelectrolyte chain length on layer-by-layer protein/polyelectrolyte assembly--an experimental study

The effect of polyelectrolyte chain length on the formation of multilayered assemblies of alternating globular proteins and linear polyanions prepared by the layer-by-layer electrostatic adsorption technique was investigated. The systems studied were albumin/sodium poly(styrenesulfonate), immunoglobulin G/sodium poly(styrenesulfonate), albumin/sodium dextran sulfate, and albumin/heparin. The formation of assemblies was followed using FTIR multiple internal reflection spectroscopy. While the amount of polyelectrolyte adsorbed on the first (primary) protein layer did not depend on its molecular weight, the effect of polyelectrolyte chain length was clearly observed in the following steps of alternating adsorption. Some short-chain polyanion molecules were removed from the surface when a next protein layer was adsorbed from solution. The short polyanion chains were not able to make a sufficient number of ion pairs for stable interaction with additional protein molecules and left the surface as soluble protein/polyanion complexes. The most pronounced effect could be seen with sodium poly(styrenesulfonate) of Mw up to ca. 2 x 10(4), but a detectable effect could be traced even up to Mw ca. 8 x 10(4). Such a pronounced effect, however, was not observed with dextran sulfate. The effect of molecular weight of heparin was clearly observed but all heparins tested, regardless of their molecular weight, effectively assembled with albumin to form multilayer.     

Sol-Gel transition of κ-Carrageenan as viewed through NMR

²³Na, ⁸⁷Rb, and ¹³³Cs NMR spectra were observed to study an interaction between these cations and κ-carrageenan. It was found that the NMR intensity of gel-forming cations undergoes a remarkable change in the vicinity of the sol-gel transition temperature, while the non-gel-forming cation which coexists together with a gel-forming cation does not show any change. These results suggest that gel-forming cations selectively interact with κ-carrageenan.Motion of water molecules in the gel and the solution of κ-carrageenan was studied by measuring the water proton spin-spin relaxation time. The results indicate three different types of water molecule: a free bulk water molecule, a water molecule strongly bound to κ-carrageenan, and a water molecule weakly bound to the gel network.     

Gold nanomaterials based pseudostationary phases in capillary electrophoresis: A brand‐new attempt at chondroitin sulfate isomers separation

In this work, a CE method with bare gold nanorods (GNRs) based pseudostationary phase was developed and applied for the separation of chondroitin sulfate (CS) isomers, CS, and dermatan sulfate (DS). The separation efficiency was investigated by varying the experimental parameters such as concentration and pH of the BGE, separation voltage, internal diameter of capillary, different size, and morphology of gold nanomaterials. Results showed that different size and morphology of gold nanomaterials had different effects on the separation of CS and DS. The best separation of CS and DS was achieved in the BGE composed of aqueous 150 mmol/L (mM) ethylenediamine + 20 mM sodium dihydrogen phosphate + 30% v/v GNRs, pH 4.5, at the separation voltage of ?10 kV. Capillary was 59.2 cm in length (effective length 49 cm), 50 μm id capillary thermostated at 25°C. CE with bare GNRs used as pseudostationary phase was shown to be a suitable technique for the separation of CS and DS mixtures with wider peaks. RSD of migration time and peak area of CS and DS were 0.13, 0.14 and 0.86, 1.07%, respectively.     

Binding of anthraquinone dyes by polyion complexes consisting of a piperidinium cationic polymer and various polyanions in aqueous solution

The extent of binding of 1-amino-4-alkylaminoanthraquinone-2-sulfonates (alkyl?methyl, ethyl, propyl, and butyl), which have different chemical structures from methyl orange derivatives, by polyion complexes consisting of a piperidinium cationic polymer and various polyanions such as sodium poly acrylate, poly methacrylate, poly styrenesulfonate, carboxymethylcellulose, and dextran sulfate, was measured in an aqueous solution. The effect of alkyl group of the anthraquinone dye on the binding behavior was investigated. Also, the resultant binding characteristics were compared with those previously observed with methyl orange and its homologs. These polyion complexes exhibited very strong binding affinity toward the anthraquinone dye. The polyion complex of the polycation and sodium poly styrenesulfonate bound the dye noncooperatively and the binding process was athermal. The first binding constant accompanying the binding is of the order of 10⁵–10⁶. In contrast, the polyion complexes composed of the polycation and the other polyanions exhibited strong cooperative binding and the binding process was exothermic. The possible mode of binding is discussed.     

Immobilization of camptothecin with surfactant into hydrogel for controlled drug release

A new strategy has been developed for the controlled release of a hydrophobic anticancer drug, camptothecin (CPT), which suffers a limited therapeutical utility because of its poor water solubility. CPT was first solubilized in the solution of a cationic surfactant, dodecyltrimethylammonium bromide (DTAB). It has been demonstrated that the presence of DTAB has increased the solubility of CPT significantly. In a 50 mM DTAB solution, the drug’s solubility was enhanced to 85 μM, 22 times of its solubility in pure water. The micellar drug solution of CPT-DTAB was subsequently used to prepare agarose hydrogels, which act as the drug carriers in the release studies. To fully take advantage of the cationic property of DTAB, negatively charged κ-carrageenan was added as a guest polymer in some hydrogel samples. The release of CPT from these hydrogel-surfactant systems was performed at 37 °C and the effects of DTAB and κ-carrageenan on the release of CPT were studied respectively. By fitting to the well-known Fickian diffusion model, the diffusion coefficients of CPT were obtained.     

Studies on Sulfation of Lycium barbarum Polysaccharides

Polysaccharides can anti-virus, such as human immunodeficiency virus (HIV-1),[1] herpes simplex virus (HSV-1,HSV-2) and cytomegalovirus. Some of them are sulfates, e.g. dextran sulfate, heparin, sulfonation of chitosan and sulfated derivatives of Lentinan. Our results showed that sulfated derivatives of Lycium barbarum polysaccharides (LBP)have anti-HIV activity. Because the anti-HIV activity of LBP was deeply dependent on the molecular weight, the sulfation pattern and glycosidic branches besides degree of sulfation (DS), so we emphasized our work on the factors of DS.     

过氧化氢在黄铁矿的溶解过程中对铼-锇信号强度及年龄的影响

对黄铁矿在高温高压密闭的Carius管溶解过程中出现的黄色沉淀物进行了定性研究。在黄铁矿中加入少量的辉钼矿及185Re和190Os混合稀释剂,在常规的逆王水溶矿过程中加入适量H2O2,用ICP-MS检测,研究了H2O2对Re、Os信号强度变化及同位素交换平衡产生的影响。结果表明,在黄铁矿溶解过程中出现的黄色沉淀物为羟基硫酸铁(FeOHSO4)而不是单质硫,它是由于密闭的Carius管内氧化性不够而生成的。H2O2的加入对ICP-MS测定Re信号强度没有影响,而对Os则有显著的改善,但这种改善并未影响到加入的185Re和190Os稀释剂与样品中的Re和Os达到同位素交换平衡,因而也不影响到样品的Re、Os含量及最终的Re-Os同位素年龄计算。     

Polyelectrolyte‐Functionalized Gold Nanoparticle Scaffold for the Sensing of Heparin and Protamine in Serum

We describe a shape‐controlled synthesis of polyelectrolyte‐functionalized flowerlike and polyhedral Au nanoparticles and the development of a nanoarchitectured platform for the selective and highly sensitive detection of protamine and heparin by voltammetric, impedimetric, and microgravimetric techniques. The functionalized Au nanoparticles were chemically synthesized in aqueous solution at room temperature in the presence of the polyelectrolyte (either protamine or heparin). The charge on the polyelectrolyte controlled the shape and surface morphology of the nanoparticles. The negatively charged heparin‐functionalized Au nanoparticles have multiple branched flowerlike shapes with an average size of 50 nm, whereas the cationic protamine‐functionalized nanoparticles are of polyhedral shape with an average size of 25 nm. Both flowerlike and polyhedral nanoparticles have (111), (200), (220), and (311) planes of a face‐centered cubic lattice of Au. Voltammetric, impedimetric, and microgravimetric sensing platforms based on functionalized Au nanoparticles have been developed for the sensing of heparin and protamine. The sensing platforms are developed by self‐assembling the functionalized nanoparticles on a thiol‐functionalized three‐dimensional silicate network. The microgravimetric sensing platform shows very high sensitivity and it can detect heparin and protamine at concentrations as low as 0.05 μg mL^?1. The selectivity of the sensing platform towards heparin was examined with potential interferents such as hyaluronic acid (HA) and chondroitin‐4‐sulfate (CS). Both HA and CS did not interfere with the measurement of heparin. The practical application of the sensing platform was demonstrated by measuring the concentration of heparin and protamine in human serum samples. The sensing platform could successfully quantify the concentration of heparin and protamine in the real serum samples with excellent recovery. The sensing platform was robust and could be used for repeated measurement without compromising the sensitivity.     

Combining size-exclusion chromatography and fully automated chip-based nanoelectrospray quadrupole time-of-flight tandem mass spectrometry for structural analysis of chondroitin/dermatan sulfate in human decorin

Chondroitin/dermatan sulfate (CS/DS) chain of decorin (DCN) from human skin fibroblasts (HSk) was released by reductive β-elimination reaction and digested with chondroitin AC I lyase. Enzymatic hydrolysis mixture of CS/DS chains was separated by size-exclusion chromatography (SEC). Collected octasaccharide fraction was subjected to fully automated chip-based nanoelectrospray (nanoESI) quadrupole time-of-flight (QTOF) MS and tandem MS (MS/MS). MS of human skin fibroblasts DCN CS/DS displayed a high complexity due to the large variety of glycoforms, which under chip-nanoESI MS readily ionized to form multiply charged ions. Except for the regularly tetrasulfated octasaccharide, the investigated fraction contained four additional octasaccharides of atypical sulfation status. Two new oversulfated glycoforms and two undersulfated species were identified. Remarkably, the series of decasaccharides discovered in the same SEC pool was found to encompass a trisulfated and a novel hexasulfated [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?] species. MS/MS by collision-induced dissociation (CID) on the [M-4H]? ion corresponding to the previously not reported [4,5-Δ-GlcAGalNAc(IdoAGalNAc)?](5S) corroborated for a novel motif in which three N-acetylgalactosamine (GalNAc) moieties are monosulfated, 4,5-Δ-GlcA and the first IdoA from the non-reducing end bear one sulfate group each, while the second N-acetylgalactosamine from the reducing end is unsulfated.