Synthesis of structure-defined branched hyaluronan tetrasaccharide glycoclusters

Hyaluronan is a glycosaminoglycan with a large number of biological activity. Hyaluronan of different molecular weight often shows different biological activity, sometimes even completely opposite, but the mechanism is not clear. Herein, the hyaluronan tetrasaccharide glycoclusters using hyaluronan tetrasaccharide obtained by enzymolysis of natural hyaluronan were firstly synthesized in high yield. The structurally determined and diverse glycoclusters were of wide molecular weight range and might be used for mimicking the biological activity of natural hyaluronan and facilitating the mechanism study.     

Molecular structure of hyaluronan: an introduction

Hyaluronan is an unbranched polysaccharide of repeating disaccharides consisting of d-glucuronic acid and N-acetyl-d-glucosamine. Its strong water-retaining ability and visco-elastic properties have been broadly utilized in medical applications. Hyaluronan is an important constituent of the extracellular matrix whose physiological functions are manifested both as the substance is by itself as well as when it is being linked to various proteins. Compared with other biopolymers, such as nucleic acids and proteins, the structural chemistry of hyaluronan is much less developed. The scarce information about the metrical aspects of its structure shows no unusual features. Its secondary structure is characterized by intramolecular hydrogen bonding that is hard to distinguish from hydrogen bonding involving water molecules when hyaluronan is in aqueous medium. The tertiary structure of hyaluronan is sensitively dependent on its environment. The relative rigidity of the glycosidic bond and the intramolecular hydrogen bonds would tend to restrict rotational freedom and thus conformational variability. This, however, seems to be overwritten by the impact of molecular environment leading to a great variability of tertiary structure. A large number of conformations are possible and may be present as witnessed by their rather small free energy differences. Of the plethora of physical techniques and computational methods, X-ray crystallography and molecular dynamics calculations have proved to be the most fruitful so far. There are untapped possibilities in NMR spectroscopy for structural studies and quantum chemical calculations are also expected to contribute substantially to the structural chemistry of hyaluronan. There are many basic data as well as structural intricacies of hyaluronan that have so far eluded the researchers of its molecular structure. Dedicated to Endre A. Balazs, pioneer in hyaluronan research.     

Layer-by-layer films from hyaluronan and amine-modified hyaluronan

Hyaluronan is a polysaccharide that is increasingly investigated for its role in cellular adhesion and for the preparation of biomimetic matrices for tissue engineering. Hyaluronan gels are prepared for application as space fillers, whereas hyaluronan films are usually obtained by adsorbing or grafting a single hyaluronan layer onto a biomaterial surface. Here, we examine the possibility to employ the layer-by-layer technique to deposit thin films of cationic-modified hyaluronan (HA+) and hyaluronan (HA) of controlled thicknesses. The buildup conditions are investigated, and growth is compared to that of other polyelectrolyte multilayer films containing either HA as the polyanion or HA+ as the polycation. The films could be formed in a low ionic strength medium but are required to be cross-linked prior to contact with a physiological medium. NIH3T3 fibroblasts were perfectly viable on self-assembled hyaluronan films with, however, a preference for hyaluronan ending films. These findings point out the possibility to tune the thickness of thin hyaluronan films at the nanometer scale. Such architectures could be employed for investigating cell/substrate interactions or for functionalizing biomaterial surfaces.     

Structure of polymer and particle aggregates in hydrogel composites

Knowledge of the structure of a biomaterial is usually vital to control its function. This article provides a structural characterization of a hyaluronan scaffold that has demonstrated good biocompatibility and is used to induce bone regeneration. Hyaluronan hydrogels are appealing materials that can function as a matrix to incorporate both organic and inorganic substances to enhance tissue growth. Because of the intrinsic properties of this swollen matrix, one needs a very sensitive technique that can be applied in situ to determine the organization of the polymers in a gel. Small-angle neutron scattering is used to determine the characteristics of the inhomogeneous structure of the hydrogel both with and without added particles. The results are interpreted using models of structure with two length scales that are beyond the traditional picture of homogeneous gels. The observed structure and the dimensions can explain the previously reported rheological properties of gels containing different amount of polymers. Hydroxyapatite nanoparticles added to the gel are frozen in the gel matrix. We are able to determine the distribution and shape of these particles as they aggregate around the polymer chains. We have also concluded, in this case, that the particle structure is concentration independent. Information about the nanostructure for an applicable biomaterial guides the formulation, preparation, and use that should lead to further understanding of its exploitation. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Pulmonary surfactant adsorption is increased by hyaluronan or polyethylene glycol

In acute lung injuries, inactivating agents may interfere with transfer (adsorption) of pulmonary surfactants to the interface between air and the aqueous layer that coats the interior of alveoli. Some ionic and nonionic polymers reduce surfactant inactivation in vitro and in vivo. In this study, we tested directly whether an ionic polymer, hyaluronan, or a nonionic polymer, polyethylene glycol, enhanced adsorption of a surfactant used clinically. We used three different methods of measuring adsorption in vitro: a modified pulsating bubble surfactometer; a King/Clements device; and a spreading trough. In addition we measured the effects of both polymers on surfactant turbidity, using this assay as a nonspecific index of aggregation. We found that both hyaluronan and polyethylene glycol significantly increased the rate and degree of surfactant material adsorbed to the surface in all three assays. Hyaluronan was effective in lower concentrations (20-fold) than polyethylene glycol and, unlike polyethylene glycol, hyaluronan did not increase apparent aggregation of surfactant. Surfactant adsorption in the presence of serum was also enhanced by both polymers regardless of whether hyaluronan or polyethylene glycol was included with serum in the subphase or added to the surfactant applied to the surface. Therefore, endogenous polymers in the alveolar subphase, or exogenous polymers added to surfactant used as therapy, may both be important for reducing inactivation of surfactant that occurs with various lung injuries.     

Polyacrylamide gel electrophoresis of fluorophore-labeled hyaluronan and chondroitin sulfate disaccharides: application to the analysis in cells and tissues

This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore-labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Delta-disaccharides. PAGEFS relies on derivatization of reducing ends of HA- and the variously sulfated CS-derived Delta-disaccharides with 2-aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris-borate and Tris-HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high-performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA- and CS-derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC-labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.     

Hydrogel of Guar Gum in Experimental Osteoarthritis in Rats

Summary: Commercial Guar Gum, a galactomannan polysaccharide, was purified by the use of two subsequent methods, including precipitation with Fehling solution in order to eliminate protein impurities. The protein content (3.6%) was totally removed. Glutaraldehyde cross-linked gels were prepared with purified (GGP) and unpurified gum (GGU). The viscosity of the gels is similar to that of Hylan G-F 20, a commercial substitute of hyaluronic acid, used in viscosupplementation in human osteoarthritis. Guar Gums (gel and solution) were injected intra-articularly into the knee joints of rats subjected to experimental OA and the effect in hypernociception and cells influx measured. GGU promoted hypernociception and cell influx in naïve rats. GGP was innocuous to naïve rats and inhibited hypernociception, both as a gel or solution, to the same extent as Hylan G-F20. GGP promotes analgesia in OA due to its carbohydrate component.     

透明质酸三糖模拟物的高效合成

设计合成了2个透明质酸(HA)模拟物1和2, 通过最小基团MeO的引入修饰, 模拟天然HA片段的特性, 用于透明质酸合成酶(HAS)催化机理与抑制剂的研究.     

Biologically modified hydrogels for chemical and biochemical analysis

This review focuses on recent advances in the use of hydrogels for chemical and biochemical analysis. Specifically, we discuss recognition elements encapsulated within synthetic hydrogels, synthetic polymers grafted with biological molecules that form freestanding gels, and gels made of naturally occurring polymers, such as proteins or polysaccharides. Gels considered here serve as a platform for the analysis or separation of chemical systems with a high degree of selectivity. Analysis or separation was achieved primarily by incorporating biologically active molecules into a gel matrix. Future developments in biologically integrated gel research for chemical analysis are promising due to the diversity of chemical systems yet to be investigated by these versatile analytical platforms.     

Physical gelation processes revealed by fluorescence technique

The structure of an isotactic polystyrene (iPS) chain in its thermoreversible gel formed with trans‐ and cis‐decalin was determined to be of 3₁‐helical form or near 3₁‐helical form by monitoring the fluorescence behavior of iPS. The fluorescence depolarization measurements of five fluorescent molecules having different molecular sizes doped into iPS in decalin clearly showed that there is enough space for decalin molecules to intercalate among the iPS helical chains. Our fluorescence data support the idea that a polymer‐solvent compound that can be described by a ladder‐like model is formed in iPS‐decalin gels.     

Gelrite‐Based In Vitro Gelation Ophthalmic Drug Delivery System of Gatifloxacin

The conventional ophthalmic solutions have demonstrated poor bioavailability and low therapeutic response due to rapid precorneal elimination of drug, which could be overcome by the use of in situ gelling systems that are instilled as drops into the eye and undergo a sol‐gel transition in the cul‐de‐sac. The present work describes the formulation of an ophthalmic delivery system of an antibacterial agent, Gatifloxacin, based on the concept of ion‐activated in situ gelation; the rheological properties of the formulation and in vitro release of the drug were evaluated. Gelrite gellan gum, a novel vehicle for ophthalmic delivery system, which gels in the presence of mono/divalent cations present in the tear fluid, was used as the gelling agent. The developed formulation showed pseudoplastic rheology and was therapeutically efficacious and provides sustained release of the drug over a 12‐hour period. The developed system is thus a viable alternative to conventional eye drops.     

Hyaluronan: from biomimetic to industrial business strategy

Hyaluronan (hyaluronic acid) is a naturally occurring polysaccharide of a linear repeating disaccharide unit consisting of beta-(1-->4)-linked D-glucopyranuronic acid and beta-(1-->3)-linked 2-acetamido-2-deoxy-D-glucopyranose, which is present in extracellular matrices, the synovial fluid of joints, and scaffolding that comprises cartilage. In its mechanism of synthesis, its size, and its physico-chemical properties, hyaluronan is unique amongst other glycosaminoglycans. The network-forming, viscoelastic and its charge characteristics are important to many biochemical properties of living tissues. It is an important pericellular and cell surface constituent; its interaction with other macromolecules such as proteins, participates in regulating cell behavior during numerous morphogenic, restorative, and pathological processes in the body. The knowledge of HA in diseases such as various forms of cancers, arthritis and osteoporosis has led to new impetus in research and development in the preparation of biomaterials for surgical implants and drug conjugates for targeted delivery. A concise and focused review on hyaluronan is timely. This review will cover the following important aspects of hyaluronan: (i) biological functions and synthesis in nature; (ii) current industrial production and potential biosynthetic processes of hyaluronan; (iii) chemical modifications of hyaluronan leading to products of commercial significance; and (iv) and the global market position and manufacturers of hyaluronan.     

Manual and automated enrichment procedures for biological samples using lipophilic gels

Aspects of the use of lipophilic gels in manual sample preparation procedures are reviewed. Neutral gels with a controlled hydrophobicity are used for sorbent extraction of non-polar and medium polarity compounds from biological fluids. Acidic amphiphilic compounds can be extracted as ion-pairs with decyltrimethylammonium ions. Solvent or detergent extracts of tissues or faeces can be mixed with hydrophobic gels for transfer of analytes from a solvent to a gel phase, permitting subsequent sample preparation in gel bed systems. Hydrophobic gels, alkyl-bonded silica and polystyrene matrices can be used in series for extraction of compounds with a wide range of polarities. Group fractionations are performed on neutral and ion-exchanging lipophilic gels to yield fractions of neutral, basic and acidic metabolites within selected polarity ranges. Selective isolation of phenolic acids on a strong anion exchanger, of ethynylic steroids on a strong cation exchanger in silver form and of oximes of ketonic steroids on a strong cation exchanger in hydrogen form is possible. A computerized system for automatic sample preparation is also described. It consists of an extraction bed, a cation-exchange column and an anion-exchange column. The pumps and switching valves are arranged so that the columns can operate in series or parallel for isolation of neutral, basic and acidic metabolites of amphiphilic compounds and for regeneration of the column beds. Fractions can be collected, or the effluent from the column beds can be diluted with water to permit sorption on a solid phase. The applicability of the automated method to the analysis of bile acids and metabolites of mono(2-ethylhexyl) phthalate is demonstrated.     

Ichthyosan in fish eye

Ichthyosan A and V are two highly elastoviscous glycan complexes present in the aqueus and vitreus [here aqueus and vitreus are used as nouns as was suggested by Balazs and Denlinger in The eye, vol 1A. Vegetative physiology and biochemistry, 3rd edn. Academic Press, New York, pp 533–589, 1984] of the fish eye. Ichthyosan A, with its high elastic properties, surrounds and stabilizes the lens of the eye. Ichthyosan V, within the collagen fibers, serves as a structure stabilizer of the gel vitreus. These two molecular complexes are non-covalent aggregates composed of hyaluronan, a chondroitin-proteoglycan (sulfate free), and a keratan-like molecule. The ratio of hyaluronan to chondroitin–proteoglycan varies in the two ichthyosans. Electrophoretic separation methods (both free and gel electrophoresis) demonstrate that the hyaluronan–proteoglycan aggregates move as one molecular entity. The average molecular weight of the ichthyosan varies from 5.2 to 13.0 million in various species. Aquatic mammals do not have ichthyosan in their eyes.     

Encapsulation of dye molecules and nanoparticles in hollow organogel fibers of a nonchiral polyurethane model compound

We present the case of a nonchiral organogelator that forms hollow fibers and encapsulates silver nanoparticles (SNP) and a dye molecule. The biscarbamate molecule (a model compound for polyurethanes), which has two hydrogen-bonding motifs symmetrically attached to n-dodecyl side chains (C(12)), gels benzonitrile with hollow fibrillar morphology. The C(12) molecules form sheets that eventually wrap into hollow fibers to form the gel network. Herein, two-component gels were prepared with C(12) as one component and SNP, phthalocyanine (Pc), or perylene (Pe) as the other. Microscopic analysis and partial melting experiments confirmed the inclusion of the silver nanoparticles and phthalocyanine into the hollow fibers. On the other hand, Pe molecules tend to form crystals at the outer surface of the C(12) fibers, which results in a significant increase in the width of the gel fibers. This difference in the behavior of Pc and Pe molecules were accounted for by their crystal geometry and significantly different crystal growth rate compared with that of C(12) fiber formation in the gels. Pc crystallizes in a needle shape that facilitates occlusion in the gel fibers, whereas Pe forms large platelets. X-ray diffraction and spectroscopic analysis of the two-component gels along with their neat components confirmed that there was no change in the packing behavior of the Pc and Pe molecules in the gels. Therefore, these are examples of two-component physical gels in which the Pc crystals are occluded within the hollow fibers of C(12) by physical mixing of the components without the aid of any inter-molecular interactions between the different components. We have thus shown that lumen-loaded gel fibers with nanoparticles and dye molecules can be prepared by the two-component gel route, provided that the above growth rate, shape, and size conditions are satisfied.     

Concentration effect in hyaluronan analysis by size exclusion chromatography

Summary The concentration effect in size-exclusion chromatographic analysis of hyaluronans of various relative molecular masses (RMM) has been studied. A critical concentration has been found that is negatively dependent on the hyaluronan molecular mass; the higher the biopolymer molecular mass, the smaller the injected sample where the concentration effect should be taken into account for accurate evaluation of molecular mass distribution. At higher temperatures, however, the concentration effect diminishes significantly.     

The Use of Polystyrene Gels in Preparative Recycle-HPLC for the Separation of Structurally Related Molecules

Abstract

This paper deals with the use of polystyrene gels in high performance liquid chromatography for preparative separations of small molecules with very similar structures. The preparative chromatograph, equipped with a recycle device and made in our laboratory, is described. The column set, consisted of three columns packed with low porosity polystyrene gel. The mobile phase was diisopropylether. Three particle sizes (10μ, 20μ, 50μ) were studied for improving the preparative performance. We checked the very high efficiency of our recycling system versus sample load. High sample loadings (up to 100 grams) can be injected when the separation does not require a large number of plates but typical sample loads are 10–20 grams for high performance separations. Some examples of separation of diastereoisomers, configurational isomers or related structure molecules are given. The advantages of our preparative system are discussed.     

Application of Fluorescence Depolarization Method to Monitor Free Volume in Gels of Stereoregular Polystyrene

Summary: A fluorescence depolarization technique was applied to get the information on free volume among polymer chains in gel form. Four fluorescent molecules with different molecular sizes were doped throughout the gels of syndiotactic polystyrene (sPS) and isotactic polystyrene (iPS) physical gel system, and their fluorescence anisotropy values were examined in detail for a range of polymer concentrations. Consequently, the free volume among sPS chains in sPS/chloroform gels is as large as the size of molecules smaller than 1,5-dimethylnaphthalene and is consistent with that of the cavity size in the δ-empty crystalline form of sPS solids. The cause to produce δ-empty crystalline form of sPS solids and to form cocrystals between sPS and guest molecules is discussed by comparing the molar size of guest molecules with the free volume among sPS chains in gel form.     

Versatile Use of Chitosan and Hyaluronan in Medicine

Chitosan is industrially acquired by the alkaline N-deacetylation of chitin. Chitin belongs to the β-N-acetyl-glucosamine polymers, providing structure, contrary to α-polymers, which provide food and energy. Another β-polymer providing structure is hyaluronan. A lot of studies have been performed on chitosan to explore its industrial use. Since chitosan is biodegradable, non-toxic, bacteriostatic, and fungistatic, it has numerous applications in medicine. Hyaluronan, one of the major structural components of the extracellular matrix in vertebrate tissues, is broadly exploited in medicine as well. This review summarizes the main areas where these two biopolymers have an impact. The reviewed areas mostly cover most medical applications, along with non-medical applications, such as cosmetics.