Amelia Gamini

Supramolecular structure of microbial polysaccharides in solution : from chain conformation to rheological properties

Abstract Literature data and unpublished results have been used to correlate the primary and secondary structures of gellan, welan, rhamsan, succinoglycan and galactoglucan in solution with their rheological properties. In dilute solution the chain conformation ranges from that of the random coil of galactoglucan to that of the stiffer double helix of gellan, and possibly a similar conformation exists for welan and rhamsan. In semidilute solution the rheological behaviour of the above microbial polysaccharides is mainly determined by their conformation in solution. Ordered regions along the polysaccharide chains seem to be the prerequisite to establish intermolecular interactions which lead to non-transient polymer networks. The lack of ordered secondary structure leads to entangled networks in which the topological constraints govern the rheological behaviour. Experimental evidence is reported to support the hypothesis that the chain conformation and rigidity can be qualitatively predicted on the basis of rheological measurements.

“The Good, the Bad and the Ugly” of Chitosans

The objective of this paper is to emphasize the fact that while consistent interest has been paid to the industrial use of chitosan, minor attention has been devoted to spread the knowledge of a good characterization of its physico-chemical properties. Therefore, the paper attempts to critically comment on the conflicting experimental results, highlighting the facts, the myths and the controversies. The goal is to indicate how to take advantage of chitosan versatility, to learn how to manage its variability and show how to properly tackle some unexpected undesirable features. In the sections of the paper various issues that relate chitosan properties to some basic features and to advanced solutions and applications are presented. The introduction outlines some historical pioneering works, where the chemistry of chitosan was originally explored. Thereafter, particular reference is made to analytical purity, characterization and chain modifications. The macromolecular characterization is mostly related to molecular weight and to degree of acetylation, but also refers to the conformational and rheological properties and solution stability. Then, the antimicrobial activity of chitosan in relation with its solubility is reviewed. A section is dedicated to the formulation of chitosan biomaterials, from gel to nanobeads, exploring their innovative application as active carrier nanoparticles. Finally, the toxicity issue of chitosan as a polymer and as a constructed nanomaterial is briefly commented in the conclusions.

Structural investigations of cross-linked hyaluronan

Structural properties of several cross-linked hyaluronan derivatives, obtained by scanning electron microscopy, monodimensional NMR microscopy and small angle X-ray scattering of synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid, used as a reference material. The experimental results, obtained in different media, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of denser polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover, the presence of polymer junction zones gave rise to the appearance of a well-defined correlation peak in the pattern of intensity of the scattered X-radiation.

Exosomal doxorubicin reduces the cardiac toxicity of doxorubicin.

AIM To test the efficacy and toxicity of exosomal doxorubicin (exoDOX) compared with free doxorubicin. MATERIALS & METHODS The cytotoxic effects of exoDOX were tested in vitro and in nude mice by measuring the tumor volume. The toxic effects were evaluated by measuring the bodyweight and through histopathologic analyses. The biodistribution of DOX was assessed by MS. RESULTS In vitro and in vivo studies showed that exosomes did not decrease the efficacy of DOX. Surprisingly, exoDOX showed no cardiotoxicity as observed in DOX-treated mice and MS studies confirmed that the accumulation of exoDOX in the heart was reduced by approximately 40%. CONCLUSION We demonstrated that exoDOX was less toxic than DOX through its altered biodistribution.

Determination of the diadic composition of alginate by means of circular dichroism: a fast and accurate improved method.

Circular dichroism (CD) is presented as a reliable and sensitive method of determining the diadic frequency composition of alginate (F(GG), F(MM) and F(GM+MG)). The availability of samples, very largely or even completely conforming to the limiting structures of polymannuronate (MM)(n), polyguluronate (GG)(n) and polyalternating MG (MG)(n), respectively, allowed the limiting CD spectra for each alginate diad to be obtained. These showed very different CD behaviour, thus pointing out the crucial importance of the neighbouring residue in chiroptical properties. Using an iterative best-fit procedure, the diadic composition of commercial alginates could be obtained from their respective CD spectra by means of a linear combination of the spectra of the three limiting diads. The results were found in excellent agreement with the composition parameters obtained by 1H NMR spectroscopy.

Hydrogen-bonding and conformation of agarose in methyl sulfoxide and aqueous solutions investigated by 1H and 13C NMR spectroscopy

Abstract The conformation of agarose in dimethyl sulfoxide (Me 2 SO) or in aqueous solutions has been studied by 1 H and 13 C NMR spectroscopy. Partial assignment of the 1 H NMR spectrum of agarose in Me 2 SO- d 6 has been obtained using 1D and 2D techniques. Coupling constant, chemical shift temperature dependence and exchange rate data for the hydroxyl protons have been used to establish the presence of intramolecular hydrogen bonds. The reported data show that the protons of HO-2 in the 3,6- anhydro-α- l -Gal p residue (A) and HO-4 in the β- d -Gal p residue (G) have a less rotational freedom and a higher thermal stability than the other hydroxyl protons. On the basis of the exchange rate data, obtained from 1D NOESY experiments with selective excitation of the residual water signal, an inter-residue hydrogen bond is proposed only for G HO-4 in Me 2 SO, while the involvement of A HO-2 in the agarose-Me 2 SO complex, recently reported, is suggested. The latter hypothesis is supported by the 13 C NMR data obtained in Me 2 SO- d 6 , in a Me 2 SO- d 6 -D 2 O mixture and in D 2 O, which also show that a relatively small conformational rearrangement of the chain segments occurs in aqueous solution. Deuterium isotope effect data indicate the persistence in water of the inter-residue G HO-4 mediated hydrogen bond with the additional involvement of G HO-6. In addition, the negligible isotope effect on the C-2 atom of A would indicate that the corresponding OH group, although free from the agarose-Me 2 SO complex, is not available for further strong interactions. The hydrogen bonding and conformation of agarose in methyl sulfoxide or in aqueous solutions were studied by NMR spectroscopy. Based on 1 H NMR data, an inter-residue H-bond is proposed for HO-4 of β- d -Gal p in Me 2 SO, while the involvement of HO-2 of 3,6- anhydro -α- l -Gal p in an agarose-Me 2 SO complex is suggested. These hypotheses are supported by the 13 C NMR data.

SYNTHESIS OF GLYCOSYLAMINES: IDENTIFICATION AND QUANTIFICATION OF SIDE PRODUCTS

The synthesis of some glycosylamines (1-amino-1-deoxy D-glucose, 1-amino-1-deoxy-D-galactose and 1-amino-1-deoxylactose) was carried out by treatment of the corresponding reducing sugars with ammonium hydrogencarbonate in concentrated ammonia. The reaction mixture was first analyzed by capillary electrophoresis with indirect absorbance detection and high performance anion-exchange chromatography with pulsed amperometric detection. Beside glycosylcarbamate, a known reaction by-product, fructose and lactulose were detected during the synthesis of 1-amino-1-deoxyglucose and 1-amino-1-deoxylactose, respectively. Quantification of glycosylamines was carried out by micellar electrokinetic chromatography with UV detection of their 9-fluorenylmethyloxycarbonyl (Fmoc) derivatives; lactulosylamine was thus detected in the synthesis mixture of 1-amino-1-deoxylactose. The Fmocglycosylamines were easily purified from the other components of the crude synthesis mixtures.

Solution conformation and properties of the galactoglucan from Rhizobium meliloti strain YE-2(S1).

The physicochemical solution properties of the galactoglucan excreted by Rhizobium meliloti strain YE-2(S1) have been investigated by capillary viscometry, potentiometric titration, isothermal mixing microcalorimetry, and circular dichroism. Potentiometric and chiro-optical data, as a function of the degree of ionisation, indicate the absence of a co-operative conformational transition. Solution properties, as a function of ionic strength and temperature, suggest that the galactoglucan adopts a disordered conformation characterised by moderate flexibility. Polyelectrolyte theory is used to fit the enthalpy of dilution data with a suitable linear charge-density parameter. Conformational calculations and chain modelling, using molecular mechanics, give an unperturbed characteristic ratio, (C infinity) of 20, which was smaller than that estimated from intrinsic-viscosity and molecular-weight data for an expanded-coil chain model.

Analysis of N-acetylaminosugars by CE: A comparative derivatization study

N‐linked or O‐linked glycans derived from glycoprotein processing carry, an N‐acetylglucosamine or an N‐acetylgalactosamine respectively, at their reducing termini. The presence of the N‐acetylamino group on C‐2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N‐acetyllactosamine, N‐acetylglucosamine, N‐acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4‐aminobenzonitrile, 2‐aminopyridine, 2‐aminobenzoic acid (2‐AA)) by reductive amination and analysed by CE with UV detection. The 2‐AA derivatives showed the lowest concentration detection limits, varying approximately in the 2–3 μM range for the saccharides tested including the N‐acetamido ones. The possibility to separate and detect with the same sensitivity ten 2‐AA‐labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate‐complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2‐AA is also shortly addressed.

NOVEL BIOMATERIALS BASED ON CROSS-LINKED HYALURONAN: STRUCTURAL INVESTIGATIONS

ABSTRACT Structural properties of several cross-linked hyaluronan derivatives, obtained by electron microscopy, monodimensional NMR microscopy and low angle X-ray scattering by synchrotron radiation, are presented and compared with those observed for non-modified hyaluronic acid used as reference material. The experimental results, obtained in different media by the different techniques, showed a consistent picture of the synthesized matrices. In particular, the presence of zones of more dense polymeric material observed by electron microscopy resulted in a higher transversal relaxation rate of the bulk water protons as well as in a decrease of the diffusion coefficient obtained by NMR microscopy. Moreover the presence of polymer junction zones gave rise to the appearance of an additional correlation peak in the pattern of the intensity of the scattered X-radiation.