Ladislav Šoltés

Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications

Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-d-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.

Role, Metabolism, Chemical Modifications and Applications of Hyaluronan

Hyaluronan (hyaluronic acid, HA) is a linear naturally occurring polysaccharide formed from repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronate. Despite its relatively simple structure, HA is an extraordinarily versatile glycosaminoglycan currently receiving attention across a wide front of research areas. It has a very high molar mass, usually in the order of millions of Daltons, and possesses interesting visco-elastic properties based on its polymeric and polyelectrolyte characteristics. HA is omnipresent in the human body and in other vertebrates, occurring in almost all biological fluids and tissues, although the highest amounts of HA are found in the extracellular matrix of soft connective tissues. HA is involved in several key processes, including cell signaling, wound repair and regeneration, morphogenesis, matrix organization and pathobiology. Clinically, it is used as a diagnostic marker for many disease states including cancer, rheumatoid arthritis, liver pathologies, and as an early marker for impending rejection following organ transplantation. It is also used for supplementation of impaired synovial fluid in arthritic patients, following cataract surgery, as a filler in cosmetic and soft tissue surgery, as a device in several surgical procedures, particularly as an anti-adhesive following abdominal procedures, and also in tissue engineering. This review will provide an overview of the structure and physiological role of HA, as well as of its biomedical and industrial applications. Recent advances in biotechnological approaches for the preparation of HA-based materials, and as a component of tissue scaffolding for artificial organs will also be presented.

Aminoglycoside antibiotics — two decades of their hplc bioanalysis†

Several reviews have been published on high-performance liquid chromatographic (HPLC) methods for the determination of aminoglycoside antibiotics (aminoglycosides) in biological fluids [e.g. Nilsson-Ehle, I. (1983). J. Liq. Chromat. 6: 251]. Of these, the paper by Maitra et al. [(1979a). Clin. Chem. 25: 1361.] briefly summarizes the early 2-3 years of experience on HPLC assaying of amikacin, gentamicin, netilmicin and tobramycin in body fluids. The reviews by Nilsson-Ehle, I. [(1983). J. Liq. Chromat. 6: 251] and by Miner, D. J. [(1985). Antibiotics. In Therapeutic Drug Monitoring and Toxicology by Liquid Chromatography, (Wong S. H. Y., ed.), ch. 10, p. 269. Marcel Dekker, New York and Basel.] devoted to the monitoring of antibiotics, also evaluated the first 6-8 years of the application of HPLC assays for the aminoglycosides amikacin, gentamicin, netilmicin, sisomicin and tobramycin. This report presents a great majority of the HPLC assay methods published during the last two decades for determining practically a dozen different aminoglycoside antibiotics in body fluids, particularly in the serum or plasma, and in urine.

High-molecular-weight hyaluronan : a valuable tool in testing the antioxidative activity of amphiphilic drugs stobadine and vinpocetine

The antioxidative activity of stobadine and vinpocetine was studied in vitro by measuring their inhibition effect on the depolymerization of the high-molecular-weight hyaluronan by hydroxyl radicals. The radicals were generated by the Cu(2+)-H2O2 system. Hyaluronan depolymerization was monitored by means of size exclusion chromatography. The antioxidative activity of stobadine and vinpocetine was compared to that of D-mannitol. A 50% inhibition of hyaluronan depolymerization was reached at stobadine and vinpocetine concentrations of 1.7 x 10(-6) and 3.0 x 10(-7) mol l-1, respectively, while a D-mannitol level of 2.6 x 10(-3) mol l-1 was needed to achieve the same inhibitory effect.

Sonication of chitin–glucan, preparation of water-soluble fractions and characterization by HPLC

A water-insoluble chitin-glucan complex, isolated from the mycelium of Aspergillus niger, was swollen in various aqueous media and treated subsequently by high-energy sonication. The concentration of the resulting water-soluble polysaccharide fractions was dependent on the swelling medium, the amount of the chitin-glucan complex in the suspension, and on the time of sonication. The yields of water-soluble chitin-glucan were within the range 13.6 to 24.4% relative to the mass of the original chitin-glucan. The nitrogen content obtained for the samples of water-soluble chitin-glucan indicated a higher content of chitin (3.45% of nitrogen in high-molecular fraction) than in the original water-insoluble chitin-glucan sample (1.8%). The distribution of the molecular weights of the water-soluble fractions prepared was determined by high-performance liquid chromatography.

Preparation of water-soluble/insoluble derivatives of hyaluronic acid by cross-linking with epichlorohydrin in aqueous NaOH/NH4OH solution

Hyaluronan (HA; 0.1 or 1 mmol) was cross-linked by using epichlorohydrin (E; 0.005-0.25 mol) in the presence of NaOH (0.005-0.25 mol) and without or with NH 4 OH (0.005 or 0.01 mol). The deacetylation of the N-acetyl-D-glucosamine units and a degradation of the product was confirmed in solution by NMR and the static light-scattering (LS) analysis. The products prepared with 0.005-0. mol of NaOH, at the presence of NH 4 OH, and with 0.005-0.05 mol of E were highly water soluble. But at 0.075 mol of E and 0. mol of NaOH/5 mol of water present, the prepared products were water-insoluble. For their analysis, the solid-state 13 C CP-MAS NMR spectroscopy was employed. By this method, the presence of both hydroxypropyl and hydroxypropylamine groups, partially overlapped with HA signals, were observed. The T 1ρ (H) relaxation experiments supported the assumption that the linkages between HA and hydroxypropylamine or hydroxypropyl groups were formed, because all the carbon signals resulted in close T 1ρ (H) values.

Size exclusion chromatographic characterization of sodium hyaluronate fractions prepared by high energetic sonication

SummaryA high molecular weight sodium hyaluronate isolated from rooster combs was degraded by ultrasonication. High-performance size exclusion chromatography allowed rapid and accurate determination of molecular weight parameters (Mz, Mw, Mn) and distributions. The time dependence of hyaluronan ultrasonication to the molecular characteristics of the polymer was investigated. A non-random nature of the degradation process was demonstrated and the reciprocal Mn value was found to be linearly proportional to the time of ultrasonication.

Pro-oxidative effect of peroxynitrite regarding biological systems: a special focus on high-molar-mass hyaluronan degradation.

Current understanding on the role of peroxynitrite in etiology and pathogenesis of some human diseases, such as cardio-vascular diseases, stroke, cancer, inflammation, neurodegenerative disorders, diabetes mellitus and diabetic complications has recently led to intensive investigation of peroxynitrite involvement in physiology and pathophysiology. Mechanism of cytotoxic effects of peroxynitrite involve its reactions with lipids, DNA/RNA, proteins, and polysaccharides, thus triggering cellular responses ranging from subtle changes of cell functioning to severe oxidative damage of the affected macromolecules leading to necrosis or apoptosis. The present work is aimed at providing a brief overview of i) peroxynitrite biosynthesis and reaction pathways in vivo, ii) its synthetic preparation in vitro, and iii) to reveal its potential damaging role in vivo, on actions studied via monitoring in vitro hyaluronan degradation. The complex biochemical behavior of peroxynitrite is determined by a number of variables, such as chemistry of the reaction itself, depending mostly on the involvement of conformational structures of different energy states, concentration of the species involved, content of reactive intermediates and trace transition metal ions, contribution of carbon dioxide, presence of trace organics, and by the reaction kinetics. Recently, in vitro studies of oxidative cleavage of hyaluronan have, in fact, been the subject of growing interest. Here we also describe our experimental set-up for studying peroxynitrite-mediated degradation of hyaluronan, a system, which may be suitable for testing prospective pharmacological substances.

Cyclodextrin derivative of hyaluronan

Conversion of hyaluronan (HA) to its β-cyclodextrin derivative (HA-β-CD) was accomplished by direct coupling of β-cyclodextrin (β-CD) molecules with carboxylic acid groups of the HA macromolecule. The intermolecular dehydration, yielding the HA-β-CD derivative, was performed by the action of diethyl azodicarboxylate and triphenylphosphine under mild, neutral conditions. The physico-chemical characteristics of the novel (bio)material, determined both in solution and solid state, were compared with those of native HA. The specific action of a hyaluronidase was exploited to advantage in studying the depolymerization kinetics of the two types (HA and HA-β-CD) of macrobiomolecules.

Gel permeation chromatographic characterization of sodium hyaluronate and its fractions prepared by ultrasonic degradation

SummaryHigh-molecular-weight sodium hyaluronate isolated from rooster combs was degraded by ultrasonication. The molecular weight of hyaluronate and its polydispersity was determined by gel-permeation chromatography. During 75-min treatment the molecular weight value decreased from 1.39×106 Da to 2.25×105 Da while the polydispersity of the molecular weight increased from 1.29 to 2.36. The reciprocal value of the square of the hyaluronates molecular weight was linearly proportional to the time of ultrasonication.