The properties of hyaluronic acid (HA) can be enhanced by targeted modification. HA oligosaccharides can be further modified at the ends of their chains. However, methods to derivatize the non-reducing terminus are underdeveloped. In this work, various thiols were attached to the non-reducing terminus of unsaturated HA oligosaccharides (ΔHA), maintaining the linear character of HA-conjugates and their reducing terminus in the native form. Esters of ΔHA were successfully used as Michael thiol acceptors, while amides of ΔHA were unreactive. Based on the thiol structure, their addition provided mixtures of l-altro, d-galacto, and an unexpected d-gluco isomer. On the other hand, photo-induced radical (thiol-ene) addition to native ΔHA, its esters or amides, provided exclusively d-galacto products. This approach allowed the synthesis of sulphur-bridged oligosaccharides with defined sulphur position and a length range of 3 to 28 saccharides. No cytotoxic effect on 3T3, NHDF, HaCaT, and HT-29 cells was found with eight selected compounds. The sulphur bridge resisted cleavage with both hyaluronan hydrolases and lyases. These findings allow a synthesis of covalent conjugates of HA-oligosaccharides and active substances with native reducing end. Higher resilience against enzymatic cleavage and tunable affinity to HA cell-receptors can be another benefit for biomedical applications.

Hyaluronan, a linear glycosaminoglycan comprising D- N -acetylglucosamine and D-glucuronic acid, is the main component of the extracellular matrix. Its influence on cell proliferation, migration, inflammation, signalling, and other functions, depends heavily on its molecular weight and chemical modification. Unsaturated HA oligosaccharides are available in defined length and purity. Their potential therapeutic utility can be further improved by chemical modification, e. g., reduction. No synthesis of such modified oligosaccharides, either stepwise or by hyaluronan cleavage, has been reported yet. Here we show a three-step synthesis (esterification, depolymerization and reduction) of unsaturated even numbered hyaluronan oligosaccharides with carboxylates and the reducing terminus reduced to an alcohol. Particular oligosaccharides were synthesised. The modified oligosaccharides are not cleaved by mammalian or bacterial hyaluronidase and do not affect the growth of mouse and human fibroblasts. Further, MTT and NRU viability tests showed that they inhibit the growth of human colon carcinoma cells HT-29 by 20 - 50 % in concentrations 500 - 1000 mu g/mL. Interestingly, this effect takes place regardless of CD44 receptor expression and was not observed with unmodified HA oligosaccharides. These compounds could serve as enzymatically stable building blocks for biologically active substances.

A mild and efficient reduction of negatively charged glucuronate units of hyaluronic acid (HA) into less polar glucose units has not been reported yet. However, this modification could significantly affect physical and chemical properties. Here we show a one-pot procedure where HA is converted into its derivate with carboxyl groups reduced to primary alcohols (HA-Red) without severe polymer degradation. Optimized synthesis aimed at aqueous solutions allowed the preparation of polysaccharides with molecular weights up to 1000 kDa. The chemical structure of HA-Red was proved by 2-dimensional NMR methodologies, FT-IR, LC-MS and SECMALLS. The final materials were exposed to a higher temperature or digested with bovine testicular hyaluronidase (BTH). Obtained data proved higher stability of HA-Red compared to HA, and significant dependence of stability on the degree of modification was observed in most cases. Preliminary in vitro studies showed no negative effects of HARed on the growth of 3T3 fibroblasts, which may be promising for applications requiring biodegradable and biocompatible HA derivatives with increased resistance to degradation.

The wide range of hydrogel applications is a result of the optimization of gelation. Herein, the various aminooxylinkers were synthesized to study the kinetics of gelation and mechanical properties of hydrogels prepared by crosslinking of aldehyde-modified N-acetylglucosamine (GlcNAc) of hyaluronan (ΔHA-CHO: 4,5-anhydro-6-(GlcNAc)-oxo hyaluronan or HA-CHO: 6-(GlcNAc)-oxo hyaluronan). Structural characteristics of the linkers (length, number of functional groups and rigidity) and the polymer (effect of the –C=C- double bond) were investigated in detail and showed decreasing mechanical stiffness with increasing hydrophobic character of linker and effect of its rigidity/flexibility on imine conversion. Besides known linear bis(aminooxy)alkanes with different lengths of the chain, new multifunctional aminooxy-linkers: 1,3,5-tris(aminooxymethyl)benzene and multikis(6-aminooxy-6-deoxy)-β-cyclodextrin were prepared and shortened the gelation time. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis showed that despite the aminooxylinkers ´ cytotoxicity at higher concentrations, the final oxime hydrogels are non-cytotoxic and therefore are suitable for medicinal applications.

Due to their large active surface, high loading efficiency, and tunable dissolution profiles, nanofibrous mats are often cited as promising drug carriers or antimicrobial membranes. Hyaluronic acid has outstanding biocompatibility, but it is hydrophilic. Nanofibrous structures made from hyaluronan dissolve immediately, making them unsuitable for controlled drug release and longer applications. We aimed to prepare a hyaluronan-based antimicrobial nanofibrous material, which would retain its integrity in aqueous environments. Self-supporting nanofibrous mats containing octenidine dihydrochloride or triclosan were produced by electrospinning from hydrophobized hyaluronan modified with a symmetric lauric acid anhydride. The nanofibrous mats required no cross-linking to be stable in PBS for 7 days. The encapsulation efficiency of antiseptics was nearly 100%. Minimal release of octenidine was observed, while up to 30% of triclosan was gradually released in 72 h. The nanofibrous materials exhibited antimicrobial activity, the fibroblast viability was directly dependent on the antiseptic content and its release.

Electron-deficient chlorine covalently immobilised on an amido group of hyaluronic acid (HA) can be potentially exceptional for applications requiring biodegradable and biocompatible polymers with enhanced antibacterial or antiviral activity. This expectation is supported by the assumption that a small amount of HA chloramide (HACl) is formed in the extracellular matrix under inflammatory conditions by a reaction of endogenous HA with hypochlorous acid (HClO) generated by a myeloperoxidase/H2O2/Cl- system. HACl synthesis optimisation showed significant limitations of HClO as an oxidative agent where only lower degrees of substitution (DS) was achieved. Commercially available oxidative agents based on chlorinated isocyanuric acid were successfully tested, producing the HA chain with almost entirely chlorinated amidic groups. The structure of the final HACl was thoroughly studied using advanced 2-dimensional NMR methodologies and LC/MS. Stability of HACl at different temperatures was monitored over 12 months. Preliminary antimicrobial and antiviral tests demonstrated the potential of HACl for applications in biomedicine.

Deacetylated hyaluronan (daHA) containing reactive free amino groups is an important intermediate for further modification. Comparing direct and indirect NMR and HPLC to characterize the degree of HA deacetylation (DD), direct NMR approach using area ratio of anomeric CH and CH-NH2 groups was the most precise one. To describe the substitution pattern, daHA was selectively cleaved by nitrous acid generated in situ or hyaluronan lyase from Streptococcus pneumoniae. The resulting oligomers were identified by LC-ESI-MS. The experimental distribution of these oligomers was compared with theoretically expected random oligomer distribution. Independently on the starting HA molecular weight and deacetylation conditions, the experimental data differed from the random distribution model and suggested that deacetylation of certain N-acetyl-D-glucosamine had reduced the probability of deacetylation at the neighbouring disaccharide. This phenomenon was explained by conformational changes of HA caused by intra- and intermolecular interactions between positively charged amino and negatively charged carboxylic groups.

Conjugates of hyaluronic acid (HA) with biologically active molecules showed great potential in many applications. Oligomers of hyaluronic acid (oliHA) as possible regulators of biological processes and potential anti-cancer therapeutics, were reversibly attached via covalent bond on HA modified with an aldehyde group (HA-CHO) using bis-hydrazido or bis-oxyamino spacers. The structure of the final conjugates (HA-spacer-oliHA) was studied by advanced 2-dimensional NMR techniques in detail. The suggested synthetic strategy is simple, straightforward and can be used for the synthesis of various types of HA or oliHA conjugates. in vitro cytotoxicity assays showed selective activity of HA-spacer-oliHA conjugates against cancer cell lines in comparison with standard human fibroblasts.

The effect of hydrazide linkers on the formation and mechanical properties of hyaluronan hydrogels was intensively evaluated. The reaction kinetics of hydrazone formation was monitored by NMR spectroscopy under physiological conditions where polyaldehyde hyaluronan (unsaturated: ΔHA-CHO, saturated: HA-CHO) was reacted with various hydrazides to form hydrogels. Linear (adipic, oxalic dihydrazide) and branched (N,N´,N´´-tris(hexanoylhydrazide-6-yl)phosphoric triamide and 4-arm-PEG hydrazide) hydrazides were compared as crosslinking agents. The mechanical properties of hydrogels were also modified by attaching a hydrophobic chain to HA-CHO; however, it was found that this modification did not lead to an increase in hydrogel stiffness. Cytotoxicity tests showed that all tested hydrazide crosslinkers reduced the viability of cells only slightly, and that the final hyaluronan hydrogels were non-toxic materials.

Chondroitin sulfate (CS) was regio-specifically modified to an unsaturated derivative (ΔCS) with a double bond in positions 4 and 5 of N-acetyl-d-galactosamine. The structure of ΔCS was elucidated in detail by two dimensional nuclear magnetic resonance, ultraviolet spectroscopy and mass spectrometry. The introduction of a nucleophilic CC double bond into a polymer backbone had no influence on biocompatibility of CS, which was demonstrated by MTT live-dead assay and enzymatic degradation in vitro. On the other hand the chemical modification significantly enhanced the reactivity of ΔCS towards numerous oxidizing agents, which might be promising for a variety of biomedical and cosmetic applications.

Hyaluronic acid (HA) modified with an aldehyde group (HA-CHO) has enormous potential for the covalent attachment of the amino compounds and for the crosslinking of HA with bis or multi-functional amino linkers under physiological conditions. Modification of HA-CHO to its α,β-unsaturated analogue (ΔHA-CHO) generally increases the stability of the reversible imino-attachment due to conjugation of the imine moiety with the adjacent CC double bound. Conjugation of a wide range of structurally different amines including the amines with enhanced biological activity were studied in detail. Results showed that the stabilities of the final conjugates ΔHACHNR significantly depend on the chemical structure of the amine and on the pH value.

Hyaluronic acid (HA) containing CC double bond in positions 4 and 5 of N-acetyl-glucosamine ring (ΔHA) is an unique material, which could be used for biomedical applications and cosmetics. The main advantage of the CC double bond is its ability to react with a wide range of oxidation agents. Location of the CC double bond directly on the glucopyranose ring allows to change the chemical capabilities and simultaneously to mimic the intrinsic physical properties of HA without introduction of linkers or other substances. The synthesis, structural analysis and basic chemical and biological characteristics of this novel biopolymer are described in details. In vitro cytotoxicity assays showed selective activity of ΔHA against cancer cell lines in comparison with standard human fibroblasts so this material has great potential in the field of anticancer drugs.

A new photopolymerizable derivative of hyaluronan (methacrylhydrazide-HA, MAHA) was prepared by carbodiimide chemistry. The reaction conditions were optimized for molecular weight (Mw), reaction time and amount of reagents with a degree of methacrylation (DM) ranging from 2% to 58%. Methacrylhydrazide-HA was hydrolytically stable (PBS, 7days, 37°C) in contrast to commonly used methacrylester analoque (23% hydrolyzed). MAHA readily photopolymerized into densely crosslinked hydrogels under physiological conditions. The varied DM, Mw, irradiation time (texp) and macromer concentration in photocrosslinking afforded hydrogels with different physical (swelling ratio, degradation rate) and mechanical properties (stiffness, toughness). Three-dimensional fabrication and surface patterning of MAHA hydrogels were demonstrated by photolithography and light mediated micromolding. A live-dead assay with skin fibroblasts showed convenient biocompatibility of MAHA (16%, 116kDa) for potential scaffolding applications in tissue engineering and regenerative medicine.

This study aims to design an optimal polyelectrolyte multilayer film of poly-l-lysine (PLL) and hyaluronic acid (HA) as an anti-inflammatory cytokine release system in order to decrease the implant failure due to any immune reactions. The chemical modification of the HA with aldehyde moieties allows self-cross-linking of the film and an improvement in the mechanical properties of the film. The cross-linking of the film and the release of immunomodulatory cytokine (IL-4) stimulate the differentiation of primary human monocytes seeded on the films into pro-healing macrophages phenotype. This induces the production of anti-inflammatory cytokines (IL1-RA and CCL18) and the decrease of pro-inflammatory cytokines secreted (IL-12, TNF-α, and IL-1β). Moreover, we demonstrate that cross-linking PLL/HA film using HA-aldehyde is already effective by itself to limit inflammatory processes. Finally, this functionalized self-cross-linked PLL/HA-aldehyde films constitutes an innovative and efficient candidate for immunomodulation of any kind of implants of various architecture and properties.

Chondroitin sulfate (CS) was chemoselectively oxidized by Tempo/NaClO to C-6 aldehyde of a d-galactosamine subunit (GalNAc). The subsequent, spontaneous desulfatation of oxidized CS gave rise to α,β-unsaturated aldehyde. A new derivative of CS was fully characterized and a degree of oxidation was determined by spectroscopic analysis. The optimization of reaction conditions showed a proportional degree of oxidation to an amount of sodium hypochlorite. The utility of α,β-unsaturated aldehyde for crosslinking and conjugation was demonstrated by a seamless condensation with various N-nucleophiles. We also demonstrated pH-dependent release of biologically active agents from oxidized CS. A live-dead assay in the presence of α,β-unsaturated aldehyde revealed unaffected viability of NIH 3T3 fibroblasts, which made this precursor promising for several biomedical applications including drug delivery and tissue engineering.

Hyaluronan (HA) was chemically modified to a photocurable derivative by the acylation with a mixed anhydride of trans-cinnamic acid and characterized by UV, IR and 2D NMR spectroscopy. Photocurable HA was processed to a microfibrous structure by wet-spinning technology. Water solubility of otherwise water-soluble HA microfibres was reduced significantly by the solid-state photocrosslinking. We also investigated that the nature of polymer structure had a great impact to a final crosslink ratio. The analysis of the mechanical properties showed higher ultimate tensile strength and increased rigidity of the photocrosslinked fibres in comparison to the untreated ones. Moreover all tested materials are regarded as biocompatible according to the tests of cell viability, phototoxicity and enzymatic degradability, which make them suitable candidates for numerous biomedical applications.