This work reveals the growing potential of novel electrochemical methods that are applicable for polysaccharides. It was shown for the first time that the molecules of hyaluronic acid (HA) exhibit electrochemical response using phase-sensitive alternating current (AC) voltammetry in phase-out mode. Adsorption and desorption processes of HA fragments at a charged interface of mercury electrode were observed in buffered HA solutions. Electrostatic and hydrophobic manners of interactions were distinguished for native hyaluronan fragments in a wide electric potential range. The AC voltammetry response depended on the temperature, concentration, and length of HA chains. Results of this work open possibilities for further structural characterization of widely used HA fragments and understanding manners of interactions with charged hydrophobic surfaces that could be useful in the future for understanding HA interactions at biological levels.
- Publikační typ
- časopisecké články MeSH
Nanofibrous materials produced from natural polymers have wide range of potential uses in regenerative medicine. This paper focuses on preparation of nanofibrous layers produced from intentionally hydrophobized derivatives of hyaluronan, which is known for its ability to promote wound healing. This structural modification of hyaluronan expands the range of potential uses of this promising material, which is otherwise limited due to the hydrophilic nature of hyaluronic acid. The aim of this research was preparation of nanofibrous material that would retain its fibrous structure and dimensional stability even after getting into contact with an aqueous medium, which is impossible to achieve with layers composed solely of native hyaluronan. As a result, such material would be able to retain its breathability and good mechanical properties when both dry and wet. Furthermore, all prepared materials were proved non-toxic for cells. This self-supporting nanofibrous matrix can be used as a scaffold, or porous wound dressing.
- Publikační typ
- časopisecké články MeSH
Hyaluronan (HA) comprises a fundamental component of the extracellular matrix and participates in a variety of biological processes. Half of the total amount of HA in the human body is present in the skin. HA exhibits a dynamic turnover; its half-life in the skin is less than one day. Nevertheless, the specific participants in the catabolism of HA in the skin have not yet been described in detail, despite the essential role of HA in cutaneous biology. A deeper knowledge of the processes involved will act to support the development of HA-based topical and implantable materials and enhance the understanding of the various related pathological cutaneous conditions. This study aimed to characterize the distribution and activity of hyaluronidases and the other proteins involved in the degradation of HA in healthy human full-thickness skin, the epidermis and the dermis. Hyaluronidase activity was detected for the first time in healthy human skin. The degradation of HA occurred in lysates at an acidic pH. HA gel zymography revealed a single band corresponding to approximately 50 kDa. This study provided the first comprehensive view of the distribution of canonic HA-degrading proteins (HYAL1 and HYAL2) in human skin employing IHF and IHC. Furthermore, contrary to previous assumptions TMEM2, a novel hyaluronidase, as well as CEMIP, a protein involved in HA degradation, were localized in the human epidermis, as well as in the dermis.
Hyaluronan (HA) is widely used for eye drops as lubricant to counteract dry eye disease. High and low molecular weight HA are currently used in ophthalmology. However, a large portion of the current literature on friction and lubrication addresses articular (joint) cartilage. Therefore, eye drops compositions based on HA and its derivatized forms are extensively characterized providing data on the tribological and mucoadhesive properties. The physiochemical properties are investigated in buffers used commonly in eye drops formulations. The tribological investigation reveals that amphiphilic HA-C12 decreases the friction coefficient. At the same time, the combination of trehalose/HA or HAC12 enhances up to eighty-fold the mucoadhesiveness. Thus, it is predicted a prolonged residence time on the surface of the eye. The incorporation of trehalose enhances the protection of human keratinocytes (HaCaT) cells, as demonstrated in an in-vitro cell-desiccation model. The presence of trehalose increases the friction coefficient. Medium molecular weight HA shows significantly lower friction coefficient than high molecular weight HA. This research represents a first, wide array of features of diverse HA forms for eye drops contributing to increase the knowledge of these preparations. The results here presented also provide valuable information for the design of highly performing HA-formulations addressing specific needs before preclinic.
- MeSH
- adhezivita MeSH
- buněčné linie keratinocytů HaCaT MeSH
- filtrace MeSH
- hlen účinky léků MeSH
- kyselina hyaluronová chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- lubrikace * MeSH
- nefelometrie a turbidimetrie MeSH
- oči účinky léků MeSH
- oční roztoky farmakologie MeSH
- protonová magnetická rezonanční spektroskopie MeSH
- reologie MeSH
- sterilizace MeSH
- systémy cílené aplikace léků * MeSH
- tření MeSH
- viskozita MeSH
- vysoušení MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lauroyl derivatives of hyaluronan are safe and biodegradable materials that seem promising for application in medicine. However, their potential in the field of drug delivery was not yet explored. We thus prepared lauroyl hyaluronan films loaded with various drugs and studied the effects of lauroyl hyaluronan properties, drug hydrophobicity and medium composition on the drug release. Since biomolecules will always be present in real clinical applications, media supplemented by albumin were also included. The amphiphilic character of lauroyl hyaluronan enabled convenient loading of the films by both hydrophilic and hydrophobic drugs. Dominant factors influencing drug release were drug hydrophobicity and the presence of albumin. Hydrophilic diclofenac was released rapidly in all cases, while triclosan with medium hydrophobicity exhibited slower release sensitive to other parameters, reaching equilibrium values in the used experimental setup. The release of hydrophobic octenidine into pure buffer was almost negligible, but the addition of albumin did promote its release. The strong effect of albumin highlights the importance of considering biomolecules in the design of release experiments.
In this work, low molecular weight (17 kDa) hyaluronan was modified by dodecanoyl substituents. The activation of dodecanoic acid was mediated by benzoyl chloride towards the preparation of a mixed anhydride, which reacts in a second step with HA in water mixed with an organic solvent. The effect of the cosolvent was studied and showed an even distribution of substituents and higher reaction rate in water: 1,4-dioxane compared to water:tert-butanol where substituents occupy adjacent positions. The chemical characterization of the prepared derivatives was elucidated by NMR, FTIR spectroscopy, thermal analyses, and gas chromatography, while the distribution of substituents was evaluated by enzymatic degradation. Molecular-dynamics simulations reveal opposite solvent separations around HA and dodecanoyl chains, that is stronger in water:tert-butanol solution. The resulting incompatibility of solvation-shells of the two entities repels the reaction intermediates from the HA chain and drives them towards the already bound substituents, explaining the observed differences in the distribution evenness. Thus, the influence of the solvent on the reaction selectivity is observed by shielding reactive sites around HA. Therefore, a control of the distribution of the substituents was obtained by defining the concentration of HA and used cosolvent.
There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.
- MeSH
- cesty eliminace léčiva MeSH
- chrupavka metabolismus MeSH
- cyklická ADP-ribosa metabolismus MeSH
- intravenózní podání MeSH
- izotopové značení metody MeSH
- izotopy uhlíku chemie metabolismus farmakokinetika MeSH
- kosti a kostní tkáň metabolismus MeSH
- kyselina hyaluronová chemie metabolismus farmakokinetika MeSH
- molekulová hmotnost MeSH
- myši inbrední BALB C MeSH
- tkáňová distribuce MeSH
- uridindifosfát-N-acetylglukosamin metabolismus MeSH
- uridindifosfátglukosa metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The aim of this paper is to review chromatographic and mass-spectrometric methods and underline the best analytical approaches for successful analysis of various hyaluronic acid species in different types of samples. Hyaluronan-degrading enzymes and chemical depolymerization produce di- or oligosaccharides suitable for hyaluronan quantification or structural characterization of hyaluronan derivatives. Efficient purification and pre-column derivatization of hyaluronan disaccharides by reductive amination allow subnanogram quantification in biological samples. The chromatographic separation is capable to distinguish all glycosaminoglycans disaccharides and to resolve hyaluronan fragments with 2-40 monomers. Using electrospray ionization or matrix assisted laser desorption ionization, hyaluronan fragments up to 8 kDa or 41 kDa, respectively, can be observed. One- or two-dimensional chromatographic separation with higly sensitive mass-spectrometric detection is an indispensable tool for revealing substituent position, extent of modification and substitution patterns of chemically modified hyaluronan derivatives. It is essential for studying structure-biological function relationships of hyaluronan and its derivatives.
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.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- antifungální látky chemie farmakologie MeSH
- antivirové látky chemie farmakologie MeSH
- Bacteria účinky léků MeSH
- chloraminy chemie farmakologie MeSH
- halogenace MeSH
- houby účinky léků MeSH
- kyselina chlorná chemie MeSH
- kyselina hyaluronová chemie MeSH
- viry účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
N-Deacetylated hyaluronan (daHA) has been widely investigated as a starting material to develop biomaterials with unique composition and performance. However, its structure elucidation remains a challenging task due to its polysaccharide nature. After the brief mention of its properties and preparation, this review critically evaluates different analytical methods and approaches to characterize this promising polysaccharide. A special attention is paid to the determination of the degree of deacetylation. Besides, the analysis of its molecular weight, primary structure, and deacetylation pattern is also described. The older procedures are compared with the advanced techniques to provide reliable description of daHA.
- MeSH
- acetylace MeSH
- biokompatibilní materiály chemická syntéza MeSH
- kyselina hyaluronová chemie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
