Enzymatically stable unsaturated hyaluronan-derived oligosaccharides with selective cytostatic properties.

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 µg/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.

Injecting hyaluronan in the thoracolumbar fascia: A model study.

Hyaluronan (HA) has been recently identified as a key component of the densification of thoracolumbar fascia (TLF), a potential contributor to non-specific lower back pain (LBP) currently treated with manual therapy and systemic or local delivery of anti-inflammatory drugs. The aim of this study was to establish a novel animal model suitable for studying ultrasound-guided intrafascial injection prepared from HA with low and high Mw. Effects of these preparations on the profibrotic switch and mechanical properties of TLF were measured by qPCR and rheology, respectively, while their lubricating properties were evaluated by tribology. Rabbit proved to be a suitable model of TLF physiology due to its manageable size enabling both TLF extraction and in situ intrafascial injection. Surprisingly, the tribology showed that low Mw HA was a better lubricant than the high Mw HA. It was also better suited for intrafascial injection due to its lower injection force and ability to freely spread between TLF layers. No profibrotic effects of either HA preparation in the TLF were observed. The intrafascial application of HA with lower MW into the TLF appears to be a promising way how to increase the gliding of the fascial layers and target the myofascial LBP.

The hyaluronan metabolism in the UV-irradiated human epidermis and the relevance of in vitro epidermal models.

Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.

Preparation of hyaluronan oligosaccharides by a prokaryotic beta-glucuronidase: Characterization of free and immobilized forms of the enzyme.

Popularity of hyaluronan (HA) in the cosmetics and pharmaceutical industries, led to the investigation and development of new HA-based materials, with enzymes playing a key role. Beta-D-glucuronidases catalyze the hydrolysis of a beta-D-glucuronic acid residue from the non-reducing end of various substrates. However, lack of specificity towards HA for most beta-D-glucuronidases, in addition to the high cost and low purity of those active on HA, have prevented their widespread application. In this study, we investigated a recombinant beta-glucuronidase from Bacteroides fragilis (rBfGUS). We demonstrated the rBfGUS's activity on native, modified, and derivatized HA oligosaccharides (oHAs). Using chromogenic beta-glucuronidase substrate and oHAs, we characterized the enzyme's optimal conditions and kinetic parameters. Additionally, we evaluated rBfGUS's activity towards oHAs of various sizes and types. To increase reusability and ensure the preparation of enzyme-free oHA products, rBfGUS was immobilized on two types of magnetic macroporous bead cellulose particles. Both immobilized forms of rBfGUS demonstrated suitable operational and storage stabilities, and their activity parameters were comparable to the free form. Our findings suggest that native and derivatized oHAs can be prepared using this bacterial beta-glucuronidase, and a novel biocatalyst with enhanced operational parameters has been developed with a potential for industrial use.

Endogenously produced hyaluronan contributes to the regulation of peritoneal adhesion development.

Peritoneal adhesions are postsurgical fibrotic complications connected to peritoneal inflammation. The exact mechanism of development is unknown; however, an important role is attributed to activated mesothelial cells (MCs) overproducing macromolecules of extracellular matrix (ECM), including hyaluronic acid (HA). It was suggested that endogenously-produced HA contributes to the regulation of different fibrosis-related pathologies. However, little is known about the role of altered HA production in peritoneal fibrosis. We focused on the consequences of the increased turnover of HA in the murine model of peritoneal adhesions. Changes of HA metabolism were observed in early phases of peritoneal adhesion development in vivo. To study the mechanism, human MCs MeT-5A and murine MCs isolated from the peritoneum of healthy mice were pro-fibrotically activated by transforming growth factor ß (TGFß), and the production of HA was attenuated by two modulators of carbohydrate metabolism, 4-methylumbelliferone (4-MU) and 2-deoxyglucose (2-DG). The attenuation of HA production was mediated by upregulation of HAS2 and downregulation of HYAL2 and connected to the lower expression of pro-fibrotic markers, including fibronectin and α-smooth muscle actin (αSMA). Moreover, the inclination of MCs to form fibrotic clusters was also downregulated, particularly in 2-DG-treated cells. The effects of 2-DG, but not 4-MU, were connected to changes in cellular metabolism. Importantly, the inhibition of AKT phosphorylation was observed after the use of both HA production inhibitors. In summary, we identified endogenous HA as an important regulator of peritoneal fibrosis, not just a passive player during this pathological process.

Molecular weight and gut microbiota determine the bioavailability of orally administered hyaluronic acid.

The ability of hyaluronan as a dietary supplement to increase skin moisture and relieve knee pain has been demonstrated in several clinical studies. To understand the mechanism of action, determining hyaluronan's bioavailability and in vivo fate is crucial. Here, we used 13C-hyaluronan combined with LC-MS analysis to compare the absorption and metabolism of oral hyaluronan in germ-free and conventional wild-type mice. The presence of Bacteroides spp. in the gut was crucial for hyaluronan absorption. Specific microorganisms cleave hyaluronan into unsaturated oligosaccharides (<3 kDa) which are partially absorbed through the intestinal wall. The remaining hyaluronan fragments are metabolized into short-chain fatty acids, which are only metabolites available to the host. The poor bioavailability (~0.2 %) of oral hyaluronan indicates that the mechanism of action is the result of the systematic regulatory function of hyaluronan or its metabolites rather than the direct effects of hyaluronan at distal sites of action (skin, joints).

Intraperitoneally administered native and lauroyl-modified hyaluronan films: Pharmacokinetic and metabolism studies.

Hyaluronan is being investigated extensively as a biocompatible and biodegradable material for use in biomedical applications. While the derivatization of hyaluronan broadens its potential therapeutic use, the pharmacokinetics and metabolization of the derivatives must be thoroughly investigated. The fate of intraperitoneally-applied native and lauroyl-modified hyaluronan films with varying degrees of substitution was investigated in-vivo employing an exclusive stable isotope-labelling approach and LC-MS analysis. The materials were gradually degraded in peritoneal fluid, lymphatically absorbed, preferentially metabolized in the liver and eliminated without any observable accumulation in the body. Hyaluronan acylation prolongs its presence in the peritoneal cavity depending on the degree of substitution. The safety of acylated hyaluronan derivatives was confirmed via a metabolic study that revealed its degradation into non-toxic metabolites, i.e. native hyaluronan and free fatty acid. Stable isotope-labelling with LC-MS tracking comprises a high-quality procedure for the investigation of the metabolism and biodegradability of hyaluronan-based medical products in-vivo.

An in vitro model that mimics the foreign body response in the peritoneum: Study of the bioadhesive properties of HA-based materials.

A cascade of reactions known as the foreign body response (FBR) follows the implantation of biomaterials leading to the formation of a fibrotic capsule around the implant and subsequent health complications. The severity of the FBR is driven mostly by the physicochemical characteristics of implanted material, the method and place of implantation, and the degree of immune system activation. Here we present an in vitro model for assessing new materials with respect to their potential to induce a FBR in the peritoneum. The model is based on evaluating protein sorption and cell adhesion on the implanted material. We tested our model on the free-standing films prepared from hyaluronan derivatives with different hydrophobicity, swelling ratio, and rate of solubilization. The proteomic analysis of films incubated in the mouse peritoneum showed that the presence of fibrinogen was driving the cell adhesion. Neither the film surface hydrophobicity/hydrophilicity nor the quantity of adsorbed proteins were decisive for the induction of the long-term cell adhesion leading to the FBR, while the dissolution rate of the material proved to be a crucial factor. Our model thus helps determine the probability of a FBR to materials implanted in the peritoneum while limiting the need for in vivo animal testing.

The Degradation of Hyaluronan in the Skin.

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.

How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy.

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.

Analysis of hyaluronan and its derivatives using chromatographic and mass spectrometric techniques.

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.

The effect of hydrazide linkers on hyaluronan hydrazone hydrogels.

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.

LC-MS/MS study of in vivo fate of hyaluronan polymeric micelles carrying doxorubicin.

A better understanding of in vivo behavior of nanocarriers is necessary for further improvement in their development. Here we present a novel approach, where both the matrix and the drug can be analyzed by LCMS/MS after one sample handling. The developed method was applied for the comparison of pharmacokinetic profile of free and encapsulated doxorubicin (DOX) in oleyl hyaluronan (HA-C18:1) polymeric micelles. The results indicated that nanocarriers were rapidly dissociated upon in vivo administration. Despite this fact, the administration of encapsulated DOX led to its longer circulation time and enhanced tumor targeting. This effect was not observed injecting blank HA-C18:1 micelles followed by unencapsulated DOX. Biodistribution studies and molecular weight estimation of the carrier matrix indicated relatively high stability of HA-C18:1 ester bond in bloodstream and complete elimination of the derivative within 72 h. The proposed methodology provides a novel strategy to elucidate the pharmacokinetic behavior of polysaccharide-based drug delivery systems.

Simple area determination of strongly overlapping ion mobility peaks.

Coupling of ion mobility with mass spectrometry has brought new frontiers in separation and quantitation of a wide range of isobaric/isomeric compounds. Ion mobility spectrometry may separate ions possessing the identical molecular formula but having different molecular shapes. The separation space in most commercially available instruments is limited and rarely the mobility resolving power exceeds one hundred. From this perspective, new approaches allowing for extracting individual compound signals out of a more complex mixture are needed. In this work we present a new simple analytical approach based on fitting of arrival time distribution (ATD) profiles by Gaussian functions and generating of ATD functions. These ATD functions well describe even distorted ion mobility peaks of individual compounds and allow for extracting their peaks from mobilograms of mixtures. Contrary to classical integration, our approach works well with irregular overlapping peaks. Using mobilograms of standards to generate ATD functions, poorly separated compounds, e.g. isomers, with identical mass spectra representing a hard to solve task for various chemometric methods can be easily distinguished by our procedure. Alternatively ATD functions can be obtained from ATD profiles of ions unique to individual mixture components (if such ions exist) and mobilograms of standards are not required. On a set of hyaluronan-derived oligosaccharides we demonstrated excellent ATD repeatability enabling the resolution of binary mixtures, including mixtures with minor component level about 5%. Ion mobility quantitative data of isomers were confirmed by high performance liquid chromatography.