Jaromír Kulhánek

All-trans-retinoic acid (atRA) is a potent ligand that regulates gene expression and is used to treat several skin disorders. Hyaluronic acid (HA) was previously conjugated with atRA (HA-atRA) to obtain a novel amphiphilic compound. HA-atRA forms micelles that incorporate hydrophobic molecules and facilitate their transport through the skin. The aim of this study was to determine the influence of HA-atRA on gene expression in skin cells and to compare it with that of unbound atRA. Gene expression was investigated using microarrays and a luciferase system with a canonical atRA promoter. HA-atRA upregulated gene expression similarly to atRA. However, HA-atRA activated the expression of cholesterol metabolism genes, unlike atRA. Further investigation using HPLC and filipin III staining suggested that the treated cells induced cholesterol synthesis to replenish the cholesterol removed from the cells by HA-atRA. HA modified with oleate (HA-C18:1) removed cholesterol from the cells similarly to HA-atRA, suggesting that the cholesterol removal stemmed from the amphiphilic nature of the two derivatives. HA-atRA induces retinoid signaling. Thus, HA-atRA could be used to treat skin diseases, such as acne and psoriasis, where the combined action of atRA signaling and anti-inflammatory cholesterol removal may be potentially beneficial.

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.

In this work, a new amphiphilic derivative made of 10-undecylenic acid grafted to hyaluronan was prepared by mixed anhydrides. The reaction conditions were optimized, and the effect of the molecular weight (Mw), reaction time, and the molar ratio of reagents was explored. Using this methodology, a degree of substitution up to 50% can be obtained. The viscosity of the conjugate can be controlled by varying the substitution degree. The physicochemical characterization of the modified hyaluronan was performed by infrared spectroscopy, Nuclear Magnetic Resonance, Size-Exclusion Chromatography combined with Multiangle Laser Light Scattering (SEC-MALLS), and rheology. The low proton motility and self-aggregation of the amphiphilic conjugate produced overestimation of the degree of substitution. Thus, a novel method using proton NMR was developed. Encapsulation of model hydrophobic guest molecules, coenzyme Q10, curcumin, and α-tocopherol into the micellar core was also investigated by solvent evaporation. HA-UDA amphiphiles were also shown to self-assemble into spherical nanostructures (about 300 nm) in water as established by dynamic light scattering. Furthermore, HA-UDA was crosslinked via radical polymerization mediated by ammonium persulphate (APS/TEMED). The cross-linking was also tested by photo-polymerization catalyzed by Irgacure 2959. The presence of the hydrophobic moiety decreases the swelling degree of the prepared hydrogels compared to methacrylated-HA. Here, we report a novel hybrid hyaluronan (HA) hydrogel system of physically encapsulated active compounds and chemical crosslinking for potential applications in drug delivery.

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed β-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-d-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved β-1,4 glycosidic bonds of HA in β-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes’ pH and temperature optima were pH 3.0 and 37–45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.