Hyaluronan (HA) and its principal receptor CD44 are known to be involved in regulating tumor cell dissemination and metastasis. It is hypothesized that the CD44-HA interaction regulates proliferation and invasion of tumor cells in dependence on the molecular weight and the presentation form of HA. To address this hypothesis, we reconstituted 3D collagen (Coll I) matrices and functionalized them with HA of molecular weight of 30-50 kDa (low molecular weight; LMW-HA) and 500-750 kDa (high molecular weight; HMW-HA). A post-modification strategy was applied to covalently immobilize HA to reconstituted fibrillar Coll I matrices, resulting in a non-altered Coll I network microstructure and stable immobilization over days. Functionalized Coll I matrices were characterized regarding topological and mechanical characteristics as well as HA amount using confocal laser scanning microscopy, colloidal probe force spectroscopy and quantitative Alcian blue assay, respectively. To elucidate tumor cell behavior, BRO melanoma cell lines with and without CD44 receptor expression were used for in vitro cell experiments. We demonstrated that only soluble LMW-HA promoted cell proliferation in a CD44 dependent manner, while HMW-HA and immobilized LMW-HA did not. Furthermore, an enhanced cell invasion was found only for immobilized LMW-HA. Both findings correlated with a very strong and specific adhesive interaction of LMW-HA and CD44+ cells quantified in single cell adhesion measurements using soft colloidal force spectroscopy. Overall, our results emphasize the importance of presentation mode and molecular weight specificity in biomaterial studies on the impact of HA on cell behavior.

This diploma thesis is focused on two parts. The first one deals with rheological behavior of sodium carboxymethylcellulose (CMC-Na) solutions in the presence of sodium and calcium ions. The solutions of various concentrations were prepared by solubilization in aqueous solutions of NaCl or CaCl2. The effect of monovalent and bivalent ions on apparent viscosity and viscoelastic properties of these solutions was studied. It was found that in the case of low concentrations of CMC-Na in the sample (up to 0.5% w/w) the elastic character increases. At the same time the stiffness of the sample is higher with higher concentration of calcium ion in the solution. This increase might indicate the formation of gel network. On the other hand the viscosity of these solutions decreases with higher concentration of calcium ions. In case of solutions with concentration higher than 1.5% w/w changes of viscosity did not happen by influence of addition ions. Rheological behavior was compared also with solutions prepared without adding an electrolyte as well as with acid form of carboxymethylcellulose (CMC-H). In terms of second part the molecular dynamic simulations of hyaluronan tetramer both sodium and calcium in aqueous medium were performed. Structure and arrangement of water molecules in the surrounding of hyaluronan chain were studied. Structure was described by formalism of radial distribution functions. In the case of system containing sodium ions only, the obtained results are in accordance with earlier published data. Presence of calcium ions does not cause qualitative changes during the radial distribution functions in comparison with the system with sodium ions.

Influence of the preparing of a biopolymer-surfactant system on its solubilization properties and stability was investigated by using fluorescence spectroscopy and dynamic light scattering methods. Investigation was made on complex of native hyaluronan with cationic surfactant cetyltrimethylammonium bromide (CTAB). System has been studied in aqueous and in saline solutions. The effect of temperature of stock solutions and freezing effect on subsequent properties were investigated. Further was examinated, what effect has a way of introducing fluorescent probe into the system on subsequent solubilization properties. It was found that the conditions of preparing of biopolymer-surfactant system have a significant effect on the solubilization properies and stability of complex.

This Master thesis investigates the interaction between the polysaccharide sodium hyaluronate (HA) and some amphiphilic molecules. It is known that the presence of the carboxylic group on HA and the aminogroup on the amphiphiles leads to electrostatic interaction between these two compounds. This supposal offers the possibility to physically modify HA and use it as a new type of a carrier of bioactive compounds, for example medicals. However, successful carrier of bioactive compound has to resist a certain value of ionic strength. The high-molecular weights HA (1.75MDa) and amphiphile lysine were chosen for the study of the influence of ionic strength on the system HA – amphiphile. Our results show that system HA – amphiphile system is suppressed even by low concentrate solution of electrolyte. Therefore the system was reinforced by protonation of the aminoacid. The results show, that the interactions were reinforced, nevertheless negative influence of chlorine anions had to be eliminated by lyophilization. The solutions with strengthened system HA – amphiphile were used for the research of ionic strength influence. The amphiphiles lysine, 6 - aminocaproic acid and arginine were selected for this study. The interactions were investigated by means of reometry and conductometry.

Diploma thesis was aimed to deal with evaluation of microrheology method in the research of biopolymer-protein. Used biopolymer was sodium hyaluronate and proteins were trypsin and chymotrypsin. For measuring of microrheology were used particles with different radius (0,5 m and 1 m). It was found, that both substances have viscous charakter. Passive microrheology is suitable for measuring the viscoelastic properties of biopolymers.

Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.

Hyaluronic acid is an organic polysaccharide with a wide range of uses in medical and cosmetic industries due to its physiological properties. Crosslinked hyaluronic acid is a commonly used filler agent because of its water retention capabilities. N-deacetylation can be performed to enable new derivatives of hyaluronic acid. Both chemical and enzymatical approaches were investigated in this literature study to find methods retaining a high molecular weight product. Chemical N-deacetylation of hyaluronic acid has significant challenges with being treated by acid or base while both preventing degradation and maintaining its molecular weight. The method that seems the most promising is treating hyaluronic acid with hydroxylamine. Another method is enzymatic N-deacetylation. It was found that an enzyme N-deacetylated hyaluronic acid in female breast skin from 69-year-olds and above. The isolated enzyme had molecular weights ranging from 63 kDa to 79 kDa. Another enzyme that was produced recombinantly proved to be efficient since it retained high molecular weight and had a degree of deacetylation of 10.1 %. Today there exists only a few methods for crosslinking deacetylated hyaluronic acid. However, for chitosan, there are multiple methods available for crosslinking. Since it uses similar reactions that could be applicable to that of deacetylated hyaluronic acid. Reacetylation of the free amino groups has proven to be possible after crosslinking with a simple and cheap method resulting in an almost complete reacetylation. NMR proved to be an adequate method for analyzing the degree of deacetylation and higher-order structures. HPLC-UV spectroscopy may be used to increase the credibility of the analysis.

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