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Derivatization of Sodium Hyaluronate as a Possible Tool for Increasing of the Stability of Model Artificial Synovial Fluid
Hrochová, Eliška ; Mravec, Filip (referee) ; Kalina, Michal (advisor)
This master thesis deals with the optimization of the procedure of modification of hyaluronic acid structure for the use in the artificial synovial liquids. Based on the literature research, the amino acid alanine was used for the modification of carboxylic group in the glucuronic acid. The main subject of study is the improvement of the stability and mechanical properties of synovial liquid. DLS microrheology, macrorheology, thermogravimetric analysis (TGA), multi-angle light scattering with flow-field flow fractionation (AF4-MALS) and infrared spectroscopy (FTIR) were used for characterization. The theoretical part of this theses submits review of the musculoskeletal system, role of hyaluronic acid in metabolism and summary of synovial liquid. The experimental part focuses on the measurement of the stability and mechanical properties of three artificial samples (first with no modification, second with modified hyaluronic acid and third with modified hyaluronic acid and chondroitin sulphate). These samples were compared with real horse synovial fluid and artificial viscosupplement Orthovisc®.
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Stability of vesicular complexes against changes in their concentration
Velichová, Veronika ; Marková, Kateřina (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on the stability of vesicular systems that consist of ion pair amphiphile. These catanionic vesicles were prepared from single-chained surfactants CTAB cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate), which were stabilized by adding double-chained surfactant DODAC (dimethyldioctadecylammonium chloride) with 43 mol.% cholesterol. The aim of the thesis was to verify the stability of vesicular systems against changes in their concentration. For the measurement, a concentration series was prepared by diluting vesicles with deionized water. The stability was evaluated by dynamic (DLS) and electrophoretic (ELS) light scattering measurements at weekly intervals for four weeks. By DLS method were determined the average values of the size of vesicles and their changes during the measurement, the method ELS provided the information about changes of charge with different concentration in time. Samples with a higher concentration, i.e. no dilution till tenfold dilution of the vesicle solution, were marked as stable, higher values of average sizes and lower values of zeta potential were measured at solution with lower concentrations, according to which such diluted vesicles can be described as unstable. There were also visual observations made, but no visible aggregation in the solutions was observed. Furthermore, the aggregation behavior of the vesicles was observed by fluorescence spectroscopy using pyrene as fluorescent probe.
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Study of the interaction of hyaluronan and biocompatible amphiphilic substances
Cigánková, Hana ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of the interactions between hyaluronan and selected phospholipids and their mixtures in water or buffer. First, the aggregation behavior of these lipids was measured, afterwards the influence of hyaluronan was studied. This behavior was investigated by fluorescence spectoscopy using pyrene as fluorescence probe. The Critical Agregation Concentration of the lipids before and after addition of hyaluronan was determined. The measurements revealed that the addition of hyaluronan has an observable influence on the aggregate behavior of the selected lipids and their mixtures.
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Study of interaction of negatively charged vesicular systems with polycations
Repová, Romana ; Klučáková, Martina (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the preparation and characterization of negatively charged catanionic vesicular systems and their combination with selected polycations. The catanionic vesicular system was prepared by mixing of two oppositely charged surfactants SDS and CTAB. The negative charge as well as the stability of the vesicular system was provided by the incorporation of phosphatidic acid. Polycations, DEAE and TMC, have been selected for use in a pharmaceutical applications. Characterization of the prepared systems was performed by measuring DLS and ELS. The results indicate that we were able to prepare stable negatively charged vesicles that were eligible to non-covalently interact with selected polycations.
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Determination of residual substances from the preparation of colloidal systems based on biopolymers
Borůvková, Tereza ; Vávrová, Milada (referee) ; Mravec, Filip (advisor)
Volatile organic compounds could be isolated and concentrated from liquid solution using methods, which are based on analysis of equilibrium gas phase, because of their volatility. The aim of this bachelor´s thesis is determination the amount of residual tert-butanol, belonging to volatile organic compounds, in the samples after lyophilisation. Samples were prepared by mixing hydrofobic sonde and hydrofilic hyaluronan. For mixing these components it is necessary to add organic solvent tert-butanol, which ensures stable environment. Thanks to its presence mixing of probe and hyaluronan occurs. Consecutive lyopfilisation of the sample leads to the disruption of a water cover of hyaluronan and nonpolar hydrogen atoms, which are hidden in it, can interact with hydrofobic probe. Bonding the probe is important in oncology, where this fact could be used in targeted distribution of pharmaceuticals. However presence of tert-butanol is unacceptable in the final product, mainly in medical applications. The object this study is the minimization of residual tert-butanol amount in final lyophilised cake. The method of equilibrium gas phase analysis (static Head-Space) by gas chromatography with flame ionization detection (GC/FID) was used for the determination of its content.
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Interaction between amphiphilic fluorescent probes and hyaluronan
Střondalová, Hana ; Vala, Martin (referee) ; Mravec, Filip (advisor)
This bachelor thesis dealt with studying the interaction of amphiphilic fluorescent probes with hyaluronan because these probes could significantly help in clarifying the links between hyaluronan and surfactants. This complex could be a great benefit in targeting drug distribution. This thesis was focused on the interaction between hyaluronan and amphiphilic fluorescent probes – specifically with dodecylacridine orange. First explored was the interaction with hyaluronan, and then with the hyaluronan – surfactant system. Cetyltrimethylamonium bromide was selected as surfactant. Samples were examined using fluorescence spectroscopy. Emission and absorption spectrum of dodecyl acridine orange was measured. This experiment was based on the hypothesis that dodecylacridine orange forms dimers as acridine orange and nonylacridine orange. It was found that in a system of hyaluronan-probe, dodecyl acridine orange was pushed out of the complex by cetyltrimethylamonium bromide, which was bounded instead. Surfactant interacts with hyaluronan binding positions - with carboxyl groups.
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Inverse FCS in colloidal systems research
Richterová, Veronika ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on the study of inverse fluorescence correlation spectroscopy, especially with the regard for the usage of different fluorescent probes and different sized analysed particles. At first, the proper concentration of fluorescent probes was determined. In this concentration is the probe considered as a medium surrounding the analysed particles. Based on this concentration, which was determined as 400 M, several sets of samples were prepared. This samples contained different concentration of polystyrene particles of 100 and 500 nm diameter and multilamellar liposomes. Then, the FCS curves of samples with different fluorescent probes were measured. Fluorescein, rhodamine 6G and Atto 488 were used as fluorescent probes. As a result from experiments, it was found, that particles with 100 nm diameter cannot be analysed with none of the fluorescent probes. Inverse FCS method can be applied to systems, that contains particles with 500 nm diameter and fluorescein. Systems with rhodamine 6G have the same behaviour as typical FCS measurement. It is caused by dimerization of this probe and it cannot be used for 500 nm particles. Liposome samples can be established with iFCS method, but the results are biased by random distribution of liposomes size.
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Studying the quality of hydrophobic domains in the phase separated hydrogels
Burešová, Natálie ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
The thesis deals with the study of hydrophobic domains in phase separated hydrogels. Two types of polyelectrolytes (hyaluronan, dextran) and oppositely charged surfactants (carbethopendecinium bromide, sodium dodecylsulphate ) are used. Phase separated hydrogels are formed by electrostatic interactions. The target of the thesis is to study the polarity of environment in phase separated hydrogeles by fluorescence spectroscopy. Fluorescence measurements is performed by using pyrene, Nile red and laurdane as fluorescence probes. All probes are sensitive to the polarity of the environment. The results showed that probes can be expected in the polar environment of hydrogels.
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Native hyaluronan as a delivery system for hydrophobic drugs
Černá, Eva ; Mravec, Filip (referee) ; Pekař, Miloslav (advisor)
The aim of this paper is to discover whether it is possible to use the native form of hyaluronic acid as a hydrophobic drug carrier for a targeted distribution in the body. In its structure, hyaluronic acid is a linear high molecular weight biopolysaccharide which is found in most living organisms. Hyaluronan is involved in many physiological processes and therefore is essential for the functionality of the human body. It is in most tissues of the human body, high concentration is in the skin, the vitreous body and is also observed in cancer cells that contain several receptors for hyaluronan. These receptors include CD44 and RHAMM. The interaction of the hyaluronic acid delivery system and the hydrophobic medicinal with these receptors could ensure a free passage for drugs to the affected tissue, where the release of the drug would destroy the affected cells. The drug would directly target the damaged tissue and did not burden the rest of the body like the cytotoxic agents do. In this paper the native form of hyaluronic acid, which we normally find in the human organism, was chosen as the carrier. Its properties do not stand above other carrier systems, but its biocompatibility and biodegradability in the body greatly exceed them. High molecular weight hyaluronic acid was used as a carrier and the hydrophobic dye sudan red G, a substance of similar properties, was used instead of a hydrophobic drug.
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Interactions between hyaluronan and cetrimide
Adamcová, Zuzana ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
Interactions between hyaluronan and cationic Cetrimide were studied using fluorescence spectroscopy and turbidimetry in model physiological solution (0,15 M NaCl) and water. Results obtained in water showed strong electrostatic interactions before reaching CMC of the surfactant, which were screened in presence of salt due to high ionic strenght of the solution. Behavior of Cetrimide-hyaluronan systems was compared with the pure TTAB in equivalent system. It was found, that changes in Cetrimide-HA system occure at lower concentrations of the surfactant than in TTAB-HA system. This was probably caused by presence of small amount of CTAB in Cetrimide.
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