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Hydrogels with incorporated enzymes
Geistová, Karolína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of incorporation of enzymes into phase separated hydrogels. The aim of this work is to determine the enzyme activity in phase separated gels. Gels were prepared by the dry-way based on the interaction of negatively charged polyelectrolyte (hyaluronan) with positively charged surfactant (Septonex). Two enzymes, bromelain and collagenase, were incorporated into the hydrogels. To determine enzyme activity, the modified albumin protein with bound sulfanilamide group (azoalbumin) was used as a substrate. The enzyme activity of the enzyme itself, the enzyme activity affected by one of the two components of the system as well as the activity of the enzyme directly in the hydrogel was determined on UV-VIS spectrophotometry. The enzyme was found to be incorporated in the hydrogel. Furthermore, a significant effect of the positively charged surfactant on the enzyme activity was detected in phase-separated hydrogels.
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Fluorescence probes to study the properties of hydrogel systems
Máčala, Jakub ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This bachelors thesis is focused on possibility of selected fluorescent probes for study of properties of hydrogel systems. Chosen probes are Prodan for polarity study, HPTS for measurement of pH of the probe environment and platinum complex [2,6-Bis(2-pyridyl)fenyl-C,N,N]chloroplatinum(II) for its unique fluorescence properties and potential of energy transfer studies in hydrogel systems. Spectral properties of fluorophores were defined in selected set of solvents with different polarity with steady state fluorescence spectroscopy method. Properties of HPTS probe were also tested in buffers with different pH. Subsequently, hydrogels based on hyaluronan-septonex and dextran-SDS with incorporated fluorescence probes were prepared. Steady state fluorescence measurement of fluorophores from hydrogel were made using fluorimeter optical fibres. It was found, that probes Prodan and HPTS are suitable for study of polarity and pH of hydrogels and probably they will be able to be used for study of hydrogel with other techniques, like fluorescence microscopy, however platinum complex is, due to the low fluorescence intensity and possible quenching by polar water environment, probably not suitable for other measurements.
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Transport properties of hyaluronan-based hydrogel systems
Káčeríková, Martina ; Smilek, Jiří (referee) ; Klučáková, Martina (advisor)
This bachelor thesis deals with the study of trasport properties of hydrogels, that were prepared by interaction of hyaluronan with karbethopendecinium bromide (Septonex), as a surfactant. Nile red is a fluorescent probe to determine the ability of the system to absorb or to release the substance. All the hydrogels were prepared by the „wet“ way, where the solutions of hyaluronan and surfactant were prepared first. Nile red was prepared in the solution of tenzide, which after the mixing with hyaluronan produced colorful gels. By the spectrophotometric measurement, the absorbance of supernatants were observed. The absorbance was measured in original but also in changed supernatatns after 24, 72 and 168 hours.
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Monitoring the pH change in the phase-separated hydrogel systems
Heger, Richard ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This thesis pursues monitoring of pH changes and description of properties in phase–separated hydrogels prepared by interaction of polyelectrolyte and oppositely charged surfactants. For the purposes of this work, all hydrogels were prepared by the interaction of hyaluronan with Septonex. All hydrogels were exposed to pH changes (4–9). Changes in pH were being monitored using spectroscopic indicators, methyl orange, methyl red, bromothymol blue and phenolphtalein. The pre– and post– pH changes in properties were compared using rheological methods. Additional information was gathered through thermogravimetric analysis. By these methods it was proved that the changes in the pH can modify the mechanical properties and partly the internal structure of the hydrogels. Rheological tests show, that from the application point of view, the most interesting hydrogels are observed at pH 9 which have the strongest bonds but have low permeability and hydrogels described at pH 7 which are much softer and are capable of absorbing large amounts of water.
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Hyaluronan-Micelle Aggregates and their Potential for Nanomedicine Applications
Pilgrová, Tereza ; Burgert, Ladislav (referee) ; Reháková, Milena (referee) ; Pekař, Miloslav (advisor)
This thesis deals with the study of preparation and characterization of hyaluronan-micelle aggregates. The theoretical part deals with drug delivery systems, characterization of used materials and methods especially fluorescence spectroscopy, dynamic light scattering and turbidimetry. Methods of determination of measured data are summarized in the experimental section. The result section is divided into two subsections dedicated to different preparation methods of hyaluronan-surfactant complexes. Induced aggregates of hyaluronan with Septonex are characterized in terms of their origin and stability, and the results are compared with previously studied surfactants CTAB. In the second part are discussed so-called decorated micelles, their formation, properties and stability.
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The electromigration methods and their use in the analysis of drugs.
Tučková, Dominika ; Tulková, Tereza (referee) ; Řezáčová, Veronika (advisor)
This bachelor thesis deals with the determination of septonex, effective component in medical agents, using capillary isotachophoresis. The main aim is optimization of this method for fast, sensitive and inexpensive determination of septonex. In bachelor thesis chemical and physical properties of septonex are introduced as well as its negative and positive properties, which can be used in medicine, and summary of possible methods of analysis. Further this thesis gathers information about drugs containing septonex and available on Czech market. Following chapters of the thesis summarize theory of electromigration methods, their principles, analytical devices and their use for septonex analysis. Finally, the thesis presents optimal conditions for analysis and results of determination of septonex in drug Septonex spray, which is available on Czech market, using capillary isotachophoresis. Content of determined septonex is compared to amount of septonex contained in this drug, which is declared by producer, in this thesis.
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Diffusion of organic molecules in the hydrogel environment
Holubová, Anna ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with study of hydrogels formed by phase separation of hyaluronan with oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and Septonex. It follows the bachelor thesis and extends the knowledge about the detailed characterisation of the inner environment of the hydrogel by determining the diffusion behaviour of the fluorescent probes Atto 488 and Nile Red using fluorescence correlation spectroscopy (FCS) technique and its modified version dual-focus fluorescence correlation spectroscopy (2f-FCS). Compared results showed that both methods show similar values and probes specifically interact with CTAB but Atto 488 shows only weak interaction with Septonex compared to Nile Red. Additionally, these interactions were not affected by the molecular weight of hyaluronan. In conclusion, it was recommended to measure this type of hydrogel in a small depth of gel using a conventional method.
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Resonance energy transfer in the environment of the hydrogel matrix
Janča, David ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.
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Characterization of coloid particles by excited-state proton transfer with advanced fluorescence techniques
Kotouček, Jan ; Mravec, Filip (referee) ; Pekař, Miloslav (advisor)
The deprotonation characteristics of fluorescent probes -naphthol and 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) were studied in this diploma thesis, using steady-state and time-resolved fluorescence spectroscopy. Two cationic surfactants, Septonex and cetyltrimethylammonium bromide (CTAB), were studied. These surfactants were measured in the complex with hyaluronan (1.75 MDa, 1 MDa and 300 kDa). Steady-state fluorescence was used for determination of critical aggregation concentration of each surfactant and pKa*. Time-resolved fluorescence decays were used to calculate the average lifetimes and the deprotonation constants of naphthol and HPTS. The measurement with hyaluronan were compared with the polystyrenesulfonate (PSS) – surfactant system. The effect of hydration shell of hyaluronan on hyaluronan – surfactant complex formation results from the comparison of above mentioned systems. Large differences were found in the deprotonation characteristic between surfactants and even between individual molecular weights of hyaluronan. The measurement shows that the hydration shell is located near to the dissociated carboxyl groups of hyaluronan chain, where the interaction with the positively charged surfactants occurs. Furthermore, the aggregation number of Septonex was determined by quenching of pyrene using cetylpyridinium chloride (CPC) as a quencher. The aggregation number for 20 mM Septonex solution was determined as a value of 104 molecules. CPC was used for confirmation of the localization of -naphthol in the micelles of CTAB and polymer – CTAB, respectively.
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Hyaluronan ion complexes
Cimalová, Jana ; Sedlařík, Vladimír (referee) ; Pekař, Miloslav (advisor)
This diploma thesis is focused on the study of physical and chemical properties of hyaluronan and cationic surfactant. As the cationic surfactant Septonex was used. The influence of the environment on the system, the effect of molecular weight of hyaluronan, and its concentration was studied. Then, the study of the influence and the effects of concentration of Septonex on the interaction of hyaluronan-surfactant followed. Different methods of measurement were chosen to characterize these ionokomplexes. The critical micelle concentration of the surfactant itself was measured, and then also with the addition of hyaluronan by spectrofluorimetry with fluorescent probe pyren. It was found, that hyaluronan forms gel with Septonex. On this basis, gels were prepared for three different molecular weights of hyaluronan – 300 kDa, 806 kDa and 1697 kDa. Gels were prepared in a ratio of hyaluronan – surfactant 1:1. In gels prepared in this way, the influence of environmental water and 0.15 M NaCl was studied and it was found that at 0.15 M NaCl clear gels are formed. Selected samples of the gels were then measured with oscillatory testing and the rheological behavior of gels of Septonex was studied. As the last method the turbidimetric measurement was chosen, which characterized the turbidity point in the gradual addition of Septonex to sodium hyaluronate solution. Again, the effect of the molecular weight of hyaluronan and its concentration in two environments - water and 0.15 M NaCl was evaluated. It was found that 0,15 M NaCl suppresses formation of turbidity and formation of precipitates.
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