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Preparation and characterization of quatsomes
Havlíčková, Anna ; Szabová, Jana (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with preparing and characterizing quatsomes and similar vesicular systems. The preparation consisted of weighing an equimolar amount of sterol and surfactant, hydration with deionized water, and subsequent sonication with an ultrasonic probe with an energy of 5, 10 and 15 kJ. In this work, two types of quatsomes were prepared, the first contained equimolar amounts of cholesterol and cetyltrimethylammonium bromide (CTAB), and the second consisted of cholesterol carbethopendecinium bromide (Septonex). The optimal dispersion energy of 10 kJ was selected for both types based on turbidity measurements, visual observation and dynamic and electrophoretic light scattering analysis. Furthermore, the observed properties were found to be constant from day 7 after sonication. In addition to quatsomes, similar vesicles were prepared, which contained a surfactant in the form of a sulfate salt. Once again, two types were prepared, the first containing sodium dodecyl sulfate (SDS) and cholesterol, the second sodium tetradecyl sulfate (STS) and cholesterol. Based on the same measurements as for quatsomes, optimal dispersion energy of 10 kJ was selected. Furthermore, mixtures with different ratios of cholesterol and a given surfactant were prepared, in which the effect of cholesterol addition was monitored. The analysis of these mixtures was performed by the same methods as the analysis of quatsomes.
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Interaction of negatively charged vesicular systems based on ionic amphiphilic pairs with polycations
Fardová, Lucie ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This work is focused on the preparation of negatively charged vesicular systems from amphiphilic pairs deprived of counterions and their interaction with the polycation. First, vesicular systems were prepared from the surfactants sodium dodecyl sulfate and cetyltrimethylammonium bromide. The addition of cholesterol and phosphatidic acid stabilized and at the same time gained a negative charge. Subsequently, there was an interaction with the polycation diethylaminoethyl-dextran hydrochloride. The systems were investigated by the measuring of particle size, zeta potential, generalized polarization and anisotropy. The measurement of generalized polarization was enabled with the laurdan probe and measurement of anisotropy was performed with the introduction of the 1,6-diphenyl-1,3,5-hexatriene probe. The results indicate the successful preparation of stable, negatively charged systems that interacted with positive diethylaminoethyl-dextran hydrochloride, which affected the behavior of the outer membrane but not the inner. The systems were still stable.
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Stability of vesicular systems using fluorescence spectroscopy techniques
Máčala, Jakub ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This thesis is focused on possibility of studying stability and fusion of catanionic vesicles with Förster resonance energy transfer. The mainly used technique was Time-Correlated single photon counting. Firstly, excitation and emission spectra of chosen probes were measured and donor-acceptor pairs were suggested: 5-hexadecanoylaminofluorescein with Octadecyl Rhodamine B, Bodipy 493/503 with rhodamine or DiI, perylene with fluorescein, DiO with DiI. Then, time-resolved measurements of suggested pairs from environment of catanionic vesicles with different content of cholesterol were made in order to track the FRET associated with fusion of vesicles. It was found out, that it is not possible to use DiO as a donor because of it’s inefficient solubilisation into vesicles. It is also not possible for Bodipy to be used as a donor, because of it‘s excimer formation. In case of using fluorescein as a donor, it was found, that there is ongoing homo-fret between fluorescein molecules. Thanks to this, fusion was tracked by addition of unstained vesicles. It was also possible to track fusion in longer period of time. Also perylene-fluorescein pair was found to be capable of tracking the fusion, but with the exception of vesicles with content of cholesterol of 43 mol. %, tracking of fusion was possible only in short period of time.
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Study of system based on phospholipids for sonographical application.
Überall, Martin ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
The aim of this thesis was to determine solubilizing capacity of surfactants and phospholipids using the UV-VIS spectrophotometry. The concentrations of solubilized substances within these systems was further determined. From the surfactant group, three representatives were selected, cetyltrimethylammonium bromide (CTAB), sodium dodecylbenzenesulfonate (SDBS) and Triton X-100. From the phospholipid group, lecithin and commercially available SonoVue® were chosen. The solubilizing capacity was determined using hydrophobic solutes Sudan Red G (SRG), Oil Red O (ORO) and 1-naphtol in order to obtain a model system of solubilized drugs or vitamins which are also hydrophobic. The behavior of solutes in surfactants was determined in a moderate polar medium of physiological saline (0.15 M NaCl), the behavior of solutes in lecithin was conversely determined in Mili-Q water medium. The determined concentration of solubilized hydrophobic solutes inside the micelles of surfactants is within the range of tens to hundreds of micromoles per liter. With increasing concentration of surfactant increases the concentration of solubilized solutes. The results obtained in this work can be used for further experiments and research focused on solubilization of drugs in contrast agents used in ultrasonography.
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Fluorescenec spectroscopy in study of aerosol-forming substances
Lippay, Josef ; Jedelský, Jan (referee) ; Mravec, Filip (advisor)
To study the spectral characteristics of a substance DEHS several experiments were suggested. Closer investigation was made on study of the effect of different environments on the spectral properties of the substance DEHS. It was found that the highest positive effect on the fluorescence of the substance DEHS have a medium with hydroxyl groups. IPA was evaluated as the best medium for further experiments. It was found that the medium xylene decreases intensity of the fluorescence to the detection limit. Spectral characterization of the nondegraded and degraded DEHS was performed. It was found that for the degraded DEHS shift to higher wavelengths occurs due to degradation. Possible contamination of the DEHS with substances causing luminescence has not been proven.
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Study of aggregation in a biopolymer-surfactant system at low surfactant concentration.
Stiborský, Filip ; Vojtová, Lucy (referee) ; Mravec, Filip (advisor)
In this bachelor thesis, the interaction between hyaluronate and surfactant using fluorescent spectroscopy and pyrene as fluorescent probe was studied. A cationic surfactant cetyltrimethylammonium bromide and a native hyaluronate with different molecular size was used. Before a detected critical micelle concentracion, there were no interactions observed in phosphate buffer. However, we could observe electrostatical interactions in the water systém measuring. At low concentration of surfactant (between critical aggregation concentration and critical micelle concentration), the production of gel phase appeared.
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Study of physical gels with hydrophobic domains
Kovářová, Lenka ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
The thesis is focused on physical hyaluronan gels. The object of study is the interaction of hyaluronan (HyA) with oppositely charged surfactants in physiologic solution (0.15 M NaCl), leading to the formation of gel. In the first part of work have been determined the solids´ contents (X) in gels and their supernatants in percentage and their correlation with molecular weight concentration of original HyA solution and the ratio of binding sites on hyaluronan chain and surfactant CTAB. To conclude, decrease in HyA concentration results in higher values of X and vice versa. On the other hand, increase in the value of X with increasing molecular weight of HyA is not so significant. Analogous conclusions have been made for supernatants and the amount of solids in gel. Drying process has been recorded by drying curves. Swelling process has been used for the characterization of gels. The percentage of water that can be absorbed by dried gel, was determined. The results are in agreement with the measurements of solids´ content in gels. In the next part, the correlation between rheological properties of gels and HyA concentration, HyA molecular weight and concentration of CTAB have been studied by the oscillation and flow tests. The samples with the highest molecular weight and concentration have the most viscoelastic character. The flow test confirmed the assumed pseudoplastic behavior of gels. A very interesting trend arose while comparing HyA concentrations and viscosity in stock solutions and gels. Whereas in stock solution viscosity (at low shear rate) is lower with increasing of HyA concentration, the situation was exactly the opposite in gels. The results are in agreement with frequency tests and observed character of gels.
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Influence of particle size on microreology experiments using fluorescence correlation spectroscopy
Valovič, Stela ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with microrheology measured via the fluorescence correlation spectroscopy. As microrheological probes, fluorescently marked nanoparticles of 5 sizes in the range of 10-100 nm, were used. The particles had been immersed in a variety of concentrated glycerol solutions and agarose gels of different concentrations, and the FCS measurement revealed a diffusion coefficient of individual particles in each environment. Based on the coefficient, the viscosity of the glycerol needed to stop the particles could be determined. Particles of 10 nm size were not stopped even by the 100 wt% glycerol. In the case of the agarose gels, a combination of higher agarose concentration and larger particles resulted in an increase in the diffusion coefficient to an unlikely high value. This was caused probably by an agarose autofluorescence and the value indicates stopping of the particles in the given agarose gel. Later, the data acquired by the FCS measurement were converted to MSD curves using MATLAB software. The thesis discusses the influence of the experimental parameters on the shape of the MSD curve. The results showed that the number of particles and autocorrelation function have the most significant effect.
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Advanced microrheological techniques in the research of hydrogels
Kábrtová, Petra ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the use of fluorescence correlation spectroscopy technique for microrheological characterization of hydrogel in a system of hyaluronate-cetyltrimethylammonium bromide. Fluorescently labelled particles were used for microrheological FCS analysis. To optimize the method the most appropriate size of particles was chosen on the basis of Newtonian glycerol solutions analysis. Among other things, the discussion was focused on the influence of refractive index change of analysed solutions on analysis results. After hyaluronate solutions analysis it was possible to assess the biopolymer concentration and molecular weight impact on the FCS microrheology results, which could then be compared with analysis results of model hydrogels of hyaluronate and CTAB. Finally, usability and limitations of FCS microrheology have been discussed.
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Incorporation of low molecular weight and high molecular weight substances into vesicular systems
Geistová, Karolína ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This master´s thesis deals with the study of the incorporation of low and high molecular weight substances into liposomal systems. The aim of the work was to determine the encapsulation efficiency (EE) of the active substance and the influence of individual components of the liposomal system on EE. Liposomes were prepared from dipalmitoylphosphatidylcholine. They were stabilized by cholesteroland and phosphatidic acid was added to give a negative charge. Stealth properties gain the binding of polyethylene glycol and other trimethyl chitosan we enabled the entry of liposomes into the bloodstream by the paracellular pathway. Vitamin C and the enzyme bromelain were used for incorporation into liposomes. UV-VIS spectrophotometry was used to determine the encapsulation efficiency of liposomes prepared by combining the individual components. It has been suggested that vitamin C and the enzyme can be incorporated into liposomes, but an enzyme with a higher EE. Furthermore, phosphatidic acid and trimethyl chitosan have been found to affect EE, which increases the EE of vitamin C and decreases the EE of the enzyme.
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