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Voltammetry and liquid chromatography of cholesterol, its precursors, and oxidation products
Kubešová, Jana ; Schwarzová, Karolina (advisor) ; Baluchová, Simona (referee)
Electrochemical detection of sterols is difficult. This thesis aims to study the voltammetric behaviour of selected oxysterols, i.e. 25-hydroxycholesterol and 7-ketocholesterol on the boron-doped diamond electrode. Further, an HPLC method with spectrophotometric and electrochemical detection was developed for separation of selected sterols. The study was performed using cyclic and differential pulse voltammetry. 0.1 mol l−1 perchloric acid in acetonitrile (water content 0.55 %) was chosen as the most suitable electrolyte. 25- hydroxycholesterol provides anodic signal at the potential of +1500 mV and 7-ketocholesterol at +1900 mV in that media. Electrochemical reduction of 7-ketocholesterol was also studied. However, the observed cathodic signal isn't reproducible. The stability of oxysterols in the presence of perchloric acid was confirmed by UV-VIS spectroscopy. Calibration dependence of oxysterols was measured using differential pulse voltammetry and resulted in the limit of detection of 0.82 µmol l−1 for 25-hydroxycholesterol and 0.66 µmol l−1 for 7-ketocholesterol. In the second part of the thesis, an HPLC method was developed for the separation of a sterol mixture on a reversed-phase C18 column using a mobile phase containing 50 mmol l−1 sodium perchlorate in acetonitrile with 0% or 6% water...
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Interaction of modified polymers with liposomes
Dimidova, Aleksandra ; Kalina, Michal (referee) ; Mravec, Filip (advisor)
The aim of this work was to investigate the interactions of modified polymers with vesicular systems, focusing on hydrophobically modified biopolymers. The work included a literature search, design of experiments to study these interactions, execution of the experiments and evaluation of the results in terms of potential medical applications. Different methods of preparing liposomes were studied in order to optimize their physicochemical properties for efficient drug delivery. Although the preparation of liposomes from DPPC faced problems due to the quality of chemicals, the ethanol addition method resulted in the formation of stable liposomes. These liposomes showed good size and stability, and their interaction with sodium palmitoyl hyaluronate indicated the potential for increasing the size of liposomes and affecting their stability.
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Study of interaction of charged catanionic vesicles with oppositely charged polyelectrolytes
Havlíková, Martina ; Lehocký, Marián (referee) ; Táborský, Petr (referee) ; Mravec, Filip (advisor)
The presented dissertation is focused on positively charged catanionic vesicles and their interactions with oppositely charged hyaluronan. The catanionic vesicles were composed of ion pair amphiphile of hexadecyltrimethylammonium-dodecyl sulfate (HTMA-DS), a positively charged double-chain surfactant that electrostatically stabilized the vesicles and cholesterol. Cholesterol may strongly affect the physico-chemical properties of the vesicles, therefore it was necessary to determine its optimal amount in the membrane. Especially, the size and stability of vesicles were studied by dynamic and electrophoretic light scattering, membrane microviscosity by fluorescence anisotropy and hydration of the outer part of the membrane by generalized polarization. It was found that vesicles show ideal properties at a cholesterol content about 40 mol.%. These catanionic vesicles interacted with hyaluronan regardless of its molecular weight. Visible aggregates were observed around their isoelectric point, but they disintegrated with increasing polymer concentration. However, this decay was different for different hyaluronan molecular weights. An increase in ionic strength, or more precisely concentration of sodium chloride above 25 mM had a destabilizing effect on both uncoated and hyaluronan coated vesicles. It was also interesting that increasing the ionic strength affected the microviscosity of the membrane, which became more fluid, but there were no significant changes in the hydration of its outer part. The presented work is also focused on the preparation and characterization of a new type of catanionic vesicles, where the cationic surfactants HTMAB was replaced by Septonex. The same methods were chosen for their characterization as in the case of the HTMA-DS vesicles. This exchange of surfactants had a major effect on the resulting properties of the vesicles, which are stable even at very low cholesterol content. In accordance with long-term stability, a suitable concentration of cholesterol was chosen from 15 mol.% and higher.
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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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Characterization catanionic vesicular system by HRUS
Kopřivová, Hana ; Sovová, Šárka (referee) ; Hurčíková, Andrea (advisor)
This bachelor thesis is about catanionic vesicular systems, which could serve as carriers of drugs or genes in the future. These systems are prepared from the cationic surfactants CTAB and DDAC, the anionic surfactant SDS and cholesterol, which serves as a stabilizer. In the experimental part, these vesicles were characterized by high-resolution ultrasonic spectroscopy. The behavior of systems with different percentages of cholesterol was monitored. As next, the temperature properties of the vesicles were studied using temperature scans between 10 °C and 65 °C. The density of the samples was examined by densitometry at temperatures of 25 °C and at a human body temperature of 37 °C.
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Ecotoxicity of cholesterol-lowering-drugs
Königsmarková, Kristýna ; Vávrová, Milada (referee) ; Zlámalová Gargošová, Helena (advisor)
This bachelor thesis is focused on the ecotoxicological testing of blood cholesterol lowering drugs. At first it considers the issue from a theoretical point of view – describes the effect of residual drugs on the environment, the distribution of hypolipidemics, the risks of elevated cholesterol, then deals with ecotoxicology and introduces the principles of ecotoxicological testing. In the experimental part, these following drugs were tested: Corsim, Atoris, Lipanthyl Supra, Lipanthyl 267M. In addition, clofibric acid – the metabolite of fibrates was tested. Residues of these drugs are found mainly in the aquatic ecosystem, that is why tests of aquatic organisms have been used to evaluate ecotoxicity. Test organisms were Thamnocephalus platyurus, Lemna minor and Vibrio fischeri. LC50, EC50 and IC50 values were determined on the basis of test results. Using these values, the ecotoxicity of the above mentioned hypolipidemics was evaluated.
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Mixed lipids and their interaction with biopolymers
Cigánková, Hana ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This thesis focuses on the study of interactions between hyaluronan and lipid DOTAP and its mixtures with DPPC in water. First, the aggregation behavior of the lipid itself and its mixtures was measured, afterwards the influence of hyaluronan in different concentration was studied. Further the fluorescence anisotropy of fluorescence probe DPH in these mixtures and influence of cholesterol was measured. These properties were investigated by fluorescence spectoscopy using pyrene and DPH as fluorescence probes. The measurements revealed that the addition of hyaluronan has observable influence on the aggregation behavior of the lipid DOTAP and its mixtures with DPPC and that cholesterol influences liposomes fluidity.
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New amphiphilic ion pairs for the preparation of vesicular systems
Strnadová, Martina ; Kalina, Michal (referee) ; Mravec, Filip (advisor)
The bachelor thesis is focused on the preparation of the new vesicular systems. The aim of this work is to create the most stable system and find out the most optimal working method for its production. The compared parameters were the energy used for dispersion, the molar percentage of cholesterol added, and the type of double-stranded positively charged surfactant. The whole system consisted of negatively charged surfactant sodium dodecyl sulfate and positively charged surfactant septonex. Each system was compared over time to determine stability based on the size of the particle, zeta potential, and turbidity of the solution. By the method of dynamic light scattering, particle size and polydispersity coefficient were measured. On the other hand, the zeta potential was determined by electrophoretic light scattering. The result of the thesis presents the most suitable parameters of ultrasonic dispersion, the molar proportion of cholesterol, and the use of cationic surfactant. Based on these parameters, it is possible to create a physically stable system for a long period of time, thanks to which such a system can be used in the future for other applications.
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Incorporation of small organic hydrophilic substances into vesicular systems
Janoušková, Vendula ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of the encapsulation efficiency of hydrophilic drugs and their releasing from the aqueous core depending on the different composition of the components of individual liposomal systems. The hydrophilic fluorescent probe called pyranine was chosen as a model drug. The aim was to prepare liposomes which would be suitable for inhalation administration in terms of their properties. These liposomes would provide passive targeting with prolonged release time without causing negative side effects on the organism. We have succeeded in developing a standard operating procedure for the incorporation of hydrophilic drugs. Liposomal systems have been prepared consisting of the addition of various components as cholesterol, phosphatidic acid, pegylated phosphatidylethanolamine and trimethylchitosan. Furthermore, we were able to characterize the individual liposomal systems in terms of size, stability and encapsulation efficiency which are important physicochemical properties for further application potential.
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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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