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Study of microviscosity of membrane systems based on ionic amphiphilic pairs
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
In this master ‘s thesis, catanionic vesicles formed by the pseudo-double-chain complex CTA – DS were investigated from the point of view of microviscosity. Samplesand of cationic vesicles contained 23, 43 and 53 mol. % of cholesterol and the double-chain surfactant DODAC. Cationic vesicles were prepared for visual observation, their stability was determined by DLS and the prepared system was further investigated. Microviscosity was determined from fluorescence anisotropy. To study the outer part of the membrane, laurdan fluorescent probes were used whereas diphenylhexatriene was used for the inner part of the membrane. This method has been proven to be suitable because it reflects the conditions of the membrane. Moreover, a 1,3-bispyrenylpropane probe forming intramolecular excimers was used to study the microviscosity in the vesicle bilayer. The dicyanovinyljulolidine (DCVJ) probe was applied in the case of the molecular rotor technique. It has been shown that in the case of the DCVJ probe, the molecular rotor technique is practically unusable, due to the fact that the probe has a low quantum yield at low temperatures. Also, the excimer formation of P3P probes does not lead to the expected results. The cationic vesicles do not seem to support this formation, as they are too closely related. This type of probe can be used for the selected system with some restrictions.
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Study of the hydration shell of vesicular systems based on ionic amphiphilic pairs
Rašticová, Barbora ; Szabová, Jana (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of hydration shell of positively charged vesicular systems. The aim of the work was to monitor the hydration shell and to find out the amount of water molecules in the environment at different conditions of the membrane. Furthermore, a comparison of two selected fluorescent probes were studied. Positively charged vesicular systems composed of two types of surfactants were used, namely negatively charged SDS and positively charged CTAB. The two-chain positively charged surfactant was also added, resulting a positively charged vesicular systems. Three different concentrations of cholesterol were selected for the study and added to the system, namely 20, 40 and 60 mol. %. The study was performed by measuring the fluorescent emission as a function of temperature. The temperature range was from 10 to 80 °C. As a fluorescent probe Laurdan and Prodan were selected. For evaluation the method of generalized polarization was used. Due to its structure, Laurdan is mainly found in the membrane environment, so the method of two-wavelength generalized polarization, which includes fluorescence contributions only from the membrane, was sufficient for its evaluation. Compared to Laurdan, Prodan has a shorter hydrocarbon chain, so its distribution is not only in the membrane but also in the aqueous environment. Therefore, the method of three-wavelength generalized polarization, which also includes the contribution of fluorescence from the aqueous environment was used. The results showed that the behaviour of both probes is very similar. In all systems, the values of generalized polarization decreased with increasing temperature. Thus, at low temperatures, the membrane is in a solid ordered phase. With increasing temperature, it transforms into a liquid disordered phase. Thus, with increasing temperature, the number of water molecules increases. As the cholesterol concentration increased, the phase transition interval increased. The results also showed that the values of Prodan’s three wavelength generalized polarization are always higher than the values of Laurdan’s two wavelength generalized polarization.
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Study of membrane properties of liposomal systems using fluorescence spectroscopy
Zbořilová, Hana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
The presented diploma thesis is focused on the preparation, characterization and study of membrane properties of liposomal systems which were composed of the neutral phosphatidylcholine (DPPC), cholesterol, negatively charged phosphatidylglycerol (DPPG), polyethylenglycol bounded to phosphatidylethanolamine (PEG5000–PE) and polycation N,N,N-trimethylchitosan (TMC). The influence of individual components and their concentrations on the average particle size, zeta potential and changes in the outer and inner part of the bilayer was investigated. In this matter, methods of dynamic and electrophoretic light scattering and fluorescence spectroscopy with the application of laurdan and DPH probes were used. Based on the above-mentioned parameters, concentrations of components that most suitably influence properties of liposomes in terms of the intended application were selected for the definite complex. It was managed to prepare a liposomal complex stealth liposome–N,N,N-trimethylchitosan, which, due to the optimized composition, could have suitable attributes as a drug delivery system for inhalation administration of biologically active substances.
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Time-resolved fluorescence study of liquid and condensed systems based on biopolymer-surfactant interactions.
Černá, Ladislava ; Žitňan, Michal (referee) ; Mravec, Filip (advisor)
This thesis studies properties of hydrogel, which arises on the basis of electrostatic and hydrophobic interactions between hyaluronan chain and micelles of cationic surfactant. A native sodium hyaluronan at molecular weight 750–1 000 kDa and a cationic surfactant CTAB (cetyltrimethylammonium bromide) were used. This hydrogel was assessed as a material for drug delivery systems. The hydrogels were made by mixing 200mM CTAB with 0.5% hyaluronan, both dissolved in 0.15M aqueous solution of NaCl simulating physiological solution. Methods used in this study were steady-state and time-resolved fluorescence spectroscopy, more accurately time-resolved emission spectra (TRES) and deconvolution of steady-state emission spectra of a whole sample by means of parameters gained from fluorescence intensity decays at a set of wavelenghts. Selected systems were investigated by three fluorescent probes, prodan, laurdan and rhodamine 6G. The first two mentioned probes were in hydrogel localized only within micelles in three different microenvironments. Rhodamine 6G pointed out that in hydrogel the aqueous environment is significantly restricted in comparison to purely micellar solution. In addition, rhodamine informed about less available micelle surfaces, caused by hyaluronan chains occupation. There were no interactions between the probes and hyaluronan chains. Freshly made hydrogels showed almost the same results as after a week of maturation under its supernatant.
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Influence of ionic strength on the properties of ionic amphiphilic pairs
Filipová, Lenka ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
The Bachelor thesis studies catanionic vesicle systems consisting of ion pair amphiphile (IPA) and their properties in the presence of ionic strength. Ion pair amphiphile was prepared from single-chained surfactants: cationic surfactant HTMAB (hexadecyltrimethylammonium bromide) and anionic surfactant SDS (sodium lauryl sulfate). Cationic double-chained surfactant DDAC (dimethyldioctadecylammonium chloride) was added in order to stabilize the vesicle system and make it positively charged. Cholesterol was also added to further stabilize the vesicle system. The system is considered to be relatively stable when consisting of 90 % IPA and 10 % DDAC with 43 mol.% cholesterol in a membrane. The stability of the system can be disrupted by external factors such as ionic strength. The Ionic strength was induced by NaCl solutions of varying concentrations (0.0, 0.5, 1.0, 2.0, 50.0, 100.0, 150.0 and 300.0 mM). The change of properties of a vesicle system, which was induced by ionic strength, was analyzed by electrophoretic and dynamic light scattering, fluorescence anisotropy, and generalized polarization. –potential was obtained by electrophoretic light scattering analysis which determines the stability of the system. Dynamic light scattering measurement resulted in the determination of the size of vesicles. The fluidity of vesicles’ membranes was examined by fluorescence anisotropy with DPH (1,6-diphenyl-1,3,5-hexatriene) as a probe. The hydration shell of vesicles was observed by generalized polarization with Laurdan (2-(dimethylamino)-6-dodecanoylnaphthalene) as a probe. As a result, it was found out that low ionic strength (0.5 to 2.0 mM) causes a decrease in the size of vesicles and high ionic strength (50.0 to 300.0 mM) causes vesicles to grow in size. –potential showed a consistent trend for the whole concentration series – its value rapidly decreasing with increasing ionic strength. The samples with the concentrations of salt 50.0 to 300.0 mM were considered unstable according to the –potential data. Fluorescence anisotropy decreases with increasing temperature and increasing ionic strength. The value of generalized polarization decreases with increasing temperature; hence there is more solvation of a vesicle membrane. The highest value of generalized polarization was measured when the concentration of sodium chloride was relatively high (from 50.0 to 300.0 mM), as a result of which the membrane was less hydrated and therefore, more organized.
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Fluorescence spectroscopy in research on the organization of biological membranes
Vítek, Ondřej ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This bachelor's thesis deals with the comparison and optimization of methods for studying biological membranes using fluorescence spectroscopy and microscopy, utilizing the bacteria Cupriavidus necator H16 (containing PHB granules) and PHB-4 (not containing PHB granules) as model organisms. The appropriate concentration of the membrane probe laurdan for staining biological membranes and the time required for its penetration into the cell were determined. Organisms stained in this way were further studied using steady-state spectroscopy, which helped examine the influence of temperature on the shape of the emission spectrum and the possibility of washing as one of the steps in sample preparation. Both of these measurements suggest the advantages of washing but do not provide definite results. However, a clear difference between the two bacterial strains was observed. Furthermore, the bacteria were studied by fluorescence microscopy. This method proved to be potentially useful for studying significant variations in the membrane order of the cell membrane and for imaging other larger structures. The distribution of laurdan within Cupriavidus necator H16 appears more uneven compared to PHB-4. The findings thus suggest the influence of granule presence when staining with fluorescent probes, but they cannot be interpreted definitively.
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Study of membrane properties of catanionic vesicles from new SEPT-DS amphiphilic ion pairs
Vajcíková, Katarína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This work describes the possibility of determining the membrane properties of vesicular systems formed by ion pair amphiphile (IPAs), which were prepared by mixing positively and negatively charged surfactants, namely the positively charged surfactant carbethopendecinium bromide (Septonex) and negatively charged surfactant sodium dodecyl sulphate with the addition of dioctadecyldimethylammonium chloride and cholesterol for stability enhancement. Such systems are potential drug carriers and hence their careful characterization is very important for further research. In this work, the possibilities of studying the membrane properties of such systems using spectrometric methods, namely fluorescence anisotropy and generalized polarization, by which the fluidity and solvation of the bilayer membrane were detected as a function of the amount of added cholesterol and the change in temperature, are described. The size and stability of the prepared vesicular systems formed from the new amphiphilic pairs were investigated by dynamic and electrophoretic light scattering, and finally, the phase transition temperature was detected using differential scanning calorimetry method.
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Study of microviscosity of membrane systems based on ionic amphiphilic pairs
Moslerová, Lenka ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
In this master ‘s thesis, catanionic vesicles formed by the pseudo-double-chain complex CTA – DS were investigated from the point of view of microviscosity. Samplesand of cationic vesicles contained 23, 43 and 53 mol. % of cholesterol and the double-chain surfactant DODAC. Cationic vesicles were prepared for visual observation, their stability was determined by DLS and the prepared system was further investigated. Microviscosity was determined from fluorescence anisotropy. To study the outer part of the membrane, laurdan fluorescent probes were used whereas diphenylhexatriene was used for the inner part of the membrane. This method has been proven to be suitable because it reflects the conditions of the membrane. Moreover, a 1,3-bispyrenylpropane probe forming intramolecular excimers was used to study the microviscosity in the vesicle bilayer. The dicyanovinyljulolidine (DCVJ) probe was applied in the case of the molecular rotor technique. It has been shown that in the case of the DCVJ probe, the molecular rotor technique is practically unusable, due to the fact that the probe has a low quantum yield at low temperatures. Also, the excimer formation of P3P probes does not lead to the expected results. The cationic vesicles do not seem to support this formation, as they are too closely related. This type of probe can be used for the selected system with some restrictions.
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|
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Study of membrane properties of liposomal systems using fluorescence spectroscopy
Zbořilová, Hana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
The presented diploma thesis is focused on the preparation, characterization and study of membrane properties of liposomal systems which were composed of the neutral phosphatidylcholine (DPPC), cholesterol, negatively charged phosphatidylglycerol (DPPG), polyethylenglycol bounded to phosphatidylethanolamine (PEG5000–PE) and polycation N,N,N-trimethylchitosan (TMC). The influence of individual components and their concentrations on the average particle size, zeta potential and changes in the outer and inner part of the bilayer was investigated. In this matter, methods of dynamic and electrophoretic light scattering and fluorescence spectroscopy with the application of laurdan and DPH probes were used. Based on the above-mentioned parameters, concentrations of components that most suitably influence properties of liposomes in terms of the intended application were selected for the definite complex. It was managed to prepare a liposomal complex stealth liposome–N,N,N-trimethylchitosan, which, due to the optimized composition, could have suitable attributes as a drug delivery system for inhalation administration of biologically active substances.
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Study of the hydration shell of vesicular systems based on ionic amphiphilic pairs
Rašticová, Barbora ; Szabová, Jana (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of hydration shell of positively charged vesicular systems. The aim of the work was to monitor the hydration shell and to find out the amount of water molecules in the environment at different conditions of the membrane. Furthermore, a comparison of two selected fluorescent probes were studied. Positively charged vesicular systems composed of two types of surfactants were used, namely negatively charged SDS and positively charged CTAB. The two-chain positively charged surfactant was also added, resulting a positively charged vesicular systems. Three different concentrations of cholesterol were selected for the study and added to the system, namely 20, 40 and 60 mol. %. The study was performed by measuring the fluorescent emission as a function of temperature. The temperature range was from 10 to 80 °C. As a fluorescent probe Laurdan and Prodan were selected. For evaluation the method of generalized polarization was used. Due to its structure, Laurdan is mainly found in the membrane environment, so the method of two-wavelength generalized polarization, which includes fluorescence contributions only from the membrane, was sufficient for its evaluation. Compared to Laurdan, Prodan has a shorter hydrocarbon chain, so its distribution is not only in the membrane but also in the aqueous environment. Therefore, the method of three-wavelength generalized polarization, which also includes the contribution of fluorescence from the aqueous environment was used. The results showed that the behaviour of both probes is very similar. In all systems, the values of generalized polarization decreased with increasing temperature. Thus, at low temperatures, the membrane is in a solid ordered phase. With increasing temperature, it transforms into a liquid disordered phase. Thus, with increasing temperature, the number of water molecules increases. As the cholesterol concentration increased, the phase transition interval increased. The results also showed that the values of Prodan’s three wavelength generalized polarization are always higher than the values of Laurdan’s two wavelength generalized polarization.
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