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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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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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Time-resolved fluorescence in investigation of colloid system
Černá, Ladislava ; Ouzzane, Imad (referee) ; Mravec, Filip (advisor)
This thesis is focused on determination of the basic spectral characteristics, such as fluorescence maximum and lifetime, of fluorescent probes prodan [6-propionyl-2-(dimethylamino)naphthalene] and perylene by steady-state and time-resolved fluorescence spectroscopy. Time-resolved measurements were realized through TCSPC method (time-correlated single photon counting). The characteristics of prodan were found in water, acetone, n-heptane and in mixtures of these, so that a polar scale of environments would arise. Both of the mentioned probes were further investigated in systems of aqueous solutions of hyaluronic acid (300 kDa) at the concentration of 10 mg•l-1 together with cationic surfactant CTAB (cetyltrimethylammonium bromide) at the concentration range from 0.01 to 0.20 mmol•l-1 (before CMC). The aim was to confirm the reach and the range of critical aggregation concentration of the surfactant (CAC), to uncover the basic spectral characteristics of the mentioned probes in this concentration range and to verify the stability of this system in the presence of NaCl at the concentration of 0.15 mol•l-1. Furthermore, by TCSPC method were obtained time-resolved emission spectra of prodan from the system of hyaluronic acid and CTAB at one concentration of CTAB in CAC interval both without salt and with salt at the concentration of 0.15 mol•l-1. The obtained time-resolved emission spectra correlated with the results of the CAC concentration range measurement. After addition of salt the system of CAC proved to be unstable.
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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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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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Advanced Fluorescence Techniques in Research on Micellar Systems and Their Interactions with Biopolymers
Holínková, Petra ; Burgert, Ladislav (referee) ; Táborský, Petr (referee) ; Pekař, Miloslav (advisor)
The dissertation thesis deals with study of advanced steady-state and time-resolved fluorescence techniques, which can be used for study of micellar systems properties. Selected fluorescence techniques were used for characterization of Septonex and CTAB cationic micellar systems and theirs interactions with hyaluronan. Fluorescent probe pyrene was used for determination of critical micelle concentration (CMC) and micellar aggregation number of these surfactants. The changes of fluorescence behaviour of fluorescein and prodan were studied in wide concentration range of Septonex. Next chapter of thesis deals with study of Förster resonance energy transfer between perylene and fluorescein in Septonex and CTAB micellar solutions and the effect of hyaluronan addition to these systems. Also steady-state and time-resolved fluorescence anisotropy studies were used for research of the effect of hyaluronan addition to micellar solutions. The last chapter of this thesis is focused on photophysical behaviour of Prodan in different solutions (water, Septonex solutions below CMC, hyaluronan solution, Septonex micellar solution and Septonex micellar solution with hyaluronan), which was discussed on the basis of time-resolved emission spectra.
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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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Time-resolved fluorescence in investigation of colloid system
Černá, Ladislava ; Ouzzane, Imad (referee) ; Mravec, Filip (advisor)
This thesis is focused on determination of the basic spectral characteristics, such as fluorescence maximum and lifetime, of fluorescent probes prodan [6-propionyl-2-(dimethylamino)naphthalene] and perylene by steady-state and time-resolved fluorescence spectroscopy. Time-resolved measurements were realized through TCSPC method (time-correlated single photon counting). The characteristics of prodan were found in water, acetone, n-heptane and in mixtures of these, so that a polar scale of environments would arise. Both of the mentioned probes were further investigated in systems of aqueous solutions of hyaluronic acid (300 kDa) at the concentration of 10 mg•l-1 together with cationic surfactant CTAB (cetyltrimethylammonium bromide) at the concentration range from 0.01 to 0.20 mmol•l-1 (before CMC). The aim was to confirm the reach and the range of critical aggregation concentration of the surfactant (CAC), to uncover the basic spectral characteristics of the mentioned probes in this concentration range and to verify the stability of this system in the presence of NaCl at the concentration of 0.15 mol•l-1. Furthermore, by TCSPC method were obtained time-resolved emission spectra of prodan from the system of hyaluronic acid and CTAB at one concentration of CTAB in CAC interval both without salt and with salt at the concentration of 0.15 mol•l-1. The obtained time-resolved emission spectra correlated with the results of the CAC concentration range measurement. After addition of salt the system of CAC proved to be unstable.
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