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Preparation and characterization of triple-IPA for the formation of vesicular systems
Novotná, Ludmila ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This thesis deals with the preparation and characterization of vesicular systems consisting of triple-IPA (ion pair amphiphile with three hydrophobic chains). For the preparation of tripleIPA were used double-chain cationic surfactants dimethyldimyristylammonium bromide (DMSAB), dimethyldipalmitylammonium bromide (DPAB), dimethyldioctadecylammonium bromide (DODAB) and dimethyldioctadecylammonium chloride (DODAC) and the singlechain anionic surfactant sodium dodecyl sulfate (SDS). Among the cationic surfactants, DMSAB and DPAB were selected to be studied more thoroughly. Triple-IPAs were prepared by mixing solutions of oppositely charged surfactants in equimolar ratio, and the resulting triple-IPA precipitate was filtered and dried. From the obtained powder, vesicular systems were prepared by rehydration and sonication. Cholesterol and cationic surfactants, from which the triple-IPAs were prepared, were used to stabilize the cationic vesicles. The properties of the vesicular systems were evaluated by dynamic and electrophoretic light scattering measurements and turbidimetry. The dispersion solutions with the addition of cholesterol were less turbid; therefore, it can be assumed that they supported the formation of cationic vesicles. The optimum cholesterol content is 20 mol. %. At this concentration, the effect of cholesterol on vesicle membrane reorganization becomes apparent and the cationic system remains monodisperse. A positive zeta potential in the stable region was determined for all prepared solutions, which was further increased by the addition of a positive surfactant. The most suitable cationic surfactant for the preparation of triple-IPA is DPAB, which is easy to work with, produces the most monodisperse systems and has the lowest measurement uncertainties.
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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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Stability of vesicular complexes against changes in their concentration
Velichová, Veronika ; Marková, Kateřina (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on the stability of vesicular systems that consist of ion pair amphiphile. These catanionic vesicles were prepared from single-chained surfactants CTAB cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate), which were stabilized by adding double-chained surfactant DODAC (dimethyldioctadecylammonium chloride) with 43 mol.% cholesterol. The aim of the thesis was to verify the stability of vesicular systems against changes in their concentration. For the measurement, a concentration series was prepared by diluting vesicles with deionized water. The stability was evaluated by dynamic (DLS) and electrophoretic (ELS) light scattering measurements at weekly intervals for four weeks. By DLS method were determined the average values of the size of vesicles and their changes during the measurement, the method ELS provided the information about changes of charge with different concentration in time. Samples with a higher concentration, i.e. no dilution till tenfold dilution of the vesicle solution, were marked as stable, higher values of average sizes and lower values of zeta potential were measured at solution with lower concentrations, according to which such diluted vesicles can be described as unstable. There were also visual observations made, but no visible aggregation in the solutions was observed. Furthermore, the aggregation behavior of the vesicles was observed by fluorescence spectroscopy using pyrene as fluorescent probe.
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Solubilization of water-insoluble vitamins into vesicular systems based on ionic amphiphilic pairs
Kolomá, Nikola ; Mravcová, Ludmila (referee) ; Mravec, Filip (advisor)
This thesis deals with the characterization and preparation of catanionic vesicular systems and their interaction with water-insoluble vitamins. Catanionic vesicles systems was prepared from two differently charged sufractants CTAB and SDS in formation ion pair amphiphile. Stability of vesicles was secured by adding cholesterol with positively charged surfactant DODAC. Were selected vitamin A and vitamin E for solubilization into these vesicles. The next part of this work was focused on determining the efficiency of incorporation these vitamins into the HTMA-DS vesicular system. The characterization of these systems was improved by measurement on a UV-VIS spectrometer, DLS and by HPLC with UV-VIS detector. During measurement by UV-VIS and HPLC the efficiency of solubilization of vitamin A and vitamin E into vesicles was determinated. In both cases higher efficiency was determinated for vesicles with incorporated vitamin A. Higher efficiency for both vitamins was determinated by HPLC. The reason is probably the higher sensitivity of the metod, work in labs with dark glass and preparing individuals samples into vials, which were inserted into the device for individual dosing by injector. The work serves primary evaluation results in view of the vesicular system for use in pharmaceutical application like carrier non-polar vitamins.
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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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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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Resonance energy transfer in the study of polymer-vesicle interaction
Jízdná, Zuzana ; Kratochvíl, Matouš (referee) ; Mravec, Filip (advisor)
The presented bachelor's thesis dealt with the subject of preparation of cationic vesicular systems, formed by the CTAB and SDS surfactants, in order to study their mutual interaction with the hyaluronan polymer using Förster resonance energy transfer measurements. First, concentration series of vesicular systems with fluorophores were prepared. DPH, perylene and fluorescein were used as fluorophores. Afterwards, the fluorescence lifetime of selected fluorophores was measured by the TCSPC method. Then, the characteristics of the prepared vesicular systems were measured using the DLS and ESL methods. For the final measurement of the interaction between the vesicle and the polymer, a vesiculation solution was chosen based on all the measurements carried out, which contained DPH with a concentration of 3·10-5 mol/l and hyaluronan. Two molecular weights of hyaluronan (300 and 900 kDa) were tested at mass concentrations of 1, 5, 10, 50 and 100 mg/l. The measurement confirmed their mutual interaction, thus this system could find application in the medical sector.
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Preparation and characterization of triple-IPA for the formation of vesicular systems
Novotná, Ludmila ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This thesis deals with the preparation and characterization of vesicular systems consisting of triple-IPA (ion pair amphiphile with three hydrophobic chains). For the preparation of tripleIPA were used double-chain cationic surfactants dimethyldimyristylammonium bromide (DMSAB), dimethyldipalmitylammonium bromide (DPAB), dimethyldioctadecylammonium bromide (DODAB) and dimethyldioctadecylammonium chloride (DODAC) and the singlechain anionic surfactant sodium dodecyl sulfate (SDS). Among the cationic surfactants, DMSAB and DPAB were selected to be studied more thoroughly. Triple-IPAs were prepared by mixing solutions of oppositely charged surfactants in equimolar ratio, and the resulting triple-IPA precipitate was filtered and dried. From the obtained powder, vesicular systems were prepared by rehydration and sonication. Cholesterol and cationic surfactants, from which the triple-IPAs were prepared, were used to stabilize the cationic vesicles. The properties of the vesicular systems were evaluated by dynamic and electrophoretic light scattering measurements and turbidimetry. The dispersion solutions with the addition of cholesterol were less turbid; therefore, it can be assumed that they supported the formation of cationic vesicles. The optimum cholesterol content is 20 mol. %. At this concentration, the effect of cholesterol on vesicle membrane reorganization becomes apparent and the cationic system remains monodisperse. A positive zeta potential in the stable region was determined for all prepared solutions, which was further increased by the addition of a positive surfactant. The most suitable cationic surfactant for the preparation of triple-IPA is DPAB, which is easy to work with, produces the most monodisperse systems and has the lowest measurement uncertainties.
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Effect of changing ionic strength on the properties of catanionic vesicles from HTMA-DS
Filipová, Lenka ; Klučáková, Martina (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on evaluating the influence of ionic strength on catanionic vesicules made of ion pair amphiphile (IPA) in the form of HTMA-DS (hexadecyltrimethylammonium-dodecyl sulphate). Catanionic vesicles were stabilized by the addition of cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and cholesterol. The ionic strength was attained by adding CaCl2 and Na2SO4 salts in the concentrations 0–300 mM. The changes influenced by ionic strength were studied by dynamic and electrophoretic light scattering (DLS and ELS), fluorescence anisotropy, generalized polarization (GP), pH measurement, and visual observation. During this experiment, the temperature was set both constant and altering in time. The measurement was performed shortly after adding salts to the catanionic vesicles and in-time measurement was performed as well. This thesis follows a bachelor thesis, where the influence of NaCl on the same type of catanionic vesicles in the same range of ionic strength was studied. The CaCl2 and Na2SO4 influences were then compared to the NaCl according to Hofmeister series. By the DLS and ELS method it was found that the size of vesicles with added CaCl2 and Na2SO4 was decreasing at first and then increasing whereas the -potential was exponentially decreasing with increasing ionic strength. The vesicle size, when the ionic strength was applied, was increasing in time while -potential was almost constant in time. The addition of both salts caused slight dehydration of the external part of the membrane which was examined by the fluorescent probe Laurdan (6-dodecanoyl-2-dimethylamino-naphthalene) and generalized polarization. By fluorescence anisotropy with the probe DPH (1,6-diphenyl-1,3,5-hexatriene), it was detected that both salts caused an increase of the fluidity of an inner part of a membrane. By both of the fluorescent techniques, it was confirmed that the increase in temperature caused a phase transition of the membrane from the solid ordered through the liquid ordered phase to the liquid disordered phase. The pH values also did not show any change with the addition of salts. When comparing the salts’ influence (CaCl2 and Na2SO4 versus NaCl) on catanionic vesicles it was found that in most cases NaCl influenced the observed properties the most. The addition of NaCl caused the largest increase in vesicle size, highest -potential values, initial dehydration of the external part of the membrane, and increased fluidity of the inner part of the membrane at I > 15 mM. These results are in agreement with the Hofmeister series. Based on the visual observation, the samples with ionic strength of more than 150 mM for CaCl2 and 75 mM for Na2SO4 were said to be unstable. This fact is in agreement with the result of ELS. The rest of the samples did not show any visual changes in time (28 days). Additionally, the influence of PBS buffer on catanionic vesicles at the same ionic strength values was studied. PBS buffer simulates the ionic environment of living organisms. Its' influence was characterized by the same methods under the same conditions as mentioned above. The effect of PBS on catanionic vesicles displayed almost the same behaviour in the observed properties. During this experiment, it was also found that the PBS influence on the studied properties was more significant than the influence of CaCl2, Na2SO4, and NaCl salts. Influence of PBS was most similar to the effect of NaCl on catanionic vesicles.
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Stability of vesicular complexes against changes in their concentration
Velichová, Veronika ; Marková, Kateřina (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on the stability of vesicular systems that consist of ion pair amphiphile. These catanionic vesicles were prepared from single-chained surfactants CTAB cetyltrimethylammonium bromide) and SDS (sodium dodecyl sulphate), which were stabilized by adding double-chained surfactant DODAC (dimethyldioctadecylammonium chloride) with 43 mol.% cholesterol. The aim of the thesis was to verify the stability of vesicular systems against changes in their concentration. For the measurement, a concentration series was prepared by diluting vesicles with deionized water. The stability was evaluated by dynamic (DLS) and electrophoretic (ELS) light scattering measurements at weekly intervals for four weeks. By DLS method were determined the average values of the size of vesicles and their changes during the measurement, the method ELS provided the information about changes of charge with different concentration in time. Samples with a higher concentration, i.e. no dilution till tenfold dilution of the vesicle solution, were marked as stable, higher values of average sizes and lower values of zeta potential were measured at solution with lower concentrations, according to which such diluted vesicles can be described as unstable. There were also visual observations made, but no visible aggregation in the solutions was observed. Furthermore, the aggregation behavior of the vesicles was observed by fluorescence spectroscopy using pyrene as fluorescent probe.
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