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Hyaluronan-CTAT hydrogels
Velcer, Tomáš ; Venerová, Tereza (referee) ; Pekař, Miloslav (advisor)
This thesis studies the properties and behaviour of phase-separated hydrogels prepared by interaction of hyaluronan with oppositely charged surfactants. Three representatives of surfactants, namely cetyltrimethylammonium bromide, chloride and p-toluensulfonate (CTAB, CTAC, CTAT), were selected for comparison. Using the method of rheology, the fact that the system of Hya-CTAT forms the most rigid hydrogels has been proved. Higher molecular weight of hyaluronan has also direct influence on the volume and stiffness of the newly formed hydrogels. Preparation methods were compared as well. Mixing the stock solutions of entry components appeard to be the most suitable. Small-angle X-ray scattering was used for determination of shape and size of surfactant's micelles, concluding that it has no effect on the volume of formed gels. The results of this study indicate that given gels are to a certain extent competitive and incorporation of hyaluronan into their structure is desirable with respect to its biological activity. This offers a potential usage of these substances in the field of medical applications.
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Characterization of coloid particles by excited-state proton transfer with advanced fluorescence techniques
Kotouček, Jan ; Mravec, Filip (referee) ; Pekař, Miloslav (advisor)
The deprotonation characteristics of fluorescent probes -naphthol and 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) were studied in this diploma thesis, using steady-state and time-resolved fluorescence spectroscopy. Two cationic surfactants, Septonex and cetyltrimethylammonium bromide (CTAB), were studied. These surfactants were measured in the complex with hyaluronan (1.75 MDa, 1 MDa and 300 kDa). Steady-state fluorescence was used for determination of critical aggregation concentration of each surfactant and pKa*. Time-resolved fluorescence decays were used to calculate the average lifetimes and the deprotonation constants of naphthol and HPTS. The measurement with hyaluronan were compared with the polystyrenesulfonate (PSS) – surfactant system. The effect of hydration shell of hyaluronan on hyaluronan – surfactant complex formation results from the comparison of above mentioned systems. Large differences were found in the deprotonation characteristic between surfactants and even between individual molecular weights of hyaluronan. The measurement shows that the hydration shell is located near to the dissociated carboxyl groups of hyaluronan chain, where the interaction with the positively charged surfactants occurs. Furthermore, the aggregation number of Septonex was determined by quenching of pyrene using cetylpyridinium chloride (CPC) as a quencher. The aggregation number for 20 mM Septonex solution was determined as a value of 104 molecules. CPC was used for confirmation of the localization of -naphthol in the micelles of CTAB and polymer – CTAB, respectively.
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Using diffusion techniques in the study of hydrogels
Holubová, Anna ; Žitňan, Michal (referee) ; Mravec, Filip (advisor)
Properties of colloidal systems, especially hydrogels, were studied using the method of fluorescence correlation spectroscopy (FCS). Hydrogels were prepared based on hyaluronan and cationic surfactant CTAB interactions. The proposed hydrophilic fluorescent probe, suitable for the study of phase-separated hydrogels in polymer-surfactant systems, was chosen ATTO 488 due to the suppression of triplet state. As a hydrophobic probe was chosen Nile Red. Individually were examined micellar systems of 10 mM CTAB and 0,5% sodium hyaluronate in water and 0,15 M NaCl in different concentrations of selected fluorescent probes. Subsequently the hydrogel systems were studied. The results showed that the measurement of hydrogels by this method is realizable. Short lifetimes were created by a complex probe and CTAB causing quenching and low levels of diffusion coefficients characterize the viscous environment of study system.
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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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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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Diffusion of organic molecules in the hydrogel environment
Holubová, Anna ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with study of hydrogels formed by phase separation of hyaluronan with oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and Septonex. It follows the bachelor thesis and extends the knowledge about the detailed characterisation of the inner environment of the hydrogel by determining the diffusion behaviour of the fluorescent probes Atto 488 and Nile Red using fluorescence correlation spectroscopy (FCS) technique and its modified version dual-focus fluorescence correlation spectroscopy (2f-FCS). Compared results showed that both methods show similar values and probes specifically interact with CTAB but Atto 488 shows only weak interaction with Septonex compared to Nile Red. Additionally, these interactions were not affected by the molecular weight of hyaluronan. In conclusion, it was recommended to measure this type of hydrogel in a small depth of gel using a conventional method.
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Time-resolved fluorescence of system polymer-surfactant
Mondek, Jakub ; Čeppan, Michal (referee) ; Mravec, Filip (advisor)
In this diploma thesis was studied time-resolved fluorescence in polymer-surfactant system. At first aggregation numbers of cationic (cetyltrimethylammonium bromide), anionic (sodium dodecylsulfate) and nonionic (Triton X-100) surfactants were studied by steady-state and time-resolved fluorescence spectroscopy. These two methods were compared. Aggregation numbers by steady-state method were always lower than aggregation numbers measured by time-resolved method. Steady-state method of determination aggregation numbers is useless for surfactants with high aggregation number and for aerated samples. Addition of hyaluronan to surfactant system was studied. There was observed change in aggregation number after addition of hyaluronan and change in percentage of dynamic quenching after addition of hyaluronan. Hyaluronan affected aggregation number of cetyltrimethylammonium bromide and Triton X-100. Hyaluronan increased percentage of dynamic quenching in cetyltrimethylammonium bromide and in Triton X-100. Pyren in sodium dodecylsulfate was quenched by sphere of action with negligible percentage of dynamic quenching and addition of hyaluronan had no effect on quenching. As next goal of this thesis, the determination of the position of fluorescence probe pyrene in cetyltrimethylammonium bromide, sodium dodecylsulfate and Triton X-100 micelles was chosen. Position of pyrene changed with charge and structure of micelles. Next was studied how percentage of dynamic quenching by iodide compounds changes with different charge of micelle. In all cases majority of dynamic quenching was calculated.
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A novel way of preparing hydrogels
Kratochvílová, Olga ; Smilek, Jiří (referee) ; Mravec, Filip (advisor)
This work is focused on testing the effect of ionic strength on the preparation of hydrogels prepared by phase separation hylauronanu polyelectrolyte solution (Hya) with cationic surfactant cetyltrimethylammonium bromide (CTAB) called „dry way“. In this case, there is a mixing of the polymer and the surfactant in the dry form and then added to the dispersion medium. With observation and rheology, the effects of the proportion of the components for the formation of the hydrogel. It was found that the higher the ionic strength causes the hydrogel has a lower viscosity and a more fluid. Tthis fact was confirmed from the results of rheological measurements. The gel with the ionic strength of 1 M is less viscous to gel the ionic strength of 0,5 M. Next, the purity of the hydrogel was observed, and it was found that hydrogels with the ionic strength of 0,15 M are clear without the use of centrifugation. In case of getting gel of the specific ionic forces of 0,15 M and 0,3 M formed by addition of multivalent cations/anions from observations we can say that the ionic strength in the case of the method of preparation of gels does not matter.
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Fluorescence quenching in study of aggregation behavior of colloids
Srholcová, Barbora ; Hrdina,, Radim (referee) ; Pekař, Miloslav (advisor)
This work focuses on examination of hyaluronan-sulfactant aggregates in term of determination of aggregate number. The value of critical micellar concentration (CMC) cetyltrimethylammonium bromide (CTAB) in three different solvents (water, phosphate buffer and physiological solution). Next the effect of the native hyaluronan supplement on the value of CMC was examined. It wasfound out that the solvent has the biggest effect on the value of CMC whilst the hyaluronan supplement affects CMC only a little. The aggregate number (Nagg) CTAB and the effect of the native hyaluronan supplement were determined out by means of fluorescence quenching. Pyrene was used as a fluorescence probe. Iodine and cetylpyridinium chloride (CPC) were used as quenchers. Sulfactant was dissolved in three different solvents (water, phosphate buffer and physiological solution). Not only the hyaluronan supplement but also the used solvent has the strong effect on the value of the aggregate number. When using 10mM CTAB dissolved in physiological solution the value of Nagg was 119 ± 4 while the value was half in buffer. Then we found out that in most cases the hyaluronan supplement reduces the value of the aggregate number.
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Diffusion of organic molecules in the hydrogel environment
Holubová, Anna ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with study of hydrogels formed by phase separation of hyaluronan with oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and Septonex. It follows the bachelor thesis and extends the knowledge about the detailed characterisation of the inner environment of the hydrogel by determining the diffusion behaviour of the fluorescent probes Atto 488 and Nile Red using fluorescence correlation spectroscopy (FCS) technique and its modified version dual-focus fluorescence correlation spectroscopy (2f-FCS). Compared results showed that both methods show similar values and probes specifically interact with CTAB but Atto 488 shows only weak interaction with Septonex compared to Nile Red. Additionally, these interactions were not affected by the molecular weight of hyaluronan. In conclusion, it was recommended to measure this type of hydrogel in a small depth of gel using a conventional method.
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