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Advanced microscopy in the research of colloidal systems
Valovič, Stela ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This bachelor thesis is focused on dual-colour fluorescence cross-correlation spectroscopy. As a suitable combinations of fluorescenct dyes were designated this combinations: perylene + nile red, perylene + DiD and DiO + DiD. The funtionality of this method was veried by tetraspeck. The experiment of FCCS was performed on micellar surfactants solitions which proved to be unsuitable for this method. Consequently, liposomes with DiD and DiO incorporated in phospholipid bilayer were measured. The cross-correlation in liposomes was succesfuly measured.
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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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Preparation of model membranes to study their interactions with biopolymers using fluorescence correlation spectroscopy
Adamcová, Zuzana ; Márová, Ivana (referee) ; Mravec, Filip (advisor)
This diploma thesis is focused on preparation and characterization of supported lipid bilayers as simplified models of cell membranes. The bilayers were prepared from source system of lecithin liposomes in phosphate buffer using the vesicle fusion method on a cover glass sufrace hydrophilized by plasma. Three fluorescent probes – Nile red, Oregon Green DHPE and DiO – were utilized to characterize diffusion within the bilayer using fluorescence correlation spectroscopy. For this purpose Z-scan FCS, which is a method developed specially for planar samples, was used. After the process of preparation and characterization of supported lipid bilayer was optimalized, interaction between this artificial membrane and solution of hyaluronic acid in phosphate buffer was studied. It was found out, that addition of this biopolymer causes slowing the diffusion of the fluorescent probe within the bilayer.
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The penetration features of the hydrofobized hyaluronic acid – based polymeric micelles.
Mischingerová, Monika ; Pekař, Miloslav (referee) ; Muthný, Tomáš (advisor)
The aim of this thesis was to investigate the penetration features of the hydrofobized hyaluronic acid – based polymeric micelles using Nile red as carried tracer. Furthermore, to implement basic characterization of polymeric micelles for potential cosmetic applications using Coenzyme Q10 (CoQ10) as carried substance. It was found that the size of the polymeric micelles with carried CoQ10 did not exceed 100 nm. Applied delivery systems based on hydrophobic hyaluronic acid were suitable for potential topical application. Delivery systems with Nile Red as carried tracer demonstrated excellent penetration features. We assume that delivery systems with CoQ10 will exhibit similar penetration features. An issue has appeared whether the carrier breaks or proceeds along with NR to the skin. Moreover, another experiments have been designed which could also verify the penetration features of these systems.
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Interactions and aggregation in hyaluronan-aminoacid-surfactant systems
Venerová, Tereza ; Velebný, Vladimír (referee) ; Pekař, Miloslav (advisor)
The effect of native hyaluronan addition on CMC of excluded surfactants (Tween 20, BETADET THC 2, SDS, CTAT a CTAB) in physiological solution (0,15 M NaCl) has been investigated by fluoresence spectroscopy with Pyrene and Nile red as probes. The greatest influence on CMC has been observed in systems with cationic surfactant. System of cetyltrimethylamonnium bromid has been closely studied. Solubilization experiments with a hydrophobic dye Sudan red has been realized and aggregation number of this system has been determinated via fluorescence quenching with cetylpyridinium chloride as quencher and Pyrene as fluorescence probe. Addition of hyaluronan (native or hydrophobicaly modified) reduces aggregation number of system.
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Transport of Biomedical Active Compounds Through Porous Membranes
Vašíčková, Kateřina ; Smilková, Marcela (referee) ; Smilek, Jiří (advisor)
This bachelor thesis deals with a study of transport of biomedical active compounds through porous membranes. The main goal is to optimize a penetration method of micellar solutions with a solubilized dye and subsequently with solubilized drug. Cetyltrimethylammonium bromide is chosen as a surfactant and a nile red dye is used as a diffusion and fluorescence probe. Selected active compound is a nonsteroidal anti-inflammatory drug ketoprofen with analgesic and antipyretic effects. The diffusion processes are realized using Franz diffusion cells with polycarbonate membrane with a 2 µm size of poruses. The samples are withdrawn in a chosen time intervals and afterwards evaluated by a fluorescence correlation spectroscopy. Using this method, it is possible to screen the diffusion in time even in nanomolar concentrations of a fluorescence probe chosen. This thesis provides an overview of nile red behaviour in the micellar solutions of surfactant during diffusion processes. The experiment can be extended using an active compound, for instance using a ketoprofen as it is mentioned above.
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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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Hyaluronan ionocomplexes for cosmetic and farmaceutical applications
Cimalová, Jana ; Obruča, Stanislav (referee) ; Pekař, Miloslav (advisor)
This bachelor thesis is focused on the study of a system of cationic surfactant – hyaluronan. Using fluorescent spectroscopy the critical micellar concentration of Carbethopendecinium bromide (Septonex) was determined in three environments – water, 0,15 M NaCl, and buffer PBS, with three fluorescent probes – pyrene, perylene, and nile red. It was found that the physiological environments (buffer and NaCl) reduce the value of CMC about one order. During the experiments with native hyaluronan, precipitate and gel formation were observed. The following part dealt with aggregate behaviour of the new biologically acceptable amphoteric surfactant tetradecylfosfocholine (TPC) in the environment of water and NaCl according to the increasing molar molecular weight of hyaluronan. As a fluorescent probe perylene was used. These measurements showed that the molar molecular weight or a change of ionic force has no effect on the value of CMC and therefore there are no interactions with hyaluronan. The last surfactant to study was 1,2-dihexadecanoyl-3-dimetylammonium-propan (DPTAP), which is water insoluble. DPTAP is soluble in chloroform, but after evaporation of chloroform and after addition of hyaluronan, it precipitated. The results so far, have confirmed that in terms of research of aggregation by fluorescence spectroscopy the new cationic surfactants TPC and DPTAP are not suitable for complexation with hyaluronan.
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Study of aggregation in a biopolymer-surfactant system at low surfactant concentration.
Stiborský, Filip ; Vojtová, Lucy (referee) ; Mravec, Filip (advisor)
In this bachelor thesis, the interaction between hyaluronate and surfactant using fluorescent spectroscopy and pyrene as fluorescent probe was studied. A cationic surfactant cetyltrimethylammonium bromide and a native hyaluronate with different molecular size was used. Before a detected critical micelle concentracion, there were no interactions observed in phosphate buffer. However, we could observe electrostatical interactions in the water systém measuring. At low concentration of surfactant (between critical aggregation concentration and critical micelle concentration), the production of gel phase appeared.
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Influence of the preparing of a biopolymer-surfactant system on its aggregation behavior.
Pilgrová, Tereza ; Sedláček, Petr (referee) ; Pekař, Miloslav (advisor)
To study the effect of the method of preparation of biopolymer-surfactant on the aggregation properties several experiments were designed. Closer investigation was made on native hyaluronan-cetyltrimethylammonium bromide (CTAB) system. The system has been studied in the phosphate buffer in aqueous and saline solutions. The effect of the order of substances addition in the samples, the influence of different methods of preparation of certain concentrations of the solutions or the effect of dissolving powdered biopolymer and surfactant on the contrary, the resulting system were investigated. Fluorescence intensity was measured for all prepared samples and its changes were investigated. Pyrene and Nile red were used as fluorescent probes. Some samples were also measured by tensiometer. Measurement of surface tension was based on the method of divorcing the ring from the surface. It was found that the order of the mixing of solutions has a significant effect on the aggregation properties of the system and its appearance. Further effects were detected on the prepared samples by dissolving powdered biopolymer surfactants and vice versa, and environmental influences of the polarity of the system. It was found that sodium chloride abolished the effects of interactions in the system. Effect of method of preparation of certain concentration of CTAB in phosphate buffer is reflected only slightly.
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