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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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Aggregation Behaviour of Polysaccharides in Aqueous Solutions
Mravec, Filip ; Valko, Marián (referee) ; Hrdlovič, Pavol (referee) ; Valko, Marián (referee) ; Pekař, Miloslav (advisor)
Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.
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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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Bigels - Preparation and Characterization
Mušková, Alexandra ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with study of bigels, which are composed of hydrogel and oleogel. The aim of this work is to prepare and characterize bigels. Preparation of hydrogels was based on interaction between hyaluronan and cationic surfactant carbethopendecinium bromide. Oleogels were prepared by mixing a non-ionic surfactant (sorbitan monopalmitate) with sunflower oil. Individual bigels were prepared by mixing the various rations of hydrogel and oleogel, and were characterized using a fluorescence microscope and rheological measurements. Fluorescence observations were done on prepared samples using Nile Red, Perylene, HPTS, ATTO 655 and fluorescein. Rheological experiments show that pure oleogel is the strongest and most solid in comparison with bigels and hydrogels. The greater amount of oleogel in system is, the stronger bigel is.
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Fluorescence spectroscopy in the research of aminoclays
Jančík Procházková, Anna ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
Aminoclay belongs to a large group of organic modified materials based on clays. These materials could find many potential practical applications and, in particular, may be used as a matrix with the ability of the organisation and stabilisation of incorporated substances. The utilisation of aminoclay in the field of medical applications seems highly prospective, but there is the need to visualise the clay system and the processes within it. This essay deals with the issue of aminoclay visualisation by means of fluorescent spectroscopy. Through fluorescent spectroscopy, aminoclay was discovered to be autofluorescent. The autofluorescence of aminoclay is not usable in practical applications because of the low fluorescence intensity. This is the reason for the research into other possibilities for modifications of the internal structure of aminoclay by europium ions. These ions in chelated form are able to be fluorescent and modifications of the external structure are possible by electrostatic implementation of fluorescent dyes. Characterisation of the prepared complexes was provided by spectrofluorimetry and fluorescent microscopy for the purpose of evaluation of the practical application of aminoclay.
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Study of aggregation process and physical stability of polymeric micelle by fluorescence probe method
Chovancová, Romana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
Amphiphilic block copolymer poly(ethylene glycol)-b-poly(–caprolactone) (PEG–PCL) was synthesized from poly(ethylene glycol) (PEG) and poly(–caprolactone) (PCL) by using ring-opening polymerization. The structure and composition of this copolymer was determined by IR spectroscopy. Polymeric micelles were prepared by membrane dialysis method and direct dissolution of copolymer in water. The process of aggregation and physical stability in water solution was studied by fluorescence spectroscopy using pyrene and perylene as fluorescent probes.The results of steady-state and time resolved fluorescence measurements indicate that system of PEG–PCL forms both unimolecular and multimolecular micelles, which depends on copolymer concentration. The critical micelle concentration (CMC) was around 0,002 g/L. Measurement of micelles size by dynamic light scattering method suggested that systems with higher concentration form bigger aggregates. In addition, copolymer behavior was explored under physiological conditions. The results show that CMC of copolymer increased in 0,15 mol/L sal solution at temperature 37°C to 0,02 g/L when compared to copolymer in water.
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Microcalorimetric and Light Scattering Methods in the Study of Interactions in Biopolymer - Surfactant System
Šojdrová, Kamila ; Mravec, Filip (referee) ; Krouská, Jitka (advisor)
Biopolymer surfactant systems have been studied by using different physico chemical methods. As the biopolymer, it was chosen high (1400–1600 kDa), medium (250–450 kDa) and low (8–15 kDa) molecular weight sodium hyaluronate. Two cationic surfactants – cetyltrimethylammonium bromide (CTAB) and carbethopendecinium bromide (Septonex) were selected to provide polyelectrolyte complexes with oppositely charged hyaluronan. The critical aggregation concentration of the surfactant in the system was monitored by dynamic light scattering (Zetasizer Nano ZS). SEC-MALS method was used for the description of the conformation of sodium hyaluronate of different molecular weights and polyelectrolytes complexes hyaluronan surfactant after reaching the aggregation point. Isothermal titration calorimetry (ITC) was used to determine the critical aggregation concentration of the surfactant by monitoring the thermal changes accompanying the aggregation of the system. It was found out that the increasing molecular weight of hyaluronan in these systems decreases the critical aggregation concentrations and conversely.
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Stability of humic acids in production of eye drops
Lišková, Kateřina ; Mravec, Filip (referee) ; Bilerová, Helena (advisor)
Glykoaminoglykans are the biological active compounds, which significantly affect the physiological processes in living organisms from under low concentrations. Marked representative of this group is hyaluronic acid. It is linear polysacharid, whose primary structure is consists of D-glucuronic acid and N-acetyl-D-glucosamin linking by glycosidic bonds (13) and (14). It is occurring connective tissues, synovial fluids and vitreous humor too, in which forms system with unique rheological and optical properties. Except of human body is its concentration especially high in the cockscombs. Hyaluronic acid has very high ability to bonding molecules of water, increasing molecular weight a then viscosity of aqueous solutions too. This fact together with characteristic viscoelasticity and lubricating properties plays an important role in preserve of organism. Acid, but its salt too form basic component of compounds using in ophthalmic surgery. It is using in extraction of cataract, operation of glaucoma, implantation of crystalline lens and corneal grafting too. Aim of this bachelor work was a determination of stability of hyaluronic acid solutions along the production of eye drops. Main objective was using by rheological measurement detect the influence of addition polymers (PVP, PACA, PEOX, PEG, PUD, PEI) and different pH on stability, degradation aqueous solution of hyaluronic acid at sterilization in the steam autoclave respectively.
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Study of interaction between hyaluronan and phospholipid in a presence of biocompatible non-ionic surfactant
Burdíková, Jana ; Klučáková, Martina (referee) ; Mravec, Filip (advisor)
This work is based on investigation of aggregation behavior of sugar surfactants and sn-glycero-dipalmitoylphosphatidylcholin (DPPC) in aqueous environment and on study of the interactions in nonionic surfactant-DPPC and DPPC-nonionic surfactant-hyaluronan systems. Sugar surfactants were used from non-ionic surfactants. The behavior of each systems was studied by fluorescence spectroscopy. Values of critical micellar concentrations (CMC) of sugar surfactants, begining of aggregation of DPPC, DPPC aggregation effect of sugar surfactants and the effect of hyaluronan on the system non-ionic surfactant-DPPC has been investigated. Also CMC of the CPC (cetylpyrimidium chloride), which was used for fluorescence quenching in the determination of aggregate numbers of sugar surfactants, has been determined. Fluorescent probes pyrene, perylene, nile red, acridine orange and hydrophobic dye sudan red were used for measurements.
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Fixation media for fluorescence microscopy of liposomes
Kvasnicová, Klára ; Sedláček, Petr (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the synthesis of fixation medias suitable for fluorescence microscopy of liposomes. The theoretical part contains a literature search focused primarily on the characterization, classification and use of chemical fixatives in chemical practice. Experimental section also deals with the selection of appropriate fixative for preparation of microscopic preparations based on liposomes.
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