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Modification of Biodegradable Polyurethanes by Biologically Active Substances
Kupka, Vojtěch ; Khunová, Viera (referee) ; Pekař, Miloslav (referee) ; Vojtová, Lucy (advisor)
Předkládaná dizertační práce se zabývá novým způsobem přípravy biodegradabilních polyuretanů (PU) a jejich modifikací biologicky aktivními celulózovými nanokrystaly. Literární rešerše se zaměřuje na bioresorbovatelné PU v tkáňovém inženýrství. Shrnuje příklady těchto PU elastomerů, skafoldů (nosičů buněk) i injektovatelných PU společně se způsoby biodegradace na netoxické produkty. Poslední část je zaměřena na nanocelulózu, která si získala pozornost díky svým pozoruhodným fyzikálním (velký specifický povrch, mechanické vlastnosti) a biologickým (biokompatibilita, biodegradabilita a nízká toxicita) vlastnostem jako materiál pro biomedicínu. V experimentální části byly charakterizovány amfifilní biodegradovatelné polyuretanové filmy (bio-PU) syntetizované bez použití rozpouštědla polyadiční reakcí z hydrofilního poly(ethylenglykolu) (PEG) a hydrofobního poly(e-kaprolaktonu) (PCL) jako makrodiolů společně s hexamethylen diizokyanátem. Připravené bio-PU filmy byly charakterizovány pro různé poměry jak mezi PEG/PCL, tak i mezi NCO/OH reagujícími skupinami (izokyanátový poměr). Bio-PU filmy projevily markantní nárůst mechanických vlastností při hmotnostním poměru PEG/PCL rovnému nebo menšímu než 20/80 díky vzniku krystalických domén PCL. Přítomnost PEGu zvyšovala schopnost bio-PU filmu absorbovat vodu i urychlila jeho hydrolytickou degradaci. Oproti tomu nižší absorpční schopnost a delší čas hydrolytické degradace materiálu způsobil vyšší izokyanátový poměr, a tedy i vyšší síťová hustota. Třetí část práce se zabývá přípravou polyuretanových nanokompozitů unikátní metodou bez použití rozpouštědla za využití bio-PU matrice a celulózových nanokrystalů buď nemodifikovaných, nebo povrchově roubovaných PEGem. Strukturní analýza prokázala, že přítomnost tyčinkovitých nanočástic způsobuje imobilizaci polymerních segmentů, v důsledku čehož se zvýšila tuhost a křehkost materiálu. Nastavením vhodného poměru mezi PEG/PCL, množstvím izokyanátu, či přídavkem modifikovaného nanoplniva může být bio-PU materiál "ušit na míru" s vhodnými mechanickými (houževnatost, tažnost) a fyzikálními (botnání, degradace) vlastnostmi. Díky přípravě bez použití rozpouštědla by mohly být připravené materiály využity v regenerativní medicíně např. jako cévní štěpy.
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Hyaluronane interactions with hydrophobic solutes
Slezáková, Dagmar ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The diploma thesis is based on the study of hydrophobic interactions of the native hyaluronan with selected solutes. On the basis of a literature search were chosen fluorescent probes and fluorescing biologically active substances, which are useful for investigation of colloids as 6-(p-toluidino)-2-naphthalenesulfonic acid (polarity probe), lipophilic vitamin (±)-alpha-tocopherol, pyrene (polarity probe) and finally hydrophilic vitamin riboflavin. In the experimental part of this thesis was studied the influence of solvents with different polarities, or more precisely dielectric constant, on the emission spectra, as well. There were investigated interactions of native hyaluronan with TNS and then interactions, which were influenced by the ionic strength. Such influenced interactions were not observed, that was probably due to the strong solvation´s wrapping of the hyaluronan. Interactions were observed after the process of lyophilisation followed-up by the rehydratation of the samples. For the next study of interactions the riboflavin was chosen and was investigated the REES effect in the native hyaluronan in different concentrations of its different molecular weights. In this case were not observed any shifts in the emission maximum with the excitation wavelenght shift and that is why the interactions of hyaluronan with riboflavin were not demonstrated in the field of chosen concentrations. By using another probe alpha-tocopherol was investigated the associative behaviour of hyaluronan and moreover was observed anisotropy of alpha-tocopherol in different concentrations of different molecular weights of native hyaluronan. The anisotropy reached high values in contrast to the reference solute that was the mixture of glycerol and ethanol. The anisotropy depended more on the molecular weight than on the concentration of hyaluronan. Interactions of hyaluronan were also studied by using the polarity probe pyrene in different concentrations of different molecular weights of the hyaluronan. The pyrene 1:3 ratio did not show the concentration dependence within the chosen concentrations except for the molecular weight 253.9 kg mol–1. Both probes alpha-tocopherol and pyrene were performed by the process of lyophilisation followed-up by the rehydratation, which improved interactions of these probes with hyaluronan.
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Utilization of thermal analysis in study on protein denaturation
Bošeľová, Miriam ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
This bachelor thesis deals with the use of techniques of thermal analysis in the study of thermal denaturation of proteins. As a model protein was chosen lysozyme. In the experimental part were used two methods of thermal analysis – differential scanning calorimetry (DSC) and temperature modulated differential scanning calorimetry (TMDSC). One of the objectives of the study was to determine the utility of DSC and TMDSC in the study of lysozyme denaturation and to futher characterize the results we can obtain from the individual methods. Another goal was the optimization of TMDSC. During optimalization of TMDSC heat-iso, heat-only and heat-cool modes were compared in 60 seconds and 100 second periods. By selected methods was observed denaturation of lysozyme. Denaturation of lysozyme is predominantly created by reversible component of the heat flow and the denaturation temperature is in the range of 71,16 °C to 75,21 °C depending on the set parameters.
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Effects of hyaluronan on properties of Opthalmo-Septonex eye drops
Chromá, Kateřina ; Němcová, Andrea (referee) ; Pekař, Miloslav (advisor)
Modification of eye drops using hyaluronan to increase retention time on the eye surface. Using modification of Mahrous method have been determined concentration of Septonex in water. Than using gradual addition of hyaluronan into Opthalmo-Septonex eye drops was investigated appropriate concentration for membrane dialysis. With membrane dialysis was observed interaction hyaluronan-septonex. Than was determined size and charge of particles.
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Bioproduction, Characterization and Application of Selected Biopolymers
Benešová, Pavla ; Pekař, Miloslav (referee) ; Čertík, Milan (referee) ; Márová, Ivana (advisor)
Proposed doctoral thesis deals with microbial production of biopolymers (polysaccharides and polyesters) using renewableble inexpensive resources as carbon and nitrogen source. At first, production of extracellular metabolites, mainly pullulan and poly-L-malic acid, by polymorphic microorganism Aureobasisium pullulans was studied. Further part of thesis was focused on production of bacterial biopolyesters polyhydroxyalkanoates (PHA). PHA are produced in the form of intracellular granules by various bacteria species. During the study of PHA production, sythesis of poly(3-hydroxybutyrate) and technologically significant copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was investigated as well with regard to waste substrate utilization in biorafinery concept. Spent coffee ground seemed to be applicable waste in biorefinery proces. One of the coffee wastes used for PHA production was spent coffee ground hydrolysate which was converted to PHA by Burkholderia cepacia. Moreover, copolymer of 3HB and 3HV was accumulated when SCGH was used as a sole carbon source, no precursor had to be added. Another coffee waste, which was utillized by Cupriavidus necator H16, is represented by oil extracted from spent coffee grounds. Hydrolysis proces of waste substrates had to be optimized, to reach the highest possible saccharide concentration. Due to the hydrolysis process several toxic compounds can be formed, hence, influence of hydrolysis procesings on polyphenols content in hydrolysates was monitored. Waste substrates were also tested as potentional complex nitrogen source for PHA production. Hydrolysates of cheese whey and chicken feather were used as inexpensive complex nitrogen source for PHA production by C. necator, when waste frying oil was used as a carbon substrate. The finnal part of the thesis is dealing with possibilities how the PHA can be processed into atractive aplication forms. Liposomes with partial content of PHB had been prepared, moreover, for improving of antioxidant activity and antimicrobial effect the waste coffee oil was added. Micro-fibrous PHB materials were prepared for the purposes of water filtration. Higher filtration effectivity was observed when composite PHB fibrous material containing active charcoal or metal oxides was used. Important part of thesis is represented by study of degradation processes of prepared PHA-based aplication forms. Various microorganisms were tested on PHA depolymerases production. Biodegradability of PHA materials was tested in of composting conditions by standard composting test IS/ISO 20200. Changes in mechanical properties of polyurethan, where polyol was partialy replaced by PHB, were tested depending on the exposure of composting condition.
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Native hyaluronan as delivery agent for hydrophobic molecules
Michalicová, Petra ; Márová, Ivana (referee) ; Pekař, Miloslav (advisor)
Hyaluronan is a chemical, which can be qualified as essential for vertebrates. It is a part of the extracellular matrix in most of tissues and also a major component of some other tissues. Besides of the mechanical functions this compound is important for many biological processes such as growth of tumor cells. The objective of this thesis was development of carrier systems containing native hyaluronan and hydrophobic drugs. For purposes of this work fluorescence probes (pyrene, prodan, perylene, DPH, mereocynine 540) instead of drugs were used. By using further mentioned sophisticated methods the properties of these systems were studied. The systems were prepared by freeze-drying. The effect of freeze-drying on support of interactions was observed by fluorescence spectrometry (steady-state and time-resolved). The stability of freeze-dried systems was determined by zeta potential, which was measured by electrophoretic light scattering. Cakes obtained by freeze-drying were analyzed by several methods. First one was effluence gas chromatography connected with FT-IR spectrometry. In this method the present of tertiary butyl alcohol in product was observed. The cakes were also analyzed by scanning electron microscopy, which can provide the information about the surface and elemental constitution of the material. The results of this work can shed light on the area of developing of drugs with targeted distribution of active compound.
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Adsorption of biologically active substances in colloidal solutions of aminoclay
Málková, Ivana ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
This paper builds on a previous study of magnesium aminoclay and its interactions with biological compounds. The theoretical part deals with the research of the usage of aminoclay as a carrier in medical applications, the method of its formulation, the selection of biologically active compounds and a suitable method of varification. The experimental part deals with the synthesis of aminoclay, samples preparation and study of the adsorption behavior of the selected bioactive compounds under different conditions. The experimental part of the paper also includes varification of the suitability of the selected UV VIS spektrometry to determine the adsorption behavior of the biologically active compounds in colloidal aminoclay solutions.
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Dynamic surface tension in study of hyaluronan-surfactant interactions
Herzog, Milan ; Žídek, Jan (referee) ; Pekař, Miloslav (advisor)
The main task was to characterize interactions between negatively charged biopolymer sodium hyaluronate and two cationic surfactants: cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium bromide (TTAB). Interactions between sodium hyaluronate and these surfactants are based on their different charge and can be detected by changes of surface tension. The influence of constant addition of sodium hyaluronate (MW = 90 and 1400 kDa) on interface tension values at increasing bubble lifetime was observed. For this purpose, the maximum bubble pressure method was choosen. The measurements were performed in water and in 0,15 mol•l-1 NaCl solution. The obtained results can be used as for description of dynamic properties of hyaluronate-surfactant systems as, e.g., in targeting drug delivery.
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Solubilizattion properties of polysaccharides
Lenartová, Radka ; Foglarová, Marcela (referee) ; Pekař, Miloslav (advisor)
In this diploma thesis were studied solubilization properties of polysaccharides by using hydrophobic solutes (Sudan Orange G, Sudan Red G, (±)-alpha-Tocopherol, Pyrene, Perylene, Nile red), which were represented by alkyl derivates of hyaluronan. At first, a behaviour of individual hydrophobic solutes was investigated in variously polar solvents (Methanol, 1 Propanol, Chloroforme, Cyklohexane, n Heptane) and in the environment of varying ionic strength (water, 0.1 M and 0.4 M NaCl). Afterwards, solubilization properties of Sodium Dodecyl Sulfate model solubilizated the hydrophobic solutes into a core of micelles was examinate. We were interested in the solubilization capacity as the mol of solubilized molecules per mol micelles of surfactant corresponding with a state of micelles saturation. In the case of the solubilization of (±)-alpha-Tocopherol into the core of micelles, it was not possible to determine the solubilization capacity. So we changed the determination of universally solubilization power. The solubilization power is defined as mol of molecules solubilized per mol surfactant relative to the quantity solubilizate at the micelles saturation. Model system of Sodium Dodecyl Sulfate as a simple surfactant carrying a negative charge as the alkyl derivates of hyaluronan was selected bacause of its characteristics.The surfactant forms unimolar micelles and its critical micelle concentrations and aggregation numbers are tabelated for the investigated microenvironment. The main aim of the study was investigating of hydrophobic domains of alkyl derivates of hyaluronan as free places for incorporation hydropbobic solutes in the microenvironment of varying ionic strength. The critical aggregation concentrations were determined by the Pyrene 1:3 ratio method. For the research of micropolarity of alkyl derivates hyaluronan’s domains were selected two concentrations of derivates for the next research of solubilization experiments - the first concentration near the critical aggregation concentration and the second concentration above it. The effect of concentration of Pyrene on a core polarity of derivates was investigated. We discovered the influence of the concentration and the other we found a stationary area of the concentration. In the end we investigated the influence of preparation of solutions of derivates of hyaluronan on the core polarity by the concentration of pyrene which corresponds to the stationary area. The study of solubilization properties of alkyl derivates of hyaluronan is not a simple case as we assumed. When we measured spectra of the absorbance, higher concentration of derivates of hyaluronan belittle absorbance of solubilizates. At the experiment of solubilization with Sudan Red G we found out that Sudan Red G is not able to solubilizate into the hydrophobic core of micelles of hyaluronan’s derivates because of lipophilic or steric effects. We had to change Perylene as a new solubilizate. From the measured emission spectra we found saturation micelles. We can express the solubilization power of hyaluronan’s derivates for the concentration of Perylene. The main aim of the diploma thesis was to determine optimal way of the preparation of hyaluronan’s derivates solutions with required degree of solubilization.
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Fluorescence study of hydrophilic domains of associating colloids
Londinová, Monika ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The properties of the hyaluronan were investigated by using different fluorescence probes, because hyaluronan is a hopeful carrier of an active matter in medicine and cosmetics. Selected fluorescence probes were: cationic acridine orange, Nile Blue A, methylene blue, amphiphilic 4-Di-2-ASP and anionic fluorescein. Except from fluorescence and absorption spectra of the probes were observed electrostatic and hydrophobic interactions as well. The probes in solvents with different polarity (MeOH, EtOH, DMSO) showed the bathochromic shift in the emission maximum and quenching of the fluorescence with the increasing polarity of the solvents. The influence of the ionic strength on fluorescence properties of the probe acridine orange and 4-Di-2-ASP was investigated in aqueous solutions of chlorides. The formation of acridine orange dimer is inhibited with increasing ionic strength. CaCl2 increased the ionic strength the most, then prevented repulsion of carboxylate groups, so it means the expansion of hyaluronan cluster into the solution. However, the emission of the probe 4-Di-2-ASP was quenched with the addition of CaCl2 the most. The first additions of COO– groups cause the formation of dimers of AO shown as decreasing in extinction coefficient and fluorescence intensity. Next addition of the hyaluronan caused a depolymerization of formed dimers and the increase of the emission intensity. The repolymerization caused the decrease and then again the increase. In case of 4-Di-2-ASP was the pattern of the fluorescence (the intensity and the position of the emission) firstly the same, but at the concentration of 1 g dm-3 the emission intensity increased. The probes MB and F were used for spectroscopic studies of the interaction between methylene blue-fluorescein complex and anionic and cationic surfactants. The absorbance of separate MB and F changed only with the addition of surfactants with the opposite electric charge. Absorbance of the mixture MB-F changed with the addition of the CTAC surfactant, while the addition of SDS into the mixture caused only the change of MB absorption spectra.
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