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Preparation and characterization of enzyme-containing wound dressings
Jurová, Bronislava ; Szotkowski, Martin (referee) ; Skoumalová, Petra (advisor)
This diploma thesis is focused on the study and characterization of nanofiber wound dressings from natural biopolymers. Proteolytic enzymes, specifically collagenase and bromelain, were incorporated into these nanofiber covers. The theoretical part deals with a brief description of skin wounds, their healing and skin covers. There are also methods for preparing these covers and the methods used to characterize them. The practical part deals with the optimization of the preparation of nanofiber coatings based on polyhydroxybutyrate, gelatin, alginate and chitosan. These nanofibers were then enriched with active substances and their gradual release into the model environment was monitored. Finally, their proteolytic activity was determined for these substances.
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Incorporation of microbial cells in hydrogel carriers
Orišková, Sofia ; Pekař, Miloslav (referee) ; Sedláček, Petr (advisor)
The presented diploma thesis focuses on the use of plant growth promoting bacteria as an ecological alternative to conventional fertilizers. The incorporation of bacterial cells into hydrogel carriers is already a well-studied topic, but due to its disadvantages it has not yet found wider application in agriculture. This work offers a novel concept of encapsulating bacteria by gelation directly from the culture. This is achieved by crosslinking the bacterial alginate produced by the model microorganism Azotobacter vinelandii. Since this process was not described before, first its optimization was needed. Alginate production was determined gravimetrically, and its parameters were further characterized using available analytical methods – infrared spectroscopy to monitor structural parameters (monomer composition and the extent of acetylation), dynamic light scattering to characterize the size distribution and AF4-MALS-dRI to obtain the molecular weight. Bacterial PHB production was also investigated using gas chromatography and infrared spectroscopy. The second part of the work is focused on the optimization of the gelling process using bacterial alginate from the culture and CaCl2 as a crosslinking agent. Rheological experiments were used as a tool in understanding the viscoelastic properties of the prepared gels. Gelation was demonstrated within the first day after inoculation. Maximum production of alginate (1,9 ± 0,3) g/l was reached on the fourth day after inoculation. It was found that the addition of 5 g/l of calcium carbonate promotes the production of alginate. Nevertheless, further addition of CaCO3 (30 g/l) showed adverse effects on the molecular weight and is therefore not recommended. Production of PHB was confirmed by both FTIR and GC measurements, with a maximum yield of (23 ± 3) % CDW. Rheological testing confirmed that the product of the crosslinking was a gel. It was found that the crosslinker concentration plays an important role at time 0 min of the gelation, forming a denser network in the structure and causing higher rigidity. Using the highest studied concentration of CaCl2, the critical strain reached values of (5,0 ± 0,7) %. Finally, the incorporation of bacterial cells into the hydrogel was confirmed using fluorescence microscope.
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Phospholipids as the basis of biodegradable delivery systems
Burdíková, Jana ; Čeppan, Michal (referee) ; Mravec, Filip (advisor)
This thesis is focused on investigation of phospholipid-hyaluronan system. First, appropriate method for preparation of bulk solution of phospholipid/lipid and suitable fluorescence probe were chosen. Sonification was selected as a method for preparation of bulk solution and pyrene was chosen as a fluorescence probe. From the group of phospholipids lecithin was selected. Next to phospholipid, lipid with no phosphate group (DPTAP) was utilized for comparison, alternatively a mixture of lipid (DPTAP) and phospholipid (DPPC). Instead of hyaluronan another polyelectrolytes (sodium polystyrene sulfonate, sodium alginate) were used too. Measurements were performed in water environment and in phosphate buffer saline (PBS). All investigation was accomplished by fluorescence spectroscopy and dynamic light scattering.
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New possibilities in wound healing
Nováková, Laura ; Szotkowski, Martin (referee) ; Skoumalová, Petra (advisor)
The diploma thesis is focused on the study of fibrous wound dressings prepared by electrospinning method from natural biopolymers. Three active ingredients were added to the dressings: ampicillin, ibuprofen and collagenase, which are responsible for relieving pain, reducing the risk of infection and selectively removing necrotic tissue in the wound. The theoretical part describes the therapeutic dressings currently available on the market and the most common methods of nanofiber production. The experimental part evaluates the optimization of the preparation of gelatin, alginate and chitosan fibrous wound dressings, which were subsequently enriched with active substances and their gradual release into the model environment was determined spectrophotometrically. Antimicrobial effects against E.coli and S. epidermidis strains andantifungal activity against C. glabrata yeast were monitored. Finally, two cytotoxicity tests on the human keratinocyte cell line HaCaT confirmed the safety of the prepared products, which can serve as bioactive skin dressings in the future.
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The Utilization of Light Scattering Techniques for the Study on Hydrogel Gelation
Candráková, Simona ; Smilek, Jiří (referee) ; Kalina, Michal (advisor)
This bachelor thesis focuses on the study of hydrogel gelation using light scattering methods. For these purposes two different biopolymers (agarose and sodium alginate), with different sol to gelation phase transition, were selected. In the case of agarose, the gelation is caused by temperature change. On the other side, the gelation of sodium alginate is initiated by addition of polyvalent cations. In the experimental part of the work, agarose gelation was studied by DLS, temperature gradients of agarose solutions (40 – 30 °C) were measured. During the measurement the particle size distributions in the solutions having different concentrations were monitored as well as the temperature influence on the diffusion coefficient. The DLS method provided the values of the gelling temperatures of the solutions at a concentration of 0,5 wt.% and 1,0 wt.%. Furthermore, the DLS microrheology method was used, where temperature dependences of viscoelastic characteristics of agarose solutions (40 – 30 °C) were also measured, from which the gelling temperatures of agarose solutions of 0,1 wt.% and 0,5 wt.% were evaluated. The classical rheology was also used in the work to compare the viscoelastic behaviour of the samples and to determine the gelation point of the agarose solutions. By this method were determined gelling temperatures for all studied concentration of agarose (0,1 wt.%, 0,5 wt.% and 1,0 wt.%). The DLS method was also used to monitor the interactions of sodium alginate with the addition of Ca2+ ions, these interactions were then also evaluated and discussed in the experimental part.
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Mechanical and Transport Properties of Hybrid Hydrogel Systems
Klímová, Eliška ; Sedláček, Petr (referee) ; Kalina, Michal (advisor)
This master´s thesis deals with the study on mechanical and transport properties of hybrid hydrogel systems. Considering applications of hydrogels, especially in chemical industry, pharmacy, or eventually medical applications, for the study gellan and alginate-based hydrogels were selected. In order to compare individual characteristics physical and hybrid hydrogels were prepared. Gellan hydrogels were prepared in deionization aqua solution, calcium chloride dihydrate and tween 80 solution. Alginate hydrogels were prepared in calcium chloride dihydrate solution as well, and polyacrylamide with N,N´–methylenbisacrylamide. For the study of mechanical properties moisture analyser and rheology measurements were selected. Transport properties were studied using the diffusion experiments combined with UV-VIS spectroscopic detection. Concluding of this thesis is summarization of measured values, which provides comprehensive review of the problematics. It was discovered that the conveniently selected concentrations of structural components of hydrogel matrix and the additives can influence both the mechanical as well as the transport properties of studied hydrogels.
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Encapsulation of selected natural extract for food application.
Vyskočilová, Terezie ; Kočí, Radka (referee) ; Márová, Ivana (advisor)
This diploma thesis deals with encapsulation of natural extracts. In the theoretical part the methods of encapsulation, materials for particle preparation, as well as application of encapsulation techniques in food industry were described. In experimental part selected natural extracts of propolis, green barley and probiotics were characterized. There substances were encapsulated into alginate and chitosan. In the total of 25 types of prepared particles long-term stability in some model physiological conditions as well as in four different model foods was evaluated. Additionally, stability of selected particles in several real milk-based products was followed too. The stability of particles was determined spectroptohometrically. In natural extract a content of polyphenols, proteins, chlorophylls, as well as total antioxidant activity were analysed. To analysis of probiotics optical and fluorescence microscopy were used. In propolis and green barley antimicrobial activity was tested too. Moreover, in the sample of propolis also cytotoxic assay was applied. Agar-chitosan was chosen as the best shell material for propolis due to its optimal stability in model physiological conditions as well as model foods. Liposomes were evaluated as unstable and were not recommended for further application. As the suitable shell material for powdered green barley starch-alginate (rate 1:4) and agar-chitosan were proposed, while the second one showed better stability for released proteins. Agar-chitosan shell material was usable for fresh green barley too. For probiotics encapsulation alginate or alginate-starch were chosen because of their porosity and possibility of nutrients diffusion. In real foods the best results were reached with application of probiotic particles into milk. Coencapsulation of powdered barley and probiotics did not confirm inhibition of culture growth. Neither the antimicrobial effect of propolis and barley nor the cytotoxic effect of propolis were confirmed.
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Encapsulation of active substances into nanofibers and possibilities of their application
Procházková, Lucie ; Pernicová, Iva (referee) ; Skoumalová, Petra (advisor)
The master thesis was based on the optimization of the production of nanofiber covers and to gaine the product for subsequent functional use. The production of nanofiber covers was made by electrospinning and forcespinning from selected materials. Polyhydroxybutyrate, gelatin, chitosan and alginate were used as starting materials. After successful optimization, these materials were enriched with active ingredients ampicillin and ibuprofen for the functionalized use of covers for more effective wound healing. The theoretical part was focused on the issue of skin, healing processes, types of wounds and nanofibers, the characterization of selected starting materials for the formation of nanofibers was also mentioned. The practical part was based on the lengthy optimization of the preparation of fiber covers and later enriched with active ingredients. Furthermore, combined covers made of different materials with contents of both active ingredients were designed. This was followed by the characterization of all prepared covers from the point of view of stability in the short and long term. The gradual release of active ingredients was determined spectrophotometrically and by hifh performance liquid chromatography. It was also important to determine the antimicrobial activity of selected active substances. At the end of all testing, combined coatings containing both active ingredients were used for safety testing with human keratinocyte cells (HaCaT). Safety testing was based on determining the viability of human cells using the MTT test, to verify the LDH test. A scratch test was also performed, a wound healing test after the application of devised combined covers.
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Preparation and characterization of wound dressings
Dzurická, Lucia ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
The diploma thesis if focused on the study of bioactive hydrogél and nanofiber wound dressings composed of natural biopolymers, which were functionalized by active compounds in the form of analgesic, antibiotics and enzymes. Hydrogél wound dressings were constituted from alginate and chitosan and nanofibers were created from polyhydroxybutyrate. The following 7 active compounds were selected to be added to the wound dressings: ampicillin, streptomycin, ibuprofen, papain, bromelain, collagenase and trypsin. In the theoretical part the structure of the skin and types of wound injuries were described. This part also talks about types of wound dressing and their applications, as well as treatment of skin wounds using enzymes and compounds with analgesic and antimicrobial properties. In addition, this section describes safety assays, in particular cytotoxicity assays on human cells. At the beginning of the experimental part, the process of preparation of hydrogél wound dressing was optimised. Subsequently, the dressings were enriched with active compounds and the rate of gradual releasing of the substances into model environment was monitored. In the case of enzymes, their proteolytic activity was also tested after their incorporation to the wound dressings. Furthermore, the prepared bioactive wound dressings were analyzed for possible cytotoxic effect on human keratinocytes. Finally, the wound dressing with combined content of active substances was created and also characterized for the rate of substance release, proteolytic activity and cytotoxicity. Antimicrobial activity of this wound dressings, against two selected strains of microorganisms: Escherichia coli and Staphylococcus epidermidis, was also evaluated.
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Development and characterization of hydrogel carriers for modern agricultural applications
Mai, Thuy Ha ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
The bachelor thesis is focused on characterization of hydrogel carriers and their application in agriculture. Entrapment of active phase within the hydrogel matrix would allow release of its content in controlled rate. The theoretical part describes encapsulation techniques and suitable biocompatible materials involved in encapsulation. For the experimental part, the release of soil bacteria Cupriavidus necator H16 from alginate matrix was examined. The focus was on the release rate of bacteria via diffusion and later, on the release rate of bacteria encouraged by chemical or enzymatic reagents. Further modification of carrier was suggested by adding hydrophobic barrier composed of polyhydroxybutyrate (PHB). The modified alginate beads seemed to be more resistant toward enzymatic or chemical agents, thus the process of the release was to some extent suppressed. This fact might suggest PHB as a possible vessel for optimization of controlled release system of hydrogel carriers.
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