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Preparation of nanoparticles and nanofibers with antimicrobial components
Kubišová, Veronika ; Slaninová, Eva (referee) ; Skoumalová, Petra (advisor)
This thesis addresses the problem of inadequate current wound therapy and presents a solution in the form of nanomaterial-based wound dressings (coverings). Specifically, it focuses on the development and characterization of various nanofibrous materials with integrated liposome particles that could serve as a source of therapeutic agents and be useful specifically in the field of a wound therapy. The review in the introductory part of the thesis first focused on the mentioned problem, which describes the shortcomings of existing conventional dressing materials. A description of human skin from an anatomical and functional point of view was not omitted, nor was the wound healing process itself. Different types of covering materials were also presented. However, a large part was focused on nanomaterials and their use in the field of the skin wound therapy. The nanomaterials mentioned were mainly liposome particles and nanofibres, as the experimental part of the work was focused on these structures. The description dealt with the characterization of these structures as well as the biopolymers used for their preparation. However, the aim was to prepare nanofibers with liposome content, so the method of forming such systems was described. Various therapeutic agents are also an integral part of the covering materials, especially those that suppress the development of infection and reduce wound pain; therefore, the search focused on the antibiotic ampicillin and the analgesic ibuprofen. The experimental part was devoted to the actual production of nanofibers with liposomes and also to the qualitative demonstration of the presence of liposomes in electrostatically prepared nanofibers. The selected polymeric components of these systems were polyhydroxybutyrate (PHB) and gelatine. However, the nanofibers and liposome particles (as well as combined liposomes with PHB) were first prepared in their own form and characterized mainly in terms of the gradual release of the drug substances. The results obtained were then compared with those of therapeutic drug delivery via combined nanofibrous structures with liposomes. For these combined structures, the aim was to achieve synergy in drug delivery between these systems. The aim of this work was to create a biomaterial covering with the controlled drug release. The drugs contained in these materials were the aforementioned ampicillin and ibuprofen. An important part of the work was then to determine the safety of the prepared materials which were tested for cytotoxicity, where the tests performed were MTT test and LDH test. And the actual wound healing ability of the nanofibers was then monitored in a scratch test or a "wound healing" test. At the end of the paper, recommendations for future work on this topic are given.
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Preparation and application of nanoparticles and nanofibres with natural UV filters
Plachá, Monika ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The presented diploma thesis is focused on preparation of nanoparticles and nanofibres with natural UV filters. Liposomes with encapsulated aqueous, ethanol and lipid extracts were prepared. Nanofibers from PHB containing lipid extract were prepared too. As a part of this work, an overview of natural sources with potential effects as UV filters were introduced. Moreover, nanoparticles and nanofibers and methods of their characterization were described. Size, polydisperse index and colloid stability of prepared nanoparticles were characterized via DLS. In experimental part aqueous, ethanol and lipid extracts were prepared from roasted coffee, green coffee and cascara. These extracts were spectrophotometrically characterized for the content of polyphenols, flavonoids, antioxidant activity, tannins and their SPF. Liposomes and liposomes containing PHB with these extracts were prepared and the encapsulation effectivity, short–term and long–term stability as well as SPF of nanoparticles were determined. Nanofibers from PHB containing lipid extracts were prepared via electrospinning and forcespinning methods. Prepared nanofibers were examined via FTIR–ATR. Antioxidant activity, short–term and long–term stability were determined spectrophotometrically. From selected nanoparticles, emulsions and gels were prepared and their SPF was also determined. Three types of emulsions with the best SPF were selected and tested on volunteers. Sedimentation stability of emulsions was tested by analytical centrifuge. Finally, cytotoxicity of selected nanoparticles and nanofibers was tested via MTT assay using human keratinocytes.
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Use of plasma jet for wound healing
Dvořáková, Eva ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
This diploma thesis was focused on the possibility of using a plasma nozzle to accelerate the wound healing process. The benefits of using low-temperature plasma in medicine or biomedical applications are known from many studies, and low-temperature plasma is already used to sterilize medical devices, materials or surgical instruments. Some studies also report a high potential of usinh plasma nozzle in the treatment of skin wounds. In the experimental part of this work, an in vitro wound healing test was performed using two different low-temperature plasma sources. Source No. 1 was a surface wave microwave discharge and source No. 2 was a torch microwave discharge. An in vitro scratch healing test was performed on a monolayer of HaCaT keratinocytes and testing was performed using various parameters. The influence of the plasma treatment time was monitored, as well as the influence of the plasma discharge power and also the influence of the argon working gas flow. Especially when using a torch microwave discharge, faster wound healing was recorded at most of the parameters used compared to the control. Thus, it can be said that this source appears to be potentially suitable for faster wound healing. Furthermore, in the work using the MTT cytotoxicity test, the viability of skin cells after their plasmination was also monitored using the same conditions as in the in vitro wound healing test. When performed in the standard MTT assay, none of the settings or sources used showed any cytotoxic effects on keratinocytes. LDH cytotoxicity tests were also performed concurrently to verify the accuracy of the MTT assays. The results of both tests agreed and the use of low-temperature plasma in skin treatment can be considered as safe. Overall, the results show that the plasma nozzle can find use in medicine in the healing of skin wounds and chronic defects as a potentially fast, inexpensive and effective method.
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Influence of some super-foods and their active components on human cells
Maslonková, Ivana ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
The presented diploma thesis is focused on the study of composition and biological effects of some super-foods. Theoretical part deals with basic information about chosen superfoods and their bioactive substances. Further, theoretical part describes the overview of vesicular systems used for encapsulation and the most common methods of particle characterization. A brief review of cell cultures and cultivation of human cells is presented as well as methods for cytotoxicity a genotoxicity testing. In the experimental section, aqueous and ethanol extracts of super-foods were prepared. These extracts were then encapsulated into liposomal and combined PHB particles. Super-food extracts were characterized by spectrophotometrical methods in order to determine the content of polyphenols, flavonoids, anthocyanins, carotenes, chlorophyll, tannins, and antioxidant activity. The physico-chemical characteristics of prepared liposomal and combined particles were determined too. The particles with encapsulated extracts were further tested using the MTT assay and SOS chromotest to describe their potential cytotoxic and genotoxic effects.
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Use of cell cultures to testing of natural substances of plant origin
Ručková, Michaela ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
The presented bachelor thesis is focused on preparing aqueous and oil extracts of plant origin, their characterization and testing of their cytotoxicity and genotoxicity. The theoretical part contains basic information about plants and kinds of spices or tea that were used. Description of used methods of liposome preparation and encapsulation of active substances, measurement of antioxidant activity, particle characterization, cultivation of cell cultures and the principle of cytotoxicity and genotoxicity tests used in this thesis is included. Phenolics and flavonoids content was determined using the spectrophotometry. Antioxidant activity was evaluated spectrophotometrically as well. By ultrasonic homogenization method, liposomes were prepared and active agents were encapsulated into these vesicles. Encapsulation efficiency and stability of active substances were measured by spectrophotometry. By DLS method, size of particles was determined. Particle stability was derived from the zeta potential value. Important part of the bachelor thesis was cultivation of human keratinocytes. The cytotoxicity of extracts and liposomes was assessed by MTT assay on human cell cultures. The genotoxicity of extracts and liposomes was defined using SOS Chromotest on model organism, genetically modified E. coli. In conclusion, the potential use of prepared liposomes in the cosmetic or pharmaceutical industry has been proposed.
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Preparation of nanoparticles and nanofibers with antimicrobial components
Sosková, Simona ; Veselá, Mária (referee) ; Skoumalová, Petra (advisor)
The presented diploma thesis is focused on the preparation of new materials with antimicrobial effect. Liposomes and nanofibers from polyhydroxybutyrate containing clotrimazole and natural extracts with good antifungal and antioxidant effects were prepared. The theoretical part contains examples and short description of using nanoparticles and nanofibers in cosmetics and medicine and the description of plants which have positive and potential antimycotic effects. Moreover, methods for particles and fibers characterisation were shortly described. In the experimental part, natural water and lipid extracts were prepared and spectrophotometrically characterised for the content of polyphenols, flavonoids and the antioxidant activity. Liposomes and liposomes containtng PHB were prepared from selected extracts and the encapsulation effectivity, shortterm and longterm stability via determination of polyphenols were determined. Prepared particles were characterized with DLS method (size) and zeta- potential (stability). PHB nanofibers functionalised with selected lipid extracts and clotrimazole were prepared via electrospinning and forcespinning, and examined via FLIM and FTIR-ATR methods and spectrophotometry was used for antioxidant activity and release of active substances determination. Antifungal properties of prepared particles, extracts and fibers using the test system Candida glabrata were studied. Finally, cytotoxicity of selected samples was tested with MTT assay using human keratinocytes.
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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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Use of cell cultures to test cytotoxicity of natural substances
Blažková, Veronika ; Skoumalová, Petra (referee) ; Márová, Ivana (advisor)
This thesis is aimed on study of cell cultures, primarily on introduction of techniques for cultivation of human cells and optimization of their usage in cytotoxicity tests. The thesis is aimed preferably on natural substances and liposomes that are suitable to be used in cosmetics. Theoretical part contains literature review focused on overview of cell cultures, appropriate cultivation conditions for in vitro testing and summary of cytotoxicity tests. Experimental part contains characteristics of natural extracts with antimicrobial effect in terms of content of polyphenols, flavonoids and antioxidant activity. Further, these extracts were encapsulated into the liposomes. Encapsulation effectivity was determined. Distribution of particles size by dynamic light scattering and finally also stability of prepared particles were also determined. Long-term stability in water and gel environment was tested too. This thesis also contains study of optimization of cell culture of human skin keratinocytes cultivation and use of those cells for testing cytotoxicity of selected natural substances by MTT method.
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Study of interactions of biomaterials with human cell cultures.
Maslonková, Ivana ; Kostovová, Iveta (referee) ; Márová, Ivana (advisor)
The bachelor thesis was focused on introducing of techniques of cultivation of human cells in vitro and optimization of their uses in variety of cytotoxicity tests. Further, in this work were studied also interactions of cells with biomaterials and prepared organic-inorganic nanoparticles. In the theoretical part the basic conditions of cell cultivation, the characteristics of keratinocytes as cell cultures, basic characteristics of particles and also short search about the most used tests of cytotoxicity on various cells systems were described. The experimental part was focused on the optimizing the preparation of silver particles, where maltose was chosen as the most appropriate reduction agent. The average size of these prepared particles was 35 nm. The possibility of preparation of combined organic-inorganic particles including liposome particles, PHB particles, alginate particles and chitosan particles was also tested in the thesis. Later, the silver nanoparticles were encapsulated into these organic-inorganic particles as an inorganic component. The liposome particles prepared by ultrasound were evaluated as the most stable of all the other prepared particles. Human keratinocytes were chosen as the cell cultures that were needed for the observation of the particle interactions with human cells. In this work basic methods of cell cultivation and passage were introduced. Conditions of experimental work in cell culture laboratory were setting and possible application of cell cultures to cytotoxicity testing by MTT was tested.
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Encapsulation of active substances and possibilities of their application in anti-aging products
Horváthová, Iveta ; Němcová, Andrea (referee) ; Skoumalová, Petra (advisor)
The aims of this thesis were encapsulation of selected active substances and their possible application in cosmetic products with anti-aging effects. 6 kinds of active substances were studied – lipophilic vitamin E and vitamin A, hydrophilic vitamin B1 and vitamin B2, and phenolic compounds ferulic acid and chlorogenic acid. In theoretocal part, the general characteristics of active compounds and their use are described. It also includes the description of used methods. Practical part was focused on characterisation of active compounds from the perspective of antioxidant activity and encapsulation into liposomes from soy and sunflower lecithin. Encapsulation effectivity, size, stability and long-term stability of liposomes after four weeks were determined. The most suitable liposomes with active compounds were also tested for safety on human skin cells using the MTT test of cytotoxicity on keratinocytes HaCaT. Furthernore, 3 types of cosmetic products were prepared – day cream, night cream and skin serum. Each kind of cosmetic product was devided into 4 types – the product without any active compounds, the product containing free active compounds, the product with liposomes with active compouds and the product containing empty liposomes. As active compounds all our tested substances were used. Final products were tested 14 days on 15 female volunteers. Before and after products usage parametres of their skin were measured to analyze effect of products on skin rejuvenation. Volunteers were also rating sensory analysis of cosmetic products. The part of this thesis was also creating an online questionnaire about anti-aging cosmetics for publicity to find out the popularity of this kind of cosmetics.
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