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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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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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Adhesion, growth and differentiation of skin cells on nanofibrous polymer membranes
Pajorová, Júlia ; Bačáková, Lucie (advisor) ; Eckhardt, Adam (referee)
Our study contributes to the tissue engineering, mainly to the construction of appropriate scaffolds for regeneration of damaged skin. Simultaneously, it brings valuable insights for basic research in the field of molecular mechanisms of adhesion, proliferation and phenotypic maturation of cells and the control of the cell behavior through the cell extracellular matrix (ECM), represented by synthetic nanofibrous material. Nanofibrous polylactic-co-glycolic acid (PLGA) membranes were prepared by needle-less electrospinning technology. These membranes were further modified with cell adhesion-mediating biomolecules, e.g. collagen, fibronectin and fibrin in order to increase their affinity to colonizing cells. Adhesion, growth and differentiation of keratinocytes (HaCaT) and fibroblasts, i.e. major cell types of epidermis and dermis, were evaluated on these nanofibrous membranes. The results show that the membrane modification using fibrin structures improved adhesion and proliferation of human dermal fibroblasts. The collagen structure on the surface of membranes improved the adhesion and proliferation of human HaCaT keratinocytes. Furthermore, fibrin structure stimulated fibroblasts to produce collagen, which is a major component of ECM in the natural skin dermis. Fibronectin enhanced cell attachment...
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Cell viability changes after interaction with TiO2 nanoparticules and anthracycline cytostatics
Kondělková, Regina ; Štenglová Netíková, Irena (advisor) ; Merta, Ladislav (referee)
The goal of this thesis is to conduct a literary research about cell viability changes after interaction with TiO2 nanoparticules and anthracycline cytostatics. Anthracycline cytotoxic agents are one of the most commonly used groups of antineoplastic drugs, particulary doxorubicin. A serious side effect of anthracyclines in para drug administration (extravasation) is necrosis of the surrounding tissue. Effective treatment for this side effect is not available as of yet. One possible way could be to use sorption and degradation characteristics of nanoparticles of TiO2, which are non-toxic to the human body. Anthracyclines are characterized by rapid adsorption to the surface of nanoparticles of TiO2 and subsequent degradation to non-toxic products. Therefore further I deal with the use of nanoparticles of TiO2, their unique chemical properties and the way they affect cell viability, especially keratinocyte cell lines in vitro. It has been shown that there is no reduction in cell viability when culturing keratinocytes together with TiO2 nanoparticles and thus it opens the door for further studies on the use of nanoparticles of TiO2 for the treatment of necrotizing anthracycline extravasation.
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Cell growth on biomaterials for skin replacements and wound dressings
Kudláčková, Radmila ; Bačáková, Lucie (advisor) ; Rösel, Daniel (referee) ; Eckhardt, Adam (referee)
Tissue engineering is an emerging interdisciplinary field developing new ways of treatment of patient's tissue defects using artificial substitutes. Skin tissue engineering is developing skin substitutes and wound dressings that would replace current treatment using autologous, allogeneic or xenogenic substitutes. There are high demands on materials which should serve as a scaffolds for dermal fibroblasts and keratinocytes. They must be non-cytotoxic and biodegradable with a rate proportional to formation of a new tissue. The materials should support adhesion and proliferation of the cells and even they could release growth factors and antimicrobial substance to enhance healing and new tissue formation. In this master thesis, the cell adhesion and proliferation were evaluated on sodium carboxymethyl cellulose (Hcel® NaT), poly-ε-caprolactone (PCL), poly-L-lactide-co-ε-caprolactone (PLA/PCL) and cellulose acetate (AC) nanofiber membranes. Primary human dermal fibroblasts and HaCaT cell line keratinocytes were selected for evaluation. The cell adhesion was observed by fluorescent microscopy, the proliferation was determined by metabolic assay (WST-1) and the material cytotoxicity was evaluated in xCELLigence® system. Materials did not show cytotoxic effects on the cells. However, the materials did...
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Molecular mechanisms of activation and modulation of TRPV3 receptor
Chvojka, Štěpán ; Vlachová, Viktorie (advisor) ; Novotný, Jiří (referee)
Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...
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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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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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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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