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Vliv proteinové korony na toxicitu částic
Jemelíková, Vendula
When foreign particles enter the organism, they come into contact with the physiological environment and subsequent adsorption of naturally occurring biomolecules on the surface of the particles. Protein corona is formed during the adsorption of proteins, which fundamentally changes the physico-chemical properties of the particles. The aim of this work was to focus on the change in properties and toxicity of particles caused by the formation of a protein corona around selected particles. The formation of protein corona around different particles based on hydroxyapatites (CaNPs), iron (FeNPs), platinum (PtNPs) and titanium (TiNPs), was characterized and proved using electrophoretic methods, and subsequently the change in size, zeta potential and cytotoxicity was monitored depending on the formation of a protein corona. Based on the results, it is possible to claim that in all cases particles with a protein corona were less toxic to breast tumor cells, than particles without a corona and CaNPs were the most cytotoxic. In general, it is possible to say that the formation of protein corona around the tested nanoparticles affects their properties and reduces cytotoxicity and oxidative stress.
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Vliv povrchových modifikací lipidových nanočástic na jejich vlastnosti
Mašková, Vendula
The aim of the thesis was to study the effect of surface modifications of liposomal nanoparticles on their physicochemical properties and to evaluate their uptake by cells in vitro. For this purpose, two types of liposomal nanoparticles (LNPs) that differed in their sensitivity to pH changes, i.e. pH-sensitive LNPs containing the lipid CPA (cholesteryl-PEG350-aminoxylipid) in their composition, were prepared for comparison, to which PEG (polyethylene glycol propionaldehyde) was attached by an oxime bond, and pH-insensitive LNPs containing DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol). The siRNAs were encapsulated into these nanoparticles and the storage stability, pH sensitivity, cleavage of pH-sensitive linkage, protein corona formation and their uptake by HepG2 cells were studied. The pH sensitivity of the linkages between PEG and CPA, or between PEG and DSPE, was verified using MALDI-TOF mass spectrometry at decreasing pH. Nanoparticle size, PDI and zeta potential were also monitored during the pH sensitivity study. According to the results, pH insensitive LNPs indeed appeared to be inert to pH change and pH sensitive LNPs showed changes in parameters at pH 6.0. Furthermore, the stability of both types of LNPs during long-term storage under different temperature conditions was assessed based on the change in size and PDI. The prepared LNPs appeared to be size uniform and stable throughout the experiment even at different temperatures. The exceptions were the fluorescently labelled LNPs, which exhibited poorer stability within the parameters studied. Finally, the effect of surface modifications on the reduction of protein corona formation was confirmed. The prepared LNPs also efficiently entered the cells, and within 24 hours there was a release of siRNA into the cytoplasm from pH-sensitive LNPs and a probable accumulation of siRNA in endosomes in the case of pH-insensitive LNPs. Based on the results of this work, it can be concluded that the prepared pH-sensitive LNPs exhibit desirable behavior in biological systems, and thus have potential as a suitable carrier for siRNA transport in biological applications.
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Study of Cellular Toxicity of Representative Nanoparticles in Tissue Cultures.
Filipová, Marcela ; Holada, Karel (advisor) ; Benson, Veronika (referee) ; Hubálek Kalbáčová, Marie (referee)
Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...
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