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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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