Silver Nanoparticles Surface-Modified with Carbosilane Dendrons as Carriers of Anticancer siRNA
Elżbieta Pędziwiatr‐WerbickaMichał GorzkiewiczKatarzyna HorodeckaViktar AbashkinBarbara Klajnert‐MaculewiczCornelia E. Peña‐GonzálezJavier Sánchez‐NievesRafael GómezF. Javier de la MataMaria Bryszewska
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Gene therapy is a promising approach in cancer treatment; however, current methods have a number of limitations mainly due to the difficulty in delivering therapeutic nucleic acids to their sites of action. The application of non-viral carriers based on nanomaterials aims at protecting genetic material from degradation and enabling its effective intracellular transport. We proposed the use of silver nanoparticles (AgNPs) surface-modified with carbosilane dendrons as carriers of anticancer siRNA (siBcl-xl). Using gel electrophoresis, zeta potential and hydrodynamic diameter measurements, as well as transmission electron microscopy, we characterized AgNP:siRNA complexes and demonstrated the stability of nucleic acid in complexes in the presence of RNase. Hemolytic properties of free silver nanoparticles and complexes, their effect on lymphocyte proliferation and cytotoxic activity on HeLa cells were also examined. Confocal microscopy proved the effective cellular uptake of complexes, indicating the possible use of this type of silver nanoparticles as carriers of genetic material in gene therapy.Keywords:
HeLa
Zeta potential
Ethidium bromide
Silver nanoparticle
Nanomaterials
Surface Modification
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Neutral petites were induced in three different [rho+] strains by treating with ethidium bromide. However, EB treatment of spontaneously generated suppressive petites, which were derived from the same [rho+] strains, produced no neutral petites and moreover had little or no effect on the suppressiveness of the petities. Genetic analysis showed that the ethidium bromide resistance of the suppressive petites was a manifestation of cytoplasmically inherited, respiratory deficiency.
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This thesis is focused on toxicity and mutagenity of ethidium bromide. Effects of ethidium bromide on life organisms are observed on the basis of its chemical, physical and biological properties. Orientation test of ethidium bromide toxicity was done by means of biological model of protozoan Paramecium caudatum. We evaluated the influence of ethidium bromide on cellular line P3HR1 using fluorescent preparation. We also observed the influence of ethidium bromide on DNA fragmentation of cell line HL60. Using electrophoresis of nucleic acids, we established the amount of ethidium bromide which eluted to electrophoretical buffer. The resistance of protective gloves, which are currently used in molecular biological laboratories, was tested against effect of ethidium bromide. Finally we evaluated the methods of decontamination. We also estimated amount of ethidium bromide which gets to effluent from molecular biological laboratories.
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The effect of ethidium bromide and actinomycin D on prelabelled mitochondrial RNA was studied in Xenopus laevis embryos. Mitochondrial rRNA was found to decay with a half-life of 2–2.5 h following an initial lag of < 2 h. There was an initial increase in incorporation into mitochondrial rRNA in the presence of actinomycin D but not in the presence of ethidium bromide. Radioactivity in mitochondrial 4 S RNA rises and then is relatively stable for the duration of the chase period. Cytoplasmic rRNA and 4 S RNA are stable under the chase conditions used, and, in the presence of ethidium bromide, cytoplasmic RNA continues to be synthesized.
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The effect of methanol on the frequency of cytoplasmic respiration-deficient (RD) mutation induced by ethidium bromide was investigated in Saccharomyces cerevisiae. When growing cells were treated with ethidium bromide at concentrations higher than 1.0μg/ml, 95 to 100% of surviving cells were RD mutants as judged by the tetrazolium overlay method. The RD mutants induced by 1.0μg/ml ethidium bromide were drastically decreased by addition of 6-8% methanol in both growing and non-growing conditions. Methanol reduced uptake of ethidium bromide by yeast cells, but it did not modify the inhibitory effect of ethidium bromide on in vitro yeast mitochondrial deoxyribonucleic acid synthesis.
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By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide. S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923EtBr were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing. Compared with its parental strain, ATCC 25923EtBr was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923EtBr could be inhibited by the same EPIs. qRT–PCR showed that norA was 35-fold over-expressed in ATCC 25923EtBr, whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923EtBr. Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.
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