Short-term (5 min) and long-term (4 days) distribution of PS-COOH and PS_PEG NPs in the embryonic and adult mouse brain and in the placenta of 14 – 17 post-conception pregnant mice were investigated after a single intravenous injection (2.1µg particle mass /g bodyweight) of FITC-labeled PS NPs into the tail vein. After 5-minute and 24-hour exposures the animals were sacrificed, and 30 µm tissue sections were investigated with fluorescence microscopy and with confocal microscopy supplemented with spectrum analysis.
The histological studies (Figure 67 A,C) revealed that 5 minutes after the injection, PS-COOH particles decorated densely the walls of the brain vessels, while PS-PEG NPs were hardly revealed in the brain by the applied methods. Four days after the injection, however, all
particles were cleared from the brain. Similarly, PS-COOH NPs were initially retained by the placenta, while PS-PEG NPs were not seen. In a 4-day period, carboxylated particles were completely cleared from the placenta as well.
Figure 67. Confocal microscopic pictures and spectral analyses (right panels) of sections made from the adult mouse forebrain (A,B) and the placenta (C,D; 17th day postconception) after 5 min of injection of PS-COOH (A,C) and PS-PEG NPs (B,D). The fluorescence spectrum of nanoparticles are shown as green curves with a maximum light emission at 485 nm. The spectra of individual spots are shown with the same colouring as their outlines on the upper pictures. Picture and analysis by K.Kenesei (Kenesei et al., Nanomedicine submitted).
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Figure 68. Confocal microscopic pictures and spectral analyses (right panels) of sections made from the adult mouse forebrain (A,B) and the placenta (C,D; 17th day postconception) 4 days after the injection of PS-COOH (A,C) and PS-PEG NPs (B,D). The fluorescence spectrum of nanoparticles are shown as green curves with a maximum light emission at 485 nm. The spectra of individual spots are shown with the same colouring as their outlines on the upper pictures. Picture and analysis by K.Kenesei (Kenesei et al., Nanomedicine submitted).
In accordance with the data on proper protective functions on the placenta, PS NPs were not revealed in the embryonic brain.
Figure 69.Confocal microscopic pictures (A,C and enlarged part of A on the upper right panel) and fluorescence spectra of encircled regions (right bottom panel) of embryonic (E17) mouse brain cortex
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The data demonstrated that in vivo, the cells of the central neural tissue are protected against the invasion of PS NPs of the 50 nm size-range. Targeted studies on the in vivo penetration and neurobiological effects of LPS contaminated NPs, however, is considered for the near future.
5.
Cellular responses to silver NPs of different shapesSilver NPs (Ag NPs) are known to be toxic to microbial and tissue cells, mainly due to the release of Ag+ ions. AgNPs are widely used as anti-infectants because of the higher sensitivity of bacteria than mammalian cells. The aim of this study was to investigate the influence of particle-shape on the mammalian cellular toxicity;
therefore Ag NPs with size about 50 nm were synthesized with different – spheroid, cubic, triangle and rod – shapes (see Chapter 1.3.), and their toxicity was measured on NE-4C embryonic neuroectodermal stem cells.
NE-4C neural stem cells were exposed for 24 hours to increasing (1–100 µg/ml) concentrations of silver NPs with different shapes. Cell viability was measured with MTT reduction tests. The metabolic activity (MTT reduction capacity) was reduced below 20% of the control by Ag rods at 1 µg/ml, and by cubes at 50 µg/ml concentrations. Ag triangles showed mild (less than 50%) toxicity at 100 µg/ml concentration, while Ag spheres were not toxic when compared to untreated control.
(Figure 70).
Figure 70. Shape-dependent effects of Ag-NPs on metabolic activity of NE-4C neural stem cells.
The data-indicated a toxicity-rank for the different shapes:
rods > cubes > triangles > spheres
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To investigate whether Ag-NP toxicity was caused by the released Ag-ions, NPs were dispersed (100 µg/ml) in cell culture medium, and after 24-hour incubation the particles were removed from the suspensions by centrifugation (30 000g; 15 min).
The culture medium of NE-4C cells was replaced with the particle-free incubation solutions and cell viability (MTT reduction capacity) was assayed after 24 hour incubation with particle-free supernatants (Figure 71).
Figure 71. The effects of particle-free supernatants of NP-suspensions on viability (MTT reduction) of NE-4C cells. Spheres PVP: Ag spheres kept in polyvinylpyrrolidon (PVP) containing buffer prior to dispersion in culture medium
The data demonstrated important toxic effects of the particle-free supernatants of Ag cubes and triangles, while, almost no toxic effects of the supernatants of spheres were detected. In the case of rods, the results need further explanation. Ag rods displayed high MTT-reduction capacity in themselves. For the time being, this interference with the assay components is not explored.
The cellular uptake of Ag-NPs was investigated by electron microscopy after exposing the cells for 1 hour to 50 µg/ml doses of NPs. The heavy cytotoxicity of Ag rods was evident on the electron microscopic pictures (Figure 72).
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Figure 72. Ag-NPs with spherical, cubical or triangle shape did not cause severe structural damages of NE-4C cells during a 1-hour exposure. In contrast, NE-4C cells were disrupted in the presence of Ag nanorods.
The extreme toxicity of Ag rods was presumably due to the mechanical damage caused by this shape.
Electron microscopic studies failed to demonstrate accumulation of Ag-NPs in intracellular vesicles, and revealed only a very few particles inside the cells. It might be due either to the low cellular penetration, or the rapid dissolution of particles outside and inside of the cells.
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