Size distribution of Rubipy-SiO₂ NPs in complete media.

<p>Rubipy-SiO₂ NPs were suspended either in A549 (left) or CaCo-2 (right) complete cell culture medium (10% of serum) at concentration 1 mg/ml and the size distribution was measured in time up to 48 hours by CLS.</p

Particle size distribution in water and serum-free cell culture media.

<p>Particle size distribution in water and serum-free cell culture media.</p

Effect of cell presence on NPs size characteristics.

<p>CLS size measurement of 80 nm Rubipy-SiO₂ NPs suspended at 1 mg/ml in a) serum free A549 medium (med I), b) serum free A549 medium pre-conditioned by the incubation with cells (med II), c) filtrated medium II (med III), after 24 h incubation at 37°C.</p

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake

<div><p>Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized <i>in vitro</i> screening methods are needed to study the mechanisms by which they can interact with biological systems. The <i>in vitro</i> evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO₂ NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO₂ NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the <i>in vitro</i> toxicological assays.</p></div

Kinetics of the cell uptake of Rubipy-SiO₂ NPs.

<p>A549 cells were exposed to 200 μg/ml of 30 nm (left column) and 80 nm (right column) Rubipy-SiO₂ NPs in complete CCM for different time intervals up to 48 h, and stained with Hoechst-33342 (nuclei; blue) and with AlexaFluor 488-conjugated phalloidin (actin; green), and observed by fluorescence microscopy (Rubipy-SiO₂ NPs, red). Scale bar: 50 μm.</p

Effect of cell presence on NPs size characteristics.

<p>Rubipy-SiO₂ NPs 30 and 80 nm were suspended in cell culture medium without serum and pre-conditioned by incubation with cells for 1, 3 and 5 hours and the size distribution was measured by CLS.</p

Quantification of the cell uptake of Rubipy-SiO₂ NPs.

<p>The cellular uptake of Rubipy-SiO₂ NPs, was quantified by flow cytometry after exposure of A549 and CaCo-2 cells to 100 μg/ml of 30 nm Rubipy-SiO₂ NPs (up) or to 200 μg/ml of 80 nm Rubipy-SiO₂ NPs (down) in complete CCM during 1 h, 3 h, 5 h, 24 h and 48 h.</p

Polymer Nanopillar–Gold Arrays as Surface-Enhanced Raman Spectroscopy Substrate for the Simultaneous Detection of Multiple Genes

In our study, 2D nanopillar arrays with plasmonic crystal properties are optimized for surface-enhanced Raman spectroscopy (SERS) application and tested in a biochemical assay for the simultaneous detection of multiple genetic leukemia biomarkers. The special fabrication process combining soft lithography and plasma deposition techniques allows tailoring of the structural and chemical parameters of the crystal surfaces. In this way, it has been possible to tune the plasmonic resonance spectral position close to the excitation wavelength of the monochromatic laser light source in order to maximize the enhancing properties of the substrate. Samples are characterized by scanning electron microscopy and reflectance measurements and tested for SERS activity using malachite green. Besides, as the developed substrate had been prepared on a simple glass slide, SERS detection from the support side is also demonstrated. The optimized substrate is functionalized with thiol-modified capture oligonucleotides, and concentration-dependent signal of the target nucleotide is detected in a sandwich assay with labeled gold nanoparticles. Gold nanoparticles functionalized with different DNA and various Raman reporters are applied in a microarray-based assay recognizing a disease biomarker (Wilms tumor gene) and housekeeping gene expressions in the same time on spatially separated microspots. The multiplexing performance of the SERS-based bioassay is illustrated by distinguishing Raman dyes based on their complex spectral fingerprints

Effect of serum depletion on the kinetics of cell uptake.

<p>A549 cells and CaCo-2 cells were exposed to 100 μg/ml of 30 nm Rubipy-SiO₂ NPs (top) or to 200 μg/ml of 80 nm Rubipy-SiO₂ NPs (bottom) either in serum-free conditions or in complete medium. Cell associated fluorescence was quantified by flow cytometry after 10 min, 30 min, 1 h, 3 h and 5 h exposure.</p

Kinetics of NPs deposition.

<p>A549 cells were incubated with 30 nm (top) and 80 nm (bottom) Rubipy-SiO₂ NPs in serum-free conditions for up to 5 h. Fluorescence intensity of the supernatant and of the cell layer was measured with a spectrophotometer at every time point.</p