Endothelial cell aging detection by means of atomic force spectroscopy.

Endothelial cell aging is related to changes not only in cell phenotype, such as luminal changes, intimal and medial thickening, and increased vascular stiffness, but encompasses different cell responses to various substances including drugs or nanomaterials. In the present work, time- and dose-dependent elasticity changes evoked by silver nanoparticles in endothelial cells in early (below 15) passages were analyzed. Silver nanoparticle concentrations of 3, 3.6, and 16 µg/mL were selected for elasticity measurements for long incubation (24 hours) and of 1 and 3 µg/mL for monitoring dynamic elasticity changes of 1-, 3-, and 6-hour incubations. Surprisingly, a significant reduction in the cells elasticity modulus at lower number of passages exposed to silver nanoparticles used at 3 µg/mL for 24 hours was demonstrated. These results are in contrast to those obtained for endothelial cells in late (33-43) passages that may result from cellular aging in response to nanosilver. Furthermore, for short incubation times (1 and 3 hours), SNP-induced significant increase in the cell elasticity modulus was detected. In current work, we also attempted to answer the question whether the changes in cell elasticity were induced by the silver nanoparticles stabilized with polyvinyl pyrrolidone or by stabilizer itself. Elasticity measurements were supplemented by observations made with transmission electron microscopy and scanning electron microscopy, which confirmed the presence of silver nanoparticles inside the cells and on the cell membrane. Additionally, activation of reactive oxygen species was detected for cells exposed to SNPs for 1 and 3 hours, which was accompanied by increased cell elasticity modulus suggesting a possible mechanism of observed phenomenon.

Sensing of silver nanoparticles on/in endothelial cells using atomic force spectroscopy.

Endothelial cells, due to their location, are interesting objects for atomic force spectroscopy study. They constitute a barrier between blood and vessel tissues located deeper, and therefore they are the first line of contact with various substances present in blood, eg, drugs or nanoparticles. This work intends to verify whether the mechanical response of immortalized human umbilical vein endothelial cells (EA.hy926), when exposed to silver nanoparticles, as measured using force spectroscopy, could be effectively used as a bio-indicator of the physiological state of the cells. Silver nanoparticles were characterized with transmission electron microscopy and dynamic light scattering techniques. Tetrazolium salt reduction test was used to determine cell viability after treatment with silver nanoparticles. An elasticity of native cells was examined in the Hanks' buffer whereas fixed cells were softly fixed with formaldehyde. Additional aspect of the work is the comparative force spectroscopy utilizing AFM probes of ball-shape and conical geometries, in order to understand what changes in cell elasticity, caused by SNPs, were detectable with each probe. As a supplement to elasticity studies, cell morphology observation by atomic force microscopy and detection of silver nanoparticles inside cells using transmission electron microscopy were also performed. Cells exposed to silver nanoparticles at the highest selected concentrations (3.6 µg/mL, 16 µg/mL) are less elastic. It may be associated with the reorganization of the cellular cytoskeleton and the "strengthening" of the cell cortex caused by presence of silver nanoparticles. This observation does not depend on cell fixation. Agglomerates of silver nanoparticles were observed on the cell membrane as well as inside the cells.

Human brain endothelial barrier cells are distinctly less vulnerable to silver nanoparticles toxicity than human blood vessel cells: A cell-specific mechanism of the brain barrier?

The blood-brain barrier (BBB) constitutes a distinctive and tightly regulated interface between the brain and the peripheral circulation. The objective of studies was to compare responses of human endothelial cells representing the model of blood vessels - EA.hy926 and HUVEC cells and the model of the brain endothelial barrier - HBEC5i cells to silver nanoparticles (SNPs). A contact of SNPs with endothelial cells resulted in a formation of SNP agglomerates. Consequently, the SNPs uptake by endothelial cells affected cell viability and membrane integrity however observed responses were different. Brain endothelial barrier HBEC5i cells were much less vulnerable to SNPs toxicity comparing to EA.hy926 and HUVEC cells. It can be ascribed to the presence of specialized cellular components of the brain barrier, protecting HBEC5i cells against toxic SNPs. Fundamental understanding of SNPs inducing the BBB dysfunction may initiate engineering novel SNPs which are safe for the BBB and thereby safe for the brain.