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

Abstract

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.

Endothelial cell aging detection by means of atomic force spectroscopy

“…Short incubation times (1, 3, and 6 hours) were added before ending of total 48 hours cell culture; thus, all samples were fixed after 48 hours and reference control cells were defined in the same way for all samples (control cells cultured 48 hours on the dish). Cells were washed with warm PBS and then fixed with formaldehyde 3.7% for 5 minutes, which was described earlier 6 as the “slight” fixation method. Subsequently, the cells were washed with PBS and left in PBS until measurement.…”

“…In order to be able to compare the results to the previous work, 6 the tests were carried out under the same conditions. Force spectroscopy measurements were performed in the Hanks' buffer using a heating stage (to keep a constant temperature of 37°C) with the use of a special wrap overlay preventing the evaporation of liquid.…”

“…A very interesting issue related to cell aging is changes in their phenotype during this process, as well as cell responses to various substances including drugs or nanomaterials after different number of cell divisions. The research issue described in this work concerns the determination of the effect of silver nanoparticles on endothelial cells at early passages and correlation of obtained results with the previous work 6 to show the endothelial aging effect on their response to exposure to silver nanoparticles.…”

“…In the previous work, 6 we investigated changes in the elasticity modulus of EA.hy926 cells for late passages from 33 to 43 after incubation with silver nanoparticles for 24 hours at concentrations of 3, 3.6, and 16 μg/mL, chosen on the basis of the cell viability studies (IC10, IC25, and IC50 values, respectively). In the present work, we continued this issue for early passages ‐ from 9 to 15 of the same line of endothelial cells.…”

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed

“…A virtual issue appeared in the Journal of Molecular Recognition and is linked with this editorial. Ten research articles composed this special issue Pleskova S. N., Gorshkova E. N., and Kriukov R. N. Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus .…”