Assessment of the influence of gold nanoparticles stabilized with PAMAM dendrimers on HUVEC barrier cells.

Civilization diseases, cancer, frequent mutations of viruses and other pathogens constitute the need to look for new drugs, as well as systems for their targeted delivery. One of the promising way of using drugs is supplying them by linking to nanostructures. One of the solution for the development of nanobiomedicine are metallic nanoparticles stabilized with various polymer structures. In this report, we present the synthesis of gold nanoparticles, their stabilization with polyamidoamine (PAMAM) dendrimers with ethylenediamine core and the characteristics of the obtained product (AuNPs/PAMAM). The presence, size and morphology of synthesized gold nanoparticles were evaluated by ultraviolet-visible light spectroscopy, transmission electron microscopy and atomic force microscopy. The hydrodynamic radius distribution of the colloids was analyzed by dynamic light scattering technique. Additionally, the cytotoxicity and changes in mechanical properties of human umbilical vein endothelial cell line (HUVEC) cells caused by AuNPs/PAMAM were assessed. The results of studies on the nanomechanical properties of cells suggest a two-step changes in cell elasticity as a response to contact with nanoparticles. When using AuNPs/PAMAM in lower concentrations, no changes in cell viability were observed and the cells were softer than untreated cells. When higher concentrations were used, a decrease in the cells viability to about 80 % were observed, as well as non-physiological stiffening of the cells. The presented results may play a significant role in the development of nanomedicine.

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.

Nanomechanical testing of drug activities at the cellular level: Case study for endothelium-targeted drugs.

BACKGROUND: The pharmacological treatment of cardiovascular diseases that may potentially be attributed to endothelial dysfunction often requires the application of endothelium-targeted drugs. Simvastatin is one of such drugs currently on the market due to its established anti-inflammatory activities. The nanomechanical response to drug treatment at the cellular level is not yet known. However, this response mechanism is promising as a prospective testing method for newly developing drugs. METHODS: Force spectroscopy was used for in vitro characterization of the elastic properties of human microvascular endothelial cells. Cell dysfunction was caused by the application of tumor necrosis factor alpha. The anti-inflammatory action of the compounds was investigated for the cells incubated with each of the following agents: simvastatin, pyridine derivatives (1,4-dimethylpyridine chloride (1,4-DMP), and 1-methylpyridinium chloride (1-MP)). Moreover, in the case of 1,4-DMP and 1-MP, the measurements were supplemented with F-actin labeling data. RESULTS: We measured the simvastatin influence on the elasticity of human microvascular endothelial cells (HMECs) for concentrations: 1, 10 and 100µM. Furthermore, we evaluated the therapeutic and preventive effects of 1µM drug on inflamed cells. Finally, the effect of pyridine derivatives 1,4-dimethylpyridine chloride (1,4-DMP) and 1-methylpyridinium chloride (1-MP) was tested using force spectroscopy. CONCLUSIONS: The anti-inflammatory activity of the simvastatin is well illustrated by the endothelium cell elasticity changes returning from the characteristic inflammation time cycle "soft-stiff-soft" to control values. Furthermore, the elasticity results and F-actin labeling data indicated a preventive effect for 1- MP, whereas 1,4-DMP does not exhibit endothelium activity even at toxic concentrations.