César Pascual García

Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.

Gold Nanoparticles Downregulate Interleukin‐1β‐Induced Pro‐Inflammatory Responses

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1β both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1β, thus opening potentially novel options for further therapeutic applications.

Effects of silver nanoparticles in diatom Thalassiosira pseudonana and cyanobacterium Synechococcus sp.

The aim of the present study was to investigate the effect of silver nanoparticles (AgNP) of different sizes toward two primary producer aquatic species. Thalassiosira pseudonana and Synechococcus sp. have been selected as representative models for the lower trophic organisms in marine and freshwater habitats, respectively. Time-dependent cellular growth was measured upon exposure to both AgNP and silver nitrate (AgNO(3)). In addition, AgNP behavior in freshwater and marine waters has been followed by CPS disc centrifuge, in the time frame of AgNP exposure studies, and the kinetic release of silver from AgNP of different sizes was measured by dialysis and inductively coupled plasma mass spectrometry (ICP-MS). The combination and interpretation of all these data suggest that a shared effect of AgNP and released silver was responsible for the toxicity in both organisms. Furthermore, the toxic effects induced by AgNP exposure in the present study seem to result from a mixture of parameters including aggregated state, size of the AgNP, stability of the preparation, and speciation of the released silver.

Silica nanoparticle uptake induces survival mechanism in A549 cells by the activation of autophagy but not apoptosis.

We report here an in vitro evaluation of silica nanoparticle uptake by lung epithelial cells (A549), the cytotoxic effect of the particles and we propose autophagy as possible survival strategy. The effect of surface charge, serum proteins and the influence of inhibitors on the uptake of 20 nm monodispersed nanoparticles with various functional groups are discussed. Uptake rate of the particles with various functional groups is demonstrated to be similar in the presence of serum proteins, while the uptake rate ranking is COOH>NH2>OH under serum free conditions. Our results suggest an actin-dependent, macropinocytotic uptake process that was also confirmed by scanning and transmission electron microscopy. In spite of the intensive active uptake, significant cytotoxic effect is detected only at relatively high concentrations (above 250 μg/mL). Blebbing of the cell surface is observed already at 5h of exposure and is shown to be related to autophagy rather than apoptotic cell death. The A549 cells display elevated levels of autophagosomes, however they do not express typical apoptosis markers such as increased amount of active caspase-3 and release of mitochondrial cytochrome C. Based on these results, we propose here an autophagic activity and cross-talk between autophagic and apoptotic pathways as a mechanism allowing the survival of A549 cells under exposure to silica nanoparticles.

Morphological transformation induced by multiwall carbon nanotubes on Balb/3T3 cell model as an in vitro end point of carcinogenic potential

Abstract In this work we investigated the toxicological effects of nude and chemically functionalised (-NH2, -OH and -COOH groups) multiwall carbon nanotubes (mwCNTs) using immortalised mouse fibroblasts cell line (Balb/3T3) as in vitro model, alternative to the use of animals, to assess basal cytotoxicity, carcinogenic potential, genotoxicity and cell interaction of nanomaterials (NM). Combining in vitro tests such as cell transformation assay and micronucleus with physicochemical and topological analysis, we obtained results showing no cytotoxicity and genotoxicity. Carcinogenic potential and mwCNTs interaction with cells were instead evident. We stressed the importance that different toxicological end points have to be considered when studying NM, therefore, assays able to detect long-term effects, such as carcinogenicity, must be taken into account together with a panel of tests able to detect more immediate effects like basal cytotoxicity or genotoxicity.

Detection of Silver Nanoparticles inside Marine Diatom Thalassiosira pseudonana by Electron Microscopy and Focused Ion Beam

In the following article an electron/ion microscopy study will be presented which investigates the uptake of silver nanoparticles (AgNPs) by the marine diatom Thalassiosira pseudonana, a primary producer aquatic species. This organism has a characteristic silica exoskeleton that may represent a barrier for the uptake of some chemical pollutants, including nanoparticles (NPs), but that presents a technical challenge when attempting to use electron-microscopy (EM) methods to study NP uptake. Here we present a convenient method to detect the NPs interacting with the diatom cell. It is based on a fixation procedure involving critical point drying which, without prior slicing of the cell, allows its inspection using transmission electron microscopy. Employing a combination of electron and ion microscopy techniques to selectively cut the cell where the NPs were detected, we are able to demonstrate and visualize for the first time the presence of AgNPs inside the cell membrane.

Silver nanoparticles induce cytotoxicity, but not cell transformation or genotoxicity on Balb3T3 mouse fibroblasts

Abstract Silver nanoparticles (Ag NPs) are one of the most common nanomaterials present in nanotechnology-based products. Here, the physical chemical properties of Ag NPs suspensions of 44 nm, 84 nm and 100 nm sizes synthesized in our laboratory were characterized. The NM-300 material (average size of 17 nm), supplied by the Joint Research Centre Nanomaterials Repository was also included in the present study. The Ag NPs potential cytotoxicity was tested on the Balb3T3 cell line by the Colony Forming Efficiency assay, while their potential morphological neoplastic transformation and genotoxicity were tested by the Cell Transformation Assay and the micronucleus test, respectively. After 24 h of exposure, NM-300 showed cytotoxicity with an IC50 of 8 µM (corresponding to 0.88 µg/mL) while for the other nanomaterials tested, values of IC50 were higher than 10 µM (1.10 µg/mL). After 72 h of exposure, Ag NPs showed size-dependent cytotoxic effect with IC50 values of 1.5 µM (1.16 µg/mL) for NM-300, 1.7 µM (1.19 µg/mL) for Ag 44 nm, 1.9 µM (0.21 µg/mL) for Ag 84 nm and 3.2 µM (0.35 µg/mL) for Ag 100 nm. None of the Ag NPs tested was able to induce either morphological neoplastic transformation or micronuclei formation.

Nanopatterned submicron pores as a shield for nonspecific binding in surface plasmon resonance-based sensing

We present a novel approach to tackle the most common drawback of using surface plasmon resonance for analyte screening in complex biological matrices--the nonspecific binding to the sensor chip surface. By using a perforated membrane supported by a polymeric gel structure at the evanescent wave penetration depth, we have fabricated a non-fouling sieve above the sensing region. The sieve shields the evanescent wave from nonspecific interactions which interfere with SPR sensing by minimizing the fouled area of the polymeric gel and preventing the translocation of large particles, e.g. micelles or aggregates. The nanopatterned macropores were fabricated by means of colloidal lithography and plasma enhanced chemical vapor deposition of a polyethylene oxide-like film on top of a polymeric gel matrix commonly used in surface plasmon resonance analysis. The sieve was characterized using surface plasmon resonance imaging, contact angle, atomic force microscopy and scanning electron microscopy. The performance of the sieve was studied using an immunoassay for detection of antibiotic residues in full fat milk and porcine serum. The non-fouling membrane presented pores in the 92-138 nm range organized in a hexagonal crystal lattice with a clearance of about 5% of the total surface. Functionally, the membrane with the nanopatterned macropores showed significant improvements in immunoassay robustness and sensitivity in untreated complex samples. The utilization of the sensor built-in sieve for measurements in complex matrices offers reduction in pre-analytical sample preparation steps and thus shortens the total analysis time.

Star-like gold nanoparticles as highly active substrate for Surface Enhanced Raman Spectroscopy

Surface Enhanced Raman Spectroscopy (SERS) is a popular method in bio-analytical chemistry and a potentially powerful enabling technology for in vitro diagnostics. SERS combines the excellent chemical specificity of Raman spectroscopy with the good sensitivity provided by enhancement of the signal that is observed when a molecule is located on (or very close to) the surface of nanostructured metallic materials. Star-like gold nanoparticles (SGN) are a new class of multibranched nanoparticles that in the last few years have attracted the attention of SERS community for their plasmonic properties. In this work we present a new method to prepare star-like gold nanoparticles with a simple one step protocol at room temperature using hydroquinone as reducing agent. Besides we compare the enhancement of Raman signal of malachite green, a dye commonly employed as label in biological studies, by star-like gold nanoparticles having different size, directly in liquid. This study shows that SGN provide good enhancement of Raman signal and that the effect of their dimension is strongly dependent on the wavelength used. Moreover preliminary results suggest that SGN produced using this method are characterized by good physical-chemical properties and they can be functionalized using the standard thiol chemistry. Overall, these results suggest that star-like gold nanoparticles produced through this method could be used for the further development of highly specific and sensitive SERS-based bio-analytical tests.