Eudald Casals

Time Evolution of the Nanoparticle Protein Corona

In this work, we explore the formation of the protein corona after exposure of metallic Au nanoparticles (NPs), with sizes ranging from 4 to 40 nm, to cell culture media containing 10% of fetal bovine serum. Under in vitro cell culture conditions, zeta potential measurements, UV-vis spectroscopy, dynamic light scattering and transmission electron microscope analysis were used to monitor the time evolution of the inorganic NP-protein corona formation and to characterize the stability of the NPs and their surface state at every stage of the experiment. As expected, the red-shift of the surface plasmon resonance peak, as well as the drop of surface charge and the increase of the hydrodynamic diameter indicated the conjugation of proteins to NPs. Remarkably, an evolution from a loosely attached toward an irreversible attached protein corona over time was observed. Mass spectrometry of the digested protein corona revealed albumin as the most abundant component which suggests an improved biocompatibility.

Evaluation of the ecotoxicity of model nanoparticles

Since society at large became aware of the use of nanomaterials in ever growing quantities in consumer products and their presence in the environment, critical interest in the impact of this emerging technology has grown. The main concern is whether the unknown risks of engineered nanoparticles (NPs), in particular their impact on health and environment, outweighs their established benefits for society. Therefore, a key issue in this field is to evaluate their potential toxicity. In this context we evaluated the effects on plants and microorganisms of model nanoparticles, in particular of a stable metal (Au, 10nm mean diameter), a well-known bactericide (Ag, 2 nm mean diameter) and the broadly used Fe(3)O(4) (7 nm mean diameter). The toxicity of these nanoparticles was assayed using standard toxicity tests. Specifically, germination (cucumber and lettuce), bioluminescent (Photobacterium phosphoreum) and anaerobic toxicity tests were performed. Germination tests were conducted at a NP dose of 62, 100 and 116 microg mL(-1) for Au, Ag, and Fe(3)O(4), respectively. The bioluminscent testing (P. phosphoreum) was conducted at a dose of 28, 45 and 52 microg mL(-1) for Au, Ag, and Fe(3)O(4), respectively. Finally, anaerobic tests were conducted at a NP dose of 10, 16 and 18 microg mL(-1) for Au, Ag, and Fe(3)O(4), respectively. In all cases low or zero toxicity was observed. However, some perturbation of the normal functions with respect to controls in germinating tests was observed, suggesting the necessity for further research in this field. At the same time, the effect of NP-solvents was sometimes more significant than that of the NPs themselves, a point that is of special interest for future nanotoxicological studies.

Physicochemical characteristics of protein-NP bioconjugates: the role of particle curvature and solution conditions on human serum albumin conformation and fibrillogenesis inhibition.

Gold nanoparticles (Au NPs) from 5 to 100 nm in size synthesized with HAuCl(4) and sodium citrate were complexed with the plasma protein human serum albumin (HSA). Size, surface charge, and surface plasmon bands of the Au NPs are largely modified by the formation of a protein corona via electrostatic interactions and hydrogen bonding as revealed by thermodynamic data. Negative values of the entropy of binding suggested a restriction in the biomolecule mobility upon adsorption. The structure of the adsorbed protein molecules is slightly affected by the interaction with the metal surface, but this effect is enhanced as the NP curvature decreases. Also, it is observed that the protein molecules adsorbed onto the NP surface are more resistant to complete thermal denaturation than free protein ones as deduced from the increases in the melting temperature of the adsorbed protein. Differences in the conformations of the adsorbed protein molecules onto small (<40 nm) and large NPs were observed on the basis of ζ-potential data and FTIR spectroscopy, also suggesting a better resistance of adsorbed protein molecules to thermal denaturing conditions. We think this enhanced protein stability is responsible for a reduced formation of HSA amyloid-like fibrils in the presence of small Au NPs under HSA fibrillation conditions.

Hardening of the Nanoparticle–Protein Corona in Metal (Au, Ag) and Oxide (Fe3O4, CoO, and CeO2) Nanoparticles

The surface modifications of metal and metal oxide nanoparticles with sizes ranging from 7 to 20 nm dispersed in commonly used cell culture medium supplemented with serum are investigated. All the tested nanoparticles adsorb proteins onto their surface, thereby forming a protein corona through a dynamic process evolving towards an irreversible coating (hard protein corona). Despite the fact that the studied nanomaterials have similar characteristics of hydrophobicity and surface charge, different temporal patterns of the protein corona formation are observed that can be considered a fingerprint for nanoparticle identification. Some of the biological and toxicological implications of the formation of the nanoparticle-protein corona are studied using the human monocytic cell line THP-1 exposed to cobalt oxide nanoparticles. Results show that production of reactive oxygen species is decreased if the nanoparticles are preincubated for 48 h with serum.

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

BackgroundWith the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.ResultsIn a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.ConclusionThe problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

Synthesis of Platinum Cubes, Polypods, Cuboctahedrons, and Raspberries Assisted by Cobalt Nanocrystals

The introduction of metallic traces into the synthesis of platinum nanocrystals (Pt NCs) has been investigated as a surfactant-independent means of controlling shape. Various nanocrystal morphologies have been produced without modification of the reaction conditions, composition, and concentration other than the presence of cobalt traces (<5%). In the presence of metallic cobalt (a strong reducer for Pt cations) cubic Pt NCs are obtained, while cobalt ions or gold NCs have no effect on the synthesis, and as a result, polypods are obtained. Intermediate shapes such as cemented cubes or cuboctahedron NCs are also obtained under similar conditions. Thus, various NC shapes can be obtained with subtle changes, which illustrates the high susceptibility and mutability of the NC shape to modification of the reaction kinetics during the early reduction process. Our studies help progress toward a general mechanism for nanocrystal shape control.

Shape matters: effects of silver nanospheres and wires on human alveolar epithelial cells

BackgroundIn nanotoxicology, the exact role of particle shape, in relation to the composition, on the capacity to induce toxicity is largely unknown. We investigated the toxic and immunotoxic effects of silver wires (length: 1.5 - 25 μm; diameter 100 - 160 nm), spherical silver nanoparticles (30 nm) and silver microparticles (<45 μm) on alveolar epithelial cells (A549).MethodsWires and nanoparticles were synthesized by wet-chemistry methods and extensively characterized. Cell viability and cytotoxicity were assessed and potential immunotoxic effects were investigated. To compare the effects on an activated and a resting immune system, cells were stimulated with rhTNF-α or left untreated. Changes in intracellular free calcium levels were determined using calcium imaging. Finally, ion release from the particles was assessed by ICP-MS and the effects of released ions on cell viability and cytotoxicity were tested.ResultsNo effects were observed for the spherical particles, whereas the silver wires significantly reduced cell viability and increased LDH release from A549 cells. Cytokine promoter induction and NF-κB activation decreased in a concentration dependent manner similar to the decrease seen in cell viability. In addition, a strong increase of intracellular calcium levels within minutes after addition of wires was observed. This toxicity was not due to free silver ions, since the samples with the highest ion release did not induce toxicity and ion release control experiments with cells treated with pre-incubated medium did not show any effects either.ConclusionsThese data showed that silver wires strongly affect the alveolar epithelial cells, whereas spherical silver particles had no effect. This supports the hypothesis that shape is one of the important factors that determine particle toxicity.

Effect of cerium dioxide, titanium dioxide, silver, and gold nanoparticles on the activity of microbial communities intended in wastewater treatment

Growth in production and use of nanoparticles (NPs) will result increased concentrations of these in industrial and urban wastewaters and, consequently, in wastewater-treatment facilities. The effect of this increase on the performance of the wastewater-treatment process has not been studied systematically and including all the microbial communities involved in wastewater treatment. The present work investigates, by using respiration tests and biogas-production analysis, the inhibitory effect of four different commonly used metal oxide (CeO(2) and TiO(2)) and zero-valent metal (Ag and Au) nanoparticles on the activity of the most important microbial communities present in a modern wastewater-treatment plant. Specifically, the actions of ordinary heterotrophic organisms, ammonia oxidizing bacteria, and thermophilic and mesophilic anaerobic bacteria were tested in the presence and absence of the nanoparticles. In general, CeO(2) nanoparticles caused the greatest inhibition in biogas production (nearly 100%) and a strong inhibitory action of other biomasses; Ag nanoparticles caused an intermediate inhibition in biogas production (within 33-50%) and a slight inhibition in the action of other biomasses, and Au and TiO(2) nanoparticles caused only slight or no inhibition for all tested biomasses.

Chromium VI adsorption on cerium oxide nanoparticles and morphology changes during the process.

In this study, suspended cerium oxide nanoparticles stabilized with hexamethylenetetramine were used for the removal of dissolved chromium VI in pure water. Several concentrations of adsorbent and adsorbate were tested, trying to cover a large range of possible real conditions. Results showed that the Freundlich isotherm represented well the adsorption equilibrium reached between nanoparticles and chromium, whereas adsorption kinetics could be modeled by a pseudo-second-order expression. The separation of chromium-cerium nanoparticles from the medium and the desorption of chromium using sodium hydroxide without cerium losses was obtained. Nanoparticles agglomeration and morphological changes during the adsorption-desorption process were observed by TEM. Another remarkable result obtained in this study is the low toxicity in the water treated by nanoparticles measured by the Microtox(®) commercial method. These results can be used to propose this treatment sequence for a clean and simple removal of drinking water or wastewater re-use when a high toxicity heavy metal such as chromium VI is the responsible for water pollution.

The suitability of different cellular in vitro immunotoxicity and genotoxicity methods for the analysis of nanoparticle-induced events

Abstract Suitable assays and test strategies are needed to analyze potential genotoxic and immunotoxic health effects caused by nanoparticle exposure. The development and validation of such methods is challenging because nanoparticles may show unexpected behavior, like aggregation or interference with optical measurements, when routine in vitro assays are performed. In our interdisciplinary study, the effects of inorganic gold (4.5 nm) and iron oxide (7.3 nm) nanoparticles with a narrow size distribution were tested on human cells using different assay systems. The results show that cytotoxicity as well as immunotoxicity and genotoxicity induced by these two inorganic nanoparticles was low or absent when using a panel of cell-based tests in different laboratories. However, several technical issues had to be tackled that were specific for working with nanoparticles. The methods used, their suitability for nanotoxicity testing, and the technical problems encountered are carefully described and discussed in this paper.