Antonio Galeone

Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering.

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe3O4/SiO2 NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe3O4/SiO2 NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe3O4/SiO2 NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.

Negligible particle-specific toxicity mechanism of silver nanoparticles: the role of Ag+ ion release in the cytosol.

Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.

Impact of nanoscale topography on genomics and proteomics of adherent bacteria.

Bacterial adhesion onto inorganic/nanoengineered surfaces is a key issue in biotechnology and medicine, because it is one of the first necessary steps to determine a general pathogenic event. Understanding the molecular mechanisms of bacteria-surface interaction represents a milestone for planning a new generation of devices with unanimously certified antibacterial characteristics. Here, we show how highly controlled nanostructured substrates impact the bacterial behavior in terms of morphological, genomic, and proteomic response. We observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM) that type-1 fimbriae typically disappear in Escherichia coli adherent onto nanostructured substrates, as opposed to bacteria onto reference glass or flat gold surfaces. A genetic variation of the fimbrial operon regulation was consistently identified by real time qPCR in bacteria interacting with the nanorough substrates. To gain a deeper insight into the molecular basis of the interaction mechanisms, we explored the entire proteomic profile of E. coli by 2D-DIGE, finding significant changes in the bacteria adherent onto the nanorough substrates, such as regulations of proteins involved in stress processes and defense mechanisms. We thus demonstrated that a pure physical stimulus, that is, a nanoscale variation of surface topography, may play per se a significant role in determining the morphological, genetic, and proteomic profile of bacteria. These data suggest that in depth investigations of the molecular processes of microorganisms adhering to surfaces are of great importance for the design of innovative biomaterials with active biological functionalities.

Concentration-Dependent, Size-Independent Toxicity of Citrate Capped AuNPs in Drosophila melanogaster

The expected potential benefits promised by nanotechnology in various fields have led to a rapid increase of the presence of engineered nanomaterials in a high number of commercial goods. This is generating increasing questions about possible risks for human health and environment, due to the lack of an in-depth assessment of the physical/chemical factors responsible for their toxic effects. In this work, we evaluated the toxicity of monodisperse citrate-capped gold nanoparticles (AuNPs) of different sizes (5, 15, 40, and 80 nm) in the model organism Drosophila melanogaster, upon ingestion. To properly evaluate and distinguish the possible dose- and/or size-dependent toxicity of the AuNPs, we performed a thorough assessment of their biological effects, using two different dose-metrics. In the first approach, we kept constant the total surface area of the differently sized AuNPs (Total Exposed Surface area approach, TES), while, in the second approach, we used the same number concentration of the four different sizes of AuNPs (Total Number of Nanoparticles approach, TNN). We observed a significant AuNPs-induced toxicity in vivo, namely a strong reduction of Drosophila lifespan and fertility performance, presence of DNA fragmentation, as well as a significant modification in the expression levels of genes involved in stress responses, DNA damage recognition and apoptosis pathway. Interestingly, we found that, within the investigated experimental conditions, the toxic effects in the exposed organisms were directly related to the concentration of the AuNPs administered, irrespective of their size.

Molecular response of Escherichia coli adhering onto nanoscale topography.

Bacterial adhesion onto abiotic surfaces is an important issue in biology and medicine since understanding the bases of such interaction represents a crucial aspect in the design of safe implant devices with intrinsic antibacterial characteristics. In this framework, we investigated the effects of nanostructured metal substrates on Escherichia coli adhesion and adaptation in order to understand the bio-molecular dynamics ruling the interactions at the interface. In particular, we show how highly controlled nanostructured gold substrates impact the bacterial behavior in terms of morphological changes and lead to modifications in the expression profile of several genes, which are crucially involved in the stress response and fimbrial synthesis. These results mainly demonstrate that E. coli cells are able to sense even slight changes in surface nanotopography and to actively respond by activating stress-related pathways. At the same time, our findings highlight the possibility of designing nanoengineered substrates able to trigger specific bio-molecular effects, thus opening the perspective of smartly tuning bacterial behavior by biomaterial design.

Ranking the in vivo toxicity of nanomaterials in Drosophila melanogaster

In this work, we propose a quantitative assessment of nanoparticles toxicity in vivo. We show a quantitative ranking of several types of nanoparticles (AuNPs, AgNPs, cadmium-based QDs, cadmium-free QDs, and iron oxide NPs, with different coating and/or surface chemistries), providing a categorization of their toxicity outcomes. This strategy may offer an innovative high-throughput screening tool of nanomaterials, of potential and broad interest to the nanoscience community.

Impact of nanomaterials on in vitro and in vivo systems: role of nanoscale features in nanotoxicology

The interactions between biological systems and nanostructured materials are attracting great interest, due to the possibility to open up novel concepts for the design of smart nano-biomaterials that actively play a functional biological role. On the other hand, the assessment of the potential toxic effects arising from such interactions is gaining increasing attention, and a new field known as nanotoxicology is strongly emerging. In this frame, we investigated the response of human neurons to gold surfaces with different levels of nanoroughness, finding out that neurons are capable to sense and actively respond to these nanotopography features. These nanostructured substrates were also investigated to explore the impact of nanotopography on morphology and genomics of adherent bacteria. A multidisciplinary approach was exploited to characterize bacteria-nanostructured surface interactions, observing that type-1 fimbriae disappear in bacteria grown onto nanorough substrates. We also show how nanoparticles interact with biomolecules in culture media and in vitro and in vivo biological systems, by investigating the toxic effects of a wide range of nanomaterials (AuNPs, QDs, SiO2 NPs), demonstrating the key role of size, shape, and surface coating.