Stefania Sabella

Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response.

The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV-visible, plasmon resonance light scattering), that proteins-NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagles medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.

A general mechanism for intracellular toxicity of metal-containing nanoparticles

We demonstrate a general mechanism for the toxicity induced by metal-containing NPs, named “lysosome-enhanced Trojan horse effect”, which provides design rules to engineer safer NPs.

Neurons sense nanoscale roughness with nanometer sensitivity

The interaction between cells and nanostructured materials is attracting increasing interest, because of the possibility to open up novel concepts for the design of smart nanobiomaterials with active biological functionalities. In this frame we investigated the response of human neuroblastoma cell line (SH-SY5Y) to gold surfaces with different levels of nanoroughness. To achieve a precise control of the nanoroughness with nanometer resolution, we exploited a wet chemistry approach based on spontaneous galvanic displacement reaction. We demonstrated that neurons sense and actively respond to the surface nanotopography, with a surprising sensitivity to variations of few nanometers. We showed that focal adhesion complexes, which allow cellular sensing, are strongly affected by nanostructured surfaces, leading to a marked decrease in cell adhesion. Moreover, cells adherent on nanorough surfaces exhibit loss of neuron polarity, Golgi apparatus fragmentation, nuclear condensation, and actin cytoskeleton that is not functionally organized. Apoptosis/necrosis assays established that nanoscale features induce cell death by necrosis, with a trend directly related to roughness values. Finally, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine-tuning their surface topography at nanometer scale. Specific and functional adhesion of cells occurred only onto flat gold stripes, with a clear self-alignment of neurons, delivering a simple and elegant approach for the design and development of biomaterials with precise nanostructure-triggered biological responses.

ITS-NANO - Prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy

BackgroundTo assess the risk of all nanomaterials (NMs) on a case-by-case basis is challenging in terms of financial, ethical and time resources. Instead a more intelligent approach to knowledge gain and risk assessment is required.MethodsA framework of future research priorities was developed from the accorded opinion of experts covering all major stake holder groups (government, industry, academia, funders and NGOs). It recognises and stresses the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling approaches as key components of the current and future risk assessment of NMs.ResultsThe framework for future research has been developed from the opinions of over 80 stakeholders, that describes the research priorities for effective development of an intelligent testing strategy (ITS) to allow risk evaluation of NMs. In this context, an ITS is a process that allows the risks of NMs to be assessed accurately, effectively and efficiently, thereby reducing the need to test NMs on a case-by-case basis.For each of the major topics of physicochemical characterisation, exposure identification, hazard identification and modelling, key-priority research areas are described via a series of stepping stones, or hexagon diagrams structured into a time perspective. Importantly, this framework is flexible, allowing individual stakeholders to identify where their own activities and expertise are positioned within the prioritisation pathway and furthermore to identify how they can effectively contribute and structure their work accordingly. In other words, the prioritisation hexagon diagrams provide a tool that individual stakeholders can adapt to meet their own particular needs and to deliver an ITS for NMs risk assessment. Such an approach would, over time, reduce the need for testing by increasing the reliability and sophistication of in silico approaches.The manuscript includes an appraisal of how this framework relates to the current risk assessment approaches and how future risk assessment could adapt to accommodate these new approaches. A full report is available in electronic format (pdf) at http://www.nano.hw.ac.uk/research-projects/itsnano.html.ConclusionITS-NANO has delivered a detailed, stakeholder driven and flexible research prioritisation (or strategy) tool, which identifies specific research needs, suggests connections between areas, and frames this in a time-perspective.

Charge transport and intrinsic fluorescence in amyloid-like fibrils.

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications.

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.

Water-Repellent Cellulose Fiber Networks with Multifunctional Properties

We demonstrate a simple but highly efficient technique to introduce multifunctional properties to cellulose fiber networks by wetting them with ethyl-cyanoacrylate monomer solutions containing various suspended organic submicrometer particles or inorganic nanoparticles. Solutions can be applied on cellulosic surfaces by simple solution casting techniques or by dip coating, both being suitable for large area applications. Immediately after solvent evaporation, ethyl-cyanoacrylate starts cross-linking around cellulose fibers under ambient conditions because of naturally occurring surface hydroxyl groups and adsorbed moisture, encapsulating them with a hydrophobic polymer shell. Furthermore, by dispersing various functional particles in the monomer solutions, hydrophobic ethyl-cyanoacrylate nanocomposites with desired functionalities can be formed around the cellulose fibers. To exhibit the versatility of the method, cellulose sheets were functionalized with different ethyl-cyanoacrylate nanocomposite shells comprising submicrometer wax or polytetrafluoroethylene particles for superhydophobicity, MnFe(2)O(4) nanoparticles for magnetic activity, CdSe/ZnS quantum dots for light emission, and silver nanoparticles for antimicrobial activity. Morphological and functional properties of each system have been studied by scanning and transmission electron microscopy, detailed contact angle measurements, light emission spectra and E. coli bacterial growth measurements. A plethora of potential applications can be envisioned for this technique, such as food and industrial packaging, document protection, catalytic cellulosic membranes, textronic (electrofunctional textiles), electromagnetic devices, authentication of valuable documents, and antimicrobial wound healing products to name a few.

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.

Gold-Nanoparticle-Based Colorimetric Discrimination of Cancer-Related Point Mutations with Picomolar Sensitivity

Point mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene are being increasingly recognized as important diagnostic and prognostic markers in cancer. In this work, we describe a rapid and low-cost method for the naked-eye detection of cancer-related point mutations in KRAS based on gold nanoparticles. This simple colorimetric assay is sensitive (limit of detection in the low picomolar range), instrument-free, and employs nonstringent room temperature conditions due to a combination of DNA-conjugated gold nanoparticles, a probe design which exploits cooperative hybridization for increased binding affinity, and signal enhancement on the surface of magnetic beads. Additionally, the scheme is suitable for point-of-care applications, as it combines naked-eye detection, small sample volumes, and isothermal (PCR-free) amplification.

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.