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Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO2 variants, differing in structure, size and surface charge. TiO2 NM-105 served as a benchmark. Responses were studied in vitro in rat lung epithelial cells (RLE-6TN) and alveolar macrophages (NR8383) and compared to in vivo responses in rat lung tissues obtained from in vivo instillation and short-term inhalation studies (STIS). Time- and concentration-dependent changes were observed in both in vitro models but with cell-type specific responses. Overall, the levels of lipids and biogenic amines (BAs) tended to increase in epithelial cells but decreased in macrophages. Many identified metabolites like Met-SO, hydroxy-Pro and spermidine were related to oxidative stress, indicating that oxidative stress contributes to the MoA for the selected NMs. Several biomarker candidates such as Asp, Asn, Ser, Pro, spermidine, putrescine and LysoPCaC16:1 were identified in vitro and verified in vivo. In this study, we successfully applied a metabolomics workflow for in vitro and in vivo samples, which proved to be well suited to identify potential biomarkers, to gain insights into NM structure–activity relationship and into the underlying MoA.

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) assay. The SOP has undergone an inter- and intra-laboratory comparison, to evaluate robustness, reliability, and reproducibility, using representative materials (ZnO, CuO, Mn2O3, and BaSO4 NMs), and a number of calibration tools to normalize data. The SOP includes an NM positive control (nanoparticle carbon black (NPCB)), a chemical positive control (SIN-1), and a standard curve of fluorescein fluorescence. The interlaboratory comparison demonstrated that arbitrary fluorescence units show high levels of partner variability; however, data normalization improved variability. With statistical analysis, it was shown that the SIN-1 positive control provided an extremely high level of reliability and reproducibility as a positive control and as a normalization tool. The NPCB positive control can be used with a relatively high level of reproducibility, and in terms of the representative materials, the reproducibility CuO induced-effects was better than for Mn2O3. Using this DCFH2-DA acellular assay SOP resulted in a robust intra-laboratory reproduction of ROS measurements from all NMs tested, while effective reproduction across different laboratories was also demonstrated; the effectiveness of attaining reproducibility within the interlaboratory assessment was particle-type-specific.

13 Pág. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology., The work leading to this article has received funding from the European Union’s Horizon 2020 Research and Innovation programme, Grant Agreements no. 646221 (NanoReg2, 2015–2019), no. 814401 (Gov4Nano, 2019–2022) and no. 814425 (RiskGONE 2019–2023). In addition, the European Union’s 7th Framework Programme projects NANoREG (2013–2017, Grant Agreement no. 310584), NanoTEST (2008–2012, no. 201335) and ENPRA (2009–2012, no. 228789), the European Union’s Health Programme Joint Action project NANOGENOTOX (2010–2013, no. 2009 21 01) and the US NIH NCI caNanoLab portal are acknowledged for providing data

Nanomaterials (NMs) can be produced in plenty of variants posing several challenges for NM hazard and risk assessment. Metabolomic profiling of NM-treated cells and tissues allows for insights into underlying Mode-of-Action (MoA) and offers several advantages in this context. It supports the description of Adverse Outcome Pathways (AOPs) and, therefore, tailored AOP-based hazard testing strategies. Moreover, it bears great potential for biomarker discovery supporting toxicity prediction. Here, we applied metabolomics profiling to cells treated with four well-selected SiO

Nanomaterials (NMs) can be manufactured in plenty of variants differing in their physicochemical properties. Functional assays can be highly useful to cope with the enormous variability by supporting prioritization and categorization. Oxidative potential (OP) seems to be in particular important in this context and different assays are available. However, their reliability and predictivity are not well-characterized. This study compares four different test methods for measuring NM OP. Reactive oxygen species (ROS) generation was measured on a set of 35 different materials, all extensively characterized with respect to physicochemical properties and most of them with respect to toxicity. Different acellular assays were applied, namely electron spin resonance (ESR) spectroscopy using CPH spin probe and DMPO spin trap, and the ferric reduction ability of serum (FRAS) assay. In addition, protein carbonylation as a marker for oxidative protein damage was analyzed in NRK-52E cells. All assays were assessed individually for their predictivity compared to established toxicological endpoints. We also aimed to identify the optimal assay combination using multivariate logistic regression and other statistical measures. BET surface area-based doses were more suitable to relate surface reactivity to toxicity. In addition, normalization to the deposited dose was advantageous for cellular assays as it improved the predictivity for in vitro as well as in vivo toxicity. The carbonylation assay, potentially in combination with ESR (DMPO spin trap) or FRAS assay, led to the best predictive performance. In summary, we propose a testing strategy for NM OP and demonstrated the applicability in an extended case study on 35 materials. This work is an important contribution towards reliable grouping and testing strategies for NMs.

The physico-chemical properties of manufactured nanomaterials (NMs) can be fine-tuned to obtain different functionalities addressing the needs of specific industrial applications. The physico-chemical properties of NMs also drive their biological interactions. Accordingly, each NM requires an adequate physico-chemical characterization and potentially an extensive and time-consuming (eco)toxicological assessment, depending on regulatory requirements. Grouping and read-across approaches, which have already been established for chemicals in general, are based on similarity between substances and can be used to fill data gaps without performing additional testing. Available data on “source” chemicals are thus used to predict the fate, toxicokinetics and/or (eco)toxicity of structurally similar “target” chemical(s). For NMs similar approaches are only beginning to emerge and several challenges remain, including the identification of the most relevant physico-chemical properties for supporting the claim of similarity. In general, NMs require additional parameters for a proper physico-chemical description. Furthermore, some parameters change during a NM's life cycle, suggesting that also the toxicological profile may change. This paper compares existing concepts for NM grouping, considering their underlying basic principles and criteria as well as their applicability for regulatory and other purposes. Perspectives and recommendations based on experiences obtained during the EU Horizon 2020 project NanoReg2 are presented. These include, for instance, the importance of harmonized data storage systems, the application of harmonized scoring systems for comparing biological responses, and the use of high-throughput and other screening approaches. We also include references to other ongoing EU projects addressing some of these challenges.

We investigated a novel bichromophoric antenna system, characterized by energy transfer between a naphthalene group acting as the donor and a benzofurazane group acting as the acceptor. We studied the spectroscopic properties (infrared, Raman, UV–vis and fluorescence) of self-assembled monolayers of this molecular antenna on Ag nanoisland films and the energy-transfer process upon irradiation at 300 nm.

We report on the preparation and spectroscopic characterization of a novel nanocomposite consisting of silver nanoparticles and a fluorescent polymer obtained by in situ ps laser ablation of a silver target. While the polymer is able to detect the fungus Paecilomyces variotii by fluorescence quenching, the composite with silver nanoparticles permits an interaction with both cell walls of spores and mycelia without any appreciable reduction of the overall fluorescence quantum yield.

A POM to remember: Hexanuclear Fe(III) polyoxometalate (POM) single-molecule magnets (see structure) can be noncovalently assembled on the surface of single-wall carbon nanotubes. Complementary characterization techniques (see TEM image and magnetic hysteresis loops) demonstrate the integrity and bistability of the individual molecules, which could be used to construct single-molecule memory devices.

Surface Enhanced Raman Scattering (SERS) was employed for studying the polymerization of self-assembled carbazolyl-diacetylene monolayers chemisorbed on smooth gold substrates by depositing silver colloidal nanoparticles. After UV irradiation two polymer phases were observed by SERS measurements, with different structural order in the polymer chain.

Silver substrates have been obtained, by depositing silver colloidal nanoparticles on a roughened silver plate treated with 1,10-phenanthroline, and checked by means of AFM microscopy and Raman spectroscopy. The ligand molecules are located between two silver substrates and undergo the SERS (Surface Enhanced Raman Scattering) enhancement of both the roughened silver plate and the silver colloidal layer deposited on it. These SERS-active substrates, which show the advantages of being stable with respect to the metal colloidal suspensions, along with an easy and reproducible preparation, can be very useful for catalytic and analytical applications of the SERS spectroscopy.

The giant Raman enhancement observed by means of the SERS (surface-enhanced Raman scattering) technique upon adsorption of molecules on nanostructured Ag, Au or Cu surfaces allows widespread applications in materials science. The Raman intensities of the adsorbed molecules can be enhanced up to 107-fold upon going to the SERS spectra. Recently, the SERS spectroscopy has been applied to single-molecule detection, showing enhancement factors up to 1014–1015. In this work, by depositing silver colloidal suspensions on different metal surfaces, dry layers of Ag nanoparticles were obtained, as detected by atomic force microscopy (AFM) measurements. Hence, substrates, unable to give rise to the Raman enhancement of the adsorbate, become SERS-active, avoiding the roughening treatment of the metal surfaces.

Recently, the organisation of magnetic molecules on carbon nanotubes has raised much interest due to their possible interesting contribution to molecular spintronics. In this paper, we describe the assembly on SWNTs of a magnetic polyoxometalate encompassing a single cobalt ion (CoPOM) and its isostructural diamagnetic zinc analogue (ZnPOM). The simple magnetic behaviour of CoPOM and the availability of its diamagnetic counterpart render these POM@NTs systems interesting model compounds for the study of molecular electronics devices based on carbon nanotubes and magnetic molecules. The success and rate of the grafting have been investigated by electron microscopy, electron energy loss spectroscopy, X-ray photoelectron spectroscopy, cyclic voltammetry, Raman scattering and magnetisation measurements. These characterisations altogether demonstrate the preservation of the structural and magnetic properties of the molecules upon functionalisation and the existence of an electronic communication between the molecules and the nanotubes.

This paper presents a novel model aimed at describing the basic phenomena that cause the fragmentation of a single Au nanoparticle (AuNP) by interaction with the second and the third harmonics of a Nd:YAG picosecond laser. In order to verify the model through a comparison with experimental results, we extended the single-AuNP fragmentation model to treat the macroscopic bleaching of a suspension of AuNPs. The sample of AuNPs is obtained by laser ablation in an aqueous solution of fifth generation ethylendiamine-core poly(amidoamine) (PAMAM-G5) with the fundamental wavelength of the same laser at 1064 nm. The dependence of photobleaching on the laser pulse energy at 355 and 532 nm is studied and hence compared with the theoretical model. In particular, we discuss the role of heating and the interplay among pure thermal processes, e.g., melting and evaporation, thermoionic emission, and photon-assisted ionization. We show that, although the AuNP temperature can overcome the evaporation threshold in our range of investigation, the experimental curves disagree with a process of fragmentation mainly driven by heating. Our results highlight the role of photon-assisted transitions in AuNP fragmentation and allow us to discuss different regimes of fragmentation at different fluences and intensities. Moreover, we show that PAMAM-G5 plays a crucial role in our experiments.

We observed Forster resonance energy transfer (FRET) with a covalently linked donor-acceptor pair D-A consisting of two naphthalene groups acting as the donors and a benzofurazan group acting as the acceptor and adsorbed onto Ag or Au nanoisland films. The use of metal nanoisland films, which caused a strong enhancement of the Raman signal, permitted description of the adsorption mechanism onto the two metals. The intense fluorescence response of molecular adsorbates and the different behavior of the antenna on Ag and Au nanoislands are partly explained in terms of the radiating plasmon model.

Fifth generation ethylendiamine-core poly(amidoamine) (PAMAM G5) is presented as an efficient capping agent for the preparation of metal and semiconductor nanoparticles by ps laser ablation in water. In particular, we describe results obtained with the fundamental, second and third harmonic of a ps Nd:YAG laser and the influence of laser wavelength and pulse energy on gold particle production and subsequent photofragmentation. In this framework, the role of the dendrimer and, in particular, its interactions with gold clusters and cations are accounted.

Fifth generation ethylendiamine-core poly(amidoamine) (PAMAM G5)-capped-gold-nanoparticles were prepared by ps laser ablation in water, by using the third harmonic of a ps Nd:YAG laser. Post-irradiation with the same wavelength causes efficient photofragmentation of the nanoparticles. We performed a comparison with analogous tests carried out with 532 nm ps pulses. We found that, while 532-nm-fragmentation is a 2-photon photoelectric process, 355-nm-fragmentation is linear.

Gold nanoparticles (AuNPs) coated with the diacetylene henicosa-10,12-diyn-1-yl (DS9) disulfide were successfully prepared by direct synthesis in toluene solutions. The average size of the nanohybrid metal core was finely adjusted by manipulation of the preparative conditions in the diameter range from 1.6 to 7.5 nm, as determined by TEM characterization. The topochemical polymerization of DS9 chemisorbed onto the gold nanoclusters of different size was carried out in colloidal suspensions by exposure to UV radiation and the process was monitored by UV-Vis and Raman spectroscopies. The results showed that in these assemblies the monomer undergoes an intra-particle polymerization and that the dominant polydiacetylene phase present is ruled by the core size. The deposition of the photoirradiated colloids onto different substrates was found to leave the polydiacetylene conjugation unaltered.

This paper reports on the fabrication of self-assembled monolayers containing disulfide-functionalized diacetylenes. To this purpose, two diacetylene derivatives were synthesized: the novel 14-(9H-9-carbazolyl)tetradeca-10,12-diyn-1-yl disulfide (CDS9) and the henicosa-10,12-diyn-1-yl disulfide (DS9). Both monomers were employed to prepare two-dimensional self-assembled monolayers (2D-SAMs) by chemisorption on gold-sputtered platforms and three-dimensional self-assembled monolayers (3D-SAMs) on gold nanoclusters by direct synthesis in organic solvent. The chemisorbed diacetylenes were then converted to the polymeric form by exposure to UV radiation, and their morphological and spectroscopic properties investigated by several techniques. From SERS data it was inferred that both monomers show the same behavior, in that the highly conjugated blue phase was observed in the 3D-SAMs, while only the less extended red conformation was found in the corresponding 2D-SAMs.

We produced gold nanoparticles in water by picosecond laser ablation at 532 nm and 1064 nm. We used the amino-terminated dendrimer PAMAM G5 as stabilizing agent. At 532 nm, the production process exhibits a threshold that is about 10 times lower than at 1064 nm and rapidly saturates. The fast saturation is due to single photon absorption of the ablating beam and a possible further energy loss due to photofragmentation of existing nanoparticles.

We grew self-assembled monolayers (SAMs) of a novel carbazolyldiacetylene, CDS9, on Ag-coated glass substrates and demonstrated the topochemical UV polymerization of CDS9 monolayers by recording the Raman spectra of the organic film before and after UV exposure. We observed coexistence of the blue and red polymeric phases, usually present in polydiacetylenes. The features of the organic/metal interfaces of our samples were properly varied, in order to achieve surface enhancement of the Raman (SERS) response of the ultra-thin molecular layers.

An innovative setup to combine electrochemical and in situ surface X-ray diffraction (SXRD) measurements is described. This electrochemical cell has a different design from the other ones commonly used for X-ray diffraction studies. It allows the sample surface to stay always completely immersed into the solution under controlled potential conditions even during the SXRD measurements. The X-ray beam crosses the liquid (about 1 cm) and the cell walls. Because of the high X-ray energy, the beam attenuation is negligible and by an appropriate positioning of the detector arm slits it is possible to minimize the diffuse scattering induced by the liquid and cell walls in order to still detect the minima of the crystal truncation rods (CTRs). The liquid solution in the cell is managed by a special device, which allows the controlled exchange of the electrolyte solutions necessary in the electrochemical atomic layer epitaxy (ECALE) growth. The whole setup can be remotely controlled from outside the experimental hutch by a dedicated computer. As an example we report measurements on S layers deposited at underpotential on the Ag(1 1 1) surface, and on CdS films of increasing thickness.

UV polymerization of self-assembled monolayers of a novel carbazolyl-diacetylene (CDS9) chemisorbed on silver films was demonstrated by surface plasmon resonance (SPR) and surface enhanced Raman scattering (SERS) experiments. SPR tests performed during UV exposure permitted one to observe the growth of the absorption coefficient, associated with the formation of the polymeric backbone. The Raman spectra of polymerized monolayers exhibited the bands associated with the C=C stretching modes of the conjugated backbone, typical of the blue and red polymeric phases usually present in polydiacetylenes, with a clear predominance of the red form. Moreover, the strong surface enhancement of the Raman band corresponding to the aromatic C=C stretching modes suggested that carbazolyl groups arrange nearly perpendicularly to the metal surface. In contrast, the absence of a SERS signal in the region of conjugated C[triple bond]C bond stretchings confirmed a polymerization scheme with conjugated triple bonds nearly parallel to the plane of the metal.

Surface Enhanced Raman Scattering experiments were performed on self-assembled carbazolyl-diacetylene monolayers chemisorbed on smooth gold substrates and UV irradiated. The Raman enhancement was ensured by depositing silver colloidal nanoparticles, which strongly increase the surface roughness. The co-existence of two polymer phases, characterized by different degree of order in the polymeric backbone, was detected by SERS results, which suggest a polymerization scheme with conjugated triple bonds nearly parallel to the plane of the metal surface.