The overall objectives of WP3 (Materials) were to provide a set of stable and well-characterized engineered nanomaterials (ENM) and to ensure the stability of ENMs in aqueous stock suspension until the toxicity experiment. A total of 31 engineered nanomaterials (ENM) was acquired for performing the toxicity studies in the end-user labs and thereby generating a complete set of data to feed and develop the prototype NanoSafety Classifier. The selection of ENM types was based on a pre-established set of selection criteria, which were:
- the selected ENM should exhibit a wide range of toxicities;
- the ENM should be used by European industry with likelihood of human exposure (intentional and non-intentional), or be relevant in the future;
- the selection should support European legislation initiatives.
Outcomes of the workpackage
The selection of ENM covered both 3D (nanoparticles) and 1D (nanotubes or nanorods) ENM. The ENMs were provided by NANOCYL or synthesized for the project by the project partners PlasmaChem, University of Bordeaux (UB), University of Manchester (UNIMAN) and CiC BiomaGUNE. Nine types of ENM selected for the project cover a wide range of relevant industrial applications of ENM:
- TiO2 particles 10-20 nm (pigment/colorant in paints, cosmetics, food);
- TiO2 rods 1:5 aspect ratio (similar applications/industries as TiO2 particles);
- CuO particles 10-20 nma (antifouling agent in paints and biocide in textiles);
- Au particles 3-5 nma (potential future use in nanomedicine for e.g. drug delivery, bioimaging);
- Au particles 10-20 nma (potential future use in nanomedicine for e.g. drug delivery, bioimaging);
- Ag particles 10-20 nma (antimicrobial agent in food packaging, biocide sprays);
- CdTe particles 3-5 nma (used in LED/solar cells/lasers, inkjet printing applications);
- Nanodiamonds’ particles 3-5 nma (additive in engine oils, used in plastic reinforcements);
- Multiwall Carbon Nanotubes (MWCNTs) 1:100 aspect ratio (composite materials, sporting goods).
The synthesis of ENM with the same core material in different sizes (3-5 and 10-20 nm Au particles) and shapes (TiO2 particles and rods) allowed investigating the role of size and shape of ENM on their toxicity. In order to study the influence of the surface properties, each ENM was available in 3 specific functionalized variants. The ENM were functionalized with carboxyl groups, ammonium groups or polyethylene glycol, thus yielding ENM with pH-dependent negative and positive charges, respectively, or improved hydrophilicity. Additionally, in the case of TiO2 particles and rods, CuO particles and MWCNTs the non-functionalized variants (i.e., with no functional groups on the particle surface) were also available, which meant an additional 4 ENM to use for toxicological testing.
The 31 ENM were distributed in powder or liquid suspension form to the project partners and dispersion procedures were developed. A range of dispersion protocols were developed optimized and standardized with respect to ultrasound power input and treatment time. They were tested as part of a quality assurance scheme under the leadership of the laboratories in charge of the work on dispersion-SOPs and basic characterisation of the ENM. Only by tightly complying with the developed protocols, it could be assured that the ENM suspensions produced in the different test laboratories of the ENM had the same characteristics. Such similar characteristics were crucial to allow integration of results from several partner-laboratories into the ENM Safety Classifier. The work conducted in the project highlights the need for standardized methodologies for dispersion of ENM in large scale toxicity studies involving several laboratories.
Physico-chemical characterization of the 31 ENMs was performed and the obtained metrics transferred to the ENM Safety Classifier. The physico-chemical characterisation of the ENM comprised basic characterisation using transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta-potential measurement for provision of particle primary size / particle shape, hydrodynamic size and surface charge, respectively.
The physico-chemical characterisation also included a more advanced characterisation stage, which was based on the combined expertise of WP3 partners. The metrics selected for advanced characterisation of the ENM comprised the study of impurities by inductively coupled plasma mass spectrometry (ICP-MS). The release of metal from the ENM was also determined by ICP-MS after dialysis. The ENM were largely non-soluble or did not release any metals within a 24 h test period, with the exception of release of Cu, Ag, Cd and Te from the CuO CdTe and Ag-PEG ENM variants, and a minute release of Fe from MWCNTs. The hydrodynamic diameters were determined by the combined size-separation/size-detection technique, differential centrifugal sedimentation (DCS), which demonstrated comparable sizes with those determined by DLS for especially the CuO ENM variants. The presence of the different functional groups was verified with different characterization techniques including X-ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA) and C,H,N elemental analysis.TGA demonstrated the total mass of functional groups/impurities represented in a few cases more than 50 % of the total mass of the ENM. This information should be taken in consideration when interpreting the toxicity studies. The results from XPS and Raman spectroscopies largely verified the presence of the functional groups on the ENM according to the planned scheme. In particular, FTIR provided highly detailed information not only on which functional groups were present, but also the side chains upon which they were bonded to the ENM cores.
Besides the acquisition and characterisation of the 31 ENM, WP3 aimed at understanding the behaviour of the dispersed ENMs in selected biological fluids and cell media. Dialysis experiments for determination of dissolution rates and nanoparticle tracking analysis (NTA) for studying changes of particle size distribution/settling were performed. In terms of dissolution, TiO2 ENM (both particles and rods) were found to be very stable in all tested media with very limited dissolution. CdTe ENM demonstrated the highest dissolution followed by the CuO nanoparticles. The role of the coatings on metal release from the test materials was difficult to determine as it was not always the same across the different media.
NTA data indicated that the nature of the coating may determine particle agglomeration in biological media. Despite this, it was difficult to describe a common agglomeration behaviour pattern for all tested ENM in the different media: the results suggested that there was a trend for the functionalized ENM to agglomerate more and form larger agglomerates compared to the core ENM. This trend was observed for most of the ENM in all media tested except for the M7 media, where the type of functionalization had no effect.
Although some patterns were observed (e.g., ENM dissolve more in the artificial saliva and artificial urine solutions; carboxylate-coated ENM form larger agglomerates in artificial urine), the occurring particle-to-particle and particle-to-media interactions are complex. From the current experiments it was concluded that the dissolution and agglomeration/aggregation of the particles is determined by the particle chemistry (both the core and the coatings), as well as the composition of the biological media. None of these factors is solely responsible for the behaviour of the ENM in biological media, and instead, both factors act together.
 Primary particle size