Advanced physico-chemical characterization of engineered nanomaterials in nanotoxicology

The extensive use of engineered nanomaterials (ENM) in both industrial and consumer products is triggering a growing attention on the potential risk of ENM posed to human health and the environment. Despite the intensive toxicological investigations, both in vitro and in vivo, only few of them have embedded a solid characterization approach, including the study of ENM before, during and after toxicological testing. Within EU-FP7 (ENPRA) and national (Toxicological and environmental behaviour of nano-sized titanium dioxide) projects activities, a comprehensive characterization of both inorganic (n-TiO2, n-ZnO, n-Ag) and organic (multiwalled carbon nanotubes, MWCNT) ENM was carried out, updating and adding primary characterization data, investigating particle size, shape, crystallite size, crystalline phases, specific surface area, pore volume as well as inorganic impurities of concern. Electron microscopy, X-ray diffraction, BET method and Inductively coupled plasma- mass spectrometry or optical spectroscopy were the employed techniques. With regard to the secondary characterization of ENM, the study was divided in: (a) assessing the engineered nanoparticles (ENP) behavior in biological (0.256 mg ENP/ml) as well as in real and synthetic waters (environmentally realistic concentrations: 0.01, 0.1, 1 and 10 mg n-TiO2 P25/l) over different time interval (24 h in biological media instead of 50 h in water media) to mimic duration of toxicological tests, by means of Dynamic Light Scattering (DLS), analytical centrifugation and nephelometry; (b) evaluating the ENM biodistribution in a secondary target organ (i.e. mice brain) after intratracheally instillation of ENM (0, 1, 4, 8, 16, 32, 64 and 128 ug ENM/animal tested), achieved by a microwave-assisted digestion method, followed by ICP-MS analysis, after selecting inorganic elements (i.e. Ti, Zn, Ag, Al and Co) as tracers of ENM presence in biological tissues. To investigate the ENP behavior in biological media and ENM biodistribution in mice, both dispersion protocols of the selected ENP and analytical protocols for ENM detection after toxicological testing were provided. The study of ENP stability in biological media highlighted that the fetal bovine serum (FBS) is the main parameter affected the ENP behavior. Among biological media tested, the largest size distributions, immediately after sample preparation, were irecorded for n-TiO2 NRCWE-003 dispersions. n-ZnO NM-111 dispersions were the most stable (12% average demixing, simulating 24 h of real sedimentation), except for Ag NM-300, originally received as dispersion (<1% average demixing). As expected, the ENP sedimentation rates investigated in the biological medium without any stabilizer (i.e. RPMI), were the highest for the whole set of ENP tested. In general, the highest sedimentation rates were recorded for n-TiO2 NM-101 and n-Ag 47MN-03 dispersions (51% average demixing, simulating 24 h of real sedimentation). The study of the n-TiO2 P25 stability in waters showed that agglomeration and sedimentation of n-TiO2 were mainly affected by the initial concentration. Sedimentation data fitted satisfactorily (R2 average: 0.90; 0.74<R2>0.98) with a first- order kinetic equation. The settling rate constant, k, increased by approx. one order of magnitude by moving from the lowest to the highest concentration, resulting very similar especially for all dispersions at 1 (k = 8•10-6 s-1) and 10 mg/l (k = 2•10-5 s-1) n- TiO2, regardless the ionic strength and composition of dispersions. The results from ENM biodistribution underlined that the chemical composition and the particle size were the main parameters that influenced the ENM partitioning into organs. Ti from n-TiO2 samples with the smallest particle size distribution tested (80-400 nm and 4-100 nm) and Al from MWCNT samples were the only inorganic tracers detected in mice brain. The whole characterization approach and the implication of these results are discussed.