CHARACTERIZATION OF NANOPARTICLES IN URINE FROM RATS EXPOSED TO TITANIUM DIOXIDE NANOPARTICLES
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1 CHARACTERIZATION OF NANOPARTICLES IN URINE FROM RATS EXPOSED TO TITANIUM DIOXIDE NANOPARTICLES Caroline Marie-Desvergne, Thomas Philippe, Muriel Dubosson, Catherine Campo, Michèle Bouchard, Denis Dième, Aurélien Viscardi, Véronique Chamel Mossuz Caroline Marie-Desvergne 1th November 216 Univ. Grenoble Alpes, CEA Tech LITEN, PNS, Medical Biology Laboratory, F-38 Grenoble, France 22 NOVEMBRE 216 CEA 1 AVRIL 212 PAGE 1
2 INTRODUCTION Adapted from Bergamaschi E. Journal of Nanomaterials 212 Biomarkers of exposure Biomarkers of effect INTERNAL DOSE - Exhaled particles in EBC - Circulating particles - Particle uptake - Elemental analysis - Protein modification - EFFECTIVE DOSE/EARLY EFFECTS - Lipid peroxidation in EBC - Oxidatively damaged DNA - Exhaled NO - Carbonyl compounds in EBC - Acute phase proteins - Clotting factors - Vascular adhesion molecules - PAGE 2
3 Characterization of nanoparticles in biological fluids Collaboration with the biomarkers/xenobitics and NP Unit from UdeM (Michèle Bouchard) Toxicokinetics studies in vivo Ag, TiO 2 Characterization of nanoparticles (NP) Better comprehension of biodistribution and elimination of NP Main question: particles/ions? Perfectly characterized exposure scenarios Case study - Intravenous exposure Sprague-Dawley rats: 1 mg/kg TiO 2 anatase 2 nm - 14-day kinetics study: focus on urine PAGE 3
4 CHARACTERIZATION OF NANOPARTICLES IN BIOLOGICAL FLUIDS Usual methods hardly combine elemental and size characterization ICP-MS: a reference method for elemental analysis No information in terms of particulate contents Evaluation of coupling strategies Single Particle ICPMS Electrothermal Vaporisation ICPMS in collaboration with PAGE 4
5 SINGLE-PARTICLE ICPMS Detection of nanoparticles and ions Discontinuous signal Constant acquisition Number of pulses Intensity of pulses Particle number concentration Size of particles PAGE 5
6 Frequency Frequency Size (nm) Particle concentration (part/ml) SP-ICPMS: from theory to practice Silver Nanoparticles 6 nm (34 part/ml,.4 µg/l) Scan time 1 s - Dwell time 1 µs LIMITATIONS for TiO 2 2 nm in urine - Various ratios ions/np 4 - Size of the NP: near the LOD ~ 2 nm 3 - Interferences of the matrix ,1,2,4,8,16,32,64 1,28 2,56 5,12 1 Increasing concentrations of ions from.1 to 1 µg/l NP Ions NP Ions Intensity (counts) Intensity (counts) PAGE 6 6
7 ELECTROTHERMAL VAPORISATION (ETV) - ICPMS - Graphite furnace up to 3 C - Selective evaporation by temperature programs - Transformation of metal-ions into volatile halogenides ICP-torch Transport tube ETV-Furnace Carrier gas + Reaction gas Bypass gas Power-Supply 4kW PAGE 7
8 Ti intensity (cps) Temperature ( C) ETV ICPMS Temperature ramp Evaporation Cleaning 1 Drying Time (sec) 25 ng Ti + TiO 2 2 nm In water - KED mode TiO Ti Ti48 Ti5 Ti Time (sec) PAGE 8
9 Ti Intensity (cps) Ti Intensity (cps) Ti Intensity (cps) ETV-ICPMS: CHARACTERIZATION OF URINARY SAMPLES ON THE BASIS OF ELEMENTAL ANALYSIS OF Ti Mean cumulative urinary excretion over 14 days: 3.6 ± 1. % ID Maximal excretion rate (µg/h) : 2 h post-iv with continuous excretion up to 14 days Ions 1 day post-iv NP Ti48 Ti days post-iv Ions Time (sec) days post-iv 2 1 Ions NP Time (sec) Time (sec) PAGE 9
10 Relative proportion (%) ETV-ICPMS: CHARACTERIZATION OF URINARY SAMPLES Composition of titanium urinary excretion Ions NP Time post-iv (days) PAGE 1
11 STEM-EDX explorations: 1 day post-iv Width ~ 1-2 nm Length 1 nm PAGE 11
12 TEM-EDS EXPLORATIONS Control urine + 1 µg/l TiO 2 2 nm anatase Shape of urinary agglomerates? - in accordance with data on the cutoff size for renal excretion (Choi et al. 27) : internal arranging to get through the endothelium? - de novo formation? As shown for silver (Van der Zande et al. 212, Juling et al. 216). PAGE 12
13 CONCLUSION METHODS - SP-ICPMS: + : size and elemental detection along with quantification, sensitivity, distinction of ions/particles under certain conditions. - : for NP > 2 nm, mono-elemental, influence of matrices. - ETV-ICPMS: + : Elemental detection along with quantification, distinction of ions/particles, no influence of the matrix, for liquid or solid samples. - : no size detection, sensitivity. IN VIVO STUDY - Application of ETV-analysis to more samples - Evaluation of the quantitative aspect of ETV-ICPMS - Exploration of samples originating from the early times of the kinetics, inhalation exposure, lower doses, different sizes.. - Comparison with a «no-particle» Ti exposure APPLICATION - Potential recommendations towards human biomonitoring PAGE 13
14 Thank you for your attention! Marie-Desvergne Caroline CEA 1 AVRIL 212 Commissariat à l énergie atomique et aux énergies alternatives Centre de Grenoble 3854 Grenoble Cedex T. +33 () F. +33 () DRT SPNS LBM 22 NOVEMBRE 216 Etablissement public à caractère industriel et commercial RCS Paris B
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