The in vitro choice_M.Dusinska_2013


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The in vitro choice_M.Dusinska_2013

  1. 1. Testing strategy for nanomaterials Maria Dusinska No Norwegian Institute for Air Research Kjeller, Norway http:www. National coordinator for NanoSafety Health Effects Laboratory, GLP certified Barcelona, November 29, 2013
  2. 2. Nanotechnology is an technology that deals with structures ranging from approximately 1-100nm in at least one dimension Nanoparticles have larger surface/volume ratio and are potentially more reactive. Barcelona, November 29, 2013
  3. 3. Oxidised lipids, thiols, ROS Respiratory chain mitochondria Surface markers Cytokines Proteins RNA Cathepsins lysosome
  4. 4. Barcelona, November 29, 2013
  5. 5. Nanomaterials regulation To ensure sustainable development of nanotechnologies, urgent needs for regulation of nanomaterials is needed. EU already framed some regulation on nano-containing products: Nano-cosmetics: Article 16 of the Cosmetic Regulation (EC) 1223/2009 from 11.1. 2013, Industry must notify the Commission of cosmetic products containing nanomaterials. The Scientific Committee on Consumer Safety SCCS is performing risk assessments on cosmetics containing nanomaterials. French Registry for Engineered Nanomaterials - From 1. 1 2013 France requires mandatory registration of “substances with nanoparticle status” that manufacturers produce, import, distribute, or formulate (Articles L. 523-1 to L. 523-5 of the French Environmental Code). Until April 2013, 457 companies have made 1991 declarations
  6. 6. Physico-chemical properties The novel size-dependent properties of nanomaterials make them desirable in technical and commercial uses. Biological interactions depend on the physico-chemical properties Primary properties Secondary properties Aglomeration/aggregation Protein corona Size distribution Stability of dispersion Barcelona, November 29, 2013 Chemical composition Crystalline structure Size, Shape Surface composition Surface charge Surface area Purity/inpurities Porosity
  7. 7. NP characterisation. Primary properties U-Fe3O4, no coating 8±3nm Fluorescent 140nm OCFe3O4, coating 8±3nm Powder Water dispersion Water dispersion Anatase/rutile Unknown Ti, O TiO2 21nm Chem compos Nano SiO2 SiO2, 25 nm Fluorescen t SiO2, 50 nm Water dispersion Water dispersion Water dispersion Powder Spinel (octahedral) Spinel (octahedral) Amorphous Amorphous Amorphous C, H, O Fe, O Fe, O Si, O Si, O Si, O Not applicable 0.33 26 2.8 -2 -2 Not applicable Irregular Not applicable Oblong Oblong Round/ Round/ oblong Phase Crystal structure PLGA oblong TEM/EDX Particle concent (%) 1 Shape-TEM Crystallite size distributionTEM (nm) Surface areaBET (m2/g) Pore volumeBET (mL/g) Surface chemistry Zeta potential milliQ pH7 (mV) Irregular, rectangular 15-60 Not applicable 5-12 5-13 15-30 25-50 5-30 61 Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Uncoated Uncoated Oleate micelle coating Uncoated Not applicable Not applicable Uncoated 226 0.13 Not applicable Not applicable Uncoated -30.2 -43.4 -31.9 -2.8 - 20 - 22 0.7 Uncoated Free oleate (960 ppm), Na (26.000 ppm), Ca (1.300 ppm), K (730 ppm)
  8. 8. Biological test system In vitro vs in vivo 2D culture representing solid tissue Blood model: cells in suspension Barcelona, November 29, 2013
  9. 9. In vitro Blood: human blood cellsleucocytes, granulocytes, monocytes, etc. TK6 Outcome Blood Automation and validation Central nervous system Barrier transport Digestive system Genotoxicity Lung Oxidative stress Liver Inflammation/Immunotoxicity Vascular system Placenta Central nervous system : HCEC, EC219, Murine N11 microglial cells Mechanism studied ex vivo Liver: hepatocytes, kupffer cells and liver sinusoidal endothelial cells (LSEC), HepG2 Vascular: HCEC, EC219, ECp23, HL1 System Kidney: monkey kidney Cos-1 cells, human HEC in vivo Placenta: Placenta perfusion, BeWo cells Kidney Digestive system : Caco 2, CacoGonlet, CacoReady TM, Colon HT29 Lung: bronchial epithelial cell lines (16HBE, NCIH and Calu-3, and human alveolar type 2 cells A549, HBEC)
  10. 10. 1 min 30 min NP characterization Size distribution and stability of oleic acid coated iron oxide in various culture media Conc.: 0.25 mg/ml) Medium (Conc.: 0.25 mg/ml) Hydrodynamic diameter (nm) DMEM Very large agglomerates, DMEM +10% FBS Trimodal distribution, DMEM-HG Very large agglomerates, DMEM-HG +10% FBS 1 > 900 Size stability with time 1 < 10 min ~ 2 days > 2000 < 5 min Bimodal distribution, 36 and 153 ~ 3 days RPMI Trimodal distribution, 18, 73 and 232 ~ 2 days RPMI +10% FBS 1 18, 86 and 237 Bimodal distribution, 39 and 165 ~ 3 days DMEM- F12-HAM by DLS DMEM-F12-HAM +10% FBS Bimodal distribution, 31 and 132 ~ 3 days Bimodal distribution, 36 and 153 ~ 3 days
  11. 11. Interaction with assay components, presence of serum and problems with exposure conditions. Our as well as result from literature show that serum content in exposure media can influence NM uptake and toxic response. Forming of protein corona. Coating Coating change the behaviour and cellular uptake of the NPs. Uncoated iron oxide - uptake Coated iron oxide poor uptake Magdolenova et al, 2013 Nanotoxicology Barcelona, November 29, 2013
  12. 12. Biological effect depends on dispersion DP1 DP2 DP 1 with serum DP2: without serum The state of aggregation of NPs is important. 25 * TiO2 DP1 Tail intensity (%) DNA damage (comet assay) after 24h exposure of EUE cells to TiO2 NPs; 2 dispersions. 30 20 TiO2 DP2 * 15 10 5 1 0 by DLS 0 Magdolenova et al, JEM, 2012 0,12 0,6 3 TiO2 NPs (μg/cm2) 15 75
  13. 13. absorbance at 540 nm 1.2 MTT assay: without cells A 1 B 0.8 PLGA TiO2 0.6 0.4 0.2 Interference Nanomaterial interference with read out systems has been observed for colorimetric/fluorescens spectrophotometric assays 0 0 0.5 5 50 NP concentration (µg/ml) 200 (A) Uncoated and (B) oleic acid coated Fe3O4 NPs, PLGA-PEO NPs and TiO2 NPs. Interference observed: WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, 3H-Tymidine incorporation, automated cell counting, proinflammatory response evaluation (ELISA for GM-CSF, IL-6 and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, NO assays). Barcelona, November 29, 2013
  14. 14. NP properties and interferences with assays NP properties: Light adsorption Light scattering Aggregation/ Agglomeration Surface reactivity Dissolution Adsorption/Reaction with Assay reagents Problematic techniques: Problematic assays: Spectrophotometry Spectrofluorometry Flow cytometry Cell counting WST-1, MTT, NR, 3H-T, Cell proliferation, mBBr, DCF, Griess reagent… PI uptake… Biomolecules Chemistry Biochemistry Immunochemistry MTT, 3H-T… ELISA, LDH… Chemistry Biochemistry MTT, LDH… Guadagnini et al: Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests, Nanotoxicology 2013, accepted
  15. 15. Adoptation of Protocol for Cytokinesis Blocked Micronucleus assay OECD 487 Interference with cytochalasin B Cytokinesis block assay detects mutagenic / clastogenic and aneugenic effects and provides a measure of both chromosome breakage and chromosome loss. MN frequencies, multinucleated cells, apoptosis/necrosis and in vitro proliferation rates scored on the slides. Cos-1 cells exposed to TiO2 NP (UP7 dispersion a. Cyt B simultaneously with NPs; b. Cyt B added 2 hr after NPs; c. Cyt B added 24 hr after NPs % MN in binucleated cells protocol) in concentration 75µg /cm2 9 8 7 6 5 4 3 2 1 0 pos. ctrl Magdolenova et al., 2012 neg. ctrl a b c
  16. 16. Adoptation of Protocol for Cytokinesis Blocked Micronucleus assay OECD 487 Exposure conditions and time of the treatment • At least 24h exposure with NPs is important to complete cell cycle Cyt B must be added 24h and allowing endocytosis • Cytochalasin B must be added separately from NPs treatment allowing NPs to enter cells Suggested treatment: 24h exposure to NPs then add Cyt B for another 24h
  17. 17. The comet assay results of all NanoTEST NPs Only TiO2 and coated Fe3O4 showed genotoxic effect 35 OC-Fe3O4 U-Fe3O4 30 PLGA-PEO Fl-25 SiO2 Strand Breaks 25 Fl-50 SiO2 TiVedisp TiUPdisp 20 15 10 5 0 0 20 40 60 80 16HBE140 BeWo b30 Cos-1 HEK293 HCEC Human Lymphocytes TK6 Rat Hepatocytes Kupffer Dose (g/cm2) Cell-line/ nanomaterial evaluation of the effect
  18. 18. Final Remarks • • • • • • Both primary and secondary characterizations are crucial Level of serum influence behavior and uptake of NPs Dispersion protocol is critical Exposure conditions should represent in vivo situation Coating influence the behaviour and cellular uptake of the NPs Relevant positive and negative control should be always included to NPs testing • Concentration used and cytotoxicity data • The experimental outcome can be affected by the cell type used, by the toxicity read out system, by the exposure time, by the dispersion method and the dispersion stability
  19. 19. REACH regulation for genotoxicity In vitro Mammalian Chromosome Aberration Test OECD 473 In vitro Mammalian Cell Gene Mutation Test OECD 476 In vitro Sister Chromatid Exchange in Mammalian Cells OECD 479 NO Bacterial Reverse Mutation Test OECD 471 NO Sacharomyces cerevisiae, Gene Mutation Assay Mitotic OECD 480, 481 NO Recombination Assay DNA Damage and Repair, Unscheduled DNA Synthesis in OECD 482 NO Mammalian Cells in vitro In vitro Mammalian Cell Micronucleus Test OECD 487 In vitro Comet assay (Single-Cell Gel Electrophoresis) JaCVAM/ECVAM Cell Transformation assay Draft Guideline
  20. 20. And NanoTEST consortium
  21. 21. @qualitynano QualityNano: A pan-European Infrastructure for Quality in NMs Safety Testing Grant agreement n°: SP4-Capacities-2010-262163 Start and end dates: 1st February 2011 – 31st January 2015 Coordinator: University College Dublin, Ireland Prof. Kenneth A. Dawson, Website: NUID UCD | NHM | IOM | JRC | BFR | KIT | FUNDP | IST | UNIVLEEDS | NILU | HMGU | LMU | CIC | UU | ICN | DLO/RIKILT | WU | DGUV | TAU | VITO | SMU | TCD | FIOH | UOE | CNRS | INERIS | UoB | HWU | RIVM
  22. 22. QualityNano overview Transnational Access (TA) Networking Activities (NA) Funded access to Training modules and 15 European Best practice for nanosafety Nano-characterisation sites assessment Joint Research Activities (JRA) Development of tools and Protocols for nanosafety assessment
  23. 23. Transnational Access Sites
  24. 24. Transnational Access process 4 TA Technological categories:  A) Nanomaterial synthesis  B) Nanomaterial labelling & pre-processing  C) Nanomaterial characterization in situ & ex situ  D) Nanomaterial exposure assessment
  25. 25. Overview of the Calls 4th call over 80 applications Number of Applications granted in 1-3 Calls Call 1 Call 2 Call 3 Submitted 37 40 41 Eligible 36 40 38 Sent to Review 35 38 38 Granted 21 19 33 Success rate 60,0 % 50,0 % 80% 5th TA Call Deadline: 20st December 2013 ~ 4 monthly calls
  26. 26. Contact Details +353 1 716 2459 @qualitynano