Carbon Nanotubes for Applications in Electronics, Catalysis, Composites and Nano-Biology (CANAPE) WP6 Health In vitro effects of nanosized particles

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Howard Preston
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1 LCVN Uni Montpellier CNRS Materials Science & Technology Carbon Nanotubes for Applications in Electronics, Catalysis, Composites and Nano-Biology (CANAPE) In vitro effects of nanosized particles P. Wick, P. Manser, J.P. Kaiser, P. Spohn S. Hasler, U. Tobler and (EMPA) S. Fiorito (Uni Montpellier) Materials Science and Technology Materials Biology nteractions

2 Objective of the Workpackage -To gain data on possible toxicity of nanotubes by in-vitro tests Tasks -Determine in how far a test like biocompatibility ISO test is valid to define NP toxicity -Assess key aspects of in vitro NP toxicity (e.g. cell-type and CNT-type specificity)

Swan) 2 batches (inclusive used catalysts) C6: distributor: Fluka MWCNT: Baytubes, Nanocyl and Cheap tubes Carbon

3 CNT evaluation Material selection (SWCNT, reference materials, additional forms of CNT) SWCNT: Yangtze tubes: 4 different qualities (incl. catalysts), Elicarb (T. Swan) 2 batches (inclusive used catalysts) C6: distributor: Fluka MWCNT: Baytubes, Nanocyl and Cheap tubes Carbon black: source S. Fiorito Reference material: Asbestos, 7 oxide particles including SiO 2 Material characterisation by partners (SEM-TEM, size distribution, Raman, etc.) Methods to disperse Np Cell tests

$CNT fractionation$ in surfactant CNTrm

4 CNT fractionation supernatant of CNT in surfactant CNTrm 1nm pellet of CNT in surfactant 2nm 2nm Surfactant: polyethylene sorbitan monoleate, Tween 8 (2µg/ml)

5 Cell cultures Montpellier macrophages monocytes endothelial cells epithelial cells A549 (lung fibroblasts MRC9) pleural mesothelium MSTO211H skin fibroblasts 3T3 EMPA

6 Material selection (SWCNT, reference materials, additional forms of CNT) SWCNT: Yangtze tubes: 4 different qualities (incl. catalysts), Elicarb (T. Swan) 2 batches (inclusive used catalysts) C6: distributor: Fluka MWCNT: Baytubes, Nanocyl and Cheap tubes Carbon black: source S. Fiorito Reference material: Asbestos, 7 oxide particles including SiO 2 Material characterisation by partners (SEM-TEM, size distribution, Raman, etc.) Methods to disperse Np Cell tests MSTO211H 3T3 Reaction: uptake early middle cell activity (MTT conversion) late proliferation (DNA) ISO1993-5

7 activity [% of control] By reducing SWCNT pellet the 28 degree of +++ dispersion the effects on 1 75 both parameters is increased SWCNT raw material DNA SWCNT purified DNA DNA MTT MTT MTT SWCNT conv. purified DNA MTT % SW-CNT wt % Ni wt % Y ++ + single, bundles + non-cnt mat. REM single, clusters clusters bundles bundled bundles appearance REM SWCNT effects on MSTO211H cells SWCNT: NIR spectroscopy Y,Ni: ICP OES μg/ml 7.5 µg/ml 15 µg/ml 3 µg/ml

8 Is a test like biocompatibility ISO test valid to define NP toxicity? DNA 3T3 2 2 Results are strongly dependent on cell type and DNA MSTO211H activity [% of control] SiO2 TiO2 TCP CNT rm CNT pellet CNT supern. SWCNT p. Elicarb ZrO2 Fe2O3 Cat (Fe) Ni Y CeO2 ZnO Asb SiO2 TiO2 TCP CNT rm CNT pellet CNT supern. SWCNT p. Elicarb ZrO2 Fe2O3 Cat (Fe) Ni Y CeO2 ZnO Asb measured parameter MTT 3T Answer on Task question: No! MTT MSTO211H activity [% of control] SiO2 TiO2 TCP CNT rm CNT pellet CNT supern. SWCNT p. Elicarb ZrO2 Fe2O3 Cat (Fe) Ni Y CeO2 ZnO Asb SiO2 TiO2 TCP CNT rm CNT pellet CNT supern. SWCNT p. Elicarb ZrO2 Fe2O3 Cat (Fe) Ni Y CeO2 ZnO Asb

9 general cell division organ sp. cell-type sp. specificity systemic secundary direct/indirect pathway acute delayed progr. Accumulation appearance (Sub)Toxicity stability activation/inactivation by cells/medium stable/unstable solubility hydro-/lipophyllic volatile insoluble/particles exposure short repeated chronic

10 Material selection (SWCNT, reference materials, additional forms of CNT) SWCNT: Yangtze tubes: 4 different qualities (incl. catalysts), Elicarb (T. Swan) 2 batches (inclusive used catalysts) C6: distributor: Fluka MWCNT: Baytubes, Nanocyl and Cheap tubes Carbon black: source S. Fiorito Reference material: Asbestos, 7 oxide particles including SiO 2 Material characterisation by partners (SEM-TEM, size distribution, Raman, etc.) Methods to disperse Np Cell tests MSTO211H 3T3, A549 Reaction: early middle late uptake NoEC, mechanism ROS, gene activity cell activity, adhesion, migration proliferation, apoptosis/necrosis

MWCNT Crude MW Catalyst SWCNT SWCNT Wetcake 1h SWCNT Crude SW Catalyst fluorescence measurement

11 Cell reaction (early): ROS formation 2,7 -Dichlorofluorescein 1 dependent 1 way Ctrl MWCNT MWCNT Wetcake cell seeding 24 h wash + CNTaddition dyeaddition ROS formation MWCNT Crude MW Catalyst SWCNT SWCNT Wetcake 1h SWCNT Crude SW Catalyst fluorescence measurement 6-24 minutes SWCNT and MWCNT induces ROS partly in a preparation Fluorescence [% of control] Sin1 6 Min 12 Min 18 Min 24 Min

12 Cell reaction (early): Gene activity SWCNTpurified ATP binding 16 7 DNA binding 8 11 GTP binding 2 1 Hydrolase 7 7 Metal ion binding Nucleid acid binding 6 8 Oxidoreductase activity 3 5 Control 1.5 fold 11 SiO 2 Asbestos SWCNTpur Tween SWCNTrm affects gene activity in a cell-type dependent way Protein and binding different from 4 12asbestos Tween8 Protein serine/threonine fold Signal transducer 3 - TFs 9 8 Transferase 12 5 Unknown Zinc ion binding p>.5; 1.5 fold 3 fold fold 3 14 MSTO / A549 # of differently expressed genes (p>.5; 32h after 15 μg/ml exposure)

13 Cell reaction (middle): cell adhesion and migration Cell A549 adhesion +CNT -CNT 3 µg/ml SWCNT rm for 3 days SWCNTrm affects cell adhesion & migration 6 velocity of MSTO211H cells with / without 15µg/ml SWCNTrm velocity (µm/15 min.) without CNT with CNT

14 Cell reaction (late): apoptosis / necrosis Apoptose A549 1% Nr Material 71 MWCNT 8% SWCNT and MWCNT affect necrosis and early apoptosis to a similar degree, but MWCNT is more toxic 72 MWCNT Wetcake 6% 73 MWCNT Crude 74 MW Catalyst 75 SWCNT 4% 76 SWCNT Wetcake 2% 77 SWCNT Crude 78 SW Catalyst % Necrosis Apoptose early living 15ug 3ug 15ug 3ug 15ug 3ug 15ug 3ug 15ug 3ug 15ug 3ug 15ug 3ug 15ug 3ug treatment period: 3 days

15 Cell reaction: NoEC, mechanism 12 MTT conversion (% of control) days contineous SWCNT treatment 6hr days SWCNT treatment 6 hr SWCNT days medium The shift of the concentration 12 effect relationship SWCNTrm (µg/ml) 1 depends on endpoint and SWCNT purity MTT conversion [% of control] hr days SWCNT treatment 6 hr SWCNT days medium SWCNT purified [µg/ml]

16 Summary -A ISO like test is not valid to define CNT toxicity -Nearly all endpoints are affected by SWCNT and MWCNT Cell tests MSTO211H 3T3, A549 Reaction: uptake early ROS, gene activity middle cell activity, adhesion, migration late proliferation, apoptosis/necrosis -The exact mechanism is still unknown

17 Summary: Recently published results of CANAPE and Thank you for your attention connected projects 1. Belyanskaya L, Manser P, Spohn P, Bruinink A, Wick P (27) The reliability and limits of the MTT reduction test for carbon nanotubes - cell interaction. Carbon 45, Bruinink A (28) In vitro toxicokinetics and dynamics: modelling and interpretation of toxicity data. In: Preclinical Development Handbook (ed. S. C. Gad), John Wiley & Sons, Inc. New York, pp Brunner TJ, Wick P, Manser P, Spohn P, Grass RN, Limbach LK, Bruinink A, Stark W (26) In vitro cytotoxicity of oxide nanoparticles: Comparison to asbestos, silica and the effect of solubility. Environ. Sci. Technol. 4, Fiorito S, Serafino A, Andreola F, Togna A, Togna G. (26) Toxicity and biocompatibility of carbon nanoparticles. J. Nanosci. Nanotechnol. 6: Helland, A.; Wick, P.; Koehler, A.; Schmid, K.; Som, C. (27) Reviewing the environmental and human health knowledge base of carbon nanotubes. Environmental health perspectives, 115, Kaiser JP, Wick P, Manser P, Spohn P, Bruinink A (27) Single walled carbon nanotubes (SWCNT) affect cell physiology and cell architecture, J Mater Sci: Mater Med, DOI 1.17/s Limbach LK, Wick P, Manser P, Grass RN, Bruinink A and Stark WJ (27) Oxidative stress in nanoparticle exposed human lung epithelial cells can be directly correlated to the materials catalytic activity. Env. Sci.Technol. 41, (Best paper in this journal in 27) 8. Wick P, Manser P, Spohn P and Bruinink A (26) In vitro evaluation of possible adverse effect of nanosized materials. Physica Status Solidi 243, Wick P, Manser P, Limbach LK, Dettlaff-Weglikowska U, Krumeich K, Roth S, Stark WJ, and Bruinink A (27) The degree and kind of agglomeration affect carbon nanotube cytotoxicity. Toxicology Lett. 168,

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