NP - NanoValid

Developing Reference Methods for Nanomaterials www.nanovalid.eu Date MARINA and NanoValid International Conference Event )tle 29 September 2015 OECD Conference Center 2, rue André Pascal, 5775 Paris Cedex 16, France Imaging and quantification of nanoparticle
uptake by ICP-MS based techniques Steﬃ Böhme Helmholtz Centre for Environmental Research (UFZ) Department Bioanalytical Ecotoxicology 1 Nanomaterial testing Developing Reference Methods for Nanomaterials •  Release into the environment inevitable, hence the hazard for environmental organisms has to be assessed •  Little consideration of nanoparticle distribution and internalisation processes Toxicity testing
Source:
www.yale.edu/
Development of suitable analytical detection, quantiﬁcation and imaging techniques 2 Nanomaterial testing Model test organisms Developing Reference Methods for Nanomaterials Danio rerio (zebraﬁsh embryo) (OECD guideline 212, 236, 305)
250µm Daphnia magna (OECD guideline 202) Nanomaterials NM Primary par/cle size Coa/ng (c) / stabilizer (s) Zeta poten/al Ag-‐NP 21 ± 8 nm PVP (c) -‐ 19 mV Au-‐NP 13 ± 1 nm Sodium citrate (s) -‐ 38 mV CuO-‐NP 22-‐25 nm Tetrasodium pyrophosphate (s) -‐ 64 mV ZnO-‐NP 10-‐15 nm -‐ + 34 mV Provided as powders or suspensions by project partners 500µm 3 Quantiﬁcation of NM uptake Developing Reference Methods for Nanomaterials + SEM/EDX Quantiﬁcation of total uptake amounts Semi-‐quantitative Imaging NP solution Control (time / concentration-‐dependent studies) •  Exposure •  Exposure •  Washing step •  Washing step •  Collection of 4-‐8 individuums / compartments for 1 sample •  Fixation of individuums •  Acid digestion •  Sectioning with a cryo-‐microtome –  AgNP, CuO-‐NP digestion with HNO3 (30%) –  40 µm thick sections –  AuNP, ZnO-‐NP digestion with aqua regia –  Placement on glass slides •  ICP-‐MS measurement •  LA-‐ICP-‐MS measurement 4 Semi-‐quantitative Imaging Laser Ablation (LSX-‐500, CETAC) Developing Reference Methods for Nanomaterials ICP-‐MS measurement (ELAN-‐DRCe, Perkin Elmer) Laser Ablation Parameters: 60 % laser energy 50 µm spot diameter 30 sec delay between spots Additional gas stream: Argon Spot raster Agarose gel External Calibration Böhme, S., Stärk, H.-‐J., Kühnel, D., Reemtsma, T. (2015): Exploring LA-‐ICP-‐MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebraﬁsh (Danio rerio) embryos. Anal. Bioanal. Chem. 407 (18), 5477 – 5485 Böhme, S., Stärk, H.-‐J., Meißner, T., Springer, A., Reemtsma, T., Kühnel, D., Busch, W. (2014): Quantiﬁcation of Al2O3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb-‐ICP-‐MS, LA-‐ICP-‐MS) and ﬂow cytometry-‐based methods. J. Nanopart. Res. 16 , 2592 5 Semi-‐quantitative Imaging Developing Reference Methods for Nanomaterials Exposure: 100 µg/l AgNP 24 hours Zebraﬁsh embryo (Danio rerio) Daphnia magna Visual image of organism section Das Bild
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LA-‐ICP-‐MS 2D-‐color plot Overlay pg / 50µm spot
250µm
500µm
Böhme, S., Stärk, H.-‐J., Kühnel, D., Reemtsma, T. (2015): Exploring LA-‐ICP-‐MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebraﬁsh (Danio rerio) embryos. Anal. Bioanal. Chem. 407 (18), 5477 – 5485 6 Quantiﬁcation of NM uptake over time Developing Reference Methods for Nanomaterials Ag-‐NP Au-‐NP Böhme, S., Baccaro, M., Schmidt, M., Potthoﬀ, A., Stärk, H.-‐J., Reemtsma, T., Kühnel D.: The chorion of the zebraﬁsh (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 7 Quantiﬁcation of NM uptake over time Developing Reference Methods for Nanomaterials Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu
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CuO-‐NP ZnO-‐NP Böhme, S., Baccaro, M., Schmidt, M., Potthoﬀ, A., Stärk, H.-‐J., Reemtsma, T., Kühnel D.: The chorion of the zebraﬁsh (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 8 Quantiﬁcation of NM uptake over time = Ratio of theoretical and experimental particle number NP r (egg) Das Bild
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Developing Reference Methods for Nanomaterials NM Coverage % ZnO-‐NP 0.8 ± 1.6 Ag-‐NP 14.8 ± 10.6 CuO-‐NP 23.7 ± 39.1 Au-‐NP 49.7 ± 14.3 Coverage is dependent on: Time Concentration Nanoparticle characteristics Böhme, S., Baccaro, M., Schmidt, M., Potthoﬀ, A., Stärk, H.-‐J., Reemtsma, T., Kühnel D.: The chorion of the zebraﬁsh (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 9 Proposed sequence of NM-‐organism interaction Developing Reference Methods for Nanomaterials Passage of NPs and ions through the chorion pores Pore blockage and formation of NP layer Sorption of NPs at the chorion surface NP and ion release into the inner mass of the ZFE by thinning process of the chorion Böhme, S., Baccaro, M., Schmidt, M., Potthoﬀ, A., Stärk, H.-‐J., Reemtsma, T., Kühnel D.: The chorion of the zebraﬁsh (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 10 Eﬀect correlation Eﬀect correlation Developing Reference Methods for Nanomaterials alive sublethal lethal AgNP pg / 50µm spot pg / 50µm spot pg / 50µm spot 24h exposure ng Ag / ZFE ng Ag / ZFE ng Ag / ZFE 15.1 ± 6.8 17.4 ± 1.7 33.3 ± 7.3 Böhme, S., Stärk, H.-‐J., Reemtsma, T., Kühnel D. (2015): Eﬀect propagation after silver nanoparticle exposure in zebraﬁsh (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Published online, DOI: 10.1039/c5en00118h 11 Eﬀect correlation Developing Reference Methods for Nanomaterials Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu wenig Arbeitsspeicher,
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The occurrence of toxicological eﬀects is connected to the internal silver concentration of the organism Böhme, S., Stärk, H.-‐J., Reemtsma, T., Kühnel D. (2015): Eﬀect propagation after silver nanoparticle exposure in zebraﬁsh (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Published online, DOI: 10.1039/c5en00118h 12 -‐  Development towards a 3D imaging intensity (cps) Outlook: -‐  Developments in the high resolution ﬁeld to visualise accumulation in ﬁne tissue structures or for single organs -‐  Method combination with SP-‐ICP-‐MS to distingish between ionic and particle forms Das Bild
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intensity (cps) Applications: -‐  Visualisation of elemental/particle distribution patterns -‐  Quantitative investigations on NM uptake -‐  Correlation to the occurrence of toxicological eﬀects Developing Reference Methods for Nanomaterials Das Bild
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Al2O3-‐NP eise über
intensity (cps) Summary -‐ Methodology Ag-‐NP Au-‐NP 13 Conclusion Developing Reference Methods for Nanomaterials •  Quantiﬁcation and visualization of Ag-‐NP, Au-‐NP, CuO-‐NP and ZnO-‐NP distribution -‐ Ag-‐NP / Au-‐NP / CuO-‐NP accumulation at or within chorion structures -‐ ZnO-‐NP higher accumulation for embryonic tissues -‐ comparable results for respective ionic substances accumulation dependent on chemical identity •  the chorion surface is not completly covered by NPs •  Sorption sequence for NP-‐chorion interaction proposed •  correlation of toxic eﬀects with particle distribution patterns possible 14 Developing Reference Methods for Nanomaterials Thank you for your attention! Joseﬁne Müller Hans-‐Joachim Stärk Rolf Altenburger Thorsten Reemtsma Dana Kühnel Matthias Schmidt Marta Baccaro Annegret Potthoﬀ Das
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E-‐mail contact: steﬃ[email protected] 15