Materials Science & Technology
Harald F. Krug Empa, Department Materials Meet Life, St. Gallen, Switzerland
Functionalised clothing for the Monitoring of Body Functions
MedTech Materials for Osteointegration
Coating technology for Nanocomposites and Fibers Flame retardant
Materials & Additives
Material safety
Biopolymere Materials & Coatings
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 3
Link between Medicine and Toxicology
Bayer scientist Felix Hoffmann synthesized aspirin and heroin in the same month, and the company marketed the two drugs together. This advertisement shows Bayer's pre-1904 logo
Thomas Hartung, Johns Hopkins University
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 5
Nano for more then 100 Years
The Tumour Tissue is heated up to a maximum of about 49°C and Cells will be destroyed
Dr. A. Jordan, Charité Berlin
Dr. A. Jordan, Charité Berlin
Aminosilane coated Ironoxide Nanoparticles in aqueous Suspension (MagForce®) at a Concentration of 100 mg/ml Injected into the Tumour Tissue
Magnetic Nanoparticles for Tumour Therapy
P. Gröning / Empa / Nanotechnologie
Magnetic Nanoparticles for Drug Delivery
Magnetic Drug Targeting
P. Gröning / Empa / Nanotechnologie
C.L. Haynes (2010): The emerging field of nanotoxicology Anal. Bioanal. Chem 398, 587-588.
2011
http://www.empa.ch/plugin/template/empa/*/113719
http://www.dechema.de/studien-path-1,123212.html
http://ihcp.jrc.ec.europa.eu/our_activities/nanotechnology/nanoreport-10-11/JRC-EASAC-report.pdf
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 10
Nanomaterials – the great Uncertainty?
NANO
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 11
Three Nanotox-Principles
The Transport-Principle Limbach LK, Wick P, et al. (2007). Environ. Sci. Technol. 41:4158-4163
The Surface-Principle Nel et al. (2006) Science 311: 622-627 Oberdörster et al., (2000) HEI-Report 96
The Material-Principle
Krug & Wick (2011) Angew. Chem. Int. Ed., 50 (6): 1260-1278
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 12
Caveoli (Ø=100nm)
Size Matters!
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 13
Small Size but very large specific Surface
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 14
One Material – different Modifications and …
Carbon many more …… V2O3
ZnO
Pd
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 15
(Material)Surface Properties!
Xia et al. (2009) Annu. Rev. Public Health 30: 137-150
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 16
- + TiO2 CB CeO2 ZrO2 ZnO AlOOH 0
2
4
6
8 10
15
20
25
IL-8
Sec
retio
n (x
-fold
)
Same Size – Different Materials
0.5, 5 and 25 g/cm2
Ø: TiO2 10-20 nm Carbon Black (CB) 15 nm CeO2 20 nm ZrO2 10-25 nm ZnO 40 nm AlOOH 40 nm
Kuhlbusch T, Krug HF, and Nau K. (2009) NanoCare: health related aspects of nanomaterials. Karlsruhe, St. Gallen, DECHEMA e.V. http://www.nanopartikel.info/files/content/dana/Dokumente/NanoCare/Publikationen/NanoCare_Final_Report.pdf
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 17
Zinc-Story – essential or toxic on the Nano-Level? No difference between nano or bulk ZnO particles
Buerki-Thurnherr T, Xiao L, Diener L, Arslan O, Hirsch C, Maeder-Althaus X, Grieder K, Wampfler B, Mathur S, Wick P, and Krug HF (2012). In vitro mechanistic study towards a better understanding of ZnO nanoparticle toxicity. Nanotoxicology, online first
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 18
Variables Associated with Nanomaterial Samples
Variables Associated with Toxicity Test
Variables Associated with Biological Sample
Sample Purification to Remove Possible Biological Interferents
NIST/USA
Depending on desired endpoint, there is a wide variety of
toxicology tests each with their own set of variables
Cell Line: - Choice of Cell Line - Cell Line Identification - Age and Storage of Cell Line - Number of Passages - etc.
Sample Characterization of Raw Nanomaterial (in manufactured form): - Composition and Purity - Size - Shape - Agglomeration etc.
Sample Characterization of Raw Nanomaterial to Determine Biological Impurities - Endotoxin etc.
Dispersion in Biological Media under Appropriate Conditions for Simulated Route of Exposure: - Temperature - Humidity - Gas Concentration - Salinity etc.
Sample Characterization in Biological Media: - Size & Shape - Agglomeration - Protein coating etc.
Set of appropriate positive and negative controls to determine possible interferences with test
etc.
Control of Conditions under which Experiments are Performed: - Temperature - Humidity - Gas Concentration - Salinity - etc.
Variables Associated with in vitro Toxicity Testing (Locascio et al., 2011 in: Nanotechnology Standards Nanostructure Science and Technology, pp: 179-208, Springer
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 19
Pulskamp, K., Diabaté, S., Krug, H.F. (2007) Toxicol. Lett. 168: 58-74
The Paradox of Viability Assays
MTT-Assay
WST-Assay
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 20
Our findings strongly suggest verifying cytotoxicity data with at least two or more independent test systems for this new class of materials (nanomaterials). Moreover, we intensely recommend standardizing nanotoxicological assays with regard to the material used: there is a clear need for reference materials.
The Paradox of Viability Assays
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 21
Fullerene Paradox: Radical Scavenger
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 22
Fullerene Paradox: Oxidative Stress
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 23
C60 Fluka
Fullerene Paradox: Small Balls (C60) – Big Pitfalls
transfer THF/water
in THF
C60 in THF susp.
Suspension
start
after weeks
in water
Toxic Effect No Effect
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 24
C60 Fluka
Fullerene Paradox: Small Balls (C60) – Big Pitfalls
transfer THF/water
in THF
C60 in THF susp.
Suspension
start
after weeks
in water
Toxic Effect No Effect
Spohn P et al. (2009) Environ. Pollut. 157:1134-1139
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 25
Wrong Methods/Missing Controls How to judge an effect
Yang H, Liu C, Yang D, Zhang H, Xi Z (2009) J. Appl. Toxicol. 29:69-78
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 26
More Pitfalls and Flaws
Toxicologist Non-Toxicologist
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 27
High Dosage/ Concentrations
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 28
Excessive Delivery of Nanostructured Matter to Submersed Cells …….
Schematic illustrations of the conceivable interaction of insoluble particles with submersed cells grown at the bottom of a well, filled with an appropriate medium of height h. (A) Previously employed picture, (B) more appropriate concept discussed in this study. The number of particles in (A) and (B) is the same.
Wittmaack, K. (2011) ACS Nano, 5: 3766-3778
TiO2(80) particles (27 μg/mL)
At the quoted heights (h) of the media, the mass concentration converts to maximum achievable areal densities of (A-C) 8.4 μg/cm2 and (D-J) 25.2 μg/cm2 (corresponding thicknesses 21.5 and 64.5 nm)
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 29
0.5 mg/rat lung: no evidence for carcinogenicity
A minimum of 5 X 3 mg/rat lung, maximum 20 x 6 mg/rat lung by instillation carcinogenicity!
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 30
Calculated Overload
Rat lung weight: 0.9 g
Macrophage count: 1.200.000 0,5mg/lung 416 pg / cell: no effects (Yokohira et al., 2009)
15mg/lung 12.500 pg / cell
120mg/lung 100.000 pg / cell tumour induction (Roller, 2009)
3mg/lung 2.500 pg / cell reflects overload conditions
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 31
SOPs
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 32
Willbe available soon at www.nanoobjects.info and www.nanosafetycluster.eu
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 33
Harmonised(Standardised) Methods
V Viability
I Inflammation
G Genotoxicity
O Oxidative stress
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 34
www.nanoobjects.info www.nanopartikel.info
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 35
Exposure & Environment Foreign Entities vs. Endogenous Entities Don’t Believe a Single Method Method Reliability In Vitro in Vivo Correlation Nano-Specific Signatures REACH Challenges for Regulatory Activities Fibre Paradigm Revisited Crowns/Coronas out of Proteins, Lipids and …? How to define “Biopersistence”?
Future Challenges in Nanotoxicology
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 36
Future Needs of Nanotoxicology
The huge amount of new chemicals and expected new nanomaterials on the market raises the question for alternative methods for a first screening
In vitro methods should be reliable, robust, sensitive and predictive
Without a responsible process of marketing “nanotechnologies” will fail within the next decade
Support of (nano)toxicological activities/projects is continuously needed; risk characterisation and assessment is crucial
Comparability of Tox-studies is mandatory and should be achieved by use of standardised methods, reference materials and the appropriate controls in each experimental set-up
Materials Sci ence & Technolog y
Materials meet Life Materials Meet Life HF Krug, 19.04.2012, NanoDiaRA 37
HF Krug, 16.09.2010, NANO2010 Rom
KIT (NanoCare & DaNa): K. Nau
Materials-Biology Interactions (Empa): A. Bruinink T. Bürki (Thurnherr) M. Gasser C. Hirsch J.-P. Kaiser B. Kopf S. Lischer K. Maniura M. Rösslein M. Rottmar A. Schipanski P. Wick
Cooperations (Selection): NIST, USA NIMS / NIHS, JAPAN Karolinska Institute, Sweden KRISS, Korea
(Nano)Materials: S. Balaban – INT-CFN, Karlsruhe C. Feldmann – Uni Karlsruhe-CFN F. Hennrich – INT-CFN, Karlsruhe H. Hofmann – EPFL, Lausanne FHI Berlin University of Cologne, Germany University of Memphis, USA Bayer AG
Alumni (Nanotox): L. Belyanskaya P. Spohn K. Fischer J. Wörle-Knirsch
Funding: BMBF BAG/BAfU CCMX NCCR EU Industry KTI SNF