Synthesis of Water Soluble Gold Nanoparticles and Evaluation of their Cytotoxicity

1. Synthesis of Water Soluble Gold Nanoparticles and Evaluation of their Cytotoxicity Annika Leifert1, Monika Fischler1, Fei Wen1, Ulrich Simon1, Yu Pan2, Willi Jahnen-Dechent2, Sabine Neuss3, Günter Schmid4, Wolfgang Brandau5 1 Inorganic Chemistry, RWTH Aachen University, Germany 2 Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University, Germany 3 Pathology, RWTH Aachen University, Germany 4 Inorganic Chemistry, University of Duisburg-Essen, Germany 5 Radiochemistry, University Hospital Essen, Germany NanoMed 2009 6th International Conference on Biomedical Applications of Nanotechnology Charité, Berlin – March 4 - 6, 2009

2. Outline State of the Art Concept Synthesis and Characterisation of Gold Nanoparticles (AuNPs) Size Dependent Cytotoxicity of AuNPs Mechanism of Cell Death Effect of Varying the Ligand Shell Oxidative Stress Gene Chip Investigations Conclusions A. Leifert

3. State of the Art Connor et al., Small2005, 1, 325-327: Cell line K562 (leukemia cells) Citrate- and biotin-capped AuNPs, 18 nm diameter: not toxic AuNPs are taken up by cells; probably in endocytic vesicles Tsoli et al., Small2005, 1, 841-844: Toxicity investigation of Au55/TPPMS on 11 different cell lines (cancer and healthy human cell lines) Toxicities in the range of cisplatin Model: interaction of AuNPs (1.4 nm) with DNA (major groove) N. Lewinski, V. Colvin, R. Drezek: Cytotoxicity of Nanoparticles, Small 2008, 4, 26-49: Few, partly non-systematically conducted and not comparable studies A. Leifert

4. I. AuNP-size II. Binding strength Au - L N P S III. Charge/Functionalities L SO3- NH3+ COO- Cytotoxicity of AuNPs depending on… Fig.: Knippers R.: "Molekulare Genetik", 6th Edition 1996, Georg Thieme Verlag Stuttgart - New York. A. Leifert

5. ii. i. AuNPs (in CH2Cl2) or ii. AuNPs (in H2O) Ligand exchange: AuNP / L* (in H2O) + L* (in H2O) Syntheses of AuNPs i. G. Schmid et al., Chem. Ber.1981, 114, 3634-3642. Ligand L*: TPPMS J. Turkevitch, P. C. Stevenson, J. Hillier, Discuss. Faraday Soc.1951, 11, 55-75. A. Leifert

6. UV/Vis spectroscopy: Plasmonic band (lmax) for NPs with  ≥ ~ 2 nm; dependent on size and surrounding of AuNPs EM: TEM, SEM Ligand characterisation: CHN, NMR, IR Evaluation of concentration: AAS Cell tests: 48 h incubation, MTT assay, determine IC50 values Cell lines: HeLa cervical cancer cells (HeLa) Sk Mel 28 melanoma cells (Sk-Mel-28) L929 mouse fibroblasts (L929) J774A.1 mouse macrophages (J774A1) Characterisation of AuNPs 20 nm 50 nm Au55(TPPMS)12Cl6 (1.4 nm) 100 nm UV/Vis spectra of TPPMS-stab. Au colloids Citrate-stab. Au colloids (15 nm) A. Leifert

7. Results M.Th. Y. Pan and Dipl.Th. A. Leifert AuNPs between 0.8 and 15 nm, phosphine-stabilised Estimation of IC50 by MTT assay after 48 h Size-dependent cytotoxicity of AuNPs: → Maximum of toxicity for AuNPs with  = 1.4 nm. Small2007, 3, 1941-1949. Ligand: TPPMS A. Leifert

8. Mechanism of Cell Death Necrosis vs. apoptosis: Double staining with propidium iodide and annexin V, flow cytometry Au1.2/TPPMS: after 48 h 90% of cells dead, ~ 60 % apoptosis (285 µM) Au1.4/TPPMS: after 48 h 100% of cells dead, > 80 % necrosis (90 µM) Untreated reference Staurosporine; 2 µM TPPMS-stab. AuNP, 1.4 nm; 90 µM TPPMS-stab. AuNP, 1.2 nm; 285 µM → Toxicity too quick for model of interaction with DNA and subsequent blocking of cell division A. Leifert

9. Mechanism of Cell Death A B C D Reversal of apoptosis by caspase inhibition with Z-VAD-fmk: • A) Reference: untreated HeLa cells • B) 500 µM Z-VAD-fmk • C) 31,25 µM Z-VAD-fmk • D) Reference: HeLa incubated with staurosporine (left panel) and Au1.4MS (right panel) • Caspase inhibitor Z-VAD-fmk inhibited staurosporine-induced apoptosis, but not Au1.4MS necrosis Fluorogenic caspase activity determination: • Caspase 3/7 activity: fluorogenic protease substrate; presented as relative fluorescence unit (RFU) • 0.2 µM staurosporine: enhanced caspase 3/7 activity • 50 µM Au1.4MS treated cells: low caspase 3/7 activity • Caspase 3/7 activity increased 6.5-fold in staurosporine treated HeLa but only 2-fold in Au1.4MS treated cells A. Leifert

10. N Cytotoxicity of AuNPs depending on… II. Binding strength Au - L II. Binding strength Au - L I. AuNP-size I. AuNP-size P S III. Charge/Functionalities L SO3- NH3+ COO- Fig.: Knippers R.: "Molekulare Genetik", 6th Edition 1996, Georg Thieme Verlag Stuttgart - New York. A. Leifert

11. Ligand Binding Strength and Oxidative Stress AuNPs with a mean diameter of 1.1 nm, GSH-stabilised (Au1.1GSH) Y. Negishi, Y. Takasugi, S. Sato, H. Yao, K. Kimura, T. Tsukuda, JACS 2004, 126, 6518-6519. 5 nm non-toxic; Au-S-binding strength or antioxidant effect of GSH? toxic Au A. Leifert

12. 3 h 37 °C Au1.4MS + GSH Au1.4GSH TPPMS Glutathione (GSH) Analytics of GSH-stabilised particles IR spectroscopy: 31P NMR spectroscopy: AuNPs (black): Signal at 2526 cm-1 missing (S-H) Au1.4MS Zeta potential measurement: Au1.4MS + GSH; centrifuged, washed, redispersed in diluted HCl A. Leifert

13. Inhibition of Toxicity with NAC, GSH and TPPMS NAC, GSH and TPPMS but not ascorbic acid can partially inhibit the cytotoxicity of 100 µM Au1.4MS: A) Untreated cells (C) B) C treated with Au1.4MS for 48 h C) C pre-treated with reducing agent (RA) for 3 h, washed and post-treated with Au1.4MS for 48 h D) Au1.4MS pre-treated with RA for 3 h, mixture added to cells for 48 h E) C pre-treated with RA for 3 h, then added Au1.4MS and incubated for 48 h F) RA mixed with Au1.4MS and mixture immediately added to cells and incubated for 48 h G) C incubated with RA for 48 h Reducing Agents: N-acetylcysteine (NAC) Glutathione (GSH) Ascorbic acid TPPMS A. Leifert

14. AuNPs cause Oxidative Stress CM-H2DCFDA fluorescent Flow cytometry determination of oxidative stress: • CM-H2DCFDA staining • Green: untreated HeLa cells (no oxidative stress) • Pink: HeLa cells treated with 0.3% H2O2 for 30 minutes (strong oxidative stress) • Violet: HeLa cells treated for 48 h with 1000 µM Au15MS • Orange: 1000 µM Au1.1GSH • Rest: HeLa cells treated with 100 µM Au1.4MS for 6, 12, 18, 24, and 48 h, respectively • Au1.4MS, but not Au15MS and Au1.1GSH, induced oxidative stress in HeLa A. Leifert

15. Gene Chip Results Hierarchical cluster analysis and heat map representation of differentially regulated genes in AuNP treated HeLa cells. • HeLa cells • Treated for 1, 6 and 12 hours with Au15MS (B1, B6, B12 for Big AuNP) or with Au1.4MS (S1, 6, 12 for Small AuNP) or left untreated (Con) • Gene expression was determined using Affymetrix Genechips • Gene array data were subjected to hierarchical cluster analysis. • Several genes differentially expressed with Au1.4MS: • stress related and inflammation related genes are up-regulated • cell cycle related genes are down-regulated Blue: transcript levels below median; white: transcript levels equal to median; red: transcript levels higher than median. A. Leifert 14

16. Conclusions Evaluation of impact of AuNPs on living organisms Parameters: NP-size, binding Au-L, functionalisation Syntheses and characterisation methods of AuNPs Size-Dependent Cytotoxicity of AuNPs Cell death mechanism (Au1.4MS): necrosis Effect of ligand shell: GSH-stab. AuNPs (1.1 nm) lot less toxic than Au1.4MS Effect of reducing agents: Modification of ligand shell (exchange for GSH & NAC, double layer for TPPMS); or Scavenger for radicals created by Au1.4MS? Au1.4MS induces oxidative stress Directly or as secondary effect in cell death? Further questions: Exact mechanism of toxicity? Toxicity controllable, e. g. inducing cancer cell selectivity? A. Leifert

17. Acknowledgements …Yu Pan, Prof. W. Jahnen-Dechent (Institute for Biomedical Engineering, UK Aachen) …Prof. U. Simon …the cooperation partners: - Prof. G. Schmid (IAC, University Duisburg-Essen) - Prof. W. Brandau (Radiochemistry, UK Duisburg-Essen) …the DFG (PAK 56) …the GK 1035 „Biointerface“ A. Leifert