From THE DEPARTMENT OF BIOSCIENCES AND NUTRITION Karolinska Institutet, Stockholm, Sweden TOXICITY OF METAL AND METAL OXIDE NANOPARTICLES

The use of nanoparticles holds great promises in many technical as well as medical applications. However, development of new technologies, such as nanotechnology, is connected with uncertainties and risks. The same properties that from a technical point of view are beneficial may in other aspects be unwanted and harmful for both humans and the environment. In order to avoid unnecessary risks and facilitate the use of safe nanotechnology there is a need for adequate toxicological research, as well as risk assessments of nanoparticles and nanotechnologies. This thesis is mainly focusing on the hazards (toxicity) of nanoparticles, and more specifically metal and metal oxide containing nanoparticles. In paper I, the ability of different nanoparticles, as well as multi-walled carbon nanotubes (MWCNT), to induce a cellular response based on their material composition, was investigated. A high variation between the different particles to induce cytotoxicity, DNA damage and oxidative DNA lesions was observed, where CuO nanoparticles were the most potent. In paper II and III, the role of particle-size on cytotoxicity, DNA damage, mitochondrial depolarization and induction of oxidative DNA lesions was studied. Amongst a number of particle types, only Cu and CuO particles displayed clear sizedependent effects where the nanoparticles were more toxic than the micro-sized particles. In paper IV, the impact of different methodological settings, such as sonication and the use of serum in the cell medium when preparing nanoparticle suspensions, was investigated. Observations revealed that sonication of Cu nanoparticles caused decreased cell viability and increased Cu release compared to non-sonicated particles. Furthermore, serum in the cell medium resulted in less particle agglomeration and increased Cu release compared with medium without serum, but no clear difference in toxicity was detected. In paper III, IV and V, the degree of metal release from Cu, CuO and Ag nanoparticles and subsequent impact on particle toxicity, was investigated. Even though a high Cu release was observed within hours after suspending the particles in cell medium, a toxic response was dependent on intracellular particle uptake, via a so-called Trojan horse type mechanism. In comparison to the high toxicity observed for Cu and CuO nanoparticles, no DNA damage or cytotoxicity was observed after exposure to the Ag nanoparticles, which is likely to depend on low Ag release from the particles. In conclusion, a key property of metal and metal oxide nanoparticles is the release of ions facilitating a toxicological response. Via a so-called Trojan horse type mechanism the solid particles can facilitate uptake into cells and subsequently release toxic ionic species. LIST OF PUBLICATIONS I. Hanna L. Karlsson, Pontus Cronholm, Johanna Gustafsson, Lennart Möller. Copper oxide nanoparticles are highly toxic: A comparison between metal oxide nanoparticles and carbon nanotubes. Chemical research in toxicology, 2009, 21(9), 1726-1732 II. Hanna L. Karlsson, Johanna Gustafsson, Pontus Cronholm, Lennart Möller. Sizedependent toxicity of metal oxide particles-A comparison between nanoand micrometer size. Toxicology letters, 2009, 188(2), 112-118 III. Klara Midander, Pontus Cronholm, Hanna L. Karlsson, Karine Elihn, Lennart Möller, Christofer Leygraf, Inger Odnevall Wallinder. Surface Characteristics, Copper Release, and Toxicity of Nanoand Micrometer-Sized Copper and Copper(II) Oxide Particles: A Cross-Disciplinary Study. Small, 2009, 5(3), 389-399 IV. Pontus Cronholm, Klara Midander, Hanna L. Karlsson, Karine Elihn, Inger Odnevall Wallinder, Lennart Möller. Effect of sonication and serum proteins on copper release from copper nanoparticles and the toxicity towards lung epithelial cells. Nanotoxicology, 2011, 5(2), 269-281 V. Pontus Cronholm, Hanna L. Karlsson, Jonas Hedberg, Troy A. Lowe, Lina Winnberg, Karine Elihn, Inger Odnevall Wallinder and Lennart Möller. Intracellular Uptake and Toxicity of Agand CuO-Nanoparticles – a Comparison Between Nanoparticles and their Corresponding Metal Ions. Submitted 2012. Additional publications: 1. Karine Elihn, Pontus Cronholm, Hanna L. Karlsson, Klara Midander, Inger Odnevall Wallinder and Lennart Möller. Cellular Dose of Partly Soluble Cu Particle Aerosols at the Air-Liquid Interface Using an In Vitro Lung Cell Exposure System. Journal of aerosol medicine and pulmonary drug delivery, 2012 [Epub ahead of print] ! " #$%%$&'"&($)*++,%-&. ,%/0+&1),%2,*3"&4$*5%&6,)%78)9-&'$0)$&:$//5;8-&<,=5$&<>8? 283&$%? & @%98)&A? %8>$**&B$**5%? 8)"&18**&3837)$%8&? $3$98&$%? &C),/85%&5%/8)$;/5,%&,=&;,CC8)& ;,%/$5%5%9&%$%,C$)/5;*8+D&+0)=$;8E&$%? &38/$*&)8*8$+8&? 8C8%? 8%/&8==8;/+"&F%? 8)&=5%$*& C)8C$)$/5,%"&&