1. Importance of surface modification of silica nanoparticles, exposure conditions and particle uptake for cytokine responses in epithelial lung cells. NANOMAT Conference 2009 Lillehammer, 18 May 2009 Maurizio Gualtieri #, Tonje Skuland #, Tore-Geir Iversen ^, Marit Låg # and Magne Refsnes # # Norwegian Institute of Public Health, Oslo ^ National Radium Hospital, Oslo

2. OUTLINE 1.Introduction Definition of NPs Application Engineered nanoparticles (NPs) and health hazard 2.Materials and methods Cell line Silica NPs Exposure Conditions 3.Results Particle characterization Inflammatory effects Particle uptake 4.Conclusions

3. 0.01 0.1 1 2.5 10 µm 101001000250010000 nm Ultrafine particles and Nanoparticles (NPs)‏ Particulate matter, PM10 and PM2.5 Suh et al. (in press)‏ Nanoparticles: engineered particles with at least one dimension lower than 100 nm. Ultrafine particles: particles aero- dispersed with one dimension lower than 100 nm Aitken particles: particles in the air lower than 100 nm (physic of the atmosphere)‏ Definition of NPs

4. Application Coated SiNPs to target and repair injured guinea pig spinal cords SiNPs to polish tooth surfaces for caries prevention (Journal of Dental Research, Vol. 87, No. 10, 980-983) Nano Cancer Detector Nanoparticles for tumor immunotherapy: SiO2-nanoparticles DNA complexes Fungus makes SiNPs from sand Cream that creates a SiNPs network, which straightens and reinforces the skin matrix from the outside Why Silica Nanoparticles (SiNPs)? SiNPs used in solar panel to improve efficiency

5. Engineered nanoparticles (NPs) and health hazard SiNPs – health hazard? Borm and Kreyling 2004 NPs in lung: different deposition according to particles dimension Lungs are thus the primary target of inhaled NPs

6. Engineered nanoparticles (NPs) and health hazard SiNPs – health hazard? Epithelial cells stress promotes release of cytokines and other mediators  recruitment and activation of macrophages (release of cytokines, ROS). Recruitment of immune cells  inflammation  epithelial cell stress  possible chronic inflammation. Possible translocation of NPs and inflammation mediators  cardiac and other organ diseases

7. Materials and methods In vivo In vitro Human bronchial epithelial cell line, BEAS-2B Maintained in LCH-9 medium, 37C 5% CO 2 Silica Nanoparticles: plain SiNPs (30, 50 and 200nm)‏ rhodamine-SiNPs (50nm)‏ NH 2 -doped SiNPs (50nm)‏ COOH-doped SiNPs (50nm)‏ Particles suspension with and without Bovine Serum Albumin (BSA)‏ Cell treatment in LCH-9 media with or without BSA Dose curve response (12.5, 25, 50, 100 and 200 µg/ml) Surface-doping effects mRNA analysis and uptake

8. Results: particles characterization SiNPs stem solution was prepared without and with BSA (1,5%)‏ SiNPs were dispersed in LCH-9 media (100µg/ml) without and with BSA (0,1%) Analyses were performed with Light Dynamic Scattering (LDS) - Aggregation of SiNPs in stem solution without BSA, - Time-dependent aggregation of SiNPs in medium without BSA, - BSA in stem solution AND in the medium reduce SiNPs aggregation Effect of BSA on SiNP dispersion

9. Results: inflammatory effects Effect of BSA on cytokines release IL-6 and IL-8 were measured after 24 h of exposure. BEAS-2B cells were treated with 100µg/ml SiNPs with and without BSA in the medium or SiNPs stem solution - BSA in particles stem solution and in medium reduces IL-6 release - BSA added in particles stem solution and in medium completely reduces IL-8 release

10. Results: inflammatory effects Effect of BSA on cytokines release IL-6 was measured after 24 h of exposure. BEAS-2B cells were treated with different concentration of Rhodamine-SiNPs with and without BSA in the medium or stem solution - BSA does not influence the pro-inflammatory effects of Rhodamine-SiNPs - What is important to determine the biological effects of NPs? P. Aggarwal et al 2009 Advanced Drug Delivery Reviews 61 (2009) 428–437 -/- no BSA in SiNPs stem solution and medium +/- BSA in SiNPs stem solution but not in medium +/+ BSA in stem solution and in medium

11. Results: inflammatory effects IL-6 and IL-8 dose-response curve IL-6 and IL-8 were measured after 24 h of exposure. BEAS-2B cells were treated with SiNPs prepared with BSA in stem solution - 200nm SiNPs induce a dose- dependent IL-6 release higher than 30 and 50nm (200 nm > 30 nm > 50 nm)‏ - 200nm SiNPs induce a dose- dependent IL-8 release higher than 30 and 50nm (200 nm > 30 nm > 50 nm)‏

12. Results: inflammatory effects Effect of surface doping on cytokine release BEAS-2B cells were treated with 100µg/ml SiNPs with BSA in SiNP stem solution. IL-6 and IL-8 were measured after 24h of exposure. - Surface-doping modified 50nm SiNPs pro-inflammatory effects. IL-8 release induced by Rhodamine- SiNPs >> Rhod-Si-NH2 > plain Si- NH2 > Rhod-Si-COOH ~ Plain SiNPs > Plain Si-COOH

13. A Results: inflammatory effects Importance of SiNP uptake on cytokine release No cellular uptake of Rhodamine-Si without (A) and with (B) BSA at 45 min B Red  Rhodamine-Si nanoparticles; Blue  early endosome; Green  late endosome IL-8 mRNA expression increases after 60 min, uptake does not seem to be a key factor for Rhod-SiNP-induced cytokine expression

14. Conclusions - NPs stem solution properties determine NP behavior also in medium - Media formulation is also an important parameter for the final outcomes  protein corona and/or aggregation state of NPs? - SiNPs tested do not support the surface area paradigm  different properties at different dimensions? - SiNPs surface doping influences the pro-inflammatory potential - If not uptake, how do SiNPs induce cytokines release in exposed cells? Conclusions and open questions…

15. Thanks for your attention!!! Acknowledgments Tonje Skuland Edel Lilleaas Per Schwarze Founding Research Council of Norway

16. Importance of surface modification of silica nanoparticles, exposure conditions and particle uptake for cytokine responses in epithelial lung cells. NANOMAT Conference 2009 Lillehammer, 18 May 2009 Maurizio Gualtieri #, Tonje Skuland #, Tore-Geir Iversen ^, Marit Låg # and Magne Refsnes # # Norwegian Institute of Public Health, Oslo ^ National Radium Hospital, Oslo

17. Results: inflammatory effects Effect of cell culture plates coating on cytokines release Cells cultured on uncoated plates seem to be more responsive to SiNPs treatment. IL-6 and IL-8 were measured after 24 h of exposure. BEAS-2B cells were treated with 100µg/ml SiNPs with BSA in SiNP stem solution. Cells were cultured on plates coated or uncoated with collagen

19. Results: inflammatory effects With other nanoparticles (polystyrene) or molecules (TPA) the difference in coating is not important in resulting cytokines release