1. Dipl. Chem. Mark Geppert Center for Biomolecular Interactions, University of Bremen, Germany Center for Environmental Research and Sustainable Technology, University of Bremen, Germany Accumulation of iron oxide nanoparticles by cultured brain astrocytes

2. 2 Content Introduction –Iron oxide nanoparticles –Astrocytes Results –Synthesis and characterization of iron oxide nanoparticles –Application of iron oxide nanoparticles to cultured astrocytes Cell viability Accumulation of iron Summary

3. 3 Iron oxides 16 different iron oxides, hydroxides and oxidohydroxides have been described. The most important iron oxides are: –Iron(II)oxide (FeO)Wüstite –Iron(II,III)oxide (Fe 3 O 4 )Magnetite –Iron(III)oxide (  -Fe 2 O 3 )Hematite (  -Fe 2 O 3 )Maghemite

4. 4 Iron oxide nanoparticles Iron oxide nanoparticles consist of an iron oxide core surrounded by a certain a ligand shell. The core consists of magnetite (Fe 3 O 4 ) or maghemite (  -Fe 2 O 3 ). The ligands can be small organic molecules, polymers or proteins and are important for the stability of the nanoparticles. Iron oxide nanoparticles are superparamagnetic. Stroh et al. (2004)

5. 5 Applications for iron oxide nanoparticles Important potential applications for iron oxide nanoparticles for medicine and neurosciences are: –Contrast agents in magnetic resonance imaging –Targeted drug delivery –Elimination of tumors by magnetic mediated hyperthermia –Labelling of cells –Magnetic separation of cells

6. 6 Brain cells PfriePfger & Steinmetz (2003) La Recherche Neuron Ependymal Cells Myelin Oligodendrocyte Astrocyte Synapse Microglia Neuron Capillary Ventricle Pfrieger & Steinmetz (2003); modified

7. 7 Astrocytes Astrocytes are the most abundant cell type in the brain. Astrocytes have a variety of functions in the brain: –Metabolic support of neurons –Neurotransmitter uptake –Detoxification of xenobiotics –Protection of neurons against oxidative stress –Regulation of metal homeostasis

8. 8 Astrocytes Immunocytochemical staining of an astroglia- rich primary culuture. The characteristic marker protein (GFAP) is stained in green, the nuclei were stained with DAPI in blue. GFAP: glial fibrillary acidic protein DAPI: 4‘,6-Diamidio-2-phenylindole

9. 9 Synthesis of iron oxide nanoparticles Iron oxide nanoparticles were synthesized by coprecipitation of ferrous and ferric iron in aqueous media (modified from Bee et al., 1995). Further treatment with nitric acid and ferric nitrate leads to a stable aqueous magnetic ferrofluid. The yield of the synthesis was 78 ± 10% Aqueous solution of ferrous and ferric iron Aqueous ammonia solution Black precipitate (magnetic Fe 3 O 4 -particles) 1.) washing with H 2 O 2.) boiling with HNO 3 and Fe(NO 3 ) 3 3.) dispersion in H 2 O Aqueous dispersion of  -Fe 2 O 3 -nanoparticles

10. 10 Characterization of iron oxide nanoparticles Behaviour of an aqueous dispersion of iron oxide nanoparticles (ferrofluid) in a magnetic field.

11. 11 Characterization of iron oxide nanoparticles TEM images of the synthesized iron oxide nanoparticles 100 nm 20 nm

12. 12 Effects of iron oxide nanoparticles on cultured astrocytes Iron oxide nanoparticles were coated with an excess of sodium citrate and dispersed in physiological media. The following parameters were investigated after exposure of astrocyte-rich primary cultures to iron oxide nanoparticles: –Cell viability –Iron accumulation: 1.Time dependency 2.Temperature dependency 3.Effects of iron chelators

13. 13 Cell viability LDH: lactate dehydrogenase; FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

14. 14 Iron accumulation FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

15. 15 Temperature dependency FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

16. 16 Transmission electron microscopy (TEM) 2 µm0.5 µm

17. 17 Energy dispersive X-ray spectroscopy (EDX) TEM Fe

18. 18 Perl‘s staining A B C Perl´s stain for iron in astrocyte-rich primary cultures Fe-NP: iron oxide nanoparticles no iron 100 µM Fe-NP; 37°C 100 µM Fe-NP; 4°C

19. 19 Effects of iron chelators FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles Iron chelators (500 µM): DFX: deferoxamine; FZ: ferrozine

20. 20 Summary Iron oxide nanoparticles (Fe-NP) were synthesized via coprecipitation of ferric and ferrous iron with yields of about 80%. Fe-NP were coated with an excess of sodium citrate and dispersed in physiological media for cell culture experiments. Fe-NP were less toxic during longer incubation periods than ferric ammonium citrate (FAC), a soluble iron source Fe-NP were accumulated by the astrocytes in a time and temperature dependent manner. The presence of ferrous or ferric iron chelators did not affect the iron accumulation of Fe-NP. These results suggest, that astrocytes in culture are able to accumulate iron oxide nanoparticles!

21. 21 Acknowledgements funding : Prof. Dr. Ralf Dringen Dipl. Chem. Michaela Hohnholt Prof. Dr. Marcus BäumerDr. Ingo Grunwald B. Sc. Linda Gätjen Thank you for your attention!

23. Superparamagnetism Paramagnets increase their internal magnetization in an external magnetic field One can imagine a paramagnetic sample as many magnetic moments (displayed as small bar magnets in pictures) They are independent of each other and arrange in an external magnetic field If the magnetic field is turned off, they randomize by temperature (≠ferromagnetism) Very small particles of ferromagnetic substances (like  - Fe 2 O 3 ) behave paramagnetic with the difference, that every particle consists only of one magnetic moment. Magnetism of the particles is important for MRT- imaging.

24. Quantification of iron content in nanoparticles Quantification of the iron content according to the method of Riemer et al. (2004); Anal. Biochem. 331:370-375 Incubation with „iron releasing reagent“ over night at 60 °C –0.7-M HCl and 2.25% KMnO 4 Reduction of iron with ascorbate and detection of ferrous iron with ferrozine (magenta- coloured complex)

25. X-Ray diffraction

26. Dynamic light scattering 10 mM Fe-NP in water: Mean diameter = 38 nm 10 mM Fe-NP + 100 mM Citrat:Mean diameter = 40 nm TEM

27. Concentration dependency