Dipl. Chem.

Mark Geppert

Center for Biomolecular Interactions, University of Bremen, GermanyCenter for Environmental Research and Sustainable Technology, University of Bremen, Germany

Accumulation of iron oxide nanoparticlesby cultured brain astrocytes

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

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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 (Fe3O4) Magnetite

– Iron(III)oxide (-Fe2O3) Hematite

(-Fe2O3) Maghemite

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Iron oxide nanoparticles

• Iron oxide nanoparticles consist of an iron oxide core surrounded by a certain a ligand shell.

• The core consists of magnetite (Fe3O4) or maghemite (-Fe2O3).

• 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)

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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

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Brain cells

PfriePfger & Steinmetz (2003) La Recherche

Neuron

Ependymal Cells

Myelin

Oligodendrocyte

Astrocyte

Synapse

Microglia

Neuron

Capillary

Ventricle

Pfrieger & Steinmetz (2003); modified

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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

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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

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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

Aqueousammonia solution

Black precipitate(magnetic Fe3O4-particles)

1.) washing with H2O

2.) boiling with HNO3 and Fe(NO3)3

3.) dispersion in H2O

Aqueous dispersion of-Fe2O3-nanoparticles

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Characterization of iron oxide nanoparticles

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

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Characterization of iron oxide nanoparticles

TEM images of the synthesized iron oxide nanoparticles

100 nm 20 nm

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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

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Cell viability

LDH: lactate dehydrogenase; FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

time of incubation (h)

6 24

extr

acel

lula

r LD

H a

ctiv

ity(%

of

initi

al L

DH

act

ivity

)

0

20

40

60

80

100

control without iron 100 µM FAC 100 µM Fe-NP1000 µM Fe-NP

***

***

*

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Iron accumulation

FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

37 °C

time of incubation (h)0 1 2 3 4 6

cellu

lar

iron

cont

ent

(nm

ol /

mg

prot

ein)

0

100

200

300

100 µM Fe-NP100 µM FACno iron

4 °C

0 1 2 3 4 6

100 µM Fe-NP100 µM FACno iron

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Temperature dependency

FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

Col 2 Col 2: -- Col 2: --

iron

accu

mul

atio

n ra

te(n

mol

/ (h

× m

g))

0

10

20

30

40

***

***

FAC Fe-NP

37°C 4°C 37°C 4°C

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Transmission electron microscopy (TEM)

2 µm 0.5 µm

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Energy dispersive X-ray spectroscopy (EDX)

TEM

FeFe

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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

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Effects of iron chelators

FAC: ferric ammonium citrate; Fe-NP: iron oxide nanoparticles

Iron chelators (500 µM): DFX: deferoxamine; FZ: ferrozine

Fe-NP

control DFX FZ

cellu

lar

iron

cont

ent

(nm

ol/m

g pr

otei

n)

0

50

100

150

200

250

300 FAC

control DFX FZ

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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!

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Acknowledgements

funding:

Prof. Dr. Ralf Dringen

Dipl. Chem. Michaela Hohnholt

Prof. Dr. Marcus Bäumer Dr. Ingo Grunwald

B. Sc. Linda Gätjen

Thank you for your attention!

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 -Fe2O3) behave paramagnetic with the difference, that every particle consists only of one magnetic moment.

• Magnetism of the particles is important for MRT-imaging.

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% KMnO4

• Reduction of iron with ascorbate and detection of ferrous iron with ferrozine (magenta-coloured complex)

X-Ray diffraction

Dynamic light scattering

Intensity weighted diameter distribution

Diameter (nm)

A(I)

1 10 100 10000.00

0.01

0.02

0.03

0.04

0.05

10 mM Fe-NP in water: Mean diameter = 38 nm10 mM Fe-NP + 100 mM Citrat: Mean diameter = 40 nm

TEM

Concentration dependency