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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
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 (Fe3O4) Magnetite
– Iron(III)oxide (-Fe2O3) Hematite
(-Fe2O3) Maghemite
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 (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)
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
Brain cells
PfriePfger & Steinmetz (2003) La Recherche
Neuron
Ependymal Cells
Myelin
Oligodendrocyte
Astrocyte
Synapse
Microglia
Neuron
Capillary
Ventricle
Pfrieger & Steinmetz (2003); modified
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
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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
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
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
10
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
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
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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
15
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
16
Transmission electron microscopy (TEM)
2 µm 0.5 µm
17
Energy dispersive X-ray spectroscopy (EDX)
TEM
FeFe
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
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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
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
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