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UUptake and Metabolism of Iron Oxide
Nanoparticles in Cultured Brain Cells
Dissertation
Zur Erlangung eines Doktors in den Naturwissenschaften (Dr. rer. nat.)
Fachbereich 2 (Biologie/Chemie)
Universität Bremen
Charlotte Petters
2015
Erster Gutachter: Professor Dr. Ralf Dringen
Zweiter Gutachter: Professor Dr. Michael Koch
Datum der Verteidigung: 20.02.2015
Hiermit erkläre ich, Charlotte Petters, dass die vorliegende Doktorarbeit selbstständig
und nur unter Verwendung der angegebenen Quellen angefertigt und nicht an anderer
Stelle eingereicht wurde.
Bremen, January 2015
(Charlotte Petters)
TABLE OF CONTENT
I. Acknowledgements ................................................................................................. I
II. Structure of the thesis .......................................................................................... III
III. Summary ............................................................................................................... V
IV. Zusammenfassung .............................................................................................. VII
V. Abbreviations and symbols .................................................................................. IX
1. Introduction .......................................................................................................... 1
1.1. Brain cells .............................................................................................................. 3
1.1.1. Neurons ...................................................................................................................... 4
1.1.2. Astrocytes .................................................................................................................... 5
1.1.3. Microglia ..................................................................................................................... 6
1.1.4. Oligodendrocytes ........................................................................................................ 7
1.2. Culture models of brain cells ................................................................................. 8
1.2.1. Neurons ...................................................................................................................... 8
1.2.2. Astrocytes .................................................................................................................. 10
1.2.3. Microglia ................................................................................................................... 10
1.2.4. Oligodendrocytes ...................................................................................................... 11
1.2.5. Co-cultures and slice models.................................................................................... 12
1.3. References ........................................................................................................... 13
1.4. Publication 1 ........................................................................................................ 19
Uptake and metabolism of iron oxide nanoparticles in brain cells
1.5. Publication 2 ........................................................................................................ 23
Handling of iron oxide and silver nanoparticles by astrocytes
1.6. Aim of the thesis .................................................................................................. 27
22. Results .................................................................................................................. 29
2.1. Publication 3 ........................................................................................................ 31
Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles
2.2. Publication 4 ........................................................................................................ 35
Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells
2.3. Publication 5 ........................................................................................................ 39
Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells
2.4. Publication 6 ........................................................................................................ 43
Accumulation of iron oxide nanoparticles by cultured primary neurons
2.5. Publication 7 (manuscript) ................................................................................... 47
Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes
3. Summarizing discussion ....................................................................................... 79
3.1. Synthesis and characterization of fluorescently labeled iron oxide
nanoparticles ........................................................................................................ 81
3.1.1. Characterization of BODIPY-labeled iron oxide nanoparticles ............................ 83
3.1.2. Fluorescent properties of BODIPY-labeled iron oxide nanoparticles .................. 83
3.1.3. Stability of BODIPY-labeled iron oxide nanoparticles .......................................... 84
3.2. Uptake and effects of iron oxide nanoparticles in cultured brain cells ................. 86
3.2.1. Accumulation of iron oxide nanoparticles by cultured brain cells ......................... 87
3.2.2. Uptake of iron oxide nanoparticles in serum-free media ....................................... 90
3.2.3. Uptake of iron oxide nanoparticles in serum-containing media ............................ 91
3.2.4. Cellular localization of iron oxide nanoparticles ..................................................... 92
3.2.5. Toxic effects of an exposure of iron oxide nanoparticles to cultured brain
cells............................................................................................................................ 93
3.3. Implications of the results for the in vivo situation ............................................... 95
33.4. Future perspectives .............................................................................................. 99
3.4.1. Synthesis of fluorescently labeled iron oxide nanoparticles.................................... 99
3.4.2. Mechanism of accumulation of iron oxide nanoparticles under serum-free conditions .................................................................................................................. 99
3.4.3. Binding, uptake and fate of iron oxide nanoparticles in cultured brain cells ...... 100
3.4.4. Effects of an iron oxide nanoparticle exposure to cultured brain cells ............... 101
3.4.5. Investigations on uptake and effects of iron oxide nanoparticles in vivo ............ 102
3.5. References ......................................................................................................... 104
4. Appendix ........................................................................................................... 113
4.1. Curriculum vitae ................................................................................................ 115
4.2. List of publications ............................................................................................ 117
Acknowledgements I
II. ACKNOWLEDGEMENTS
At first and particularly, I want to thank Professor Dr. Ralf Dringen. Sincere thanks for
your constant support, help, advice and reliability throughout my thesis which made
working with you so comfortable. Your enthusiasm for science was impressive and I
really appreciate that your door was always open for all kind of questions and that
conversations with you made problems look far less dramatic.
Secondly, I want to thank Professor Dr. Michael Koch for being my second reviewer and
for the nice cooperation while writing the manuscript for the review.
I also want to thank the co-authors of my publications. Thank you for all your
collaboration and valuable comments and discussions. Especially I would like to thank
Dr. Ulf Bickmeyer for the opportunity to use the confocal laser scanning microscope.
Many thanks to all former and recent members of the Dringen working group. I want to
thank all of you for discussions of scientific and non-scientific subjects which made some
stressful days more enjoyable. Maria, it was great sharing the office with you. Thank you
for your open ear and the amusing conversations. Felix, thanks for your great
groundwork on the fluorescent nanoparticles but more importantly for being such a
pleasant and cheerful colleague who always spreads joy. Yvonne, I am grateful to all your
advice in the lab and for the talks which we could squeeze in between. Eva, thank you for
tolerating my visits in your office and for cheering me up. I also want to thank Dr. Maike
Schmidt and Dr. Eva M. Luther for all the fun at the microscope.
Furthermore, I want to thank my family for all the support throughout my life. And last
but not least, I sincerely thank Björn Peter. Thank you for all your encouragements and
mental distraction which made me going on.
Structure of the thesis III
III. STRUCTURE OF THE THESIS
The thesis contains the three main chapters introduction, results and summarizing
discussion. The introduction (chapter 1) is composed of two publications and chapters on
brain cells. The results (chapter 2) include four publications and one manuscript. The
publications are embedded as portable network graphics file made from portable
document format files while the manuscript was not accepted at time of submission of the
thesis and hence, is adapted to the style of the thesis. The results are discussed in
summary (chapter 3).
Figures and tables which are not part of a publication are numbered according the
respective main chapter followed by the running number.
Summary V
IIII. SUMMARY
Iron oxide nanoparticles (IONPs) are used in various biomedical applications and are
already applied in human therapy. Since IONPs can reach the brain, detailed knowledge
on uptake and effects of IONPs on neural cells is required. In the present thesis, IONPs
were synthesized and fluorescently labeled by attaching the green fluorescent dye
BODIPY to the coating material dimercaptosuccinate. When 1.5% of the thiol groups of
dimercaptosuccinate were functionalized, IONPs had identical physicochemical
properties to the non-fluorescent version of the IONPs and were therefore considered as
suitable fluorescent tool to study uptake and intracellular localization of
dimercaptosuccinate-coated IONPs.
Cellular uptake, localization and potential toxic effects of IONPs were investigated in cell
culture models of the four major neural cell types (neurons, astrocytes, microglia,
oligodendrocytes). In general, neural cell cultures efficiently accumulated IONPs which
led to an increase of the specific cellular iron contents to maximal levels of up to 3000
nmol iron per mg protein. The uptake of IONPs in cultured brain cells strongly
depended on experimental conditions such as time of incubation, IONP concentration,
temperature and on the absence or presence of serum. In the presence of serum, the
accumulation of IONPs was decreased by 80-90% in all cell types investigated compared
to serum-free conditions. Dependent on the cell type investigated, IONP uptake in
presence of serum was strongly lowered by known inhibitors of endocytotic processes
suggesting involvement of clathrin-mediated endocytosis and/or macropinocytosis. In
contrast, for IONP uptake in absence of serum the pathways involved remain to be
elucidated.
A direct comparison of cultured astrocytes, neurons and microglia revealed that microglia
were most efficient in IONP accumulation but also highly vulnerable to IONP exposure.
Microglial cell death was prevented by neutralizing lysosomes or by chelating iron ions,
suggesting that toxicity is mediated by rapid transfer of IONPs to lysosomes and fast
IONP degradation in the acidic environment which resulted in microglial death by iron-
mediated oxidative stress. In contrast to microglia, primary astrocytes, neurons and
oligodendroglial OLN-93 cells were not acutely damaged within hours upon IONP
exposure. However, at least neurons which had accumulated substantial amounts of
VI Summary
IONPs during a short time exposure suffered from delayed toxicity after removal of
exogenous IONPs.
The data presented in this thesis reveal that brain cells deal well with low amounts of
IONPs. However, higher iron contents after IONP exposure cause acute or delayed
toxicity in some neural cell types. Among the different cell types, especially microglia
were vulnerable to IONPs. Hence, concerning biomedical application of IONPs to the
brain one should consider protecting microglia from IONP-derived stress to reduce or
prevent potential adverse effects to the brain.
Zusammenfassung VII
IIV. ZUSAMMENFASSUNG
Eisenoxidnanopartikel (IONPs) finden eine breite Anwendung in der Biomedizin und
werden bereits für Therapien eingesetzt. Da IONPs das Gehirn erreichen können, ist
eine detaillierte Kenntnis über ihre Aufnahme in und Wirkung auf Gehirnzellen wichtig.
In der vorliegenden Arbeit wurden IONPs synthetisiert und fluoreszenzmarkiert. Dazu
wurde der grün-fluoreszierende Farbstoff BODIPY an das Hüllmaterial
Dimercaptobernsteinsäure angehängt. Die Menge an BODIPY in der Hülle hat einen
großen Einfluss auf die Stabilität. Nach Markierung von 1.5% der Thiolgruppen der
Dimercaptobernsteinsäure in der Hülle hatten diese IONPs identische physikochemische
Eigenschaften wie die nicht-markierten IONPs. Daher sind BODIPY-markierte IONPs
geeignet um die intrazelluläre Lokalisierung von Dimercaptobernsteinsäure-ummantelten
IONPs zu untersuchen.
Zelluläre Aufnahme, Lokalisierung und mögliche toxischen Effekte von IONPs wurden
in Zellkulturmodellen der vier Hauptzelltypen des Gehirns (Neurone, Astrozyten,
Microglia, Oligodendrozyten) untersucht. Im Allgemeinen akkumulierten neurale
Zellkulturen IONPs effizient, was zu einem Anstieg ihres spezifischen zellulären
Eisengehalts auf maximale Werte von bis zu 3000 nmol Eisen pro mg Protein führte. Die
IONP-Aufnahme in Gehirnzellkulturen hing stark von den experimentellen Bedingungen
wie Inkubationszeit, verwendete IONP-Konzentration, Temperatur und von der An-
oder Abwesenheit von Serum ab. In der Anwesenheit von Serum nahm die
Akkumulierung von IONPs im Vergleich zu serumfreien Bedingungen in allen
untersuchten Zellkulturen um 80-90% ab. Die IONP-Aufnahme in Anwesenheit von
Serum wurde durch bekannte Inhibitoren für endozytotische Wege stark verringert. Dies
weist darauf hin, dass abhängig vom Zelltyp Clathrin-vermittelte Endozytose und/oder
Macropinozytose an der IONP-Aufnahme in Gehirnzellen beteiligt sind. Im Gegensatz
dazu müssen die für die IONP-Aufnahme verantwortlichen Wege in Abwesenheit von
Serum noch aufgeklärt werden.
Ein direkter Vergleich von kultivierten Astrozyten, Neuronen und Microglia zeigte, dass
Microglia am effizientesten IONPs akkumulierten, aber auch besonders anfällig für
IONP-vermittelte Toxizität waren. Ihr Zelltod konnte durch eine Neutralisierung der
Lysosomen oder durch Chelatierung von Eisenionen verhindert werden. Dies lässt darauf
schließen, dass die Toxizität durch schnellen Transport der IONPs zu Lysosomen und
VIII Zusammenfassung
schnellen IONP-Auflösung im sauren Milieu, was zu eisenvermittelten oxidativen Stress
führt. Im Gegensatz zu Microglia wurden primäre Astrozyten, Neurone oder
oligodendrogliale OLN-93-Zellen durch IONP-Exposition nicht akut innerhalb von
wenigen Stunden geschädigt. Allerdings zeigten zumindest Neurone, welche substanzielle
Mengen IONPs während einer kurzen Inkubationszeit akkumulierten, eine verzögerte
Toxizität nach dem Entfernen von exogenen IONPs.
Die Daten der vorliegenden Arbeit zeigen, dass Gehirnzellen gut mit geringen Mengen an
IONPs umgehen können. Höhere Eisengehalte nach IONP-Behandlung können jedoch
zu akuter oder verzögerter Toxizität führen. Unter den verschiedenen Gehirnzelltypen
waren besonders Microglia anfällig für IONP-vermittelten Stress. Daher sollte für
biomedizinische Anwendungen von IONPs ein Schutz der Microglia in Betracht gezogen
werden um mögliche Schädigungen im Gehirn zu verhindern oder zu verringern.
Abbreviations and symbols IX
VV. ABBREVIATIONS AND SYMBOLS
°C degree Celsius % percent ζ zeta ADP adenosine diphosphate ANOVA analysis of variance APCs astroglia-rich primary cultures ASCs astroglia-rich secondary cultures ATP adenosine triphosphate ATPase adenosine triphosphatase Baf1 Bafilomycin A1 BBB blood-brain barrier bipy bipyridyl BP, BODIPY (FL C1-IA) N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-
3-yl)methyl)iodoacetamide BP-(D)-IONPs BP-labeled DMSA-coated IONPs CME clathrin-mediated endocytosis CNS central nervous system CS citrate synthase CvME caveolin-mediated endocytosis DAPI 4’,6-diamidino-2-phenylindole D-IONPs dimercaptosuccinate-coated IONPs DMEM Dulbecco’s modified Eagles medium DMSA dimercaptosuccinic acid DMT1 divalent metal transporter 1 Eds. editors EDX energy dispersive X-ray spectroscopy e.g. for example (Latin: exempli gratia) EIPA 5-(N-ethyl-N-isopropyl)amiloride etc. and so on (Latin: et cetera) FCS fetal calf serum FT-IR Fourier transform infrared spectroscopy G6PDH glucose-6-phosphate dehydrogenase GAPDH glyceraldehyde-3-phosphate dehydrogenase GCM glia-conditioned medium GFAP glial fibrillary acidic protein GR glutathione reductase GS glutamine synthetase GSH glutathione GSSG glutathione disulfide GSx total glutathione (GSH + 2 GSSG) GTPase guanosine triphosphatase
X Abbreviations and symbols
HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid HO-1 heme oxygenase-1 IB incubation buffer i.e. that is (Latin: id est) IgG immunoglobulin G IL interleukin IONPs iron oxide nanoparticles LDH lactate dehydrogenase LT lysotracker MAG myelin-associated glycoprotein MEM minimal essential media MFH magnetic fluid hyperthermia MP macropinocytosis MRI magnetic resonance imaging MT metallothionein MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide NAD(P)H nicotinamide adenine dinucleotide (phosphate) NPs nanoparticles P phagocytosis PBS phosphate-buffered saline PEG polyethylene glycol PVP polyvinylphenol PI propidium iodide ppm parts per million Rab Ras (Rat sarcoma) related in brain RME receptor mediated endocytosis RNA ribonucleic acid ROS reactive oxygen species RT room temperature SD standard deviation T temperature TEM transmission electron microscopy TNF-α tumor necrosis factor-α w/v weight per volume
1. IIntroduction
1.1. Brain cells .............................................................................................................. 3
1.1.1. Neurons ...................................................................................................................... 4
1.1.2. Astrocytes .................................................................................................................... 5
1.1.3. Microglia ..................................................................................................................... 6
1.1.4. Oligodendrocytes ........................................................................................................ 7
1.2. Culture models of brain cells ................................................................................. 8
1.2.1. Neurons ...................................................................................................................... 8
1.2.2. Astrocytes .................................................................................................................. 10
1.2.3. Microglia ................................................................................................................... 10
1.2.4. Oligodendrocytes ...................................................................................................... 11
1.2.5. Co-cultures and slice models.................................................................................... 12
1.3. References ........................................................................................................... 13
1.4. Publication 1 ........................................................................................................ 19
Petters C, Irrsack E, Koch M, Dringen R (2014) Uptake and metabolism of iron oxide nanoparticles in brain cells. Neurochem Res 39:1648-1660
1.5. Publication 2 ........................................................................................................ 23
Hohnholt MC, Geppert M, Luther EM, Petters C, Bulcke F, Dringen R (2013) Handling of iron oxide and silver nanoparticles by astrocytes. Neurochem Res 38:227-239
1.6. Aim of the thesis .................................................................................................. 27
Introduction 3
11. Introduction
1.1. Brain cells
Brain cells can be divided into neurons and glial cells (Peters and Connor, 2014; Figure
1). The glial cells are subdivided into astrocytes, oligodendrocytes and microglia. The
ratio of glia to neurons in the human brain is a matter of debate. While some reviews
state that glia, especially astrocytes, outnumber neurons by 10-fold without mentioning the
source (Allen and Barres, 2009; Sofroniew and Vinters, 2010; Verkhratsky and Parpura,
2010), other reports claim that the number of neuronal and non-neuronal cells are almost
equal with around 86 billion neurons versus 84 billion non-neuronal cells (Azevedo et al.,
2009; Herculano-Houzel, 2014). However, the glia/neuron ratio is quite diverse in
different parts of the brain such as cerebellum or cerebral cortex (Herculano-Houzel,
2014).
The following chapters will describe the physiology and function of neurons, astrocytes,
microglia and oligodendrocytes and the commonly used cell culture models of these cell
types.
Figure 1.1 The four major cell types in the brain: neurons (yellow), oligodendrocytes (blue), astrocytes (green) covering a blood vessel (red), microglia (violet).
4 Introduction
11.1.1. Neurons
Neurons were firstly described by Jan Evangelista Purkinje in 1839 (Raine, 2006) and
make up around 50% of the human brain (Azevedo et al., 2009; Herculano-Houzel,
2014). They are composed of a perikaryon or soma, a varying number of branched
dendrites and one axon which can be branched, thereby forming many synapses (Raine,
2006). The neuronal somata are located in the grey matter whereas the white matter
contains the axons (Raine, 2006). In general, neurons have the distinct function to
transmit electrochemical signals. The action potential is built at the axon hillock by the all-
or-none-law by the influx of sodium ions through voltage gated sodium channels, resulting
in a depolarization of the plasma membrane (Hille and Catterall, 2006). The signal is
transferred along the axon to the axon terminal where neurotransmitters stored in vesicles
are released by exocytosis into the synaptic cleft (Scalettar, 2006; Stevens and Williams,
2000). Subsequently, neurotransmitters bind to receptors at the postsynaptic membrane
of neighboring neurons and are cleaved (Chang et al., 1985; Silman and Sussman, 2005)
or taken up to prevent permanent excitation (Balcar and Johnston, 1972; Palmer et al.,
2003; Kondratskaya et al., 2010). The binding of the neurotransmitter to the receptor
leads to an either excitatory or inhibitory response of the postsynaptic neuron, thereby
facilitating or hinder formation of action potentials, respectively (Holz and Fisher, 2006;
Peters and Connor, 2014). The signal transduction is a highly energy consuming process
but the axon can be very elongated (up to 1 m). Hence, there are complex trafficking
mechanisms to transport mitochondria retrograde or anterograde along the axon (Sheng,
2014).
Neurons can differ regarding their morphology, location, function (motor, sensory) or
effect (excitatory, inhibitory) (Holz and Fisher, 2006; Guerout et al., 2014; Pasca et al.,
2014). For example, cerebellar granule neurons possess a small soma (6-8 μm) (Monteiro
et al., 1998; Raine, 2006) while the diameter of the soma of Purkinje cells is about 30 μm
(Floeter and Greenough, 1979; Takacs and Hamori, 1994). Beside the size, the number
and complexity of processes can differ extensively (Raine, 2006). Moreover, neurons can
specialize to sensory neurons possessing specific receptors which are activated by a certain
stimulus (Holz and Fisher, 2006) or to motor neurons which innervate muscles und
stimulate muscle cells via the neuronal end plate (Grumbles et al., 2012; Tanaka et al.,
2014). The effect of neurotransmitters depends on the receptor in the postsynaptic
membrane. Herein, one neurotransmitter can either excite or inhibit the postsynaptic
neuron (Holz and Fisher, 2006).
Introduction 5
11.1.2. Astrocytes
In the beginning of glial research, astrocytes were thought to give only structural support.
In the meanwhile, a variety of important astrocytic functions was discovered (Sofroniew
and Vinters, 2010) and due to their morphological and physiological heterogeneity it is
quite difficult to define what an astrocyte is (Kettenmann and Verkhratsky, 2011).
Astrocytes are named by their often star-like morphology (Oberheim et al., 2012). The
astrocytic end-feet cover the majority of the blood-brain barrier (BBB) which makes them
the first neural cell type to get in contact with compounds passing the BBB (Virgintino et
al., 1997; De Bock et al., 2014). They not only provide other neural cells with energy
substrates, they also play a key role in the homeostasis of ions, pH and water (Kimelberg,
2010; Sofroniew and Vinters, 2010) and they regulate the vasopressure in brain (De Bock
et al., 2014; Howarth, 2014). Moreover, they interact with neurons and modulate their
signal transmission. Considering the “tripartite synapse hypothesis”, the synapse is
composed of the axonal terminal, the synaptic cleft, the postsynaptic membrane and
additionally astrocytes in close proximity to the synapse. Due to expression of
transporters astrocytes can actively take up neurotransmitters released from the synaptic
cleft (Kettenmann and Verkhratsky, 2011; Roberts et al., 2014; Verkhratsky et al., 2014)
to prevent neurons from excitotoxicity (Oberheim et al., 2012) or they can release
gliotransmitters such as ATP, glutamate, serine or γ-amino butyric acid to modulate
signaling (Verkhratsky and Parpura, 2010; Zhuang et al., 2011; Lee et al., 2013; De Bock
et al., 2014). Astrocytes communicate with each other via intercellular calcium waves
through gap junctions and hence, can modulate synaptic activity of distinct synapses (De
Bock et al., 2014; Lallouette et al., 2014).
Due to their location, astrocytes are the first neural cell type which comes into contact
with xenobiotics crossing the BBB. Thus, they have also defense systems to metabolize
and export xenobiotics (Dringen et al., 2015) and to protect neurons from stress (Wilson
et al., 2000; Barreto et al., 2011; Genis et al., 2014). In case of cerebral injury, a glial scar
is formed by reactive astrocytes which are characterized by strong expression of glial
fibrillary acidic protein (GFAP) (Cregg et al., 2014; Liu et al., 2014) which is used as
specific astrocytic marker although not all astrocytes express GFAP (Kettenmann and
Verkhratsky, 2011).
6 Introduction
11.1.3. Microglia
The brain is described as immune privileged which means immune competent cells from
the blood are excluded from the central nervous system (CNS) (Kettenmann et al., 2011).
During early development mesodermal precursor cells enter the brain and differentiate
into microglia which are macrophage-like cells and represent the immune competent cells
of the CNS (Neumann and Wekerle, 2013; Nayak et al., 2014; Peters and Connor,
2014). Due to their phagocytic properties, they incorporate pathogens (bacteria, viruses,
fungi, parasites) invading the brain or they take up cell debris in case of apoptosis/necrosis
of neural cells (Eyo and Dailey, 2013; Nayak et al., 2014). In the healthy CNS, microglia
are “resting” and possess a ramified morphology while under pathological conditions they
get activated and turn into an amoeboid shape which was firstly described by Pío del Río-
Hortega (Wilms et al., 1997; Biber et al., 2014). This activation might be induced by
binding of certain compounds such as purines, released from injured neurons or glia, to
specific receptors in the microglial membrane (Eyo and Dailey, 2013; Bernier et al.,
2013). In the activated state, microglia secrete anti- and pro-inflammatory cytokines such
as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) or nitric oxide (Kettenmann et
al., 2011; Matsui et al., 2014; Pisanu et al., 2014).
An important feature of microglia is their mobility, including migration of the whole cells
and motility of the processes, thereby scanning their environment (Eyo and Dailey, 2013;
Nayak et al., 2014). Microglia make 5% of glial cells in the grey matter (Pelvig et al., 2008)
and they are in general regularly distributed within the brain possessing certain territories
(Milligan et al., 1991; Neumann and Wekerle, 2013). However, if microglia become
activated they guide surrounding microglia to the site of interference and start to
proliferate to increase the number of microglia at the damaged location (Nayak et al.,
2014; Marlatt et al., 2014). Besides their neuroprotective nature, microglia play also a role
during brain development, supporting the angiogenesis and controlling neuronal
apoptosis and synaptic homeostasis (Eyo and Dailey, 2013; Nayak et al., 2014).
Moreover, the activated state of microglia is discussed to be neurotoxic and associated
with neurodegeneration and aging of the brain, though this is still a matter of debate
(Biber et al., 2014).
Introduction 7
11.1.4. Oligodendrocytes
Oligodendrocytes are the most abundant glial cell type in the cerebral cortex (Pelvig et al.,
2008) and derive from oligodendrocyte precursor cells (Guerout et al., 2014; Pedraza et
al., 2014). The differentiation of these precursor cells into mature oligodendrocytes is
complex, involving loss of migration and proliferation and formation of processes (Bauer
et al., 2009; Bradl and Lassmann, 2010). The unique function of mature
oligodendrocytes is the myelination of neuronal axons by enwrapping the axons with
several layers of membrane (Derobertis et al., 1958; Aggarwal et al., 2011). A single
oligodendrocyte can ensheath 50 axons (Guerout et al., 2014) and completes its myelin
sheaths within few days (Watkins et al., 2008). The production of the myelin which is
composed of more than 70% lipids of the dry weight (Norton and Poduslo, 1973;
Aggarwal et al., 2011) consumes a lot of energy and oxygen which results in generation of
reactive oxygen species (ROS) (Bradl and Lassmann, 2010). As enzymes involved in
myelination depend on iron as cofactor, oligodendrocytes are the neural cells with the
highest iron content which makes oligodendrocytes vulnerable to oxidative stress caused
by elevated ROS production via the Fenton reaction (Thorburne and Juurlink, 1996;
Bradl and Lassmann, 2010).
The myelin sheath is interrupted in regular intervals of 100 μm (Kettenmann and
Verkhratsky, 2011), leaving the axon uncovered. These areas are called nodes of Ranvier.
Since the myelin sheaths electrically isolate the axon, no action potential can form but at
the nodes of Ranvier. This leads to saltatory signal conduction (Tasaki, 1939; Bradl and
Lassmann, 2010) and to a dramatic increase in the speed which can otherwise only be
achieved with very large unmyelinated axons with a diameter of 1 mm as in the giant
squid (Kettenmann and Verkhratsky, 2011). Besides the isolation of the axons, myelin
sheaths also influence neuronal structure and physiology such as axon diameter and
transport rates along the axon (Bradl and Lassmann, 2010) and provide the axon with
energy substrates such as lactate which is crucial for axonal health (Lee et al., 2012;
Funfschilling et al., 2012; Morrison et al., 2013; Rinholm and Bergersen, 2013).
8 Introduction
1.2. CCulture models of brain cells
Studying the metabolism of neural cells or effects of xenobiotics on brain is crucial to
understand the function and pathology of the brain but is simultaneously complicated due
to the complexity of the brain. Here, cell culture systems provide a convenient tool to
investigate a variety of metabolic parameters in the cell type of interest also from certain
brain regions under controlled culture conditions without the need of animal experiments
(Hansson and Thorlin, 1999; Lange et al., 2012). Yet, in vitro studies lack of the three
dimensional organization of the tissue, of cell cell interactions mainly between different
cell types and the environment within the living organism (Hansson and Thorlin, 1999).
Additionally, these systems are quite artificial such as culture medium which often has a
completely different composition than the cerebrospinal fluid. This divergence from the
in vivo situation has to be considered when interpreting the results. Nevertheless,
analyzing the effect of compounds on brain cells in culture give a hint to what might
happen in vivo (Sunol et al., 2008) and a lot of knowledge about brain cells was obtained
by cultured neural cells (Lange et al., 2012; Tulpule et al., 2014).
Most of brain cell cultures are obtained from rodents. There are diverse model systems
for the different brain cells, most of them are primary (deriving directly from brain tissue)
or secondary cultures (reseeded cells obtained from primary cultures). However, some
research is also performed on cell lines which have some advantages such as no need of
animals, easy culture techniques and nearly unlimited supply of cells due to cell division.
On the other hand, cell lines are immortalized, having often cancerous origin which might
imply an altered metabolism and different response to stimuli. Due to the different
culturing techniques (tissue derived cells, cell lines, days in vitro, medium composition,
coating of the plates etc.) it is important to use a cell culture system which is appropriate
for the question or purpose (Lange et al., 2012).
1.2.1. Neurons
Primary neuron cultures are mainly obtained from distinct regions of the brain (e.g.
hippocampus, cerebellum or cortex) rather than from the whole brain and might be
either prepared from embryonal or from 7 d old rodents which depends on the brain
region due to varying differentiation states (Sunol et al., 2008). Cultures of cortical and
cerebellar granule neurons have been shown to not differ from each other with respect to
respiration (Jameson et al., 1984) or chloride uptake (Sunol et al., 2008). In the present
Introduction 9
thesis, cultures of cerebellar granule neurons (Figure 2a) were used. They express
neuronal markers such as microtubule-associated protein 2 (Tulpule et al., 2014). Like
the majority of neurons in vivo, cultured primary neurons do not divide after seeding but
elongate their processes. As the yield of preparation of primary neuron cultures is
relatively low (Sunol et al., 2008), many research groups use the cell line PC12 or to a
lesser extent SH-SY5Y instead. They have rat phaeochromocytoma (Westerink and
Ewing, 2008) and human neuroblastoma (Xie et al., 2010) as origin, respectively and are
often used as a model for differentiating neurons but also to study metabolism and
toxicity in neurons (Xie et al., 2010; Westerink and Ewing, 2008).
FFigure 2.1 Cell cultures of brain cells used in the present thesis: primary cerebellar granule neurons (a), OLN-93 cells (b), astroglia-rich primary culture (c; from chapter 2.1) and primary microglial cells (d). Pictures were captured using an Eclipse TE-2000U (a, b, c) or TS-100 (d) microscope from Nikon (Düsseldorf, Germany). The scale bars represent 100 μm.
10 Introduction
11.2.2. Astrocytes
Astrocytes are frequently used as primary or secondary culture (Lange et al., 2012) while
C6 glioma cells are used a model for astroglioma cells (Barth and Kaur, 2009). Astroglia-
rich primary cultures (Figure 2b) are prepared from neonatal rodents (Hamprecht and
Löffler, 1985) and are contaminated by other glial cells (chapter 2.1; Saura, 2007; Yoo
and Wrathall, 2007). The heterogeneity of astrocytes within the living brain is challenging
for culturing astrocytes and interpreting the experimental outcome (Lange et al., 2012).
Already astroglia-rich primary cultures prepared from rats and mice respond differently
under the same culture conditions (Ahlemeyer et al., 2013). Cultured astrocytes possess
the same properties as astrocytes in brain such as glycogen formation (Dringen et al.,
1993), glutamate transport (Hertz et al., 1978) or storage of metal ions (Jones et al., 2012).
While astrocytes in vivo express the astrocytic marker GFAP only in activated state (Cregg
et al., 2014), cultured astrocytes are mainly GFAP-positive (Du et al., 2010). However,
this depends on culturing conditions (chapter 2.1; Goetschy et al., 1986; Codeluppi et al.,
2011). To explore astrocyte behavior in diseases, different in vitro models are used such
as scratch injury for trauma or oxygen glucose deprivation for ischemia (Yu et al., 1993;
Wu and Schwartz, 1998; Giffard and Swanson, 2005; Lange et al., 2012).
1.2.3. Microglia
There are two major preparation techniques described to obtain microglial cultures.
Mixed glial cultures contain besides astrocytes a certain amount of other glial cell such as
oligodendrocytes, ependymal cells and microglia and these contaminating cells are either
sitting on top of the astrocyte layer or underneath it (Saura et al., 2003; Yoo and Wrathall,
2007). Hence, microglia can be isolated by shaking the culture for a certain time to detach
the top layer microglia and subsequently, the microglia-containing medium is removed
and used for seeding into new culture plates (Tamashiro et al., 2012; Deierborg, 2013). In
the other method, the astrocytic layer is detached by trypsination and removed while the
bottom layer microglia remain in the plate (Saura et al., 2003; Figure 2d). As their in vivo
counterparts, cultured microglia are mobile (Jeon et al., 2012), express proteins for
immune response such as CD11b (Szabo and Gulya, 2013) and release nitric oxide if they
become activated (Saura et al., 2003). However, isolated microglia display in general
amoeboid morphology as seen for activated microglia in vivo while microglia in mixed
glial cultures are more ramified (Saura et al., 2003). Besides the described primary and
Introduction 11
secondary microglial cultures, cell lines such as BV2 or N9 which are derived from
murine microglia and immortalized by viruses are used as microglial model systems
(Stansley et al., 2012; Rodhe, 2013). These cell lines have the advantage that they
overcome the low yield problem of the primary cultures while possessing the same
microglial characteristics. Nevertheless, unlimited cell division might alter differentiation
and properties (Rodhe, 2013).
11.2.4. Oligodendrocytes
Cultured oligodendrocytes are obtained similarly to microglia by shaking off top layer
cells from astroglia-rich primary cultures (McCarthy and de Vellis, 1980; Barateiro and
Fernandes, 2014). While microglia are more easily detaching than oligodendrocytes,
there are often two shaking steps used to lower microglial contamination in the
oligodendrocyte culture (Barateiro and Fernandes, 2014). As observed for
oligodendroglial cells in vivo, also in culture these cells can be divided into
oligodendrocyte progenitor cells, pre-oligodendrocytes, immature and mature
oligodendrocytes, as characterized by morphology and expression of specific proteins
(Jarjour et al., 2012; Barateiro and Fernandes, 2014). Oligodendrocytic differentiation can
be provoked by e.g. thyroid hormone (Barateiro et al., 2013) or by the presence of axons
in co-cultures (Barateiro and Fernandes, 2014). Compared to oligodendrocytes in vivo,
cultured oligodendrocytes produce 500-times less area of myelin membrane (Jarjour et
al., 2012). Oligodendrocyte culturing methods are limited by a very low cell yield and the
fact that mature oligodendrocytes do not divide in vitro (Buntinx et al., 2003). Also here,
cell lines are helpful. Herein, human clonal cell lines such as HOG, MO3.13 and KG-1C
(Buntinx et al., 2003) as well as the rat cell line OLN-93 (Richter-Landsberg and
Heinrich, 1996) are available. Advantageously, OLN-93 cells (Figure 2b) are not derived
from a tumor as HOG and KG-1C but they are spontaneously transformed cells from
primary glial cultures (Richter-Landsberg and Heinrich, 1996). OLN-93 cells possess
markers for differentiated oligodendroglial cells such as galactocerebroside, myelin-
associated glycoprotein or myelin-basic protein though they resemble morphologically
more oligodendrocyte progenitors and are able to proliferate (Richter-Landsberg and
Heinrich, 1996).
12 Introduction
11.2.5. Co-cultures and slice models
Besides the conventional cell culture techniques described above there is emphasis to
mimic the in vivo situation more realistically. To allow and investigate interactions
between different neural cell types, co-cultures can be prepared (Jones et al., 2012;
Welser and Milner, 2012; Jarjour et al., 2012). This type of cell culture was very useful to
examine different aspects of interactions between cell types. For example, the release of
glutathione and extracellular degradation by astrocytes supplies neurons with the
precursors to synthesize glutathione intracellularly (Dringen et al., 1999). While
cerebellar granule neurons in vitro express glutamine synthetase, an enzyme in vivo solely
expressed in astrocytes (Norenberg and Martinez-Hernandez, 1979; Albrecht et al.,
2007), when deprived in glutamine, co-culturing with astrocytes reduced or abolished
glutamine synthetase expression in neurons (Fernandes et al., 2010). Moreover, the
formation of myelin by oligodendrocytes is enhanced by presence of astrocytes (Watkins
et al., 2008).
Additionally, ex vivo cultures of brain slices of around 200-500 μm thickness preserve the
three dimensional organization of the brain and have been successfully used to identify
the myelinating cell type in the CNS (Jarjour et al., 2012). To overcome the two
dimensional culture model, there are approaches to culture brain cells three
dimensionally on scaffolds (Kang et al., 2014; Lau et al., 2014; Weightman et al., 2014) or
as neurospheres which are free floating aggregates of neural stem cells (Brito et al., 2012;
Ghate et al., 2014). However, there will always be the drawback that human brain cell
cultures are rare and the conclusions based on results from rodent cell cultures to human
brain are hard to draw.
Introduction 13
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Introduction 17
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18 Introduction
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Introduction 19
1.4. PPublication 1
Uptake and metabolism of iron oxide nanoparticles in brain cells
Charlotte Petters, Ellen Irrsack, Michael Koch, Ralf Dringen
Neurochem Res (2014) 39:1648-1660
Contributions of Charlotte Petters
1st draft of chapters “Synthesis and properties of iron oxide nanoparticles” and “Uptake, metabolism and toxicity of iron oxide nanoparticles in cultured brain cells”
Preparation of figures 1-5 and table 1 Experimental work for figure 5 Improvement of the manuscript
1.4 Publication 1
Introduction 21
The pdf-document of this publication is not displayed due to copyright reasons. The publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-014-1380-5; DOI: 10.1007/s11064-014-1380-5.
22 Introduction
Introduction 23
1.5. PPublication 2
Handling of iron oxide and silver nanoparticles by astrocytes
Michaela C. Hohnholt, Mark Geppert, Eva M. Luther, Charlotte Petters,
Felix Bulcke, Ralf Dringen
Neurochem Res (2013) 38:227-239
Contributions of Charlotte Petters
First draft of “Conclusions and Perspectives” Table 2: Information on particle characteristics, culture, and research aspects
Improvement of the maunscript
1.5 Publication 2
Introduction 25
The pdf-document of this publication is not displayed due to copyright reasons. The
publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-012-
0930-y; DOI: 10.1007/s11064-012-0930-y.
Introduction 27
1.6. AAim of the thesis
The aim of this thesis is to investigate IONP uptake and localization of internalized
IONPs in brain cell cultures by using fluorescent IONPs. Firstly, fluorescently labelled
IONPs will be prepared and characterized for their physicochemical properties including
parameters such as size, stability and fluorescence. Furthermore, these fluorescent IONPs
will be compared with their non-fluorescent counterpart for their physicochemical
properties but also for their uptake into brain cells. Finally the characterized IONPs will
be used to compare the accumulation, localization and potential adverse effects of IONPs
on different types of culture brain cells.
2. RResults
2.1. Publication 3 ........................................................................................................ 31
Petters C, Dringen R (2014) Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles. Neurochem Res 39:46-58
2.2. Publication 4 ........................................................................................................ 35
Petters C, Bulcke F, Thiel K, Bickmeyer U, Dringen R (2014) Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells. Neurochem Res 39:372-383
2.3. Publication 5 ........................................................................................................ 39
Luther EM, Petters C, Bulcke F, Kaltz A, Thiel K, Bickmeyer U, Dringen R (2013) Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells. Acta Biomater 9:8454-8465
2.4. Publication 6 ........................................................................................................ 43
Petters C, Dringen R (2015) Accumulation of iron oxide nanoparticles by cultured primary neurons. Neurochem Int 81:1-9
2.5. Publication 7 (manuscript) ................................................................................... 47
Petters C, Dringen R Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes. Submitted
Results 31
2.1. PPublication 3
Comparison of primary and secondary rat astrocyte
cultures regarding glucose and glutathione
metabolism and the accumulation of iron oxide
nanoparticles
Charlotte Petters, Ralf Dringen
Neurochem Res (2014) 39:46–58
Contributions of Charlotte Petters
Design of the study Experimental work
Preparation of the first draft of the manuscript
2.1 Publication 3
Results 33
The pdf-document of this publication is not displayed due to copyright reasons. The publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-013-1189-7; DOI: 10.1007/s11064-013-1189-7.
Results 35
2.2. PPublication 4
Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells
Charlotte Petters, Felix Bulcke, Karsten Thiel, Ulf Bickmeyer, Ralf Dringen
Neurochem Res (2014) 39:372-383
Contributions of Charlotte Petters
Design of the study Experimental work for figures 1g,h, 2-6, table 1 and 2
First draft of the manuscript
2.2 Publication 4
Results 37
The pdf-document of this publication is not displayed due to copyright reasons. The
publication can be assessed at: http://link.springer.com/article/10.1007%2Fs11064-013-
1234-6; DOI: 10.1007/s11064-013-1234-6.
38 Results
Results 39
2.3. PPublication 5
Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells
Eva M. Luther, Charlotte Petters, Felix Bulcke, Achim Kaltz, Karsten Thiel, Ulf Bickmeyer, Ralf Dringen
Acta Biomater (2013) 9:8454-8465
Contributions of Charlotte Petters
Experimental work for figure 1 and table 1 First draft of text concerning synthesis and characterization of IONPs
Preparation of figures S1 and S2
2.3 Publication 5
Results 41
The pdf-document of this publication is not displayed due to copyright reasons. The
publication can be assessed at:
http://www.sciencedirect.com/science/article/pii/S1742706113002717; DOI:
10.1016/j.actbio.2013.05.022.
42 Results
Results 43
2.4. PPublication 6
Accumulation of iron oxide nanoparticles by cultured primary neurons
Charlotte Petters, Ralf Dringen
Neurochem Int 81:1-9
Contributions of Charlotte Petters
Design of the study Experimental work
First draft of the manuscript
2.4 Publication 6
Results 45
The pdf-document of this publication is not displayed due to copyright reasons. The
publication can be assessed at:
http://www.sciencedirect.com/science/article/pii/S0197018614002526; DOI:
10.1016/j.neuint.2014.12.005.
46 Results
Results 47
2.5. PPublication 7 (manuscript)
Lysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide
nanoparticles: comparison with neurons and astrocytes
Charlotte Petters, Ralf Dringen
submitted
Contributions of Charlotte Petters
Design of the study Experimental work
First draft of the manuscript
2.5 Publication 7 (manuscript)
Results 49
LLysosomal iron liberation is responsible for the high vulnerability of brain microglial cells to iron oxide nanoparticles: comparison with neurons and astrocytes
Charlotte Petters1, 2, Ralf Dringen1, 2
1Center for Biomedical Interactions Bremen, University of Bremen, PO. Box 330440, D-
28334 Bremen, Germany.
2Center for Environmental Research and Sustainable Technology, Leobener Strasse, D-
28359 Bremen, Germany.
Address correspondence to: Dr. Ralf Dringen Center for Biomolecular Interactions Bremen University of Bremen PO. Box 330440 D-28334 Bremen Germany Tel: +49-421-218- 63230 Fax: +49-421-218-63244 Email: [email protected] homepage: http://www.fb2.uni-bremen.de/en/dringen
Keywords: Nanotoxicology, Biocompatibility, Biochemistry
50 Results
AAbstract
Iron oxide nanoparticles (IONPs) are used for various biomedical and neurobiological
applications. Thus, detailed knowledge on the accumulation and toxic potential of IONPs
for the different types of brain cells is highly warranted. Literature data suggest that
microglial cells are more vulnerable towards IONPs exposure than other types of brain
cells. To investigate the mechanisms involved in IONP-induced microglial toxicity we
applied fluorescent dimercaptosuccinate-coated IONPs to primary cultures of microglial
cells. Exposure to IONPs for 6 h caused a strong concentration-dependent increase in the
microglial iron content which was accompanied by a substantial generation of reactive
oxygen species (ROS) and by cell toxicity. In contrast, hardly any ROS staining and no
loss in cell viability were observed for cultured primary astrocytes and neurons although
these cultures accumulated similar specific amounts of IONPs than microglia. Co-
localization studies with lysotracker revealed that in microglial cells, but not in astrocytes
and neurons, most IONP fluorescence was localized in lysosomes. ROS formation and
toxicity in IONP-treated microglial cultures were prevented by neutralizing lysosomal pH
by application of NH4Cl or Bafilomycin A1 and by application of the iron chelator 2,2’-
bipyridyl. These data demonstrate that rapid iron liberation from IONPs at acidic pH
and iron-catalyzed ROS generation are involved in the IONP-induced toxicity of
microglia and suggest that the relative resistance of astrocytes and neurons against acute
IONP toxicity is a consequence of a slow mobilization of iron from IONPs in the
lysosomal degradation pathway.
Results 51
IIntroduction
Iron oxide nanoparticles (IONPs) have gained substantial attention for biomedical
applications and are already used as contrast agent in magnetic resonance imaging (MRI),
for cancer treatment by hyperthermia or for targeted drug delivery (Li et al., 2013;
Mahmoudi et al., 2011; Maier-Hauff et al., 2011). Nanoparticles can enter the brain via
the intact or damaged blood-brain barrier, via the olfactory nerve or by direct injection
into brain in the case of hyperthermia treatment (Petters et al., 2014b). By such
applications all types of brain cells are likely to encounter IONPs in vivo.
In addition to neurons which are responsible for signal transduction, transmit information
along their axon and release neurotransmitters to other neurons (Peters and Connor,
2014; Scalettar, 2006), the brain contains various types of glial cells in substantial numbers
(Peters and Connor, 2014). Microglial cells are the immune competent cells of the brain
and clear the brain from cell debris and intruders (Nayak et al., 2014; Eyo and Dailey,
2013). In case of incident, microglial cells become activated, transform from their resting
ramified into their amoeboid form and migrate to the site of interference (Kettenmann et
al., 2011; Nayak et al., 2014). Astrocytes cover with their processes most of the brain
capillaries which form the blood-brain barrier (De Bock et al., 2014), supply other brain
cell types with nutrients (Bouzier-Sore and Pellerin, 2013; Stobart and Anderson, 2013),
are responsible for the homeostasis of ions, water and metals (Verkhratsky et al., 2014;
Dringen et al., 2013; Hohnholt and Dringen, 2013) and have important detoxifying
functions in brain (Fernandez-Fernandez et al., 2012; Dringen et al., 2015).
Several studies have investigated the uptake and biocompatibility of IONPs in culture
models for the different types of brain cell, including primary cultures of astrocytes,
neurons and microglial cells (for overview see: (Petters et al., 2014b)). However, data on a
quantitative comparison of the uptake and cytotoxicity of IONPs in different types of
brain cells are scarce. In brain cell culture systems, microglial cells have been reported to
take up fluorescent nanospheres and IONPs more efficiently than astrocytes and neurons
(Jenkins et al., 2013; Pinkernelle et al., 2012; Fernandes and Chari, 2014). In addition, in
co-cultures IONP uptake into astrocytes and oligodendrocyte precursor cells was lowered
in the presence of microglial cells (Pickard and Chari, 2010). In vivo, mainly macrophage-
like cells were found IONP-positive after injection of IONPs into human glioma (van
Landeghem et al., 2009), supporting the hypothesis that microglial cells have also in vivo a
high potential to accumulate IONPs. Macropinocytosis and clathrin-mediated endocytosis
52 Results
have been identified as molecular mechanisms involved in the IONP uptake by microglial
cells which direct the accumulated IONPs into the lysosomal degradation pathway
(Luther et al., 2013).
The strong accumulation of IONPs by microglia has been connected with a high
vulnerability of these cells towards IONPs (Jenkins et al., 2013; Luther et al., 2013) while
astrocytes (Geppert et al., 2012; Geppert et al., 2011; Jenkins et al., 2013) and neurons
(Sun et al., 2013; Petters and Dringen, 2015) appear to be relative resistant against acute
IONP toxicity. However, the mechanisms involved in the toxicity of IONPs for microglial
cells and the reason for the apparent resistance of astrocytes and neurons against IONPs
are currently unknown. To address such questions, we have exposed primary cultures of
microglia, neurons and astrocytes under identical experimental conditions to fluorescent
IONPs and directly compared the potential of the different neural cells for IONP
accumulation as well as the potential of IONPs to compromise cell viability. Here, we
report that cultured microglial cells accumulated low concentrations of fluorescent IONP
more efficiently than astrocytes and neurons while high concentrations of IONPs caused
severe ROS production and toxicity in microglial cells but not in astrocytes and neurons.
IONP-induced damage of microglial cells was prevented by neutralization of the acidic
lysosomal pH or by chelation of iron ions, suggesting that in microglial cells the rapid
lysosomal mobilization of iron from internalized IONPs causes accelerated ROS
formation and oxidative cell damage.
Results 53
MMaterial and methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) and minimal essential media (MEM)
were from Gibco (Karlsruhe, Germany). Fetal calf serum (FCS) and
penicillin/streptomycin solution were purchased from Biochrom (Berlin, Germany).
Bovine serum albumin and NADH were from Applichem (Darmstadt, Germany) and
Bafilomycin A1 was purchased from Invivogen (Toulouse, France). BODIPY FL C1-IA
(BP) [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
yl)methyl)iodoacetamide] and lysotracker red DND-99 were from Life Technologies
(Darmstadt, Germany). Other chemicals of the highest purity available were obtained
from Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland) or Sigma-Aldrich
(Steinheim, Germany). 96-well microtiter plates and 6-well cell culture plates were
purchased from Sarstedt (Nümbrecht, Germany) and Nunc (Wiesbaden, Germany),
respectively.
Synthesis and characterization of fluorescent IONPs
Fluorescent dimercaptosuccinate (DMSA)-coated IONPs were synthesized and
characterized as previously reported (Petters et al., 2014a). Briefly, maghemite IONPs
were generated by an alkaline co-precipitation method (Geppert et al., 2009) and the
IONPs were coated with BP-functionalized DMSA (Petters et al., 2014a). Single
fluorescent IONPs were spherical and had a size of 4-20 nm as seen by transmission
electron microscopy (Petters et al., 2014a). The fluorescent BP-DMSA-IONPs emit light
at 510 nm after excitation at 490 nm (Petters et al., 2014a).
The hydrodynamic diameter and the ζ-potential of IONPs in suspension were
determined by dynamic and electrophoretic light scattering in a Beckman Coulter
(Krefeld, Germany) DelsaTM Nano Particle analyzer at 25°C at scattering angle of 165° and
15°, respectively. Concentrations of IONPs refer to the total concentration of iron in
dispersion and not to the concentration of nanoparticles.
Cell culture
Microglial cultures were generated by tryptic removal of the astrocyte layer of astrocyte-
rich primary cultures in 6-well plates one day prior to experiments as described previously
(Luther et al., 2013). The remaining attached microglia were washed once with culture
54 Results
medium (90% DMEM, 10% fetal calf serum, 25 mM glucose, 1 mM pyruvate, 20
units/mL penicillin G and 20 μg/mL streptomycin sulfate) and incubated in 2 mL glia-
conditioned medium (GCM) before incubations with IONPs were started.
Immunocytochemical characterization of the microglial cultures revealed that more than
98% of the cells in these cultures are positive for the microglial marker protein CD11b
(Luther et al., 2013).
Astrocyte-rich primary cultures were prepared from newborn Wistar rats as described
recently in detail (Tulpule et al., 2014). Cells were seeded at a density of 375,000
cells/well in 6-well plates and used at day five in culture for experiments. Astrocyte-rich
cultures are strongly enriched in cells positive for the astrocyte marker protein glial
fibrillary acidic protein, contain some microglial and oligodendroglial cells but no neurons
(Petters and Dringen, 2014; Tulpule et al., 2014).
Cerebellar granule neuron cultures were prepared from brains of 7 d-old Wistar rats as
described recently in detail (Tulpule et al., 2014). The cells were seeded in neuron
culture medium (90% MEM, 10% heat-inactivated FCS, 25 mM KCl, 30 mM glucose and
2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) in a density of
1,875,000 cells/well in 6-well plates. Neurons were used for experiments after seven days
in culture. Immunocytochemical characterization of the neuron cultures revealed that
99% of the cells expressed the neuronal marker protein microtubule-associated protein-2
(Tulpule et al., 2014).
Experimental incubations
GCM is required to avoid toxicity of microglial cells in cultures (Saura et al., 2003). In
order to establish comparable incubation conditions for primary microglial cells, neurons
and astrocytes, all primary cultures were incubated in GCM. GCM was prepared as
previously described (Luther et al., 2013) by incubating astrocyte-rich primary cultures in
175 cm2 flasks for 1 d with 50 mL culture medium. The medium was harvested, filtered
through a 0.2 μm sterile filter and stored at 4°C for not more than three weeks (Luther et
al., 2013). In case of experiments on cerebellar granule neurons, the GCM had to be
supplemented with 25 mM KCl to prevent neuronal apoptosis (Contestabile, 2002).
To investigate acute consequences of IONPs on cultured primary neural cells, the
cultures were incubated at 37°C in a humidified atmosphere with 10% CO2 in 1 mL GCM
Results 55
without or with IONPs and/or other compounds as indicated in the legends of the figures
and tables.
After incubation, cells were washed once with 1 mL phosphate-buffered saline (PBS, 10
mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl) and lysed in 1 mL
1% (w/v) Triton X-100 in PBS to measure cellular lactate dehydrogenase (LDH) activity
or in 700 μL 50 mM NaOH to determine cellular protein and iron contents.
For cellular localization of fluorescent IONPs, cultures were incubated with 0.1 mM
IONPs for 6 h followed by 90 min incubation with 100 nM lysotracker DND-99 as
previously described (Luther et al., 2013). Cells were fixed with 3.5% (w/v)
paraformaldehyde in PBS and washed twice prior to analysis of cellular fluorescence by
microscopy.
Determination of cell viability and ROS production
The cell viability was investigated by determining the cellular LDH activity and the
cellular MTT reduction capacity as well as by propidium iodide (PI) staining. The cellular
LDH activity was determined as previously described (Tulpule et al., 2014; Dringen et al.,
1998). Aliquots of the Triton X-100 lysates were mixed with pyruvate and NADH and the
decline in absorbance at 340 nm was measured using a Sunrise-Basic microtiter plate
reader (Tecan, Grödig, Austria). As the presence of millimolar concentrations of IONPs
in the incubation medium strongly disturbed the photometric LDH measurement (data
not shown), cellular LDH activity and not LDH release from damaged cells was used to
investigate loss of membrane integrity. Incubation of cells with up to 3 mM IONPs did
not interfere with the measurement of the LDH activity in cell lysates while higher
concentrations did (data not shown).
The MTT reduction capacity was determined after a given incubation as described
previously (Scheiber et al., 2010). Briefly, the cells were incubated with 1 mL 1 mg/mL
MTT in GCM for 90 min, the formazan crystals dissolved in 1 mL dimethyl sulfoxide
and the absorbance at 540 nm was determined. Under the conditions used, the presence
of IONPs in cells did not affect the analysis of MTT reduction capacity (data not shown).
For PI staining, the cells were incubated with 1 mL GCM containing 5 μM PI and 10 μM
H33342 (Tulpule et al., 2014) for 15 min, washed once with PBS and monitored
immediately by fluorescence microscopy.
56 Results
Presence of cellular ROS was investigated by rhodamine staining. The fluorescent
rhodamine 123 is generated in cells by the ROS-dependent oxidation of
dihydrorhodamine 123 (Geppert et al., 2012). The cultures were washed with 1 mL
incubation buffer (20 mM HEPES, 1.8 mM CaCl2, 1 mM MgCl2, 5.4 mM KCl, 145 mM
NaCl, pH 7.4) and stained for 15 min with 1 mL 5 μg/mL dihydrorhodamine 123 and 10
μM H33342 in incubation buffer. After the incubation cells were washed once with PBS
and monitored immediately for fluorescence.
Determination of protein and iron content
The protein content of the cultures was determined by the Lowry method (Lowry et al.,
1951) with bovine serum albumin as a standard. The iron content of IONP-treated
cultures was measured by a modified ferrozine-based iron assay (Geppert et al., 2009).
Briefly, 100 μL cell lysate was mixed with 100 μL 10 mM HCl and 100 μL of a solution
containing 2.25% (w/v) KMnO4 and 0.7 M HCl. After incubation at 60°C for 16 h, 30 μL
of a mixture of 1 M ascorbate, 2.5 M ammonium acetate, 6.5 mM ferrozine and 6.5 mM
neocuproine in water was added and the absorbance at 540 nm was measured after 1 h.
The iron content determined for a sample was normalized on the protein content of the
respective sample.
Fluorescence microscopy
Stained cells were monitored for fluorescence using an Eclipse TE-2000U fluorescence
microscope with a DS-QilMc camera (Nikon, Düsseldorf, Germany). The following filter
sets were used for monitoring the BP-signal of fluorescent IONPs (excitation at 465-495
nm, emission at 505-515 nm, dichromatic mirror at 505 nm), for the H33342 staining
(excitation at 330-380 nm, emission at 420 nm, dichromatic mirror at 400 nm) and for
visualizing the staining by PI, rhodamine and lysotracker DND-99 (excitation at 510-560
nm, emission at 575 nm, dichromatic mirror at 590 nm).
Presentation of data
Quantitative data shown in figures and tables are means ± SD of values that were obtained
in experiments that had been performed on n independently prepared cultures.
Microscopic images are from representative experiments that were reproduced twice on
independently prepared cultures with similar results. Signal intensities were increased
using Adobe Photoshop 7.0 for better contrast. Significance of differences between
groups of data was analyzed by ANOVA followed by the Bonferroni post-hoc test.
Results 57
Analysis of significance of differences between two groups of data was done by the
unpaired t-test. p>0.05 was considered as not significant.
58 Results
RResults
1. Characterization of iron oxide nanoparticles
Dispersions of BP-DMSA-coated IONPs in GCM which contained 10% FCS were
colloidally stable. Compared to water dispersions, the IONPs dispersed in GCM had an
increased hydrodynamic diameter, a broader size distribution as demonstrated by the
increased polydispersity index, and a reduced negative surface charge as demonstrated by
the less negative ζ-potential (table 1). This was expected as serum proteins are considered
to form a protein corona around DMSA-coated IONPs which is accompanied by an
increase in diameter and a lowering of the negative ζ-potential (Geppert et al., 2013;
Petters et al., 2014a). The average hydrodynamic diameter of dispersions of 3 mM
IONPs in GCM was around 160 nm and significantly increased compared to GCM
dispersion of 1 mM IONPs (90 nm) which is consistent with the concentration-dependent
alteration in particle size reported for citrate-coated IONPs in serum-containing culture
medium (Safi et al., 2011). In contrast, polydispersity index and ζ-potential did not differ
for IONPs dispersions in GCM containing 1 mM or 3 mM iron (table 1). Hydrodynamic
diameter, polydispersity, and ζ-potential of IONPs were not affected if GCM was
supplemented with additional 25 mM KCl for incubation of neurons (data not shown).
2. Uptake and toxicity of iron oxide nanoparticles by microglial cells
Previously was shown that exposure of cultured microglial cells to IONPs for up to 6 h
caused a time-dependent increase in cellular iron content and compromised cell viability
(Luther et al., 2013). In order to investigate the mechanisms involved in this toxicity, we
applied fluorescent IONPs in concentrations between 0.1 mM and 3 mM to cultured
microglial cells and determined the cell viability and specific cellular iron contents after 6
h of incubation. As expected, the viability of the IONP-treated cells was compromised in
a concentration-dependent manner (figure 1a). While microglial cells incubated without
IONPs maintained a high cellular LDH activity and a high MTT reduction capacity
during the 6 h of incubation, the presence of 3 mM iron in form of IONPs significantly
lowered the cellular LDH activity (figure 1a, table 2) and the cellular MTT reduction
capacity (table 2) to around 50% of the values determined for control cells. In addition,
microglial cultures exposed for 6 h in the absence of IONPs did not contain cells which
were stained by the membrane-impermeable fluorescent dye PI (figure 2b) and hardly
any cells characterized by elevated ROS formation (figure 3b, figure 4b). In contrast, most
Results 59
cells in microglial cultures that had been incubated with 3 mM IONPs were positive for
PI (figure 2l) and ROS (figure 3l, figure 4d).
The incubation of microglial cultures with IONPs was accompanied by a concentration-
dependent increase in the specific cellular iron content from 55 ± 44 nmol/mg protein of
untreated cultures (n = 4) to around 1000 and 2500 nmol/mg for cells that had been
exposed to 1 mM and 3 mM IONPs, respectively (figure 1b).
3. Comparison of the effects of iron oxide nanoparticles on different types of brain cell
cultures
To compare microglial cells with other types of brain cells regarding the consequences of
an exposure to IONPs, primary cultures of astrocytes and neurons were incubated with
IONPs under experimental conditions that were identical (with the exception of the
essential presence of additional 25 mM KCl in the GCM for neurons) to those applied
for microglial cells. In contrast to microglial cells, the presence of IONPs in
concentrations of up to 3 mM did neither lower the cellular LDH activity of cultured
astrocytes and neurons (figure 1a,c) nor did it cause any severe increase in the cellular
ROS production (figure 4h,l).
Incubation of cultured astrocytes or neurons with IONPs caused a concentration-
dependent increase in the cellular iron contents (figure 1b). After 6 h incubation with
IONPs in concentrations of 0.1 mM or 0.3 mM, the specific cellular iron contents were
significantly lower in astrocytes and neurons compared to the respective values
determined for microglial cultures while the iron contents did not significantly differ
between the three types of neural cell cultures after incubations with 1 mM or 3 mM
IONPs (figure 1b). Direct correlation of the cellular LDH activity to the specific cellular
iron content confirmed that the loss in microglial viability was almost proportional to the
increase in specific cellular iron content while no cellular LDH loss was observed from
IONP-treated astrocytes or neurons, despite of similar high specific iron contents in all
three types of neural cell cultures after exposure to millimolar concentrations of IONPs
(figure 1c).
4. Cellular localization of fluorescent IONPs in cultured brain cells
Fluorescence microscopy was used to investigate the cellular localization of accumulated
fluorescent IONPs. As application of millimolar concentration of fluorescent IONPs
60 Results
caused toxicity in microglia, the neural cell cultures were incubated for 6 h with only 0.1
mM IONPs and subsequently stained with lysotracker. Fluorescence microscopy revealed
for all three types of IONP-treated neural cultures dotted fluorescence patterns for both
BP-labeled IONPs and lysotracker (figure 5). However, the cellular distribution of
fluorescence differed strongly between the culture types. In cultured microglial cells the
majority of IONP fluorescence was found co-localized with lysotracker and displayed a
mainly perinuclear staining (figure 5a-d). In contrast, in astrocytes (figure 5e-h) or neurons
(figure 5i-l) at best little co-localization of IONP fluorescence and lysotracker was
observed. IONPs were localized near the nucleus in cultured astrocytes (figure 5e-h) while
the small somata of neurons do not allow conclusions on potential perinuclear
localization of IONPs in cultured neurons (figure 5i-l).
5. Mechanism of IONP-mediated toxicity to microglia
Lysosomal localization of IONPs (figure 5a-d) as well as the increased ROS formation
(figure 3l, 4d) and compromised viability (figure 1b, 2l) of IONP-treated microglial cells
suggests that rapid iron liberation from IONPs in lysosomes and subsequent iron-
mediated ROS formation are involved in the observed IONP-mediated microglial
toxicity. To test for this hypothesis, microglia were treated with NH4Cl or the vacuolar H+-
ATPase inhibitor Bafilomycin A1 to neutralize lysosomes (Poole and Ohkuma, 1981;
Teplova et al., 2007) and thereby preventing the release of iron ions from the
nanoparticles. Alternatively, the cells were incubated with 2,2’-bipyridyl to chelate the
ferrous iron ions released from IONPs (Hohnholt et al., 2010). As a control for potential
iron-independent effects of the chelator, also the non-chelating isomer 4,4’-bipyridyl was
applied as control substance.
Microglia were treated in presence and absence of 3 mM IONPs for 6 h with the above
mentioned compounds and the cell viability was investigated by determination of cellular
LDH activity and MTT reduction capacity (table 2) as well as by PI (figure 2) and ROS
staining (figure 3). None of the compounds applied to modulate IONP-induced toxicity
altered significantly the specific cellular iron content of microglial cells after incubation for
6 h with 3 mM IONPs (table 2). In the absence of IONPs, incubation for 6 h with
NH4Cl, Bafilomycin A1, 2,2’-bipyridyl or 4,4’-bipyridyl did not cause any loss in cellular
LDH or MTT reduction capacity (table 2) nor was any staining of PI-permeable (figure
2d,f,h,j) or ROS-positive (figure 3d,f,h,j) microglial cells observed. The H33342 staining
Results 61
demonstrated that none of the conditions used lowered obviously the number of cells
attached to the cell culture dish (figures 2, 3).
Exposure of microglial cells to 3 mM IONPs caused a loss in cellular LDH activity by
around 50% which was completely prevented by the presence of NH4Cl, Bafilomycin A1
or the iron chelator 2,2’-bipyridyl while the non-chelating 4,4’-bipyridyl did not rescue
microglial cells from IONP-induced loss in cellular LDH activity (table 2). Also the
substantial IONP-induced loss in microglial MTT reduction capacity was prevented by
the presence of NH4Cl and at least partially prevented by the presence of Bafilomycin A1
or 2,2’-biypyridyl, whereas application of 4,4’-bipyridyl did not rescue the MTT reduction
capacity of IONP-treated microglial cells (table 2). PI-staining of IONP-treated microglial
cells (figure 2) confirmed the results obtained on LDH loss and MTT reduction capacity
of IONP-treated microglial cells (table 2). While nearly all nuclei in microglial cultures
were PI-positive after incubation with 3 mM IONPs (figure 2l), hardly any PI-positive
cells were found if the cells were incubated with IONPs in the presence of NH4Cl,
Bafilomycin A1 or 2,2’-biypyridyl (figure 2n,p,r). In contrast, presence of 4,4’-bipyridyl
did not lower the number of PI-positive cells in IONP-treated microglial cultures (figure
3t). Also the strong ROS-staining observed for IONP-treated microglial cells (figure 3l)
was completely prevented by the presence of Bafilomycin A1 (figure 3n) and at least
partially prevented by NH4Cl and 2,2’-bipyridyl (figure 3p,r) while presence of 4,4’-
bipyridyl hardly affected ROS-staining of IONP-treated cells (figure 3t).
62 Results
c
iron content (nmol/mg)0 1000 2000 3000
microgliaastrocytesneurons
a
cellu
lar L
DH
act
ivity
(% o
f con
trol)
0
20
40
60
80
100
120
b
micro- astro- neurons
iron
cont
ent (
nmol
/mg)
0
1000
2000
3000
4000
0.3 mM0.1 mM0.3 mM1.3 mM3.3 mM
glia cytes
*
#
***
******
## ##
#####
***
##
####
Figure 1 Cell viability and iron accumulation in IONP-treated cultured microglial cells,
astrocytes and neurons. The cultures were incubated with the indicated concentrations of
IONPs for 6 h and the cellular LDH activity (a) and the specific cellular iron content (b)
were determined. In panel c, the cellular LDH activity is plotted against the respective
cellular iron content. The data represent means ± SD of results obtained in five to twelve
(microglia) or three (astrocytes, neurons) experiments on individually prepared cultures.
The 100% values for the cellular LDH activity of microglial cells, astrocytes and neurons
are 505 ± 163 nmol/(min x mg) (n=20), 402 ± 81 nmol/(min x mg) (n=3) and 718 ± 215
nmol/(min x mg) (n=3), respectively. Asterisks indicate significant differences of data
compared to the respective control (incubation without IONPs) for one type of neural
culture (*p<0.05, ***p<0.001). Significant differences of data obtained for astrocytes or
neurons to those recorded for microglial cells are indicated by hashes (#p<0.05, ##p<0.01, ###p<0.001).
Results 63
FFigure 2 Propidium iodide staining of IONP-treated microglia. Microglia were incubated
for 6 h without (a-j) or with 3 mM IONPs (k-t) in the absence (a,b,k,l) or the presence of
100 nM Bafilomycin A1 (c,d,m,n), 10 mM NH4Cl (e,f,o,p), 100 μM 2,2’-bipyridyl (g,h,q,r)
or 100 μM 4,4’-bipyridyl (I,j,s,t). After the subsequent 15 min incubation with propidium
iodide (PI) and H33342, cell with impaired membrane integrity were PI-positive, while
the nuclei of all cells present are indicated by H33342-staining . The scale bar in panel t
refers to 100 μm and applies to all panels.
64 Results
FFigure 3 Staining of reactive oxygen species in IONP-treated microglia. Microglia were
incubated for 6 h without (a-j) or with 3 mM IONPs (k-t) in the absence (a,b,k,l) or the
presence of 100 nM Bafilomycin A1 (c,d,m,n), 10 mM NH4Cl (e,f,o,p), 100 μM 2,2’-
bipyridyl (g,h,q,r) or 100 μM 4,4’-bipyridyl (i,j,s,t) before the cells were incubated for 15
min with dihydrorhodamine to test for ROS generation. The nuclei of all cells present
were visualized by H33342. The scale bar in panel t refers to 100 μm and applies to all
panels.
Results 65
FFigure 4 Staining of reactive oxygen species in IONP-treated neural cell cultures. Primary
microglia, astrocytes and neurons were incubated without (a,b,e,f,i,j) or with 3 mM
IONPs (c,d,g,h,k,l) for 6 h before the cells were incubated for 15 min with
dihydrorhodamine to test for ROS generation. The nuclei of all cells present were
visualized by H33342. The scale bar in panel l represents 100 μm and applies to all
panels.
66 Results
FFigure 5 Localization of fluorescent IONPs in cultured neural cells. Microglial cells (a-d),
astrocytes (e-h) and neurons (i-l) were incubated with 0.1 mM IONPs for 6 h followed by
an incubation with 100 nM lysotracker DND-99 for further 90 min. Cells were washed
with PBS and fixed prior to monitoring by fluorescence microscopy for IONP
fluorescence (a,c,i) and lysotracker fluorescence (b,f,j). The overlays of the IONP and
lysotracker fluorescences (c,g,k) show co-localization of green IONPs and red lysotrackers
in yellow. The scale bar in l represents 25 μm and applies to all panels. Full color images
can be found in the online version of the article.
Results 67
TTable 1 Physicochemical properties of IONPs
hydrodynamic polydispersity ζ-potential [IONP] diameter (nm) index (mV) H2O 1 mM 44 ± 2 0.190 ± 0.017 -39 ± 8
3 mM 47 ± 2 0.186 ± 0.031 -50 ± 3 GCM 1 mM 90 ± 15*** 0.297 ± 0.029** -10 ± 4**
3 mM 158 ± 28**,## 0.263 ± 0.025* -10 ± 2***
Asterisks indicate significant differences between dispersion of IONPs in glia conditioned
medium (GCM) and water (*p<0.05, **p<0.01, ***p<0.001) and hashes between 1 and 3
mM IONPs (##p<0.01). Measurements were performed thrice with different batches of
IONPs.
68 Results
TTab
le 2
Neu
tral
izat
ion
of ly
soso
mes
and
che
latio
n of
iron
ions
pre
vent
IO
NP
-indu
ced
toxi
city
in c
ultu
re m
icro
glia
l cel
ls
Iron
con
tent
(n
mol
/mg
prot
ein)
n 6 3 5 5 5
Mic
rogl
ia w
ere
incu
bate
d or
with
3 m
M I
ON
Ps
in th
e ab
senc
e (c
ontr
ol)
or t
he p
rese
nce
of 1
00 n
M B
afilo
myc
in A
1 (B
af1)
, 10
mM
NH
4Cl,
100 μM
2,2
’-bip
yrid
yl o
r 10
0 μM
4,4
’-bip
yrid
yl fo
r 6
h be
fore
the
cellu
lar
LD
H a
ctiv
ity (1
00%
= 5
69 +
- 184
nm
ol/(
min
*mg)
), th
e ce
llula
r M
TT
redu
ctio
n ca
paci
ty (
100%
= a
bsor
banc
e of
1.2
3 +-
0.2
/wel
l) an
d th
e sp
ecifi
c ce
llula
r ir
on c
onte
nt w
ere
dete
rmin
ed. T
he ir
on c
onte
nt o
f con
trol
cells
that
had
bee
n in
cuba
ted
for
6 h
with
out I
ON
Ps w
as 5
5 +-
44
(n =
4)
nmol
/mg
prot
ein.
Ast
eris
ks in
dica
te s
igni
fican
t diff
eren
ces
betw
een
cells
tha
t ha
d be
en e
xpos
ed t
o th
e in
dica
ted
com
poun
ds a
nd t
he r
espe
ctiv
e co
ntro
l (*p
<0.0
5, *
*p<0
.01,
***
p<0.
001)
. Sig
nific
ant
diffe
renc
es
betw
een
data
obt
aine
d fo
r in
cuba
tions
in th
e ab
senc
e an
d pr
esen
ce o
f IO
NP
s ar
e in
dica
ted
by h
ashe
s (# p<
0.05
, ##p<
0.01
, ### p<
0.00
1).
3 m
M I
ON
P
1175
724
789
865
918
± ± ± ± ±
2834
1746
2152
2492
2578
MT
T r
educ
tion
capa
city
(% o
f con
trol
) n 5 4 5 5 5
3 m
M I
ON
P
14##
6*
5***
16**
*
12
± ± ± ± ±
46
71
96
77
48
n 4 4 4 4 4
0 m
M I
ON
P
0 24
15
8 23
± ± ± ± ±
100 99
98
107 95
Cel
lula
r L
DH
act
ivity
(% o
f con
trol
)
n 6 3 5 5 4
3 m
M I
ON
P
24#
9***
6***
11**
*
9###
± ± ± ± ±
52
99
102
101 55
n 8 4 4 4 3
0 m
M I
ON
P 0 3 1 3 2
± ± ± ± ±
100
101
100
103 97
none
Baf
1
NH
4Cl
2,2’
-bip
yrid
yl
4,4’
-bip
yrid
yl
Results 69
DDiscussion
Application of IONPs has previously been reported to compromise the viability of
cultured microglial cells (Jenkins et al., 2013; Luther et al., 2013; Pickard and Chari,
2010). This vulnerability was confirmed for microglial cells treated with fluorescent
DMSA-coated IONPs as demonstrated by a loss of cellular LDH activity and MTT
reduction capacity as well as by increased permeability of the cell membrane for PI. The
increase in cellular ROS-staining suggests that iron-catalyzed radical formation is involved
in the damage caused by IONPs to microglial cells as previously also shown for
peripheral macrophages and other cell types (Hanini et al., 2011; Dwivedi et al., 2014;
Lee et al., 2014). The most likely pathway involved in the ROS generation and toxicity in
IONP-treated microglial cells involves endocytotic uptake of IONPs, lysosomal
degradation of IONPs to iron ions, iron-catalyzed ROS formation by the Fenton reaction
and oxidative cell damage, as previously suggested (Jenkins et al., 2013; Luther et al.,
2013).
Endocytotic uptake of IONPs and trafficking of the internalized IONPs to the lysosomal
compartment have previously been demonstrate for cultured microglial cells (Luther et
al., 2013) and was confirmed for the conditions used here by the co-localization of
fluorescent IONPs with lysotracker in microglial cells. The ability of NH4Cl and
Bafilomycin A1 which neutralize the lysosomal pH by diffusion of ammonia to the acidic
lysosome (Poole and Ohkuma, 1981) and by inhibition of the lysosomal proton pump
(Teplova et al., 2007), respectively, to lower or even prevent ROS formation and cell
toxicity in IONP-treated microglial cells demonstrates that the low pH of lysosomes is
involved in the IONP-induced microglial toxicity. This is consistent with literature data
from cell-free experiments showing that iron ions are liberated from IONPs at lysosomal
pH (Levy et al., 2010; Malvindi et al., 2014). Also the presence of reducing substances
such as glutathione in lysosomes (Kurz et al., 2010) may accelerate the liberation of iron
ions from the IONPs and also helps to maintain iron in the ferrous state which is
required for export from the acidic compartment into the cytosol by the divalent metal
transporter 1 (Lawen and Lane, 2013). This transporter has been reported to be
expressed in microglial cells (Rathore et al., 2012; Urrutia et al., 2013). Thus, rapid
lysosomal liberation of internalized IONPs in microglial cells is likely to generate large
amounts of ferrous iron which causes ROS formation and cell damage. This view is
strongly supported by the ability of the membrane permeable ferrous iron chelator 2,2’-
70 Results
bipyridyl, but not its non-chelating analog 4,4’-bipyridyl (Kinnunen et al., 2002; Ma et al.,
2006), to lower ROS generation and prevent toxicity in IONP-treated microglial cells.
The residual ROS formation in microglial cells in the presence of 2,2’-bipyridyl might
result from ROS production on the IONP-surface (Voinov et al., 2011) as the Fe2+-2,2’-
bipyridyl complex is not Fenton reactive (Pierre and Fontecave, 1999). However,
microglial cells appear to cope with a low rate of ROS production found under such
conditions as their high antioxidative potential (Dringen, 2005; Hirrlinger et al., 2000) is
likely to protect the cells against toxicity by a moderate amount of ROS.
Cultured astrocytes and neurons accumulated similar amounts of IONPs under the
conditions used. The specific iron contents determined were in the range of values
previously reported for these cultures in serum-containing media (Geppert et al., 2011;
Geppert et al., 2013; Petters and Dringen, 2015). Comparison of microglial cultures with
astrocytes and neurons revealed that microglial cells accumulated IONPs more efficiently
when the different types of cultures were treated with low concentrations of 0.1 mM or
0.3 mM IONPs as demonstrated by the 2- to 5-fold higher specific cellular iron contents
in microglial cells. This stronger IONP accumulation by microglial cells is consistent with
literature data for other types of IONPs (Jenkins et al., 2013; Pinkernelle et al., 2012).
Different types of endocytotic pathways involved in IONP uptake are unlikely to explain
the observed differences in IONP accumulation between the three types of neural cells
investigated as in serum-containing media clathrin-mediated endocytosis and
macropinocytosis have been reported to mediate IONP uptake into both cultured
microglial cells and astrocytes (Geppert et al., 2013; Luther et al., 2013) while neurons
appear to take up IONPs predominantly by clathrin-mediated endocytosis (Petters and
Dringen, 2015). However, as microglia are the "phagocytotic cells" in the brain and as
microglial cells, but not astrocytes and neurons, have the prominent function to
incorporate and digest cell debris from their environment (Eyo and Dailey, 2013; Nayak
et al., 2014), microglial cells are likely to be equipped with a strong endocytotic transport
capacity which could explain the higher efficiency of microglial cells to accumulate iron
from low concentrations of IONPs. For incubations with 3 mM IONPs similar specific
iron contents were determined for all three types of neural cultures. However, this
apparent inability of microglial cells to accumulate more IONPs than astrocytes and
neurons under these conditions is likely to be a direct consequence of the developing
microglial toxicity which will impair IONP accumulation while astrocytes and neurons
remained viable during the incubation and continued to accumulate IONPs.
Results 71
The relative resistance of astrocytes and neurons towards high concentrations of IONPs is
most likely caused by a slow transfer of internalized IONPs into the lysosomal
compartment which is required for liberation of iron ions from IONPs. This view is
supported by the apparent absence of a co-localization of fluorescent IONPs with
lysotracker in IONP-treated cultured astrocytes and neurons. In contrast, IONP-treated
microglial cultures showed under identical conditions strong co-localization of fluorescent
IONPs with lysotracker. Thus, a more rapid internalization and lysosomal degradation of
IONPs to ROS-generating iron ions in microglial cells is likely to cause the severe toxicity
observed for this cell type after exposure to IONPs. Such processes appear to be slower
in astrocytes and neurons, explaining the relative resistance of these cell types against
potential IONP-induced toxicity (Geppert et al., 2012; Geppert et al., 2011; Geppert et
al., 2013; Petters and Dringen, 2015). However, also astrocytes and neurons have the
potential to slowly liberate iron from accumulated IONPs. At least for other experimental
conditions some ROS formation has been reported for IONP-treated astrocytes and
neurons (Geppert et al., 2012; Petters et al., 2014b) and depending on the type of IONPs
applied and the incubation conditions used also delayed toxicity was observed for these
cell types (Sun et al., 2013; Rivet et al., 2012; Petters and Dringen, 2015).
CConclusions
In the present study we show that microglia are more susceptible to IONP exposure as
astrocytes and neurons which is likely to be caused by fast lysosomal iron liberation from
IONPs in microglial cells which causes severe iron-mediated ROS production and
oxidative cell damage. This suggests that also in vivo microglia may be harmed more
strongly by IONPs than other brain cells. However, microglial toxicity in brain as
consequence of an IONP exposure is likely to depend on the route which mediates entry
of IONPs into the brain. Little harm to microglial cells is expected, if IONPs are applied
to the blood, e.g. as MRI contrast agent, as IONPs which pass the intact BBB via
transcytosis (Yan et al., 2013) encounter astrocytes. These cells cover almost completely
the brain capillaries (De Bock et al., 2014) and have at least in culture the capacity to
accumulate large amounts of IONPs without being damaged by the accumulated IONPs
(Geppert et al., 2012; Jenkins et al., 2013; present study). In contrast microglial cells are
very likely to encounter blood-derived IONPs, if the BBB is damaged by injury or tumors
(Nayak et al., 2014). For such conditions predominantly microglial cells have been
72 Results
reported to take up IONPs (Neuwelt et al., 2004; Taschner et al., 2005). Also intranasal
application of IONPs (Wang et al., 2011) as well as direct injection of IONPs into brain
tumors for magnetic fluid hyperthermia (van Landeghem et al., 2009) establish direct
contact of microglial cells with IONPs which causes microglial IONP accumulation and
microglial activation (Wang et al., 2011). Such an IONP-induced activation of microglial
cells might result in neurotoxicity (Correale, 2014). However, IONP-induced microglial
toxicity which will lower the number of these cells and will subsequently also impair the
neuroprotective functions of microglia in brain including the defense against pathogens
and the support in repair processes (Kettenmann et al., 2011; Nau et al., 2014). For such
conditions, strategies to prevent the fast IONP internalization, the liberation of iron ions
from internalized IONPs and/or iron-mediated ROS production should be considered in
order to prevent IONP-induced microglial toxicity.
DDeclaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the
content and writing of the paper.
Results 73
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3. SSummarizing
discussion
3.1. Synthesis and characterization of fluorescently labeled iron oxide nanoparticles ........................................................................................................ 81
3.1.1. Characterization of BODIPY-labeled iron oxide nanoparticles ............................. 83
3.1.2. Fluorescent properties of BODIPY-labeled iron oxide nanoparticles ................... 83
3.1.3. Stability of BODIPY-labeled iron oxide nanoparticles ........................................... 84
3.2. Uptake and effects of iron oxide nanoparticles by cultured brain cells ................ 86
3.2.1. Accumulation of iron oxide nanoparticles in cultured brain cells .......................... 87
3.2.2. Uptake of iron oxide nanoparticles in serum-free media ....................................... 90
3.2.3. Uptake of iron oxide nanoparticles in serum-containing media ............................. 91
3.2.4. Cellular localization of iron oxide nanoparticles ..................................................... 92
3.2.5. Toxic effects of an exposure of iron oxide nanoparticles to cultured brain
cells ............................................................................................................................ 93
3.3. Implications of the results for the in vivo situation .............................................. 95
3.4. Future perspectives .............................................................................................. 99
3.4.1. Synthesis of fluorescently labeled iron oxide nanoparticles.................................... 99
3.4.2. Mechanism of accumulation of iron oxide nanoparticles under serum-free conditions .................................................................................................................. 99
3.4.3. Binding, uptake and fate of iron oxide nanoparticles in cultured brain cells ...... 100
3.4.4. Effects of an iron oxide nanoparticle exposure to cultured brain cells ............... 101
3.4.5. Investigations on uptake and effects of iron oxide nanoparticles in vivo ............ 102
3.5. References ......................................................................................................... 104
Summarizing discussion 81
33. Summarizing discussion
Dimercaptosuccinate (DMSA)-coated IONPs have been frequently used to study
accumulation and biocompatibility of IONPs on neural cells in vitro (Geppert et al.,
2011; Geppert et al., 2012; Geppert et al., 2013; Hohnholt et al., 2010; Hohnholt and
Dringen, 2011; Hohnholt et al., 2011) and in vivo (Mejias et al., 2010; Mejias et al., 2013)
but their cellular localization could not be monitored by light microscopy. To overcome
this problem, fluorescent versions of DMSA-coated IONPs were synthesized,
characterized, compared to non-fluorescent DMSA-coated IONPs and applied to cell
culture models of the four major brain cell types to investigate and compare uptake
capacities and potential adverse consequences of an IONP exposure of different types of
brain cells.
3.1. Synthesis and characterization of fluorescently labeled iron
oxide nanoparticles
The preparation of fluorescently labeled IONPs was a three-step process: i) the bare
maghemite NPs were synthesized by an alkaline co-precipitation of ferrous and ferric salts
(Geppert et al., 2009), ii) the coating material DMSA was labeled at alkaline conditions
with the fluorescent dye BODIPY (BP) by coupling thiol groups of DMSA with the thiol-
reactive iodoacetamide function of BP, forming a thioether (Kaltz, 2011), and iii) coating
of IONPs at pH 3 with BP-DMSA (chapters 2.2 and 2.3). The initial method for
fluorescence labeling of DMSA was introduced by Felix Bulcke in a research project in
2011.
For this thesis project, three versions of BP-labeled IONPs were prepared which differed
in the amounts of BP used and in the numbers of coating steps (Table 3.1). For labeling,
DMSA and BP were mixed in a molar ratio of 1:0.03 (BP-IONPs) or 1:0.15 (BP(5x)-
IONPs). Thus, either 1.5% or 7.5% of the total number of thiol groups in the DMSA
used for coating was modified with BP. BP(5x)-IONPs were also surrounded with a
second layer of pure DMSA, resulting in D-BP(5x)-IONPs. Non-labeled DMSA-coated
IONPs are referred to as D-IONPs.
82 Summarizing discussion
T
able
3.1
Phy
sico
chem
ical
pro
pert
ies
of D
MSA
-coa
ted
and
thre
e ty
pes
of B
P-la
bele
d IO
NPs
.
cont
act t
o ce
lls
n
n.d.
3 3
n.d.
3
n.d.
3 3
n.d.
3
n.d.
3 3
n.d.
3
n.d.
3 3
n.d.
3
DM
SA-c
oate
d IO
NP
s ar
e ab
brev
iate
d by
D-I
ON
Ps. D
-IO
NPs
of
whi
ch 1
.5%
or
7.5%
of
the
thio
l gro
ups
of D
MSA
was
mod
ified
with
BO
DIP
Y a
re
nam
ed a
s B
P-
and
BP(
5x)-I
ON
Ps,
resp
ectiv
ely.
BP
(5x)
-IO
NP
s su
rrou
nded
with
a s
econ
d co
at o
f D
MSA
are
cal
led
D-B
P(5
x)-I
ON
Ps.
IO
NPs
at
a co
ncen
trat
ion
of 1
mM
wer
e di
sper
sed
in p
ure
wat
er, i
ncub
atio
n bu
ffer
(IB
), IB
with
10%
FC
S or
DM
EM
with
out o
r w
ith 1
0% F
CS
and
inve
stig
ated
fo
r th
eir
aver
age
hydr
odyn
amic
dia
met
er a
nd ζ
–po
tent
ial b
efor
e an
d af
ter
4 h
incu
batio
n at
37°
C o
f O
LN
-93
cells
. Dat
a hi
ghlig
hted
in r
ed a
re ta
ken
from
cha
pter
2.2
but
sta
tistic
al a
naly
sis
was
rec
alcu
late
d as
add
ition
ally
dat
a fr
om D
-BP
(5x)
-IO
NPs
wer
e in
clud
ed.
Ast
eris
ks i
ndic
ate
sign
ifica
nt
diffe
renc
es o
f da
ta f
or f
luor
esce
ntly
lab
eled
IO
NP
s co
mpa
red
to D
-IO
NPs
(A
NO
VA
, *p
<0.0
5, *
*p<0
.01,
***
p<0.
001)
. H
ashe
s in
dica
te s
igni
fican
t di
ffere
nces
of d
ata
for
one
type
of I
ON
Ps
befo
re a
nd a
fter
incu
batio
n w
ith c
ells
(t-te
st, # p<
0.05
, ##p<
0.01
, ### p<
0.00
1). n
.d.,
not d
eter
min
ed. ζ-po
tent
ial
(mV
) 1**
0 7 1# 1 1 1*
, ##
1 2 4 3 1
± ± ± ± ± ± ± ± ± ± ± ±
-25
-10
-15
-20 -7
-9
-23
-10 -8
-16 -6
-9
n
n.d.
3 3
n.d.
3
n.d.
3 3
n.d.
3
n.d.
3 3
n.d.
3
n.d.
4 4
n.d.
3
hydr
odyn
amic
di
amet
er (n
m)
17#
12
31
302#
3 5 639
9#
31
9###
2 3
± ± ± ± ± ± ± ± ± ± ± ±
108
111
109
1998
97
90
887
113
123 87
104 73
no c
onta
ct to
cel
ls
n 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
ζ-po
tent
ial
(mV
)
18
2 1 1 3 2 3 5 0 1 12
1 3 4**
1 4 10
1 5 2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
-58
-28
-10
-22 -9
-49
-25 -9
-18 -9
-66
-27 -8
-15
-10
-58
-20 -9
-24 -9
n 3 3 4 4 4 5 4 4 4 4 4 3 4 4 4 5 4 4 4 4
hydr
odyn
amic
di
amet
er (n
m)
5**
5 24
283
38
4***
14
4***
31
332*
* 54
2*
**
46
22*
93*
77*
1 2 23
259
19
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
63
61
138
1851
13
4 65
1488
12
2 22
21
128 67
128
165
2096
18
4 53
52
109
1464
78
med
ium
H2O
IB
IB
+ 1
0% F
CS
DM
EM
D
ME
M +
10%
FC
S H
2O
IB
IB +
10%
FC
S D
ME
M
DM
EM
+ 1
0% F
CS
H2O
IB
IB
+ 1
0% F
CS
DM
EM
D
ME
M +
10%
FC
S H
2O
IB
IB +
10%
FC
S D
ME
M
DM
EM
+ 1
0% F
CS
BP
-IO
NP
s B
P(5
x)-I
ON
Ps
D-B
P(5
x)-I
ON
Ps
D-I
ON
Ps
Summarizing discussion 83
33.1.1. Characterization of BODIPY-labeled iron oxide nanoparticles
After coating, single IONPs were spherical with a diameter of 4-20 nm. They displayed a
clear crystalline structure seen by electron microscopy (chapters 2.2 and 2.3) as reported
previously for D-IONPs (Geppert et al., 2011). Moreover, the synthesized IONPs
possessed the for superparamagnetic properties characteristic for IONPs as demonstrated
by a strong magnetization on application of a magnetic field but the absence of
remanescence after removal of the magnetic field (chapter 1.3).
When dispersed in media, IONPs form small aggregates. The initially synthesized IONPs
had hydrodynamic diameter of 60 nm and a ζ-potential of around -50 mV when
dispersed in water (chapters 2.2-2.4) as previously described (Geppert et al., 2011)
whereas later preparations generated reproducibly smaller IONPs of around 30 nm with
the same ζ-potential (chapter 2.5). The reason for this is unclear but slight variations
between the initial and later syntheses might have caused these alterations. The used
experimental set up did not allow very controlled conditions to achieve exact pH values
and temperatures between the syntheses. Indeed, the alkaline co-precipitation method is
described as simple and effective to generate large volumes of IONP dispersions but has
the disadvantage of generating IONPs of a broad size distribution since already slight
variations of the preparation conditions such as pH, temperature, iron salt species or ratio
of ferrous to ferric ions strongly affect the outcome of the IONP synthesis (Massart, 1981;
Gnanaprakash et al., 2007; Hasany et al., 2012).
3.1.2. Fluorescent properties of BODIPY-labeled iron oxide nanoparticles The labeling of DMSA with BP prior to coating was successful as confirmed by
fluorescence and mass spectrometry as well as by energy dispersive X-ray spectroscopy
(chapters 2.2 and 2.3; Kaltz, 2011). The fluorescence of BP-labeled IONPs depends on
the amount of BP within the coat but also on the pH and on presence of uncoated
IONPs which caused quenching (Kaltz, 2011). By lowering the pH, the intensity of BP-
fluorescence is decreased. Since this was not seen in BP-DMSA solution alone (data not
shown), IONPs mediate this pH effect probably by IONP destabilization with higher
proton concentration since also precipitated BP-IONPs had strongly decreased
fluorescence (data not shown). Moreover, fluorescence intensities of lysates of OLN-93
cells after incubation at 37°C were similar to those after incubation at 4°C although the
iron contents differed by a factor of two (unpublished data). The pH-dependent
84 Summarizing discussion
modulation of BP-labeled IONP fluorescence has to be considered for analysis of BP-
IONP uptake by fluorescence microscopy as fluorescence of BP-labeled IONPs within
acidic compartments might underestimate and not represent the real amount of BP-
labeled IONPs in these compartments.
Since the fluorescence of BP(5x)- and BP-IONPs principally co-localized with staining for
iron in microglial cells (chapter 2.3) and OLN-93 cells (data not shown), respectively,
these fluorescent IONPs are considered as suitable as tool for visualization of IONPs in
cells. However, also non-dotted BP-fluorescence occurred outside from IONP-containing
vesicles, e.g. in the nucleus, which hinds towards a liberation of the coat over time in
endosomal/lysosomal environments with low pH and reducing conditions and/or towards
free dye in the IONP dispersion. This elution of fluorescent dye was reported previously
for N-isopropylacrylamide NPs containing rhodamine B under cell-free conditions as well
as after exposure to A49 cells (Tenuta et al., 2011). Although BP-IONPs were washed
once after coating, the presence of a residual amount of unbound coating material cannot
be fully excluded. This might explain some variations in non-vesicular stainings in BP-
and BP(5x)-IONP-treated cells irrespective of cell type, incubation medium and time
(data not shown).
33.1.3. Stability of BODIPY-labeled iron oxide nanoparticles
The knowledge on stability of IONPs in physiological media is fundamental for
applications. Hence, the stability of IONPs in dispersion was investigated by
determination of the hydrodynamic diameter and the ζ-potential which correspond to size
and charge of IONPs, respectively (Table 3.1; chapters 2.2-2.5). These two parameters
are most crucial for interaction of NPs with bio-interfaces (Nel et al., 2009) and depended
on the media used for dispersion. Table 3.1 lists for the non-fluorescent and the three
versions of BP-labeled IONPs the hydrodynamic diameter and ζ-potential in various
media before and after contact to cells.
All four types of IONPs were colloidally stable in water as seen by a hydrodynamic
diameter between 50-70 nm and a ζ-potential of -50 to -66 mV (Table 3.1). The increased
diameter of the modified IONPs compared to the non-fluorescent D-IONPs might be
caused by the different coating procedure. D-IONPs were prepared by solving DMSA
resulting in an acidic solution to which IONPs were added and stirred for coating. In
contrast, the BP-DMSA solution was in an alkaline buffer and was acidified after IONP
Summarizing discussion 85
application. Moreover, the batch scales differed strongly for synthesis of D-IONPs and
BP-labeled IONPs. While IONPs were coated with DMSA in a larger volume of around
100 mL, BP-labeled IONPs had to be prepared in small batches of around 1 mL to
establish better reproducibility.
Presence of salts such as in incubation buffer (IB) did not affect size of D- and BP-IONPs
but the charge was altered towards less negative values (Table 3.1) which might be caused
by binding of cations to the carboxylate groups of DMSA. While in water dispersions the
introduction of BP into the coat had only marginal effects on the hydrodynamic diameter,
the stability of IONPs in IB depended strongly on the amount of dye within the coat.
BP(5x)-IONPs which had a 5-fold higher BP content than BP-IONPs agglomerated in
serum-free IB which was prevented by a second coat of DMSA (D-BP(5x)-IONPs)
though also D-BP(5x)-IONPs tend to aggregate (Table 3.1). D-IONPs are stabilized due
to formation of a DMSA cage generated by disulfide bridges between the molecules
(Fauconnier et al., 1997). The prevention of disulfide bridges between DMSA molecules
due to binding of BP as well as the steric hindrance by the dye probably result in a
destabilization of the coat when a certain amount of SH-groups is modified and hence,
leads to the observed destabilization of BP(5x)- and D-BP(5x)-IONPs. Thus, BP-IONPs
which contain only 1.5% BP in the coat are suitable for applications in IB under serum-
free conditions but not BP(5x)-IONPs or D-BP(5x)-IONPs. All four types of IONPs
agglomerated in DMEM without serum (Table 3.1) most likely due to presence of
destabilizing compounds such as phosphate (Dr. Mark Geppert, personal
communication).
Presence of 10% serum in physiological media caused the formation of a protein corona
around IONPs and thereby drastically changed interactions between NPs, as expected
(Nel et al., 2009). Hence, fluorescent and non-fluorescent IONPs aggregated in presence
of serum to agglomerates of around 160 nm (Table 3.1) or 80 nm (chapter 2.5)
diameters, dependent on IONP batches and concentration, and IONPs possessed a ζ-
potential of -10 mV as described previously (Geppert et al., 2013). While D-, BP- and
BP(5x)-IONPs were very similar in size, D-BP(5x)-IONPs were significantly larger (Table
3.1). Binding of proteins towards D-IONPs is probably mediated by ionic interaction and
perhaps by disulfide bridges between DMSA and proteins. Although a protein corona
masks the actual nature of NPs (Monopoli et al., 2012), the composition of the corona
might depend on the coat as reported for IONPs with various coatings (Jedlovszky-Hajdu
86 Summarizing discussion
et al., 2012). Since introduction of BP into the coat might change possible interactions
between proteins and coat, the composition of the proteins attached to IONPs might
differ in absence and presence of BP. However for D-IONPs and BP-IONPs, no altered
physicochemical properties and uptake by OLN-93 were observed (chapter 2.2)
suggesting no substantial differences between D- and BP-IONPs in the interaction
between protein corona and cell surface.
Contact to OLN-93 cells led to an increased hydrodynamic diameter of the four types of
IONPs when dispersed in IB in absence of serum (Table 3.1) most likely because of
compounds released from cells stimulate agglomeration as reported previously at least for
primary astrocytes (Geppert et al., 2013). While D- and BP-IONPs remained stably
dispersed, D-BP(5x)-IONPs agglomerated after contact with cells (Table 3.1),
strengthening the trend to stronger aggregation seen prior to incubation. In the presence
of serum, contact to cells did not result in a destabilization of IONPs but rather lowered
the average hydrodynamic diameter (Table 3.1) which may be a consequence of cellular
uptake of larger aggregates during incubation with IONPs.
Importantly, BP-IONPs possessed the same characteristics in morphology, size of single
NPs (chapter 2.2), hydrodynamic diameter and ζ-potential compared to D-IONPs (Table
3.1). BP-IONPs are accumulated by OLN-93 cells to similar amounts as D-IONPs and
by the same mechanism (chapter 2.2). Hence, BP-IONPs are suitable as fluorescent
version of D-IONPs to investigate localization of D-IONPs within cells for both serum-
free and serum-containing conditions. In the presence of serum, also BP(5x)-IONPs have
similar characteristics as D-IONPs and can be considered as tool to study uptake and
cellular localization of D-IONPs with the advantage of higher fluorescent signals of
BP(5x)-IONPs compared to BP-IONPs.
3.2. Uptake and effects of iron oxide nanoparticles in cultured
brain cells
Fluorescent and non-fluorescent versions of DMSA-coated IONPs were studied in this
thesis for uptake and potential adverse effects on cell culture models of brain cells. The
most important results obtained for such IONPs on neural cell cultures from this thesis
or reported in literature are compared in Table 3.2. Important in this context is that the
Summarizing discussion 87
presence of BP within the coat had no influence on the physicochemical properties and
cellular accumulation of D-IONPs (chapter 2.2; Table 3.1).
33.2.1. Accumulation of iron oxide nanoparticles by cultured brain cells
Comparable concentrations of IONPs and incubation conditions resulted in maximal
iron contents that were similar between astrocytes, microglia and neurons while OLN-93
cells seemed to have a lower accumulation capacity (Table 3.2). Also exposure to other
types of IONPs resulted in similar iron contents in cultured astrocytes and neurons (Sun
et al., 2013). Concerning microglia, it should be considered that maximal iron contents
were determined for at least partially toxic conditions whereas viable microglia were more
efficient in IONP accumulation compared to astrocytes and neurons (chapter 2.5),
supporting literature data (Pinkernelle et al., 2012; Jenkins et al., 2013). In general,
exposure to IONPs in the absence of serum in the incubation medium resulted in higher
specific cellular iron contents of neural cell cultures compared to serum-containing
conditions at least for astrocytes, neurons and OLN-93 cells (chapters 2.2 and 2.4;
Geppert et al., 2013) while no data could be obtained for primary microglia under serum-
free conditions due to some toxicity (Dr. Eva M. Luther, personal communication).
IONP accumulation by all investigated cultured brain cells slowed down with extended
incubation time (chapter 2.4; Hohnholt and Dringen, 2011; Geppert et al., 2013).
Differences in uptake kinetics between types of cultured brain cells might result from
diversity in protein or lipid composition of the cell membranes or expression of proteins
involved in endocytosis such as members of the small GTPase Rab family which are
differentially expressed in neurons and glia (Ng and Tang, 2008; DeKroon and Armati,
2001). The decreasing velocity of IONP uptake over time might result from a change in
coat/corona due to secretion of biomolecules by cells which was recently reported for
gold NPs exposed to several cell lines (Albanese et al., 2014) or from alterations in lipid
and protein composition of the membrane over time due to binding and uptake of
IONPs (Dawson et al., 2009; Wu et al., 2013b), thereby changing requirements for
endocytotic pathways. Moreover, the uptake process of IONPs might be down-regulated
and/or IONPs or iron liberated from internalized IONPs may be exported though no
export of iron was observed at least in cultured astrocytes which were loaded with IONPs
and incubated 7 d in IONP-free medium (Geppert et al., 2012).
88 Summarizing discussion
TTab
le 3
.2 U
ptak
e of
BP
-labe
led
or n
on-la
bele
d D
MSA
-coa
ted
ION
Ps
in c
ultu
red
brai
n ce
lls u
nder
diff
eren
t con
ditio
ns a
nd th
eir
effe
cts.
ref.
4 5 4 4 4 5 4,5
4 6 5
Dat
a hi
ghlig
hted
in r
ed d
eriv
e fr
om c
hapt
ers
of th
is th
esis
. Ref
eren
ces:
1, c
hapt
er 2
.1; 2
, cha
pter
2.2
; 3, c
hapt
er 2
.3; 4
, cha
pter
2.4
; 5, c
hapt
er 2
.5; 6
, ch
apte
r 1.
4; 7
, Gep
pert
et a
l., 2
011;
8, G
eppe
rt e
t al.,
201
3; 9
, Hoh
nhol
t an
d D
ring
en, 2
011;
10,
Hoh
nhol
t et a
l., 2
011;
11,
Gep
pert
et a
l., 2
012.
A
bbre
viat
ions
: IB
, inc
ubat
ion
buffe
r; I
B+F
CS,
IB
with
10%
feta
l cal
f ser
um; n
.d.,
not d
eter
min
ed.
neur
ons
2500
nm
ol/m
g (4
h, 1
mM
)
1790
nm
ol/m
g (6
h, 3
mM
)
70
47-6
3
clat
hrin
- med
iate
d en
docy
tosi
s
vesi
cula
r
no (6
h, 2
- 3 m
M)
yes
(2 h
0.5
mM
(I
B)
or
2 m
M
(IB
+FC
S) +
24
h re
cove
ry)
min
or
no
ref.
2 2 9,10
2 2 2 2 2 9 9
olig
oden
droc
ytes
1700
nm
ol/m
g (4
h, 1
mM
)
300
nmol
/mg
(4 h
, 1m
M)
650
nmol
/mg
(48
h, 1
mM
)
50
67
clat
hrin
-med
iate
d en
docy
tosi
s,
(mac
ropi
nocy
tosi
s)
vesi
cula
r,
part
ially
ly
soso
mal
no
(4 h
,1 m
M)
no (7
2 h,
1 m
M)
tran
sien
t
ref.
n.d.
5
n.d.
3 3 3,5
3,5
n.d.
5
mic
rogl
ia
2830
nm
ol/m
g (6
h, 3
mM
)
20
mac
ropi
nocy
tosi
s,
clat
hrin
-med
iate
d en
docy
tosi
s
lyso
som
al
yes
(6 h
, 0.
15 o
r 2
mM
)
Stro
ng
ref.
1 5 1,7
8 7 5 ,7,
11
5,7,
8 11
5 11
astr
ocyt
es
2265
nm
ol/m
g (4
h, 1
.5 m
M)
2340
nm
ol/m
g (6
h, 3
mM
)
46-5
0
50
mac
ropi
nocy
tosi
s,
clat
hrin
-med
iate
d en
docy
tosi
s
vesi
cula
r
no (6
h, 3
-4 m
M)
no (
4 h
1mM
, +
7 d
reco
very
)
min
or
tran
sien
t
inve
stig
ated
par
amet
er
max
imal
iron
con
tent
se
rum
-free
10
% s
erum
extr
acel
lula
r bi
ndin
g (%
of t
otal
iron
con
tent
)
se
rum
-free
10
% s
erum
endo
cyto
tic p
athw
ay
(10%
ser
um)
intr
acel
lula
r lo
caliz
aito
n
acut
e to
xici
ty
dela
yed
toxi
city
RO
S pr
oduc
tion
Summarizing discussion 89
FFigure 3.1 Uptake and effects of IONPs in cultured brain cells. IONPs are taken up in presence of serum (protein corona) via clathrin-mediated endocytosis (all cell types) and macropinocytosis (astrocytes, microglia, oligodendroglial cells) whereas in absence of serum the pathway for uptake is unknown. The identity of intracellular IONP-containing vesicles is unknown for neurons, astrocytes and oligodendrocytes whereas in microglia IONPs are localized in lysosomes. IONPs differ in their toxic potential on the four cell types. Acute exposure of IONPs causes cell death in microglia due to Fe2+-mediated ROS but not in the other cell types whereas delayed toxicity was observed in neurons. Astrocytes and oligodendroglial cells store the iron released from internalized IONPs in ferritin and are only transiently exposed to some ROS.
Immunocytochemical staining for astrocytic markers (glutamine synthetase (GS) and glial
fibrillary acidic protein (GFAP)) after treatment with BP-IONPs revealed stronger BP
staining in GS-positive than in GFAP-positive cells in astrocyte cultures (Figure 3.2).
However, a decreased GFAP expression in astrocytes did not lead to a significantly
increased accumulation of IONPs (chapter 2.1). Thus, besides differences between
neural cell types, there were alterations among individual types of astrocytes in one
culture which may reflect the heterogeneity of astrocytes in brain (chapter 2.1; Zhang and
Barres, 2010; Oberheim et al., 2012; Bayraktar et al., 2014).
90 Summarizing discussion
FFigure 3.2 IONP accumulation by GFAP- and GS-positive astrocytes. Primary cultures of astrocytes were incubated with 0.5 mM BP-IONPs in incubation buffer (see chapters 2.1 and 2.2) for 4 h at 37°C. After incubation, cells were washed thrice with cold phosphate-buffered saline, fixed with 3.5% (w/v) paraformaldehyde and stained for glial fibrillary acidic protein (GFAP; a,c) or glutamine synthetase (GS; b,d) as described in chapter 2.1. The overlay of BP fluorescence and Cy3-labeled secondary antibody is depicted in yellow. The scale bar in b represents 50 μm and applies for panels a and b, the scale bar in d represents 20 μm and applies to panels c and d.
3.2.2. Uptake of iron oxide nanoparticles in serum-free media
In cultured astrocytes, IONPs were intracellularly localized in vesicles as shown by
electron microscopy (Geppert et al., 2012; Geppert et al., 2011). This suggests that one or
more endocytotic processes are involved. Endocytosis has been divided into phagocytosis
for large particles and pinocytosis which is further subdivided in macropinocytosis,
clathrin-mediated and clathrin-independent endocytosis (Canton and Battaglia, 2012).
The latter pathway is far less understood than the formers but is described to require
cholesterol and to be mediated by caveolin, ADP-ribosylation factor 6, RhoA or flotillin
(Blouin, 2013; Maldonado-Baez et al., 2013). Depending on the mechanism and proteins
Summarizing discussion 91
involved in the invagination, also the generated endosomes vary in size and shape, such as
spherical or tubular, between the different pathways and such endosomes might traffic
differently within the cell (Blouin, 2013; Canton and Battaglia, 2012; Doherty and
McMahon, 2009). One commonly used approach to test for pathways responsible for
IONP uptake is application of inhibitors although such inhibitors target more or less
specifically a given endocytotic pathway (Ivanov, 2008).
For none of the cell culture models of brain cells used, the uptake mechanism for IONPs
in absence of serum was identified. There are several options why IONP uptake was not
inhibited in absence of serum such as rapid switch to other pathways (Doherty and
McMahon, 2009) or the involvement of a pathway for which the inhibitors used were not
useful. Furthermore, a vesicle-forming but non-endocytotic pathway might contribute to
the IONP uptake by solely physical processes. At least for silica NPs it was reported that
endocytotic proteins are not necessary for uptake. These NPs are internalized into
endocytosis-incapable red blood cells by mediating deformation and invagination of
membrane because of adhesive forces between NPs and membrane (Zhao et al., 2011).
Such a process might be facilitated especially in the absence of a protein corona due to
strong binding of the strongly charged IONPs to the cell surface.
3.2.3. Uptake of iron oxide nanoparticles in serum-containing media
In contrast to serum-free conditions in presence of 10% serum, endocytotic IONP uptake
by the different cultured brain cells was mainly clathrin-mediated endocytosis and
macropinocytosis (Table 3.2). Importantly, the inhibitors which were effective to inhibit
IONP uptake varied between the cell types investigated (chapters 2.2-2.4; Geppert et al.,
2013) as well as between different types of IONPs. For cultured astrocytes Pickard et al.
showed inhibition of uptake of carboxyl-modified SPHERO Nile Red fluorescent
magnetic particles by tyrphostin 23, dynasore, amiloride and EIPA (Pickard et al., 2010)
whereas D-IONP uptake was blocked by chlorpromazine and wortmannin (Geppert et
al., 2013). Such a variation of the effectiveness of endocytosis inhibitors was also reported
for polystyrene NPs in several cell lines (dos Santos et al., 2011). While the uptake of
IONPs by oligodendroglial cells and cerebral granule neurons was inhibited completely
by application of two or three endocytosis inhibitors (chapters 2.2 and 2.4), the uptake of
IONPs by primary astrocytes and microglia could not be blocked to the level of the 4°C
binding control suggesting the involvement of multiple pathways in IONP uptake. The
92 Summarizing discussion
different neural cell types might express certain endocytotic pathways and/or some
isoforms of proteins involved differently and hence, might strongly differ in their
sensitivity to endocytosis inhibitors.
In general, the accumulation of IONPs in presence of serum was lowered by 80-90% as
compared to incubation without serum (chapters 2.2 and 2.4; Geppert et al., 2013). This
is in accord with reports on other cell types and other types of NPs (Petri-Fink et al.,
2008; Lesniak et al., 2013). Likely, reason for this are the lower binding capacity of
IONPs with protein corona to cell membranes compared to IONPs without a protein
corona. At least for lung epithelial cells it was shown by atomic force microscopy that the
detachment force per bond of IONPs to the cells as well as the number of bonds is
higher for IONPs in serum-free media than in presence of 10% serum (Pyrgiotakis et al.,
2014). Moreover, the decreased iron content after incubation with IONPs in presence of
serum might also be caused by the involvement of different endocytotic pathways under
serum-free and serum-containing conditions.
33.2.4. Cellular localization of iron oxide nanoparticles
Cell associated IONPs are present extracellularly attached and intracellular after
internalization. Incubations at 4°C are frequently used as binding control to investigate the
amount of material attached extracellularly to the membrane (Kim et al., 2006; Geppert
et al., 2011; Smith et al., 2012b). Except for microglia which displayed only 20% of total
iron content as IONPs sticking to the cell surface, probably due to fast internalization of
IONPs, all other investigated neural cell types contained after incubation at 4°C around
50% of iron content as compared to incubations at 37°C irrespective of serum-free or
serum-containing media. This implies lower binding of protein-coated IONPs to cell
membranes compared to serum-free conditions (chapters 2.2 and, 2.4; Geppert et al.,
2013). Moreover, one should consider that at 4°C, compared to 37°C, Brownian
movement is decreased (He and Hui, 1985) and that IONPs and components of the
plasma membrane have decreased velocity and fluidity, respectively which in turn might
lower the chance for interactions between IONPs and membrane. As recently described,
also the composition of the protein corona depends on incubation temperature
(Mahmoudi et al., 2013) and hence, different interactions of IONPs with the cell surface
might be possible so that 4°C binding controls might not exclusively represent the cell
adsorbed part of cellular IONPs in presence of serum.
Summarizing discussion 93
After uptake, D- and BP-IONPs were found in vesicles in all neural cell types investigated
(Table 3.2; Figure 3.1) as seen by fluorescence microscopy (chapters 2.2-2.5) or TEM
(Geppert et al., 2011; Geppert et al., 2012) and resided mainly perinuclear (chapter 2.5),
supporting literature data for secondary astrocytes and microglia, for the neuronal cell line
PC12 and for OLN-93 cells (Pisanic et al., 2007; Pickard et al., 2010; Pickard and Chari,
2010; Hohnholt et al., 2010; Jenkins et al., 2013). This suggests that IONP-containing
vesicles are transported along microtubules as described for IONP-encapsulated silicon
particles in human microvascular vein endothelial cells (Ferrati et al., 2010). Indeed,
immunocytochemical staining for α-tubulin showed co-localization with BP-IONPs in
secondary astrocytes as observed during a research project by Nimesha Tadepalle in
2012. Only in microglia there was evident rapid lysosomal localization of the IONPs
(Figure 3.1; chapters 2.3 and 2.5) suggesting different endosomal trafficking within the
four neural cell types investigated. The fate of endosomes depends on the endocytotic
pathway from which they derive. While phagosomes fuse directly with late endosomes or
lysosomes, endosomes coming from macropinocytosis or from clathrin-, caveolin- or
ADP-ribosylation factor 6-mediated endocytosis evolve first to early endosomes which are
only slightly acidic (Doherty and McMahon, 2009). From early endosomes they can
either traffic to late endosomes and lysosomes or be sorted to the Golgi apparatus or to
recycling endosomes (Doherty and McMahon, 2009). Different intracellular trafficking of
endosomes plays probably a role the observed for differences in intracellular localization
of IONP-containing vesicles within cultured astrocytes, microglia and neurons.
3.2.5. Toxic effects of an exposure of iron oxide nanoparticles to cultured brain cells
Acute exposure of IONPs to cultured brain cells compromised microglial viability but not
that of astrocytes, oligodendroglial cells and neurons (Table 3.2; Figure 3.1). The likely
reason for this observation is the rapid transfer of IONP-containing vesicles to lysosomes
in microglia, accompanied by a rapid liberation of iron ions from IONPs in acidic
environments (chapter 2.5). The liberated Fenton-reactive ferrous iron causes extensive
ROS generation and thereby cell death of microglia (chapter 2.5; Figure 3.1). In addition,
these cells displayed a round shape (unpublished data) and clustering of cells (chapter
2.5) suggesting a harmed cytoskeleton in IONP-treated microglia. An impairment of the
cytoskeleton was shown for PC12 cells after IONP exposure by immunocytochemical
staining (Soenen et al., 2011; Dadras et al., 2013) whereas the microtubule network at
94 Summarizing discussion
least in secondary astrocytes was not altered after IONP treatment as observed during a
research project by Nimesha Tadepalle in 2012.
The slow acidification of IONP-containing vesicles in astrocytes, oligodendroglial cells
and neurons protected them from acute damage by iron-induced oxidative stress. It
seems that astrocytes are the neural cell type which is able to tolerate most IONPs
(chapter 1.4). Recently it was shown that toxicity was only observed for astrocytes when in
addition to IONP exposure an alternating magnetic field was applied which led to
mechanical damage (Schaub et al., 2014).
Delayed toxicity was observed for cultured neurons which were treated with IONPs
followed by a 24 h IONP-free recovery period when neuronal iron contents were higher
than 400 nmol/mg (chapter 2.4). For similar concentrations of carboxylated aminosilane
coated IONPs, a decreased vitality in primary cortical neurons after 24 h was reported
(Sun et al., 2013). Some ROS occurrence in neurons was seen after 6 h treatment with
2 mM D-IONPs under serum-free conditions (chapter 1.4), suggesting that the observed
delayed toxicity in cerebellar granule neurons (chapter 2.4) was probably mediated by
ROS. However, it cannot be excluded that IONPs may cause changes in
electrophysiological properties of neurons as previously reported for primary frontal
cortex cell culture (Gramowski et al., 2010). In contrast to neurons, no delayed toxicity
was reported for primary astrocytes even after 7 d post IONP exposure and for OLN-93
cells treated for up to 3 d with IONPs though also in these two cell types transient ROS
formation was observed (Hohnholt and Dringen, 2011; Geppert et al., 2012). However,
in these cells iron liberated from IONPs might have been safely stored in ferritin which
was upregulated after IONP treatment in astrocytes and OLN-93 cells (Geppert et al.,
2012; Hohnholt et al., 2011).
Cultured neural cells appear to suffer from IONP-derived stress only when cellular IONP
levels cross a certain threshold which differs between the cell types and might depend on
iron content, IONP type and time of incubation (Table 3.2). This view is supported by
literature data on cultured neurons, astrocytes, microglia and oligodendrocytes (Pickard
and Chari, 2010; Rivet et al., 2012; Sun et al., 2013; Jenkins et al., 2013). Also presence
of a protein corona might reduce toxicity by protecting IONPs from degradation for a
certain time. At least for astrocytoma 1321N1 cells the corona (or a portion of it) is
preserved during intracellular trafficking of polystyrene NPs (Wang et al., 2013). In
general, results of toxicity in cultured brain cells are difficult to compare due to varying
Summarizing discussion 95
experimental conditions and IONP types between the studies (Table 3.2; Pickard and
Chari, 2010; Wu et al., 2013a; Sun et al., 2013). In addition, different types of viability
assays have been used in such studies which strongly differ in their potential to
demonstrate acute and/or delayed toxicity (Hohnholt et al., 2014).
3.3. IImplications of the results for the in vivo situation
The data obtained on cultured brain cells in the present thesis and by others are likely to
have implications for the in vivo situation. IONPs can enter the brain via different routes
(Figure 3.3). When IONPs are applied intravenously, e.g. as contrast agent (Weinstein et
al., 2010) or for treatment of anemia (McCormack, 2012), they are rapidly coated with a
corona of serum proteins (Sakulkhu et al., 2014) and may enter the brain through an
intact BBB via transcytosis (Figure 3.3a; Yan et al., 2013). However, due to low cellular
uptake of IONPs in presence of a protein corona (chapters 2.2 and 2.4; Geppert et al.,
2013; Fleischer et al., 2013), the amount of IONPs which translocates into the brain is
likely to be quite low and may not cause adverse effects. Indeed, in vitro models of BBB
describe a low transfer of IONPs from endothelial to brain cells (Kenzaoui et al., 2013;
Thomsen et al., 2013) and little transfer of IONPs into the brain was reported in mice
and rats compared to liver and spleen (Jain et al., 2008; Wang et al., 2010).
Astrocytes cover the blood vessels (De Bock et al., 2014) and would be the first cells
coming in contact with the IONPs. After IONP uptake, astrocytes which are the least
IONP-vulnerable neural cell type, will store IONP-derived iron safely as ferritin (Figure
3.3a). However, it should be considered that astrocytes do not cover the entire BBB
(Virgintino et al., 1997) and that the BBB at the pineal gland or hypophysis is more
permeable than at other regions (Wislocki and Leduc, 1952; Arvidson and Tjalve, 1986;
Stehle et al., 2011). In such areas, more IONPs may escape the astrocytes and might
come in contact with microglia, oligodendrocytes or neurons.
Compared to physiological situations with intact BBB, higher doses of IONPs might
occur in brain at sites of injuries with a damaged BBB (Figure 3.3b) or when the BBB is
made permeable on purpose by treatment with mannitol (Mejias et al., 2010; Kozler et
al., 2013; Blanchette et al., 2014). Then, IONPs would have contact to all brain cell types
(Figure 3.3b). In this case, microglia are likely to accumulate most of the IONPs as
96 Summarizing discussion
observed also in humans and mice after intravenous administration of IONPs for
diagnostics of pathological conditions (Neuwelt et al., 2004; Taschner et al., 2005; Tysiak
et al., 2009;).
FFigure 3.3 Exposure of brain cells to IONPs in vivo. a) IONPs are transcytosed through the intact blood-brain barrier (BBB) and are likely to be taken up predominantly by astrocytes which have the capacity to store IONP-derived iron as ferritin. b) IONPs can enter the brain easily in large amounts if the BBB is impaired. Under such conditions IONPs will be taken up predominantly by microglia and to lesser extent by astrocytes, neurons and oligodendrocytes. c) After inhalation, IONPs invade the brain through sensory neurons of the olfactory bulb and may thereby directly harm these neurons. The enhanced levels of IONPs in the brain will be mostly taken up by microglia. d) For hyperthermia treatment, IONPs are injected into brain tumors. In close proximity to the injection, they may be mainly accumulated by microglia. Adapted and modified from chapter 1.4.
Summarizing discussion 97
In case of direct injection of IONPs for magnetic fluid hyperthermia (Wankhede et al.,
2012) very high concentrations (around 2 M) of IONPs are applied (Jordan et al., 2006;
van Landeghem et al., 2009). Post mortem studies have revealed that besides tumor cells
mainly macrophage-like cells took up the administered IONPs (van Landeghem et al.,
2009). The nature of the macrophages was not described and might be microglia and/or
peripheral macrophages entering the brain through a damaged BBB (Tysiak et al., 2009).
FFigure 3.4 Potential effects of IONP exposure of microglia in vivo. Exposure of microglia to IONPs may have different consequences depending on the amount of accumulated IONPs. Below a threshold level, IONPs might not damage the cells but may result in alterations in iron content and thereby facilitating neurodegenerative disorders. Above a threshold level, accumulated IONPs may cause microglial release of pro-inflammatory cytokines which are neurotoxic and/or lead to cell death of microglia which compromise brain microglial functions in neuroprotection or phagocytosis.
98 Summarizing discussion
The currently available data on IONP toxicity suggest that in brain predominant IONP
uptake by microglia has adverse effects. An overview of possible consequences of an
IONP exposure to microglia in brain is shown in Figure 3.4. In the best case, the amount
of IONPs is below a threshold and no toxicity occurs (Figure 3.4). However, dependent
on the iron content, ROS might be generated in microglia and the extent of oxidative
stress might trigger apoptosis or necrosis (Burlacu et al., 2001). Apoptotic vesicles would
be cleared by other microglia (Neumann et al., 2009; Napoli and Neumann, 2009) but
necrosis would also stimulate a pro-inflammatory response in microglia (Pais et al., 2008)
which is connected to neurodegeneration (Figure 3.3; Smith et al., 2012a). Cell death of
microglia in general would decrease the number of microglia in the region of incident
leading to a reduced microglial potential to defend against intruders or to help in
neuroprotection. Moreover, microglia can become activated after IONP application as
reported in vivo and in vitro (Wang et al., 2011a; Xue et al., 2012) and thereby might
release neurotoxic cytokines under such conditions (Figure 3.4; Correale, 2014) though
there are contradictory reports (chapter 2.3; Pickard and Chari, 2010). Activated
microglia have in addition been reported to discriminate poorly between viable and
apoptotic neurons and can promote neuronal death by so called phagoptosis which
describes killing viable cells by phagocytosis (Brown and Neher, 2012). This process is
even stimulated if neurons suffer from oxidative stress (Brown and Neher, 2012) which
might be also promoted by IONP exposure (chapter 1.4; Deng et al., 2014).
So far it is not clear how much IONPs enter the brain via the different routes under
physiological or pathological conditions and if the local concentration might be
sufficiently high to cause adverse effects in humans. However, alterations in iron
concentrations in brain have been reported to be involved in neurodegenerative disorders
such as Alzheimer’s, Parkinson’s or Huntington’s disease (Batista-Nascimento et al.,
2012; Urrutia et al., 2014; Wong and Duce, 2014). Hence, accumulation of IONPs,
gradually release of iron ions and their distribution in brain might also under non-
cytotoxic conditions alter the neural iron metabolism (Figure 3.4) and facilitate
progression of such disorders over time. However, the direct role of iron in these diseases
is unclear and it remains to be elucidated whether iron is a source or a consequence of
these diseases (Batista-Nascimento et al., 2012).
Summarizing discussion 99
3.4. FFuture perspectives
3.4.1. Synthesis of fluorescently labeled iron oxide nanoparticles
The fluorescently labeled IONPs have been proven to be a useful tool for visualization of
IONPs in cultured brain cells by fluorescence microscopy. However, further
improvement of BP-IONPs might be beneficial to prevent non-BP-IONP-associated
fluorescence as described above (chapter 3.1.2). For instance, more washing steps after
coating IONPs with BP-labeled DMSA might be helpful as reported in a similar labeling
protocol in which also DMSA was covalently modified to rhodamine and fluorescein.
Therein, no artifacts such as staining of nuclei were seen (Bertorelle et al., 2006).
Alternatively, fluorescent dyes could be embedded into a stable silica shell around IONPs
(Peng et al., 2014) from which the dye cannot elute.
3.4.2. Mechanism of accumulation of iron oxide nanoparticles under serum-free
conditions
In order to analyze the unknown uptake mechanism of IONPs in absence of serum, cells
may be depleted in specific proteins of a given endocytotic process such as clathrin,
caveolin or ADP-ribosylation factor 6 by small interfering RNA (Canton and Battaglia,
2012; Sahay et al., 2010). This approach would be more specific than usage of
endocytosis inhibitors (Ivanov, 2008). Moreover, immunocytochemical techniques, either
by the use of fluorescence microscopy or TEM, should be exploited for visualization of
certain pathways. For example, co-localization studies with known cargos of the single
endocytotic pathways or transfection of a certain protein fused with green fluorescent
protein are established methods to investigate the uptake mechanism of NPs (Sahay et al.,
2010). In the former case, transferrin (Qaddoumi et al., 2003; Barua and Rege, 2009) and
cholera toxin B or Simian virus 40 (Qaddoumi et al., 2003; Wang et al., 2011b) are used
for studying clathrin- and caveolin-mediated endocytosis, respectively. Similar studies
could be performed for macropinocytosis, for example by co-localizing fluorescently
labeled IONPs with ricinB-quantum dots which are internalized via macropinocytosis
(Iversen et al., 2012).
In addition, uptake of IONPs by endocytotic-independent invagination of the plasma
membrane would be very interesting to study. A straightforward approach would be to
100 Summarizing discussion
knock-down or inhibit all possible endocytotic pathways. However, this is difficult to
achieve since the available inhibitors are not sufficiently specific, the total incapability of
endocytosis might be harmful to cells and/or there might be unknown pathways which are
involved in IONP uptake. As alternative, to at least prevent energy-dependent endocytosis
cells could be depleted of energy by glucose deprivation (Mattson et al., 1993; Almeida et
al., 2001) and/or application of the respiratory chain inhibitor sodium azide or the
mitochondrial ATP-synthase inhibitor oligomycin A as described in astrocytes and
neurons (Swanson et al., 1997; Pamenter et al., 2012).
33.4.3. Binding, uptake and fate of iron oxide nanoparticles in cultured brain cells
The reason for differences or similarities of different types of neural cells regarding IONP
accumulation and cellular localization of IONPs should be further investigated. The first
event in uptake is the attachment of IONPs to the plasma membrane which should be
investigated in more detail and quantified. Atomic force microscopy is able to reveal the
force of interaction between IONPs and the cell surface and could be used to test for
differences in IONP adsorption between astrocytes, microglia, oligodendrocytes and
neurons as well as between incubation conditions as reported recently for IONPs and
lung epithelial cells in absence and presence of serum (Pyrgiotakis et al., 2014).
Moreover, a quantification of IONPs attached to the cell membrane can be achieved by
biotinylation of the plasma membrane, separation of the biotinylated membrane by
neutravidin beads after cell homogenization and subsequent determination of the iron
content for the obtained fractions (deBlaquiere and Burgess, 1999). Attachment of
IONPs to the cell surface could also be visualized with scanning electron microscopy
which would also reveal morphological responses of the cells to IONPs on the
ultrastructural level as seen for primary cortical neurons (Rivet et al., 2012).
Using TEM and immunocytochemical approaches, IONPs should be localizes within
astrocytes, oligodendroglial cells and neurons as in these cells IONPs were not
translocated rapidly to lysosomes, in contrast to microglia. Incubation times with IONPs
should be elongated to examine the fate of IONPs by immunocytochemical staining for
stages of endosome differentiation such as early/late endosomes, lysosomes or
caveosomes which would help to identify the IONP-containing vesicles. Alternatively,
IONPs could be functionalized with a pH-responsive fluorescent dye such as 8-
hydroxypyrene-1,3,6-trisulfonic acid, Oregon Green, fluorescein and/or rhodamine B
Summarizing discussion 101
(Overly et al., 1995; Benjaminsen et al., 2011) but also with the pH-dependent Ageladine
A (Bickmeyer et al., 2008; Bickmeyer et al., 2010) revealing at least information on the
pH of IONP-containing vesicles. When IONPs enter lysosomes, iron is released
progressively from IONPs (Levy et al., 2010; Malvindi et al., 2014; Shukla et al., 2014).
To reveal the time frame and the amount of iron liberated from internalized IONPs, iron
ions could be visualized by using iron selective fluorescent probes such as an alanine
substituted rhodamine derivative or a BODIPY-cyanine-pyridine derivative (Li et al.,
2011a; Saleem et al., 2014).
Moreover, it would be interesting to study the size dependency of IONP uptake and
toxicity in neural cells. The IONPs used in the present thesis as well as in former studies
of our group were not monodisperse i.e. the dispersion were composed of IONPs with
varying sizes of single NPs as well as of IONP aggregates in dispersion. As endocytotic
pathways are size-dependent, IONPs with a single size might be taken up by a selected
pathway (Canton and Battaglia, 2012) which would enlighten the role of certain pathways
in accumulation of IONPs.
3.4.4. Effects of an iron oxide nanoparticle exposure to cultured brain cells
So far, the vast majority of studies on consequences of IONPs on cultured brain cells
tested only for cell viability. However, sub-toxic doses of IONPs still might influence cell
type-specific functionality. Electrophysiology techniques could be used to study neuronal
function as it was recently reported that the burst rate of cultured neurons was decreased
by IONPs in primary cortical neurons (Gramowski et al., 2010). Astrocytes should be
studied for potential effects of IONPs on astrocytic functions including calcium waves,
release of gliotransmitters and supporting other neural cells (Verkhratsky and Parpura,
2010; Peters and Connor, 2014). In addition, it would be interesting to test for ability of
astrocytes to protect IONP-harmed neural cells. Preliminary experiments suggested less
vulnerability of microglia in astroglia-rich primary cultures as compared to primary
microglia cultures (data not shown). The effect of IONPs on mobility of microglia by
migration assays (Yu et al., 2014) and release of cytokines (Stenken and Poschenrieder,
2015) should be tested. Migration assays are usually performed by scratching the cell layer
and monitoring the relocation of cells (Yu et al., 2014). Such experiments revealed at least
for carbon nanotubes no change in microglial mobility (Cyrill Bussy, personal
communication). Also the reported phagoptosis of viable but stressed neurons by
102 Summarizing discussion
microglia (Brown and Neher, 2012) could be investigated by using co-cultures of
microglia and IONP-treated neurons. To study effects of IONPs on oligodendroglial
functionality, the formation and maintenance of myelin sheaths could be tested in co-
cultures with neurons (Barateiro and Fernandes, 2014).
Toxic events might be reduced or even prevented by functionalization of IONPs. Herein,
it is possible to target IONPs towards certain cell types by introducing a ligand or antibody
to a cell type specific receptor or membrane protein into the coat in order to overcome
the predominant microglial IONP uptake. For this, IONPs could be functionalized e.g.
with antibodies or ligands against the astrocytic excitatory amino acid transporter 2
(Roberts et al., 2014) or CD44 (Grabrucker et al., 2011), oligodendroglial myelin-specific
proteins such as myelin-basic protein and myelin-associated glycoprotein (Peters and
Connor, 2014) or neuronal proteins such as neural cell adhesion molecule 1 (Grabrucker
et al., 2011). To support cells against oxidative stress, the coat could be modified with
antioxidants as it was previously reported for gold and selenium NPs which were
functionalized with Trolox (an analogue of vitamin E), salvianic acid and 11-mercapto-1-
undecanol, respectively and which could scavenge ROS in cell-free attempts as well as in
vitro and in vivo (Du et al., 2013; Li et al., 2011b; Nie et al., 2007).
3.4.5. Investigations on uptake and effects of iron oxide nanoparticles in vivo
The most important aspect for risk assessment and investigations on therapeutic potential
of IONPs in biomedical application are in vivo studies. Though some publications
suggest entry of IONPs into brain through the intact BBB (Jain et al., 2008; Wang et al.,
2010; Yan et al., 2013), the data are not so clear due to limited resolution of the
published microscopic images. Hence, different application modes should be used to
investigate possible routes of entry, the amount of iron reaching the brain, and a time
dependent distribution of IONPs within the brain. For such studies, fluorescent IONPs
should be applied intravenously, intracerebrally or intranasally in rodents and the IONPs
in brain and other tissues could be visualized in slices by fluorescence microscopy
coupled with immunocytochemical staining, by Perls’ iron staining and/or by TEM after
certain incubation periods.
For the brain, parameters of toxicity such as ROS generation or apoptosis as well as
changes in morphology of the neural cell types should be analyzed after an IONP
exposure, e.g. if microglia turned into the activated state as reported in mice after
Summarizing discussion 103
intranasal application (Wang et al., 2011a). Moreover, behavioral studies on rats or mice
are highly warranted to investigate whether IONP exposure may have a long-term effect
on cognition since data on that issue are scarce (chapter 1.4). In order to study whether an
altered neural iron content in IONP-treated brains promote neurodegenerative disorders,
application of IONPs to animal models for Alzheimer’s and Parkinson’s disease (Bove
and Perier, 2012; Dujardin et al., 2014) would be an option. In this context, analysis of
the brain from IONP-treated animals for pathological alterations and characteristics of the
neurodegenerative disorders should be performed.
In addition to potential adverse effects, IONPs might also be beneficial when applied as
drug delivery system. As vehicle for therapeutics, IONPs may be used to treat disorders
such as Alzheimer’s or Parkinson’s disease as reported recently for functionalized carbon
nanotubes (Professor Dr. Kostas Kostarelos, personal communication). Iron deficiency in
brain which causes disorders like restless legs syndrome is not easy to treat by peripheral
application of low molecular weight iron due to poor permeability of the brain for iron
(Hare et al., 2013). Due to the possibility to functionalize IONPs for good permeation of
the BBB into the brain, IONPs might also be a valuable tool to treat iron deficiency.
104 Summarizing discussion
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4. AAppendix
4.1. Curriculum vitae ................................................................................................ 115
4.2. List of publications ............................................................................................ 117
Appendix 115
44. Appendix
4.1. Curriculum vitae
Personal Information
Name Charlotte Petters Birthday 23/08/1984 in Braunschweig
Education
Since 04/2011 PhD thesis on “Uptake and metabolism of iron oxide nanoparticles in cultured brain cells” in the research group of Professor Ralf Dringen, University of Bremen.
10/2008 – 01/2011 University of Bremen, Biochemistry and Molecular Biology Master of Science Title of the Master thesis: Investigation on the subcellular
localization of iron oxide nanoparticles. 10/2005 – 09/2008 Universität Bremen, Biologie und Chemie Bachelor of Science Title of the Bachelor thesis: Kupferbestimmung mit Hilfe des
Kupfer(I)-spezifischen Chelators Bathocuproindisulfonat. 09/2004 – 08/2005 Volunteer at the Bund für Umwelt und Naturschutz Deutschland
in Braunschweig. 06/2004 Abitur, Neue Oberschule in Braunschweig
Workshops and Conferences
09/2009 Participitation and presentation at a dutch-german congress on liposomes, Ameland (Netherlands). Title of the presentation: Synthesis of boron-containing lipids for the formation of liposomes.
03/2012 Three-week Workshop “Submicroscopic anatomy and preparation
technique“ for preparation and analysis of biological samples by scanning and transmission electron microscopy, University of Vienna (Austria).
06/2013 Participitation, poster and “Blitz”-presentation at the conference of
the European Society of Neurochemistry on “Advances in molecular mechanisms underlying neurological disorders” in Bath (Great Britan). Title of the poster: Uptake of iron oxide nanoparticles by oligodendroglial cells.
116 Appendix
04/2014 Participiation and poster at the conference “7th International
Nanotoxicology Congress 2014” in Antalya (Turkey). Title of the poster: Accumulation of iron oxide nanoparticles by cultured primary neurons.
06/2014 Participation and poster at the conference “Nanobio Europe” in
Münster. Title of the poster: Accumulation of iron oxide nanoparticles by cultured primary neurons.
07/2014 Participation and poster at the conference “9th FENS Forum of
Neuroscience“ in Milano (Italy). Title of the poster: Accumulation of iron oxide nanoparticles by cultured primary neurons.
TTeaching Experiences
10/2006 – 09/2010 Rechenmethoden für Naturwissenschaftler I + II: tutorial and correction of the exercises
04/2008 – 09/2008 Pflanzenphysiologie: tutorial 2011 - 2014 Practical course Biochemistry (Bachelor of Science): seminars,
supervision of students and correction of protocols 01/2013, 01/2014 Practical course Bioorganic Chemistry (Master of Science):
supervision of students Language Skills
German native language English fluent French very good knowledge Japanese good knowledge Swedish basic knowledge
Appendix 117
4.2. LList of publications
Hohnholt, M. C., Geppert, M., Luther, E. M., PPetters, C., Bulcke, F. & Dringen, R. (2013). Handling of iron oxide and silver nanoparticles by astrocytes. Neurochem Res 38:227-239 Geppert, M., PPetters, C., Thiel, K. & Dringen, R. (2013). The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes. J Nanopart Res 15:1349 Luther, E. M., PPetters, C., Bulcke, F., Thiel, K., Kaltz, A., Bickmeyer, U. & Dringen, R. (2013). Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells. Acta Biomater 9:8454-8465 Tietje, K., Rivera-Ingraham, G., PPetters, C., Abele, D., Dringen, R. & Bickmeyer, U. (2013). Reporter dyes demonstrate functional expression of multidrug resistance proteins in the marine flatworm Macrostomum lignano: the sponge-derived dye Ageladine A is not a substrate of these transporters. Mar Drugs 11:3951-3969 Petters, C. & Dringen R (2014). Comparison of primary and secondary rat astrocyte cultures regarding glucose and glutathione metabolism and the accumulation of iron oxide nanoparticles. Neurochem Res 39:46-58 Petters, C,, Bulcke, F., Thiel, K., Bickmeyer, U. & Dringen, R. (2014). Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells. Neurochem Res 39:372-383 Petters, C., Irrsack, E., Koch, M. & Dringen, R. (2014). Uptake and metabolism of iron oxide nanoparticles in brain cells. Neurochem Res 39:1648-1660 Bollhorst, T., Shahabi, S., Wörz, K., PPetters, C., Dringen, R., Maas, M. & Rezwan, K. (2015). Bifunctional submicron colloidosomes co-assembled from fluorescent and superparamagnetic nanoparticles. Angew Chem Int Ed Engl 54:118-123 Brandmann, M., Hohnholt, M. C., PPetters, C. & Dringen, R. (2014). Antiretroviral protease inhibitors accelerate glutathione export from viable cultured rat neurons. Neurochem Res 39:883-892 Petters, C. & Dringen, R. (2015). Accumulation of iron oxide nanoparticles by cultured primary neurons. Neurochem Int 81:1-9