uptake and metabolism of iron oxide nanoparticles in cultured brain cells · 2015-03-05 · uptake...

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

1.3. RReferences

Aggarwal, S., Yurlova, L. & Simons, M. (2011). Central nervous system myelin: structure, synthesis and assembly. Trends Cell Biol 21: 585-593.

Ahlemeyer, B., Kehr, K., Richter, E., Hirz, M., Baumgart-Vogt, E. & Herden, C. (2013). Phenotype, differentiation, and function differ in rat and mouse neocortical astrocytes cultured under the same conditions. J Neurosci Methods 212: 156-164.

Albrecht, J., Sonnewald, U., Waagepetersen, H. S. & Schousboe, A. (2007). Glutamine in the central nervous system: function and dysfunction. Front Biosci 12: 332-343.

Allen, N. J. & Barres, B. A. (2009). Neuroscience: glia - more than just brain glue. Nature 457: 675-677.

Azevedo, F. A., Carvalho, L. R., Grinberg, L. T., Farfel, J. M., Ferretti, R. E., Leite, R. E., Jacob Filho, W., Lent, R. & Herculano-Houzel, S. (2009). Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J Comp Neurol 513: 532-541.

Balcar, V. J. & Johnston, G. A. (1972). The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J Neurochem 19: 2657-2666.

Barateiro, A. & Fernandes, A. (2014). Temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination. Biochim Biophys Acta 1843: 1917-1929.

Barateiro, A., Miron, V. E., Santos, S. D., Relvas, J. B., Fernandes, A., Ffrench-Constant, C. & Brites, D. (2013). Unconjugated bilirubin restricts oligodendrocyte differentiation and axonal myelination. Mol Neurobiol 47: 632-644.

Barreto, G. E., Gonzalez, J., Torres, Y. & Morales, L. (2011). Astrocytic-neuronal crosstalk: implications for neuroprotection from brain injury. Neurosci Res 71: 107-113.

Barth, R. F. & Kaur, B. (2009). Rat brain tumor models in experimental neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J Neurooncol 94: 299-312.

Bauer, N. G., Richter-Landsberg, C. & Ffrench-Constant, C. (2009). Role of the oligodendroglial cytoskeleton in differentiation and myelination. Glia 57: 1691-1705.

Bernier, L. P., Ase, A. R., Boue-Grabot, E. & Seguela, P. (2013). Inhibition of P2X4 function by P2Y6 UDP receptors in microglia. Glia 61: 2038-2049.

Biber, K., Owens, T. & Boddeke, E. (2014). What is microglia neurotoxicity (not)? Glia 62: 841-854.

Bradl, M. & Lassmann, H. (2010). Oligodendrocytes: biology and pathology. Acta Neuropathol 119: 37-53.

Brito, C., Simao, D., Costa, I., Malpique, R., Pereira, C. I., Fernandes, P., Serra, M., Schwarz, S. C., Schwarz, J., Kremer, E. J. & Alves, P. M. (2012). Generation and genetic modification of 3D cultures of human dopaminergic neurons derived from neural progenitor cells. Methods 56: 452-460.

Buntinx, M., Vanderlocht, J., Hellings, N., Vandenabeele, F., Lambrichts, I., Raus, J., Ameloot, M., Stinissen, P. & Steels, P. (2003). Characterization of three human oligodendroglial cell lines as a model to study oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression. J Neurocytol 32: 25-38.

Chang, C. C., Hong, S. J., Lin, H. L. & Su, M. J. (1985). Acetylcholine hydrolysis during neuromuscular transmission in the synaptic cleft of skeletal muscle of mouse and chick. Neuropharmacology 24: 533-539.

14 Introduction

Codeluppi, S., Gregory, E. N., Kjell, J., Wigerblad, G., Olson, L. & Svensson, C. I. (2011). Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes. J Neurosci Methods 197: 118-127.

Contestabile, A. (2002). Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro. Cerebellum 1: 41-55.

Cregg, J. M., DePaul, M. A., Filous, A. R., Lang, B. T., Tran, A. & Silver, J. (2014). Functional regeneration beyond the glial scar. Exp Neurol 253: 197-207.

De Bock, M., Decrock, E., Wang, N., Bol, M., Vinken, M., Bultynck, G. & Leybaert, L. (2014). The dual face of connexin-based astroglial Ca2+ communication: a key player in brain physiology and a prime target in pathology. Biochim Biophys Acta 1843: 2211-2232.

Deierborg, T. (2013). Preparation of primary microglia cultures from postnatal mouse and rat brains. Methods Mol Biol 1041: 25-31.

Derobertis, E., Gerschenfeld, H. M. & Wald, F. (1958). Cellular mechanism of myelination in the central nervous system. J Biophys Biochem Cyt 4: 651-656.

Dringen, R., Gebhardt, R. & Hamprecht, B. (1993). Glycogen in astrocytes: possible function as lactate supply for neighboring cells. Brain Res 623: 208-214.

Dringen, R., Pfeiffer, B. & Hamprecht, B. (1999). Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19: 562-569.

Dringen, R., Brandmann, M., Hohnholt, M. C. & Blumrich, E. M. (2015). Glutathione-dependent detoxification processes in astrocytes. Neurochem Res, in press.

Du, F., Qian, Z. M., Zhu, L., Wu, X. M., Qian, C., Chan, R. & Ke, Y. (2010). Purity, cell viability, expression of GFAP and bystin in astrocytes cultured by different procedures. J Cell Biochem 109: 30-37.

Eyo, U. B. & Dailey, M. E. (2013). Microglia: key elements in neural development, plasticity, and pathology. J Neuroimmune Pharmacol 8: 494-509.

Fernandes, S. P., Dringen, R., Lawen, A. & Robinson, S. R. (2010). Neurones express glutamine synthetase when deprived of glutamine or interaction with astrocytes. J Neurochem 114: 1527-1536.

Floeter, M. K. & Greenough, W. T. (1979). Cerebellar plasticity: modification of Purkinje cell structure by differential rearing in monkeys. Science 206: 227-229.

Funfschilling, U., Supplie, L. M., Mahad, D., Boretius, S., Saab, A. S., Edgar, J., Brinkmann, B. G., Kassmann, C. M., Tzvetanova, I., D., Möbius, W., Diaz, F., Meijer, D., Suter, U., Hamprecht, B., Sereda, M. W., Moraes, C. T., Frahm, J., Goebbels, S. & Nave, K. A. (2012). Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity. Nature 485: 517-521.

Genis, L., Davila, D., Fernandez, S., Pozo-Rodrigalvarez, A., Martinez-Murillo, R. & Torres-Aleman, I. (2014). Astrocytes require insulin-like growth factor I to protect neurons against oxidative injury. F1000Res 3: 28.

Ghate, P. S., Sidhar, H., Carlson, G. A. & Giri, R. K. (2014). Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain. Springerplus 3: 161.

Giffard, R. G. & Swanson, R. A. (2005). Ischemia-induced programmed cell death in astrocytes. Glia 50: 299-306.

Goetschy, J. F., Ulrich, G., Aunis, D. & Ciesielski-Treska, J. (1986). The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture. J Neurocytol 15: 375-387.

Introduction 15

Grumbles, R. M., Almeida, V. W., Casella, G. T., Wood, P. M., Hemstapat, K. & Thomas, C. K. (2012). Motoneuron replacement for reinnervation of skeletal muscle in adult rats. J Neuropathol Exp Neurol 71: 921-930.

Guerout, N., Li, X. & Barnabe-Heider, F. (2014). Cell fate control in the developing central nervous system. Exp Cell Res 321: 77-83.

Hamprecht, B. & Löffler, F. (1985). Primary glial cultures as a model for studying hormone action. Methods Enzymol 109: 341-345.

Hansson, E. & Thorlin, T. (1999). Brain primary cultures and vibrodissociated cells as tools for the study of astroglial properties and functions. Dev Neurosci 21: 1-11.

Herculano-Houzel, S. (2014). The glia/neuron ratio: how it varies uniformly across brain structures and species and what that means for brain physiology and evolution. Glia 62:1377-1391.

Hertz, L., Schousboe, A., Boechler, N., Mukerji, S. & Fedoroff, S. (1978). Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem Res 3: 1-14.

Hille, B. & Catterall, W. A. (2006). Electrical excitability and ion channels. In Basic Neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 95-109.

Holz, R. W. & Fisher, S. K. (2006). Synaptic transmission and cellular signaling: an overview. In Basic neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 167-183.

Howarth, C. (2014). The contribution of astrocytes to the regulation of cerebral blood flow. Front Neurosci 8: 103 (9 p.).

Jameson, N., Olson, J., Nguyen, H. & Holtzman, D. (1984). Respiration in primary cultured cerebellar granule neurons and cerebral cortical neurons. J Neurochem 42: 470-474.

Jarjour, A. A., Zhang, H., Bauer, N., Ffrench-Constant, C. & Williams, A. (2012). In vitro modeling of central nervous system myelination and remyelination. Glia 60: 1-12.

Jeon, H., Kim, J. H., Kim, J. H., Lee, W. H., Lee, M. S. & Suk, K. (2012). Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity. J Neuroinflammation 9: 149 (22 p.).

Jones, E. V., Cook, D. & Murai, K. K. (2012). A neuron-astrocyte co-culture system to investigate astrocyte-secreted factors in mouse neuronal development. Methods Mol Biol 814: 341-352.

Kang, K. S., Lee, S. I., Hong, J. M., Lee, J. W., Cho, H. Y., Son, J. H., Paek, S. H. & Cho, D. W. (2014). Hybrid scaffold composed of hydrogel/3D-framework and its application as a dopamine delivery system. J Control Release 175: 10-16.

Kettenmann, H., Hanisch, U. K., Noda, M. & Verkhratsky, A. (2011). Physiology of microglia. Physiol Rev 91: 461-553.

Kettenmann, H. & Verkhratsky, A. (2011). Neuroglia, der lebende Nervenkitt. Fortschr Neurol Psychiatr 79: 588-597.

Kimelberg, H. K. (2010). Functions of mature mammalian astrocytes: a current view. Neuroscientist 16: 79-106.

Kondratskaya, E., Shin, M. C. & Akaike, N. (2010). Neuronal glutamate transporters regulate synaptic transmission in single synapses on CA1 hippocampal neurons. Brain Res Bull 81: 53-60.

Lallouette, J., De Pitta, M., Ben-Jacob, E. & Berry, H. (2014). Sparse short-distance connections enhance calcium wave propagation in a 3D model of astrocyte networks. Front Comput Neurosci 8: 45 (18 p.).

16 Introduction

Lange, S. C., Bak, L. K., Waagepetersen, H. S., Schousboe, A. & Norenberg, M. D. (2012). Primary cultures of astrocytes: their value in understanding astrocytes in health and disease. Neurochem Res 37: 2569-2588.

Lau, C. L., Kovacevic, M., Tingleff, T. S., Forsythe, J. S., Cate, H. S., Merlo, D., Cederfur, C., Maclean, F. L., Parish, C. L., Horne, M. K., Nisbet, D. R. & Beart, P. M. (2014). 3D electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes. J Neurochem 130: 215-226.

Lee, H. U., Yamazaki, Y., Tanaka, K. F., Furuya, K., Sokabe, M., Hida, H., Takao, K., Miyakawa, T., Fujii, S. & Ikenaka, K. (2013). Increased astrocytic ATP release results in enhanced excitability of the hippocampus. Glia 61: 210-224.

Lee, Y., Morrison, B. M., Li, Y., Lengacher, S., Farah, M. H., Hoffman, P. N., Liu, Y., Tsingalia, A., Jin, L., Zhang, P. W., Pellerin, L., Magistretti, P. J. & Rothstein, J. D. (2012). Oligodendroglia metabolically support axons and contribute to neurodegeneration. Nature 487: 443-448.

Liu, T., Xue, C. C., Shi, Y. L., Bai, X. J., Li, Z. F. & Yi, C. L. (2014). Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro. Neurosci Lett 579: 24-29.

Marlatt, M. W., Bauer, J., Aronica, E., van Haastert, E. S., Hoozemans, J. J., Joels, M. & Lucassen, P. J. (2014). Proliferation in the Alzheimer hippocampus is due to microglia, not astroglia, and occurs at sites of amyloid deposition. Neural Plast 2014: 693851 (12 p.).

Matsui, T., Kida, H., Iha, T., Obara, T., Nomura, S., Fujimiya, T. & Suzuki, M. (2014). Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice. Folia Neuropathol 52: 151-158.

McCarthy, K. D. & de Vellis, J. (1980). Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85: 890-902.

Milligan, C. E., Cunningham, T. J. & Levitt, P. (1991). Differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain. J Comp Neurol 314: 125-135.

Monteiro, R. A., Henrique, R. M., Rocha, E., Marini-Abreu, M. M., Oliveira, M. H. & Silva, M. W. (1998). Age-related changes in the volume of somata and organelles of cerebellar granule cells. Neurobiol Aging 19: 325-332.

Morrison, B. M., Lee, Y. & Rothstein, J. D. (2013). Oligodendroglia: metabolic supporters of axons. Trends Cell Biol 23: 644-651.

Nayak, D., Roth, T. L. & McGavern, D. B. (2014). Microglia development and function. Annu Rev Immunol 32: 367-402.

Neumann, H. & Wekerle, H. (2013). Brain microglia: watchdogs with pedigree. Nat Neurosci 16: 253-255.

Norenberg, M. D. & Martinez-Hernandez, A. (1979). Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161: 303-310.

Norton, W. T. & Poduslo, S. E. (1973). Myelination in rat brain: changes in myelin composition during brain maturation. J Neurochem 21: 759-773.

Oberheim, N. A., Goldman, S. A. & Nedergaard, M. (2012). Heterogeneity of astrocytic form and function. Methods Mol Biol 814: 23-45.

Palmer, M. J., Taschenberger, H., Hull, C., Tremere, L. & von Gersdorff, H. (2003). Synaptic activation of presynaptic glutamate transporter currents in nerve terminals. J Neurosci 23: 4831-4841.

Pasca, S. P., Panagiotakos, G. & Dolmetsch, R. E. (2014). Generating human neurons in vitro and using them to understand neuropsychiatric disease. Annu Rev Neurosci 37: 479-501.

Introduction 17

Pedraza, C. E., Taylor, C., Pereira, A., Seng, M., Tham, C. S., Izrael, M. & Webb, M. (2014). Induction of oligodendrocyte differentiation and in vitro myelination by inhibition of rho-associated kinase. ASN Neuro 6 (17 p.).

Pelvig, D. P., Pakkenberg, H., Stark, A. K. & Pakkenberg, B. (2008). Neocortical glial cell numbers in human brains. Neurobiol Aging 29: 1754-1762.

Peters, D. G. & Connor, J. R. (2014). Introduction to cells comprising the nervous system. Adv Neurobiol 9: 33-45.

Pisanu, A., Lecca, D., Mulas, G., Wardas, J., Simbula, G., Spiga, S. & Carta, A. R. (2014). Dynamic changes in pro- and anti-inflammatory cytokines in microglia after PPAR-gamma agonist neuroprotective treatment in the MPTPp mouse model of progressive Parkinson's disease. Neurobiol Dis 71: 280-291.

Raine, C. S. (2006). Neurocellular anatomy. In Basic Neurochemistry: molecular, cellular, and medical aspects 7 (Eds. Siegel, G. J., Albers, R. W., Brady, S. T. & Price, D. L.), Academic Press, Elsevier, pp. 3-19.

Richter-Landsberg, C. & Heinrich, M. (1996). OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. J Neurosci Res 45: 161-173.

Rinholm, J. E. & Bergersen, L. H. (2013). White matter lactate - does it matter? Neuroscience 276: 109-116.

Roberts, R. C., Roche, J. K. & McCullumsmith, R. E. (2014). Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: a light and electron microscopic study. Neuroscience 277: 522-540.

Rodhe, J. (2013). Cell culturing of human and murine microglia cell lines. Methods Mol Biol 1041: 11-16.

Saura, J. (2007). Microglial cells in astroglial cultures: a cautionary note. J Neuroinflammation 4: 26 (11 p.).

Saura, J., Tusell, J. M. & Serratosa, J. (2003). High-yield isolation of murine microglia by mild trypsinization. Glia 44: 183-189.

Scalettar, B. A. (2006). How neurosecretory vesicles release their cargo. Neuroscientist 12: 164-176.

Sheng, Z. H. (2014). Mitochondrial trafficking and anchoring in neurons: new insight and implications. J Cell Biol 204: 1087-1098.

Silman, I. & Sussman, J. L. (2005). Acetylcholinesterase: 'classical' and 'non-classical' functions and pharmacology. Curr Opin Pharmacol 5: 293-302.

Sofroniew, M. V. & Vinters, H. V. (2010). Astrocytes: biology and pathology. Acta Neuropathol 119: 7-35.

Stansley, B., Post, J. & Hensley, K. (2012). A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease. J Neuroinflammation 9: 115 (8 p.).

Stevens, C. F. & Williams, J. H. (2000). "Kiss and run" exocytosis at hippocampal synapses. Proc Natl Acad Sci U S A 97: 12828-12833.

Sunol, C., Babot, Z., Fonfria, E., Galofre, M., Garcia, D., Herrera, N., Iraola, S. & Vendrell, I. (2008). Studies with neuronal cells: from basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity. Toxicol In Vitro 22: 1350-1355.

Szabo, M. & Gulya, K. (2013). Development of the microglial phenotype in culture. Neuroscience 241: 280-295.

Takacs, J. & Hamori, J. (1994). Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex. J Neurosci Res 38: 515-530.

Tamashiro, T. T., Dalgard, C. L. & Byrnes, K. R. (2012). Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue. J Vis Exp: e3814 (5 p.).

18 Introduction

Tanaka, T., Wakabayashi, T., Oizumi, H., Nishio, S., Sato, T., Harada, A., Fujii, D., Matsuo, Y., Hashimoto, T. & Iwatsubo, T. (2014). CLAC-P/collagen type XXV is required for the intramuscular innervation of motoneurons during neuromuscular development. J Neurosci 34: 1370-1379.

Tasaki, I. (1939). The electro-saltatory transmission of the nerve impulse and the effect of narcosis upon the nerve fiber. Am J Physiol 127: 211-227.

Thorburne, S. K. & Juurlink, B. H. (1996). Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress. J Neurochem 67: 1014-1022.

Tulpule, K., Hohnholt, M. C., Hirrlinger, J. & Dringen, R. (2014). Primary cultures of astrocytes and neurons as model systems to study the metabolism and metabolite export from brain cells. In Neuromethods 90: Brain energy metabolism (Eds. Waagepetersen, H. & Hirrlinger, J.), Heidelberg, Springer, pp. 45-72.

Verkhratsky, A., Nedergaard, M. & Hertz, L. (2014). Why are astrocytes important? Neurochem Res, in press.

Verkhratsky, A. & Parpura, V. (2010). Recent advances in (patho)physiology of astroglia. Acta Pharmacol Sin 31: 1044-1054.

Virgintino, D., Monaghan, P., Robertson, D., Errede, M., Bertossi, M., Ambrosi, G. & Roncali, L. (1997). An immunohistochemical and morphometric study on astrocytes and microvasculature in the human cerebral cortex. Histochem J 29: 655-660.

Watkins, T. A., Emery, B., Mulinyawe, S. & Barres, B. A. (2008). Distinct stages of myelination regulated by γ-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60: 555-569.

Weightman, A., Jenkins, S., Pickard, M., Chari, D. & Yang, Y. (2014). Alignment of multiple glial cell populations in 3D nanofiber scaffolds: toward the development of multicellular implantable scaffolds for repair of neural injury. Nanomedicine 10: 291-295.

Welser, J. V. & Milner, R. (2012). Use of astrocyte-microglial cocultures to examine the regulatory influence of astrocytes on microglial activation. Methods Mol Biol 814: 367-380.

Westerink, R. H. & Ewing, A. G. (2008). The PC12 cell as model for neurosecretion. Acta Physiol (Oxf) 192: 273-285.

Wilms, H., Hartmann, D. & Sievers, J. (1997). Ramification of microglia, monocytes and macrophages in vitro: influences of various epithelial and mesenchymal cells and their conditioned media. Cell Tissue Res 287: 447-458.

Wilson, J. X., Peters, C. E., Sitar, S. M., Daoust, P. & Gelb, A. W. (2000). Glutamate stimulates ascorbate transport by astrocytes. Brain Res 858: 61-66.

Wu, V. W. & Schwartz, J. P. (1998). Cell culture models for reactive gliosis: new perspectives. J Neurosci Res 51: 675-681.

Xie, H. R., Hu, L. S. & Li, G. Y. (2010). SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease. Chin Med J (Engl) 123: 1086-1092.

Yoo, S. & Wrathall, J. R. (2007). Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors. Developmental Neurobiology 67: 860-874.

Yu, A. C., Lee, Y. L. & Eng, L. F. (1993). Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis. J Neurosci Res 34: 295-303.

Zhuang, Z., Huang, J., Cepero, M. L. & Liebl, D. J. (2011). Eph signaling regulates gliotransmitter release. Commun Integr Biol 4: 223-226.

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


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


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


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


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


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


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


Neurochem Int 81:1-9




2.4 Publication 6

Results 45


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


submitted




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

RReferences

Bouzier-Sore AK, Pellerin L. 2013. Unraveling the complex metabolic nature of astrocytes. Front Cell Neurosci 7:179.

Contestabile A. 2002. Cerebellar granule cells as a model to study mechanisms of neuronal apoptosis or survival in vivo and in vitro. Cerebellum 1:41-55.

Correale J. 2014. The role of microglial activation in disease progression. Mult Scler 20:1288-1295.

De Bock M, Decrock E, Wang N, Bol M, Vinken M, Bultynck G, Leybaert L. 2014. The dual face of connexin-based astroglial Ca2+ communication: a key player in brain physiology and a prime target in pathology. Biochim Biophys Acta 1843:2211-2232.

Dringen R. 2005. Oxidative and antioxidative potential of brain microglial cells. Antioxid Redox Signal 7:1223-1233.

Dringen R, Brandmann M, Hohnholt MC, Blumrich EM. 2015. Glutathione-dependent detoxification processes in astrocytes. Neurochem Res, in press.

Dringen R, Kussmaul L, Hamprecht B. 1998. Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Brain Res Protoc 2:223-228.

Dringen R, Scheiber IF, Mercer JF. 2013. Copper metabolism of astrocytes. Front Aging Neurosci 5:9.

Dwivedi S, Siddiqui MA, Farshori NN, Ahamed M, Musarrat J., Al-Khedhairy AA. 2014. Synthesis, characterization and toxicological evaluation of iron oxide nanoparticles in human lung alveolar epithelial cells. Colloids Surf B Biointerfaces 122:209-215.

Eyo UB, Dailey ME. 2013. Microglia: key elements in neural development, plasticity, and pathology. J Neuroimmune Pharmacol 8:494-509.

Fernandes AR, Chari DM. 2014. A multicellular, neuro-mimetic model to study nanoparticle uptake in cells of the central nervous system. Integr Biol (Camb) 6:855-861.

Fernandez-Fernandez S, Almeida A, Bolanos JP. 2012. Antioxidant and bioenergetic coupling between neurons and astrocytes. Biochem J 443:3-11.

Geppert M, Hohnholt M, Gaetjen L, Grunwald I, Baumer M, Dringen R. 2009. Accumulation of iron oxide nanoparticles by cultured brain astrocytes. J Biomed Nanotechnol 5:285-293.

Geppert M, Hohnholt MC, Nurnberger S, Dringen R. 2012. Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles. Acta Biomater 8:3832-3839.

Geppert M, Hohnholt MC, Thiel K, Nurnberger S, Grunwald I, Rezwan K, Dringen R. 2011. Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes. Nanotechnology 22:145101.

Geppert M, Petters 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.

Hanini A, Schmitt A, Kacem K, Chau F, Ammar S, Gavard J. 2011. Evaluation of iron oxide nanoparticle biocompatibility. Int J Nanomedicine 6:787-794.

Hirrlinger J, Gutterer JM, Kussmaul L, Hamprecht B, Dringen R. 2000. Microglial cells in culture express a prominent glutathione system for the defense against reactive oxygen species. Dev Neurosci 22:384-392.

Hohnholt M, Geppert M, Dringen R. 2010. Effects of iron chelators, iron salts, and iron oxide nanoparticles on the proliferation and the iron content of oligodendroglial OLN-93 cells. Neurochem Res 35:1259-1268.

74 Results

Hohnholt MC, Dringen R. 2013. Uptake and metabolism of iron and iron oxide nanoparticles in brain astrocytes. Biochem Soc Trans 41:1588-1592.

Jenkins SI, Pickard MR, Furness DN, Yiu HH, Chari DM. 2013. Differences in magnetic particle uptake by CNS neuroglial subclasses: implications for neural tissue engineering. Nanomedicine (Lond) 8:951-968.

Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. 2011. Physiology of microglia. Physiol Rev 91:461-553.

Kinnunen TJJ, Haukka M, Pesonen E, Pakkanen TA. 2002. Ruthenium complexes with 2,2'-, 2,4'- and 4,4'-bipyridine ligands: the role of bipyridine coordination modes and halide ligands. J Organomet Chem 655:31-38.

Kurz T, Eaton JW, Brunk UT. 2010. Redox activity within the lysosomal compartment: implications for aging and apoptosis. Antioxid Redox Signal 13:511-523.

Lawen A, Lane DJ. 2013. Mammalian iron homeostasis in health and disease: uptake, storage, transport, and molecular mechanisms of action. Antioxid Redox Signal 18:2473-2507.

Lee JH, Ju JE, Kim BI, Pak PJ, Choi EK, Lee HS, Chung N. 2014. Rod-shaped iron oxide nanoparticles are more toxic than sphere-shaped nanoparticles to murine macrophage cells. Environ Toxicol Chem 33:2759-66.

Levy M, Lagarde F, Maraloiu VA, Blanchin MG, Gendron F, Wilhelm C, Gazeau F. 2010. Degradability of superparamagnetic nanoparticles in a model of intracellular environment: follow-up of magnetic, structural and chemical properties. Nanotechnology 21:395103.

Li L, Jiang W, Luo K, Song H, Lan F, Wu Y, Gu Z. 2013. Superparamagnetic iron oxide nanoparticles as MRI contrast agents for non-invasive stem cell labeling and tracking. Theranostics 3:595-615.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275.

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.

Ma YM, De Groot H, Liu ZD, Hider RC, Petrat F. 2006. Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes. Biochem J 395:49-55.

Mahmoudi M, Stroeve P, Milani AS, Arbab AS. (2011). Superparamagnetic iron oxide nanoparticles: synthesis, surface engineering, cytotoxicity and biomedical applications. New York, USA: Nova Science Publishers.

Maier-Hauff K, Ulrich F, Nestler D, Niehoff H, Wust P, Thiesen B, Orawa H, Budach V, Jordan A. 2011. Efficacy and safety of intratumoral thermotherapy using magnetic iron-oxide nanoparticles combined with external beam radiotherapy on patients with recurrent glioblastoma multiforme. J Neurooncol 103:317-324.

Malvindi MA, De Matteis V, Galeone A, Brunetti V, Anyfantis GC, Athanassiou A, Cingolani R, Pompa PP. 2014. Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering. PLoS One 9:e85835.

Nau R, Ribes S, Djukic M, Eiffert H. 2014. Strategies to increase the activity of microglia as efficient protectors of the brain against infections. Front Cell Neurosci 8:138.

Nayak D, Roth TL, McGavern DB. 2014. Microglia development and function. Annu Rev Immunol 32:367-402.

Neuwelt EA, Varallyay P, Bago AG, Muldoon LL, Nesbit G, Nixon R. 2004. Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours. Neuropathol Appl Neurobiol 30:456-471.

Results 75

Peters DG, Connor JR. 2014. Introduction to cells comprising the nervous system. Adv Neurobiol 9:33-45.

Petters C, Bulcke F, Thiel K, Bickmeyer U, Dringen R. 2014a. Uptake of fluorescent iron oxide nanoparticles by oligodendroglial OLN-93 cells. Neurochem Res 39:372-383.

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, Dringen R. 2015. Accumulation of iron oxide nanoparticles by cultured primary neurons. Neurchem Int, in press.

Petters C, Irrsack E, Koch M, Dringen R. 2014b. Uptake and Metabolism of Iron Oxide Nanoparticles in Brain Cells. Neurochem Res 9:1648-60.

Pickard MR, Chari DM. 2010. Robust uptake of magnetic nanoparticles (MNPs) by central nervous system (CNS) microglia: implications for particle uptake in mixed neural cell populations. Int J Mol Sci 11:967-981.

Pierre JL, Fontecave M. 1999. Iron and activated oxygen species in biology: the basic chemistry. Biometals 12:195-199.

Pinkernelle J, Calatayud P, Goya GF, Fansa H, Keilhoff G. 2012. Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia. BMC Neurosci 13:32.

Poole B, Ohkuma S. 1981. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol 90:665-669.

Rathore KI, Redensek A, David S. 2012. Iron homeostasis in astrocytes and microglia is differentially regulated by TNF-alpha and TGF-beta1. Glia 60:738-750.

Rivet CJ, Yuan Y, Borca-Tasciuc DA, Gilbert RJ. 2012. Altering iron oxide nanoparticle surface properties induce cortical neuron cytotoxicity. Chem Res Toxicol 25:153-161.

Safi M, Courtois J, Seigneuret M, Conjeaud H, Berret JF. 2011. The effects of aggregation and protein corona on the cellular internalization of iron oxide nanoparticles. Biomaterials 32:9353-9363.

Saura J, Tusell JM, Serratosa J. 2003. High-yield isolation of murine microglia by mild trypsinization. Glia 44:183-189.

Scalettar BA. 2006. How neurosecretory vesicles release their cargo. Neuroscientist 12:164-176.

Scheiber IF, Mercer JF, Dringen R. 2010. Copper accumulation by cultured astrocytes. Neurochem Int 56:451-460.

Stobart JL, Anderson CM. 2013. Multifunctional role of astrocytes as gatekeepers of neuronal energy supply. Front Cell Neurosci 7:38.

Sun Z, Yathindranath V, Worden M, Thliveris JA, Chu S, Parkinson FE, Hegmann T, Miller DW. 2013. Characterization of cellular uptake and toxicity of aminosilane-coated iron oxide nanoparticles with different charges in central nervous system-relevant cell culture models. Int J Nanomedicine 8:961-970.

Taschner CA, Wetzel SG, Tolnay M, Froehlich J, Merlo A, Radue EW. 2005. Characteristics of ultrasmall superparamagnetic iron oxides in patients with brain tumors. AJR Am J Roentgenol 185:1477-1486.

Teplova VV, Tonshin AA, Grigoriev PA, Saris NE, Salkinoja-Salonen MS. 2007. Bafilomycin A1 is a potassium ionophore that impairs mitochondrial functions. J Bioenerg Biomembr 39:321-329.

Tulpule K, Hohnholt MC, Hirrlinger J, Dringen R. (2014). Primary cultures of astrocytes and neurons as model systems to study the metabolism and metabolite export

76 Results

from brain cells. In: Waagepetersen HS, Hirrlinger J, eds. Neuromethods: brain energy metabolism. Volume 90. Heidelberg: Springer, 45-72.

Urrutia P, Aguirre P, Esparza A, Tapia V, Mena NP, Arredondo M, Gonzalez-Billault C, Nunez MT. 2013. Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells. J Neurochem 126:541-549.

van Landeghem FKH, Maier-Hauff K, Jordan A, Hoffmann KT, Gneveckow U, Scholz R, Thiesen B, Bruck W, von Deimling A. 2009. Post-mortem studies in glioblastoma patients treated with thermotherapy using magnetic nanoparticles. Biomaterials 30:52-57.

Verkhratsky A, Nedergaard M, Hertz L. 2014. Why are astrocytes important? Neurochem Res.

Voinov MA, Pagan JOS, Morrison E, Smirnova TI, Smirnov AI. 2011. Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity. J Am Chem Soc 133:35-41.

Wang Y, Wang B, Zhu MT, Li M, Wang HJ, Wang M, Ouyang H, Chai ZF, Feng WY, Zhao YL. 2011. Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure. Toxicol Lett 205:26-37.

Yan F, Wang Y, He SZ, Ku ST, Gu W, Ye L. 2013. Transferrin-conjugated, fluorescein-loaded magnetic nanoparticles for targeted delivery across the blood-brain barrier. J Mater Sci Mater Med 24:2371-2379.

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


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


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


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


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


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).


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

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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).


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


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


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.


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


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


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


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.


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.


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).


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


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


(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


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


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.


3.5. RReferences

Albanese, A., Walkey, C. D., Olsen, J. B., Guo, H. B., Emili, A. & Chan, W. C. W. (2014). Secreted biomolecules alter the biological identity and cellular interactions of nanoparticles. ACS Nano 8: 5515-5526.

Almeida, A., Almeida, J., Bolanos, J. P. & Moncada, S. (2001). Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection. Proc Natl Acad Sci USA 98: 15294-15299.

Arvidson, B. & Tjalve, H. (1986). Distribution of Cd-109 in the nervous-system of rats after intravenous-injection. Acta Neuropathol 69: 111-116.

Barateiro, A. & Fernandes, A. (2014). Temporal oligodendrocyte lineage progression: in vitro models of proliferation, differentiation and myelination. Biochim Biophys Acta 1843: 1917-1929.

Barua, S. & Rege, K. (2009). Cancer-cell-phenotype-dependent differential intracellular trafficking of unconjugated quantum dots. Small 5: 370-376.

Batista-Nascimento, L., Pimentel, C., Menezes, R. A. & Rodrigues-Pousada, C. (2012). Iron and neurodegeneration: from cellular homeostasis to disease. Oxid Med Cell Longev 2012: 128647 (8 p.).

Bayraktar, O. A., Fuentealba, L. C., Alvarez-Buylla, A. & Rowitch, D. H. (2014). Astrocyte development and heterogeneity. Cold Spring Harb Perspect Biol 7: a020362 (17 p.).

Benjaminsen, R. V., Sun, H. H., Henriksen, J. R., Christensen, N. M., Almdal, K. & Andresen, T. L. (2011). Evaluating nanoparticle sensor design for intracellular pH measurements. ACS Nano 5: 5864-5873.

Bertorelle, F., Wilhelm, C., Roger, J., Gazeau, F., Menager, C. & Cabuil, V. (2006). Fluorescence-modified superparamagnetic nanoparticles: intracellular uptake and use in cellular imaging. Langmuir 22: 5385-5391.

Bickmeyer, U., Grube, A., Klings, K. W. & Kock, M. (2008). Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye. Biochem Biophys Res Commun 373: 419-422.

Bickmeyer, U., Heine, M., Podbielski, I., Mund, D., Kock, M. & Karuso, P. (2010). Tracking of fast moving neuronal vesicles with ageladine A. Biochem Biophys Res Commun 402: 489-494.

Blanchette, M., Tremblay, L., Lepage, M. & Fortin, D. (2014). Impact of drug size on brain tumor and brain parenchyma delivery after a blood-brain barrier disruption. J Cereb Blood Flow Metab 34: 820-826.

Blouin, C. M. (2013). Endocytose sans clathrin – La voie est libre! Med Sci (Paris) 29: 890-896.

Bove, J. & Perier, C. (2012). Neurotoxin-based models of Parkinson's disease. Neuroscience 211: 51-76.

Brown, G. C. & Neher, J. J. (2012). Eaten alive! Cell death by primary phagocytosis: 'phagoptosis'. Trends Biochem Sci 37: 325-332.

Burlacu, A., Jinga, V., Gafencu, A. V. & Simionescu, M. (2001). Severity of oxidative stress generates different mechanisms of endothelial cell death. Cell Tissue Res 306: 409-416.

Canton, I. & Battaglia, G. (2012). Endocytosis at the nanoscale. Chem Soc Rev 41: 2718-2739.

Correale, J. (2014). The role of microglial activation in disease progression. Mult Scler 20: 1288-1295.


Dadras, A., Riazi, G. H., Afrasiabi, A., Naghshineh, A., Ghalandari, B. & Mokhtari, F. (2013). In vitro study on the alterations of brain tubulin structure and assembly affected by magnetite nanoparticles. J Biol Inorg Chem 18: 357-369.

Dawson, K. A., Salvati, A. & Lynch, I. (2009). Nanoparticles reconstruct lipids. Nat Nanotechnol 4: 84-85.

De Bock, M., Decrock, E., Wang, N., Bol, M., Vinken, M., Bultynck, G. & Leybaert, L. (2014). The dual face of connexin-based astroglial Ca2+ communication: a key player in brain physiology and a prime target in pathology. Biochim Biophys Acta 1843: 2211-2232.

deBlaquiere, J. & Burgess, A. W. (1999). Affinity purification of plasma membranes. J Biomol Tech 10: 64-71.

DeKroon, R. M. & Armati, P. J. (2001). The endosomal trafficking of apolipoprotein E3 and E4 in cultured human brain neurons and astrocytes. Neurobiol Dis 8: 78-89.

Deng, M., Huang, Z., Zou, Y., Yin, G., Liu, J. & Gu, J. (2014). Fabrication and neuron cytocompatibility of iron oxide nanoparticles coated with silk-fibroin peptides. Colloids Surf B Biointerfaces 116C: 465-471.

Doherty, G. J. & McMahon, H. T. (2009). Mechanisms of endocytosis. Annu Rev Biochem 78: 857-902.

dos Santos, T., Varela, J., Lynch, I., Salvati, A. & Dawson, K. A. (2011). Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines. PLoS One 6: e24438 (10 p.).

Du, L. B., Suo, S. Q. G. W., Wang, G. Q., Jia, H. Y., Liu, K. J., Zhao, B. L. & Liu, Y. (2013). Mechanism and cellular kinetic studies of the enhancement of antioxidant activity by using surface-functionalized gold nanoparticles. Chemistry 19: 1281-1287.

Dujardin, S., Colin, M. & Buee, L. (2014). Animal models of tauopathies and their implications for research/translation into the clinic. Neuropathol Appl Neurobiol, in press.

Fauconnier, N., Pons, J. N., Roger, J. & Bee, A. (1997). Thiolation of maghemite nanoparticles by dimercaptosuccinic acid. J Colloid Interface Sci 194: 427-433.

Ferrati, S., Mack, A., Chiappini, C., Liu, X. W., Bean, A. J., Ferrari, M. & Serda, R. E. (2010). Intracellular trafficking of silicon particles and logic-embedded vectors. Nanoscale 2: 1512-1520.

Fleischer, C. C., Kumar, U. & Payne, C. K. (2013). Cellular binding of anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition. Biomater Sci 1: 975-982.

Geppert, M., Hohnholt, M., Grunwald, I., Baumer, M. & Dringen, R. (2009). Accumulation of iron oxide nanoparticles by cultured astrocytes. J Neurochem 110: 71-71.

Geppert, M., Hohnholt, M. C., Nurnberger, S. & Dringen, R. (2012). Ferritin up-regulation and transient ROS production in cultured brain astrocytes after loading with iron oxide nanoparticles. Acta Biomater 8: 3832-3839.

Geppert, M., Hohnholt, M. C., Thiel, K., Nurnberger, S., Grunwald, I., Rezwan, K. & Dringen, R. (2011). Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes. Nanotechnology 22: 145101 (11 p.).

Geppert, M., Petters, 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: 1349-1363.

Gnanaprakash, G., Mahadevan, S., Jayakumar, T., Kalyanasundaram, P., Philip, J. & Raj, B. (2007). Effect of initial pH and temperature of iron salt solutions on formation of magnetite nanoparticles. Mater Chem Phys 103: 168-175.


Grabrucker, A. M., Garner, C. C., Boeckers, T. M., Bondioli, L., Ruozi, B., Forni, F., Vandelli, M. A. & Tosi, G. (2011). Development of novel Zn2+ loaded nanoparticles designed for cell-type targeted drug release in CNS neurons: in vitro evidences. PLoS One 6 (11 p.).

Gramowski, A., Flossdorf, J., Bhattacharya, K., Jonas, L., Lantow, M., Rahman, Q., Schiffmann, D., Weiss, D. G. & Dopp, E. (2010). Nanoparticles induce changes of the electrical activity of neuronal networks on microelectrode array neurochips. Environ Health Perspect 118: 1363-1369.

Hare, D., Ayton, S., Bush, A. & Lei, P. (2013). A delicate balance: iron metabolism and diseases of the brain. Front Aging Neurosci 5: 34 (19 p.).

Hasany, S., Ahmed, I., Rajan, J. & Rehman, A. (2012). Systematic review of the preparation techniques of iron oxide magnetic nanoparticles. Nanosci Technol 2: 148-158.

He, N. B. & Hui, S. W. (1985). Electron-microscopic observation of domain movement in reconstituted erythrocyte-membranes. Proc Natl Acad Sci USA 82: 7304-7308.

Hohnholt, M., Geppert, M. & Dringen, R. (2010). Effects of iron chelators, iron salts, and iron oxide nanoparticles on the proliferation and the iron content of oligodendroglial OLN-93 cells. Neurochem Res 35: 1259-1268.

Hohnholt, M. C. & Dringen, R. (2011). Iron-dependent formation of reactive oxygen species and glutathione depletion after accumulation of magnetic iron oxide nanoparticles by oligodendroglial cells. J Nanopart Res 13: 6761-6774.

Hohnholt, M. C., Blumrich, E. M. & Dringen, R. (2014). Multiassay analysis of the toxic potential of hydrogen peroxide on cultured neurons. J Neuroscr res, in press.

Hohnholt, M. C., Geppert, M. & Dringen, R. (2011). Treatment with iron oxide nanoparticles induces ferritin synthesis but not oxidative stress in oligodendroglial cells. Acta Biomater 7: 3946-3954.

Ivanov, A. I. (2008). Pharmacological inhibition of endocytic pathways: is it specific enough to be useful? Methods Mol Biol 440: 15-33.

Iversen, T. G., Frerker, N. & Sandvig, K. (2012). Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism. J Nanobiotechnology 10: 33 (11 p.).

Jain, T. K., Reddy, M. K., Morales, M. A., Leslie-Pelecky, D. L. & Labhasetwar, V. (2008). Biodistribution, clearance, and biocompatibility of iron oxide magnetic nanoparticles in rats. Mol Pharm 5: 316-327.

Jedlovszky-Hajdu, A., Bombelli, F. B., Monopoli, M. P., Tombacz, E. & Dawson, K. A. (2012). Surface coatings shape the protein corona of SPIONs with relevance to their application in vivo. Langmuir 28: 14983-14991.

Jenkins, S. I., Pickard, M. R., Furness, D. N., Yiu, H. H. & Chari, D. M. (2013). Differences in magnetic particle uptake by CNS neuroglial subclasses: implications for neural tissue engineering. Nanomedicine (Lond) 8: 951-968.

Jordan, A., Scholz, R., Maier-Hauff, K., van Landeghem, F. K. H., Waldoefner, N., Teichgraeber, U., Pinkernelle, J., Bruhn, H., Neumann, F., Thiesen, B., von Deimling, A. & Felix, R. (2006). The effect of thermotherapy using magnetic nanoparticles on rat malignant glioma. J Neurooncol 78: 7-14.

Kaltz, A. (2011). Herstellung und Charakterisierung von fluoreszenzmarkierten Eisenoxid-Nanopartikeln. Bachelor thesis, University of Bremen, Germany.

Kenzaoui, B. H., Angeoni, S., Overstolz, T., Niedermann, P., Bernasconi, C. C., Liley, M. & Juillerat-Jeanneret, L. (2013). Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells. ACS Appl Mater Interfaces 5: 3581-3586.

Kim, J. S., Yoon, T. J., Yu, K. N., Noh, M. S., Woo, M., Kim, B. G., Lee, K. H., Sohn, B. H., Park, S. B., Lee, J. K. & Cho, M. H. (2006). Cellular uptake of magnetic


nanoparticle is mediated through energy-dependent endocytosis in A549 cells. J Vet Sci 7: 321-326.

Kozler, P., Riljak, V. & Pokorny, J. (2013). Both water intoxication and osmotic BBB disruption increase brain water content in rats. Physiol Res 62: S75-S80.

Lesniak, A., Salvati, A., Santos-Martinez, M. J., Radomski, M. W., Dawson, K. A. & Aberg, C. (2013). Nanoparticle adhesion to the cell membrane and its effect on nanoparticle uptake efficiency. J Am Chem Soc 135: 1438-1444.

Levy, M., Lagarde, F., Maraloiu, V. A., Blanchin, M. G., Gendron, F., Wilhelm, C. & Gazeau, F. (2010). Degradability of superparamagnetic nanoparticles in a model of intracellular environment: follow-up of magnetic, structural and chemical properties. Nanotechnology 21: 395103 (11 p.).

Li, P., Fang, L. B., Zhou, H., Zhang, W., Wang, X., Li, N., Zhong, H. B. & Tang, B. (2011a). A new ratiometric fluorescent probe for detection of Fe2+ with high sensitivity and its intracellular imaging applications. Chemistry 17: 10520-10523.

Li, Y. H., Li, X. L., Wong, Y. S., Chen, T. F., Zhang, H. B., Liu, C. R. & Zheng, W. J. (2011b). The reversal of cisplatin-induced nephrotoxicity by selenium nanoparticles functionalized with 11-mercapto-1-undecanol by inhibition of ROS-mediated apoptosis. Biomaterials 32: 9068-9076.

Mahmoudi, M., Abdelmonem, A. M., Behzadi, S., Clement, J. H., Dutz, S., Ejtehadi, M. R., Hartmann, R., Kantner, K., Linne, U., Maffre, P., Metzler, S., Moghadam, M. K., Pfeiffer, C., Rezaei, M., Ruiz-Lozano, P., Serpooshan, V., Shokrgozar, M. A., Nienhaus, G. U. & Parak, W. J. (2013). Temperature: the "ignored" factor at the nanobio interface. ACS Nano 7: 6555-6562.

Maldonado-Baez, L., Williamson, C. & Donaldson, J. G. (2013). Clathrin-independent endocytosis: a cargo-centric view. Exp Cell Res 319: 2759-2769.

Malvindi, M. A., De Matteis, V., Galeone, A., Brunetti, V., Anyfantis, G. C., Athanassiou, A., Cingolani, R. & Pompa, P. P. (2014). Toxicity assessment of silica coated iron oxide nanoparticles and biocompatibility improvement by surface engineering. PLoS One 9: e85835 (11 p.).

Massart, R. (1981). Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Trans Magn 17: 1247-1248.

Mattson, M. P., Zhang, Y. & Bose, S. (1993). Growth-factors prevent mitochondrial dysfunction, loss of calcium homeostasis, and cell injury, but not ATP depletion in hippocampal-neurons deprived of glucose. Exp Neurol 121: 1-13.

McCormack, P. L. (2012). Ferumoxytol: in iron deficiency anaemia in adults with chronic kidney disease. Drugs 72: 2013-2022.

Mejias, R., Gutierrez, L., Salas, G., Perez-Yague, S., Zotes, T. M., Lazaro, F. J., Morales, M. P. & Barber, D. F. (2013). Long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles support their use in biomedical applications. J Control Release 171: 225-233.

Mejias, R., Perez-Yague, S., Roca, A. G., Perez, N., Villanueva, A., Canete, M., Manes, S., Ruiz-Cabello, J., Benito, M., Labarta, A., Batlle, X., Veintemillas-Verdaguer, S., Morales, M. P., Barber, D. F. & Serna, C. J. (2010). Liver and brain imaging through dimercaptosuccinic acid-coated iron oxide nanoparticles. Nanomedicine (Lond) 5: 397-408.

Monopoli, M. P., Aberg, C., Salvati, A. & Dawson, K. A. (2012). Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 7: 779-786.

Napoli, I. & Neumann, H. (2009). Microglial clearance function in health and disease. Neuroscience 158: 1030-1038.


Nel, A. E., Madler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., Klaessig, F., Castranova, V. & Thompson, M. (2009). Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 8: 543-557.

Neumann, H., Kotter, M. R. & Franklin, R. J. M. (2009). Debris clearance by microglia: an essential link between degeneration and regeneration. Brain 132: 288-295.

Neuwelt, E. A., Varallyay, P., Bago, A. G., Muldoon, L. L., Nesbit, G. & Nixon, R. (2004). Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours. Neuropathol Appl Neurobiol 30: 456-471.

Ng, E. L. & Tang, B. L. (2008). Rab GTPases and their roles in brain neurons and glia. Brain Res Rev 58: 236-246.

Nie, Z., Liu, K. J., Zhong, C. J., Wang, L. F., Yang, Y., Tian, Q. & Liu, Y. (2007). Enhanced radical scavenging activity by antioxidant-functionalized gold nanoparticles: a novel inspiration for development of new artificial antioxidants. Free Radic Biol Med 43: 1243-1254.

Oberheim, N. A., Goldman, S. A. & Nedergaard, M. (2012). Heterogeneity of astrocytic form and function. Methods Mol Biol 814: 23-45.

Overly, C. C., Lee, K. D., Berthiaume, E. & Hollenbeck, P. J. (1995). Quantitative measurement of intraorganelle pH in the endosomal lysosomal pathway in neurons by using ratiometric imaging with pyranine. Proc Natl Acad Sci USA 92: 3156-3160.

Pais, T. F., Figueiredo, C., Peixoto, R., Braz, M. H. & Chatterjee, S. (2008). Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway. J Neuroinflammation 5: 43 (12 p.).

Pamenter, M. E., Perkins, G. A., McGinness, A. K., Gu, X. Q., Ellisman, M. H. & Haddad, G. G. (2012). Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra. PLoS One 7: e51469 (12 p.).

Peng, Y. K., Lui, C. N., Lin, T. H., Chang, C., Chou, P. T., Yung, K. K. & Tsang, S. C. (2014). Multifunctional silica-coated iron oxide nanoparticles: a facile four-in-one system for in situ study of neural stem cell harvesting. Faraday Discuss, in press.

Peters, D. G. & Connor, J. R. (2014). Introduction to cells comprising the nervous system. Adv Neurobiol 9: 33-45.

Petri-Fink, A., Steitz, B., Finka, A., Salaklang, J. & Hofmann, H. (2008). Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): colloidal stability, cytotoxicity, and cellular uptake studies. Eur J Pharm Biopharm 68: 129-137.

Pickard, M. R. & Chari, D. M. (2010). Robust uptake of magnetic nanoparticles (MNPs) by central nervous system (CNS) microglia: implications for particle uptake in mixed neural cell populations. Int J Mol Sci 11: 967-981.

Pickard, M. R., Jenkins, S. I., Koller, C. J., Furness, D. N. & Chari, D. M. (2010). Magnetic nanoparticle labeling of astrocytes derived for neural transplantation. Tissue Eng Part C Methods 17: 89-99.

Pinkernelle, J., Calatayud, P., Goya, G. F., Fansa, H. & Keilhoff, G. (2012). Magnetic nanoparticles in primary neural cell cultures are mainly taken up by microglia. BMC Neurosci 13: 32-48.

Pisanic, T. R., 2nd, Blackwell, J. D., Shubayev, V. I., Finones, R. R. & Jin, S. (2007). Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 28: 2572-2581.

Pyrgiotakis, G., Blattmann, C. O. & Demokritou, P. (2014). Real-time nanoparticle-cell interactions in physiological media by atomic force microscopy. ACS Sustain Chem Eng 2: 1681-1690.


Qaddoumi, M. G., Gukasyan, H. J., Davda, J., Labhasetwar, V., Kim, K. J. & Lee, V. H. L. (2003). Clathrin and caveolin-1 expression in primary pigmented rabbit conjunctival epithelial cells: role in PLGA nanoparticle endocytosis. Mol Vis 9: 559-568.

Rivet, C. J., Yuan, Y., Borca-Tasciuc, D. A. & Gilbert, R. J. (2012). Altering iron oxide nanoparticle surface properties induce cortical neuron cytotoxicity. Chem Res Toxicol 25: 153-161.

Roberts, R. C., Roche, J. K. & McCullumsmith, R. E. (2014). Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: a light and electron microscopic study. Neuroscience 277: 522-540.

Sahay, G., Alakhova, D. Y. & Kabanov, A. V. (2010). Endocytosis of nanomedicines. J Control Release 145: 182-195.

Sakulkhu, U., Maurizi, L., Mahmoudi, M., Motazacker, M., Vries, M., Gramoun, A., Ollivier Beuzelin, M. G., Vallee, J. P., Rezaee, F. & Hofmann, H. (2014). Ex situ evaluation of the composition of protein corona of intravenously injected superparamagnetic nanoparticles in rats. Nanoscale 6: 11439-11450.

Saleem, M., Abdullah, R., Ali, A., Park, B. J., Choi, E. H., Hong, I. S. & Lee, K. H. (2014). Facile synthesis, cytotoxicity and bioimaging of Fe3+ selective fluorescent chemosensor. Bioorg Med Chem 22: 2045-2051.

Schaub, N. J., Rende, D., Yuan, Y., Gilbert, R. J. & Borca-Tasciuc, D. A. (2014). Reduced astrocyte viability at physiological temperatures from magnetically activated iron oxide nanoparticles. Chem Res Toxicol 27: 2023-2035.

Shukla, S., Jadaun, A., Arora, V., Sinha, R. K., Biyani, N. & Jain, V. K. (2014). In vitro toxicity assessment of chitosan oligosaccharide coated iron oxide nanoparticles. Toxicol Rep, in press.

Smith, J. A., Das, A., Ray, S. K. & Banik, N. L. (2012a). Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull 87: 10-20.

Smith, P. J., Giroud, M., Wiggins, H. L., Gower, F., Thorley, J. A., Stolpe, B., Mazzolini, J., Dyson, R. J. & Rappoport, J. Z. (2012b). Cellular entry of nanoparticles via serum sensitive clathrin-mediated endocytosis, and plasma membrane permeabilization. Int J Nanomedicine 7: 2045-2055.

Soenen, S. J. H., Himmelreich, U., Nuytten, N. & De Cuyper, M. (2011). Cytotoxic effects of iron oxide nanoparticles and implications for safety in cell labelling. Biomaterials 32: 195-205.

Stehle, J. H., Saade, A., Rawashdeh, O., Ackermann, K., Jilg, A., Sebesteny, T. & Maronde, E. (2011). A survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases. J Pineal Res 51: 17-43.

Stenken, J. A. & Poschenrieder, A. J. (2015). Bioanalytical chemistry of cytokines - a review. Anal Chim Acta 853C: 95-115.

Sun, Z. Z., Yathindranath, V., Worden, M., Thliveris, J. A., Chu, S., Parkinson, F. E., Hegmann, T. & Miller, D. W. (2013). Characterization of cellular uptake and toxicity of aminosilane-coated iron oxide nanoparticles with different charges in central nervous system-relevant cell culture models. Int J Nanomedicine 8: 961-970.

Swanson, R. A., Farrell, K. & Stein, B. K. (1997). Astrocyte energetics, function, and death under conditions of incomplete ischemia: a mechanism of glial death in the penumbra. Glia 21: 142-153.


Taschner, C. A., Wetzel, S. G., Tolnay, M., Froehlich, J., Merlo, A. & Radue, E. W. (2005). Characteristics of ultrasmall superparamagnetic iron oxides in patients with brain tumors. AJR Am J Roentgenol 185: 1477-1486.

Tenuta, T., Monopoli, M. P., Kim, J., Salvati, A., Dawson, K. A., Sandin, P. & Lynch, I. (2011). Elution of labile fluorescent dye from nanoparticles during biological use. PLoS One 6: e25556 (6 p.).

Thomsen, L. B., Linemann, T., Pondman, K. M., Lichota, J., Kim, K. S., Pieters, R. J., Visser, G. M. & Moos, T. (2013). Uptake and transport of superparamagnetic iron oxide nanoparticles through human brain capillary endothelial cells. ACS Chem Neurosci 4: 1352-1360.

Tysiak, E., Asbach, P., Aktas, O., Waiczies, H., Smyth, M., Schnorr, J., Taupitz, M. & Wuerfel, J. (2009). Beyond blood brain barrier breakdown - in vivo detection of occult neuroinflammatory foci by magnetic nanoparticles in high field MRI. J Neuroinflammation 6: 20 (8 p.).

Urrutia, P. J., Mena, N. P. & Núñez, M. T. (2014). The interplay between iron accumulation, mitochondrial dysfunction, and inflammation during the execution step of neurodegenerative disorders. Front Pharmacol 5: 38 (12 p.).

van Landeghem, F. K. H., Maier-Hauff, K., Jordan, A., Hoffmann, K. T., Gneveckow, U., Scholz, R., Thiesen, B., Bruck, W. & von Deimling, A. (2009). Post-mortem studies in glioblastoma patients treated with thermotherapy using magnetic nanoparticles. Biomaterials 30: 52-57.

Verkhratsky, A. & Parpura, V. (2010). Recent advances in (patho)physiology of astroglia. Acta Pharmacol Sin 31: 1044-1054.

Virgintino, D., Monaghan, P., Robertson, D., Errede, M., Bertossi, M., Ambrosi, G. & Roncali, L. (1997). An immunohistochemical and morphometric study on astrocytes and microvasculature in the human cerebral cortex. Histochem J 29: 655-660.

Wang, F. J., Yu, L., Monopoli, M. P., Sandin, P., Mahon, E., Salvati, A. & Dawson, K. A. (2013). The biomolecular corona is retained during nanoparticle uptake and protects the cells from the damage induced by cationic nanoparticles until degraded in the lysosomes. Nanomedicine 9: 1159-1168.

Wang, J., Chen, Y., Chen, B., Ding, J., Xia, G., Gao, C., Cheng, J., Jin, N., Zhou, Y., Li, X., Tang, M. & Wang, X. M. (2010). Pharmacokinetic parameters and tissue distribution of magnetic Fe3O4 nanoparticles in mice. Int J Nanomedicine 5: 861-866.

Wang, Y., Wang, B., Zhu, M. T., Li, M., Wang, H. J., Wang, M., Ouyang, H., Chai, Z. F., Feng, W. Y. & Zhao, Y. L. (2011a). Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure. Toxicol Lett 205: 26-37.

Wang, Z. J., Tiruppathi, C., Cho, J., Minshall, R. D. & Malik, A. B. (2011b). Delivery of nanoparticle-complexed drugs across the vascular endothelial barrier via caveolae. IUBMB Life 63: 659-667.

Wankhede, M., Bouras, A., Kaluzova, M. & Hadjipanayis, C. G. (2012). Magnetic nanoparticles: an emerging technology for malignant brain tumor imaging and therapy. Expert Rev Clin Pharmacol 5: 173-186.

Weinstein, J. S., Varallyay, C. G., Dosa, E., Gahramanov, S., Hamilton, B., Rooney, W. D., Muldoon, L. L. & Neuwelt, E. A. (2010). Superparamagnetic iron oxide nanoparticles: diagnostic magnetic resonance imaging and potential therapeutic applications in neurooncology and central nervous system inflammatory pathologies, a review. J Cereb Blood Flow Metab 30: 15-35.

Wislocki, G. B. & Leduc, E. H. (1952). Vital staining of the hematoencephalic barrier by silver nitrate and Trypan Blue, and cytological comparisons of the


neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J Comp Neurol 96: 371-413.

Wong, B. X. & Duce, J. A. (2014). The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders. Front Pharmacol 5: 81 (10 p.).

Wu, H. Y., Chung, M. C., Wang, C. C., Huang, C. H., Liang, H. J. & Jan, T. R. (2013a). Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells. Part Fibre Toxicol 10: 46-56.

Wu, Y. L., Putcha, N., Ng, K. W., Leong, D. T., Lim, C. T., Loo, S. C. J. & Chen, X. D. (2013b). Biophysical responses upon the interaction of nanomaterials with cellular interfaces. Acc Chem Res 46: 782-791.

Xue, Y., Wu, J. & Sun, J. (2012). Four types of inorganic nanoparticles stimulate the inflammatory reaction in brain microglia and damage neurons in vitro. Toxicol Lett 214: 91-98.

Yan, F., Wang, Y., He, S. Z., Ku, S. T., Gu, W. & Ye, L. (2013). Transferrin-conjugated, fluorescein-loaded magnetic nanoparticles for targeted delivery across the blood-brain barrier. J Mater Sci Mater Med 24: 2371-2379.

Yu, X. W., Hu, Z. L., Ni, M., Fang, P., Zhang, P. W., Shu, Q., Fan, H., Zhou, H. Y., Ni, L., Zhu, L. Q., Chen, J. G. & Wang, F. (2014). Acid-sensing ion channels promote the inflammation and migration of cultured rat microglia. Glia, in press.

Zhang, Y. & Barres, B. A. (2010). Astrocyte heterogeneity: an underappreciated topic in neurobiology. Curr Opin Neurobiol 20: 588-594.

Zhao, Y. N., Sun, X. X., Zhang, G. N., Trewyn, B. G., Slowing, I. I. & Lin, V. S. Y. (2011). Interaction of mesoporous silica nanoparticles with human red blood cell membranes: size and surface functionality effects. ACS Nano 5: 1366-1375.

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

uptake and metabolism of iron oxide nanoparticles in cultured brain cells · 2015-03-05 · uptake...

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