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Interactions of human primary immune cells with nanoparticles, two-dimensional micropatterns, hydrogels and three-dimensional nanofibres [Elektronische Ressource] / vorgelegt von Matthias Bartneck

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125 pages
Ajouté le : 01 janvier 2010
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Interactions of human primary immune cells with
nanoparticles, two-dimensional micropatterns,
hydrogels and three-dimensional nanofibres


Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen
Grades eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Diplom-Biologe Matthias Bartneck
aus Bielefeld


Berichter: Prof. Apl. Dr. rer. nat. G. Zwadlo-Klarwasser
Universitätsprofessor Dr. rer. nat. L. Elling

Tag der mündlichen Prüfung: 14. September 2010

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Table of Contents

1.INTRODUCTION................................................................................................................ 1
1.1 Biomaterials................................................................................................................... 1
1.1.1 Metals........................................................................................................................... 2
1.1.1.2 Gold nanoparticles..................................................................................................... 2
1.1.2 Polymers ...................................................................................................................... 3
1.1.2.1 Nanofibres................................................................................................................. 3
1.2 Biocompatibility ............................................................................................................ 4
1.3 Inflammation.................................................................................................................. 5
1.4 Human immune cells..................................................................................................... 6
1.4.1 Lymphocytes ................................................................................................................ 7
1.4.2 Granulocytes ................................................................................................................ 8
1.4.3 Monocytes.................................................................................................................... 9
1.4.4 Macrophages...............................................................................................................10
1.4.5 Dendritic cells ..............................................................................................................11
1.5 Cytokines ......................................................................................................................13
1.6 Aims of the thesis.........................................................................................................14
2. MATERIALS AND METHODS .........................................................................................16
2.1 Materials........................................................................................................................16
2.1.1 Gold nanoparticles.......................................................................................................16
2.1.2 Perfluoropolyether and polyvinylidenefluoride substrates ............................................16
2.1.3 Hydrogel coated substrates .........................................................................................17
2.1.4 Nanofibers...................................................................................................................17
2.1.5 Chemicals....................................................................................................................18
2.1.6 Enzymes and inhibitors................................................................................................19
2.1.7 Kits, antibodies and cytokines .....................................................................................20
2.1.8 Consumables ..............................................................................................................21
2.1.9 Instruments..................................................................................................................21
2.1.10 Software ....................................................................................................................22
2.2 Methods ........................................................................................................................23
2.2.1 Contact angle measurement........................................................................................23
2.2.2 Zeta-potential measurement........................................................................................23
I ITable of Contents

2.2.3 UV-vis spectrophotometry ...........................................................................................23
2.2.4 Endotoxin testing.........................................................................................................24
2.2.5 Generation of autologous human serum......................................................................24
2.2.6 Isolation and purity control of human blood cells .........................................................24
2.2.6.1 Isolation of peripheral blood mononuclear cells ........................................................24
2.2.6.2 Isolation of monocytes..............................................................................................25
2.2.6.3 Isolation of lymphocytes ...........................................................................................25
2.2.6.4 Isolation of granulocytes ...........................................................................................25
2.2.6.5 Generation of monocyte-derived macrophages ........................................................25
2.2.6.6 Generation of monocyte-derived dendritic cells ........................................................26
2.2.6.7 Analysis of cell population purity...............................................................................26
2.2.6.8 Cell lines and fibroblasts...........................................................................................27
2.2.7 Cell culture, leukocyte counting and viability tests .......................................................27
2.2.8 Visualization of gold nanoparticles for light microscopy ...............................................28
2.2.9 Determination of cell-nanoparticles interactions using light microscopy .......................29
2.2.10 Inhibition of the intracellular uptake of gold nanoparticles..........................................29
2.2.11 Inhibition of extracellular trapping of gold nanoparticles.............................................29
2.2.12 Transmission electron microscopy.............................................................................30
2.2.13 Flow cytometry and fluorescence microscopy............................................................31
2.2.14 Cytokine detection.....................................................................................................31
2.2.15 Quantification of mRNA expression ...........................................................................32
2.2.16 Hierarchical clustering analysis of gene expression data...........................................33
2.2.17 Statistical analysis .....................................................................................................33
3. RESULTS ........................................................................................................................34
3.1 Interactions of human immune cells with gold nanoparticles ..................................34
3.1.1 Nanoparticle characterization and cytotoxicity .............................................................34
3.1.2 Uptake of gold nanoparticles by human immune cells .................................................37
3.1.3 Concentration and time-dependent uptake of nanoparticles by phagocytes.................40
3.1.4 Investigation of the nanoparticle uptake mechanism using inhibitors ...........................46
3.1.5 Extracellular trapping of nanoparticles by human immune cells...................................48
3.1.6 Inhibition studies on extracellular traps........................................................................51
3.1.7 Expression of function-associated surface markers.....................................................52
3.1.8 Effects of nanorod chemistry on macrophage mediator expression .............................54


II ITable of Contents

3.2. Macrophage responses to different perfluoropolyether micropatterns ..................56
3.2.2 Effects of topography on macrophage morphology and viability ..................................56
3.2.3 Expression of function associated surface markers by macrophages ..........................58
3.2.4 Topographical control of cytokine release....................................................................60
3.2.5 Effect of topography on macrophage inflammatory gene expression...........................62
3.2.6 Relationships between micropatterns and macrophage function .................................64
3.3. Macrophage response to hydrogel-coated surfaces ................................................66
3.3.1 Hydrogel characterization, cell attachment and viability...............................................66
3.3.2 Expression of function-associated surfaces antigens...................................................67
3.3.3 Effects of hydrogels on the expression of inflammatory mediators...............................70
3.4. Macrophage responses to three-dimensional nanofibers........................................73
3.4.1 Nanofiber characterization and macrophage attachment, morphology and viability .....73
3.4.2 Macrophage-mediated degradation of nanofibers........................................................75
3.4.3 Effects of nanofibers on the expression of macrophage function-associated antigens.77
3.4.4 Cytokine release of macrophages in response to nanofibers.......................................78
4. DISCUSSION...................................................................................................................80
4.1 Interactions of human immune cells with gold nanoparticles ..................................80
4.1.1 Nanoparticle uptake and extracellular trapping ............................................................80
4.1.2 Inhibition of nanoparticle uptake ..................................................................................82
4.1.3 Effects of nanoparticle chemistry on the response of innate immune cells...................83
4.2 Effects of micropatterned substrates on macrophages ............................................84
4.2.1 Topographical patterning modulates macrophage phenotype......................................85
4.2.2 Relationships between pattern geometry and cell activation........................................85
4.2.3 Indications for a complex cellular response .................................................................87
4.2.4 Relationships between geometry and mediator profile.................................................87
4.2.5 Comparison of the effects of PFPE and PVDF on macrophage response ...................88
4.3 Interactions of macrophages with hydrogels.............................................................90
4.3.1 Effects of Star PEG based hydrogels on macrophage attachment and response ........90
4.4 Interaction of macrophages with three-dimensional nanofibers ..............................92
4.4.1 Rapid degradation of nanofibers by macrophages.......................................................92
4.4.2 Effects of three-dimensional nanofibers on macrophage response..............................93
4.5 Optimization of the immunomodulatory properties of biomaterials.........................95
IV Table of Contents

5. ABSTRACT .....................................................................................................................97
6. REFERENCES...............................................................................................................103
ACKNOWLEDGEMENTS ..................................................................................................113


V Table of Contents

List of Abbreviations

Abbreviation Meaning
A.D. Anno Domini
ANOVA Analysis of variance
APC Antigen presenting cells
AuNP Gold nanoparticles
AuNR Gold nanorods
AuNS Gold nanospherules
HCA Hierarchical clustering analysis
B cell B lymphocyte
BCR B cell receptor
BSA Bovine serum albumin
CC Cysteine-cysteine
CCL CC chemokine-ligand
CCR CC chemokine-receptor
CD Cluster of differentiation
cDNA Complementary DNA
CSF Colony stimulating factor
CTAB Cetyl-trimethylammoniumbromide
CXC Cysteine-other amino acid-cysteine chemokine
CXCL CXC chemokine ligand
CXCR CXC chemokine receptor
c Nanoparticle concentration based on cellular uptake Pmax
DAPI 4',6-diamino-2-phenylinole
DC Dendritic cell
DMSO Dimethyl sulfoxide
V I Table of Contents

DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-linked immunosorbent assay
FITC Fluorescein isothiocyanate
G-CSF Granulocyte colony stimulating factor
GM-CSF Granulocyte-macrophage colony stimulating factor
HCA Hierarchical clustering analysis
HLA-DR Human leukocyte antigen
IFN-γ Interferon-γ
IL Interleukin
LAL Limulus polyphemus lysate
MHC Major histocompatibility complex
M-CSF Macrophage colony stimulating factor
MIG Monocine induced by IFN gamma
NET Neutrophil extracellular trap
NH Amine group 2
NK cells Natural killer cells
NKT cells Natural killer T cells
O.D. Optical density
OH Hydroxy group
PBS Phosphate buffered saline
PBMC Peripheral blood mononuclear cells
PCR Polymerase chain reaction
PE Phycoerythrin
V II Table of Contents

PEG Poly(ethylene glycol)
PLGA Ploy(lactid-co-glycolic) acid
Q-PCR Quantitative real time polymerase chain reaction
RNA Ribonucleic acid
RNase Ribonuclease
Rpm Revolutions per minute
RPMI Roswell Park Memorial Institute
T cells T helper cells H
TNA α Tumor necrosis factor
U Enzyme units
UV-vis Ultraviolet-visible spectrophotometry


VI II Table of Contents

List of Figures
Figure 1.4: Human immune cell lineage
Figure 2.4.1: Nanofibres immobilized to Star PEG coated substrates
Figure 2.2.6.7: Human primary immune cells after DiffQuik staining
Figure 3.1.1: Characterization of gold nanoparticles
Figure 3.1.2.A: Cytospin preparations of humane immune cells after incubation with gold
and seedless deposition
Figure 3.1.2.B: TEM-studies of CTAB-coated AuNR uptake by primary human monocytes
Figure 3.1.3.A: Concentration- and time-dependent uptake of gold nanoparticles by human
monocytes and macrophages
Figure 3.1.3.B: Uptake of gold nanorods by unprofessional phagocytes
Figure 3.1.3.C: Time and concentration dependent uptake of CTAB- and PEG-coated
nanorods
Figure 3.1.4: Studies on the uptake mechanism for gold nanorods by human
macrophages
Figure 3.1.5.A: Different human immune cells located in cell-gold networks after 15 minutes
of incubation with gold nanoparticles
Figure 3.1.6: Neutrophil granulocytes after incubation with CTAB coated gold nanorods
Figure 3.1.7.A: Alteration of macrophage phenotype after seven days of culture with
nanoparticles
Figure 3.1.7.B: Alteration of dendritic cell phenotype after three days of culture with gold
nanorods
Figure 3.1.8.A: Changes in the expression of inflammation relevant genes in macrophages
after 24 hours of incubation with gold nanorods
Figure 3.1.8.B: Release of pro-inflammatory cytokines by macrophages in response to
different nanorod surface chemistries
Figure 3.2.1.A: Morphology of macrophages induced by different topographies
Figure 3.2.1.B: Characterization of nuclei shape and viability of macrophages attached to
the large posts
Figure 3.2.2.A: Expression of the function associated macrophage surface markers CD163
and 27E10 in response to microstructures
Figure 3.2.2.B: Flow cytometric analysis of macrophages cultured on different PFPE
substrates co-expressing CD163 and 27E10
Figure 3.2.3: Cytokine, chemokine and growth factor release from macrophages in
response to different structures after 7 days of culture
Figure 3.2.4: Gene expression heatmap of Real-Time PCR data (log 2 ratios) as induced
by the different PFPE structures
IX Table of Contents

Figure 3.2.5: Mathematical relations of macrophage response with micropattern geometry
shown by nonlinear and linear fit with regression curves
Figure 3.3.1: Adherence of macrophages to Star PEG-coated substrates
Figure 3.3.2.A: Alteration of the expression of the macrophage function-associated surface
antigens CD163 and 27E10 after seven days of culture on hydrogels
Figure 3.3.2.B: Fluorescence microscopical staining of CD163, 27E10 and nuclei staining of
cells attached to Star PEG
Figure 3.3.2.C: Alteration of macrophage size and granularity by Star PEG-coated glass
slides compared to polystyrol
Figure 3.3.3.A: Gene expression in response to Star PEG-coated hydrogels compared to
alternative and classical stimulation
Figure 3.3.3.B: Cytokine release of macrophage after culture on Star PEG-coated
substrates compared to alternative and classical stimulation
Figure 3.4.1: Adherence of macrophages to Star PEG-coated glass slides and nanofibres
Figure 3.4.2.A: Macrophage--mediated degradation of nanofibres composed of different
materials
Figure 3.4.2.B: Comparison of hydrolytic degradation in medium and degradation by
macrophages
Figure 3.4.3: Expression of the function-associated surface antigens CD163 and 27E10
by macrophages in response to nanofibres
Figure 3.4.4: Cytokine release of macrophages after seven days of culture on nanofibres












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