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

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-coa