Interactions of inhaled nanoparticles with the alveolar-capillary barrier of the human respiratory tract [Elektronische Ressource] / Jennifer Yvonne Kasper

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• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2011
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Interactions of inhaled nanoparticles
with the alveolar-capillary barrier of the
human respiratory tract






Dissertation
zur Erlangung des Grades
„Doktor der Naturwissenschaften“


am Fachbereich Biologie
der Johannes Gutenberg-Universität in Mainz

Jennifer Yvonne Kasper
Geb. in Saarbrücken



Mainz, der 22.08.2011
































Dekan:
Tag der mündlichen Prüfung: 13. 12. 2011

Partial results of this thesis have been published in the
following publications



Kasper J, Hermanns MI, Bantz C, Maskos M, Stauber R, Pohl C, Unger RE, Kirkpatrick CJ:
Inflammatory and cytotoxic responses of an alveolar-capillary coculture
model to silica nanoparticles: Comparison with conventional monocultures.
Part Fibre Toxicol 2011, 8(1):6.

Hermanns MI, Kasper J, Dubruel P, Pohl C, Uboldi C, Vermeersch V, Fuchs S, Unger RE,
Kirkpatrick CJ: An impaired alveolar-capillary barrier in vitro: effect of
proinflammatory cytokines and consequences on nanocarrier interaction. J
RSocInterface, 2010, 7 Suppl 1:S41-S54.

Contents 1
Contents

Contents ................................................................................................................. 1
List of Abbreviations ............................... 4
List of Figures ......................................................................................................... 6
1 Introduction ...16
1.1 The alveolar-capillary barrier ..................................................................16
1.1.1 The alveolar capillary barrier in detail ..............16
1.2 Barrier integrity: Cell-cell contacts ..........................................................22
1.3 Nano-sized material ...............................................23
1.4 Nanoparticles encounter the human lung ...............................................24
1.4.1 Pulmonary diseases caused by nanoparticles .25
1.4.2 Deposition, translocation of NPs and systemic effects ....................27
1.4.3 Intracellular trafficking of nanomaterial in lung cells and its
application to nanomedicine ............................................................29
1.5 In vitro cell culture models of the alveolar-capillary barrier .....................32
1.6 Inflammatory and cytotoxic responses of an alveolar-capillary coculture
model to aSNPs .....................................................................................34
1.7 Aim of the study 35
2 Material and Methods ....................36
2.1 Chemicals, Material and Devices ...........................................................36
2.1.1 List of Chemicals .............................................36
2.1.2 Devices ...........................37
2.1.3 Material ................................................................38
2.1.4 Cell culture ......................38
2.1.5 Kits and assays ...............................................................................40
2.1.6 Antibodies .......................40 2 Contents
2.1.7 Molecular biology ........................................................................... 41
2.2 Methods ................................ 43
2.2.1 Cell culture and cell isolation .......................................................... 43
2.2.2 Nanoparticle characterization ......................... 47
2.2.3 Nanoparticle effects on cells .......................... 48
2.2.4 Cell staining ................................................................................... 53
2.3 Co-localisation experiments of internalized NPs with endosomal markers
54
2.3.1 Comparison of inflammatory responses between conventional
monocultures cocultures ................................................................ 55
2.3.2 Quantification of internalised nanoparticles .... 57
2.3.3 Cytotoxicity and membrane integrity after aSNP exposure on flotillin-
1 and-2 depleted cells .................................................................... 57
2.3.4 Triple coculture model of the alveolar-capillary barrier ................... 60
2.3.5 Statistical analysis .......................................................................... 60
3 Results ......................................... 61
3.1 Nanoparticle characterisation ................................................................ 61
3.2 Endotoxin remnants in NP dispersions .................. 63
3.3 Effect of nanoparticles on cells of the alveolar-capillary barrier ............. 64
3.3.1 Viability of cells in different culture systems after aSNP exposure .. 64
3.3.2 Membrane integrity upon aSNP-treatment ..................................... 64
3.4 Morphological evaluation of aSNP treated cells..... 66
3.4.1 aSNP effect on conventional monoculture and coculture in comparison
66
3.4.2 Morphological alterations in conventional monocultures and coculture
after aSNP treatment ........................................................................... 67
3.5 Comparison of inflammatory responses on aSNPs in different culture
systems ................................................................................................. 72 Contents 3
3.6 Effect of aSNPs on apoptosis markers in conventional mono- and
coculture ................................................................................................78
3.7 Quantification of internalized nanoparticles (aSNP, AmorSil, PEI ...........81
3.7.1 NP uptake by conventional monoculture and coculture of the
alveolar-capillary barrier ..................................................................81
3.7.2 Transport of NPs across the alveolar-capillary barrier in vitro ..........82
3.8 Examination of endosomal uptake routes of NPs ...85
3.9 Cytotoxicity and uptake of different sized aSNPs ................................. 100
3.10 aSNP exposure on flotillin-1- and-2-depleted cells ............................... 104
3.11 Development of a triple coculture model of the alveolar-capillary barrier
110
4 Discussion ................................................................................................... 118
4.1 Inflammatory and cytotoxic responses of an alveolar-capillary coculture
model to aSNPs: comparison with conventional monocultures ............. 118
4.2 Endocytosis pathways as cellular uptake routes for NPs ...................... 121
4.3 Size-dependent cytotoxicity and uptake of aSNPs ............................... 125
4.4 aSNP exposure to flotillin-1 and -2 depleted cells................................. 126
4.5 Approaching more closely the in vivo situation with a triple culture by
adding the alveolar macrophage .......................................................... 128
5 Summary..................................................................... 132
5.1 Summary ............................. 132
5.2 Zusammenfassung ............................................................................... 134
6 References .................................. 136
Curriculum vitae................. Fehler! Textmarke nicht definiert.

4 List of Abbreviations
List of Abbreviations

AJ adherens junctions
AJC apical junctional complex
ALI acute lung injury
AM alveolar macrophage
AmorSil amorphous Organosiloxane particles
ANOVA analysis of variance
ARDS acute respiratory distress syndrome
aSNP amorphous silica nanoparticles
ATI alveolar type I cell
ATII alveolar type II cell
BB biological barriers
Bcl-2 B-cell lymphoma 2
BFGF basic fibroblast growth factor
CatD cathepsin-D
Cav caveolin-1
CCP clathrin-coated pits
CCV clathrin-coated vesicles
CHC clathrin heavy chain
COPD chronic obstructive pulmonary disease
DNA desoxyribonucleic acid
DIC differential interference contrast
DLS dynamic light scattering
dNTP desoxyribonukleosidtriphosphate
DPPC dipalmytoylphosphatidyl-cholin
ECBM endothelial cell basal medium
EEA1 early endosome antigen 1
ELISA enzyme-linked immunosorbent assay
ER endoplasmatic reticulum
F12 siRNA for flotillin-1 and -2 in combination
FCS fetal calf serum
Flot1 flotillin-1
Flot2 flotillin-2
G-CSF granulocyte-colony stimulating factor
HIF-1a hypoxia-inducible factor alpha
HPMEC primary human pulmonary microcascular endothelial cells
ICAM-1 intercellular adhesion molecule-1
IF immunofluorescence
IL-1 interleukin-1
IL-1ß interleukin-1 beta
IL-6 interleukin-6
IL-8 interleukin-8
Lamp-1 lysosomal-associated membrane protein 1 List of Abbreviations 5
LDH lactate dehydrogenase
LPS lipopolysaccharide
M6PR mannose-6-phosphate receptor
MCP-1 monocyte chemoattractant protein-1
MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt
Neg non-trageted siRNA
NP nanoparticle
OG oregon green
PCR polymerase chain reaction
PECAM-1 platelet endothelial cell adhesion molecule
PEI polyethyleneimine