Assessing the toxicity of gold nanoparticles in vitro and in vivo [Elektronische Ressource] / Yu Pan-Bartneck

Yu Pan

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 28
• Langue: English
• Poids de l'ouvrage: 43 Mo

Extrait

Assessing the Toxicity of Gold Nanoparticles
In Vitro and In Vivo


Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Master of Science
Bachelor of Medicine
Yu Pan-Bartneck geb. Pan

aus Shanghai, China




Berichter: Univ.-Prof. Dr.rer.nat, Willi Jahnen-Dechent
Univ.-Prof. Dr.rer.nat, Ulrich Simon




Tag der mündlichen Prüfung: 23. November 2010

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.Table of Contents

1. Introduction .............................................................................................................1
1.1 Background of the study....................1
1.2 Nomenclature, composition and quantification of gold nanoparticles ................2
1.2.1 Nanoparticles, clusters and colloids............................................................2
1.2.2 Composition of gold nanoparticles..............................2
1.2.3 Quantification of AuNPs and its influence on toxicity evaluation .................3
1.3 Difference between bulk and AuNPs .................................................................4
1.3.1 Gold is the noblest of all metals..4
1.3.2 Size dependent properties of AuNPs..........................4
1.3.3 Au1.4MS .....................................................................................................4
1.4 Potential risk of AuNPs......................................................................................5
1.5 Apoptosis and necrosis5
1.5.1 Caspases....................................................................................................6
1.5.2 Staurosporine..............................7
1.5.3 DNA condensation and fragmentation ........................................................7
1.5.4 Cell cycle and DNA content ........................................................................8
1.6 Cellular redox balance.......................................................9
1.6.1 Reactive oxygen species and oxidative stress............9
1.6.2 Glutathione................................................................................................10
1.7 Gene expression microarray............11
1.8 Different models to screen the toxicity of AuNPs.............................................12
1.8.1 Cell based toxicity screening test..............................................................12
1.8.2 Zebrafish model to test the influence of AuNPs on development..............12
1.9 hERG channel .................................................................13
1.9.1 Structure and function of the hERG channel.............13
1.9.2 Resting potential, action potential, depolarization and repolarization........14
1.9.3 hERG: two–gate model.............................................................................14
2 Materials.................................................16
2.1 Chemicals........16
2.2 Materials and instruments................................................................................17
2.3 Cell lines and zebrafish....................17
2.4 Formulations....................................................................................................18
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Table of Contents

3 Methods .................................................................................................................19
3.1 Measurement of physical and chemical properties of AuNPs..........................19
3.1.1 AuNPs synthesis and characterization......................19
3.1.2 Osmolality measurement ..........................................................................20
3.1.3 pH measurement.......................20
3.1.4 Stability of AuNPs in various media ..........................................................20
3.2 Cell culture.......................................................................21
3.2.1 Cell line culture21
3.2.2 IC50 measurement with MTT test .............................................................21
3.3 Viability and death pathway determination......................22
3.3.1 Fluorogenic caspase activity determination ..............................................22
3.3.2 Detection of the nuclear fragmentation with the DNA laddering test .........22
3.3.3 Inhibition of apoptosis with Z-VAD-fmk.....................23
3.3.4 Cell cycle and DNA content measurement using flow cytometry ..............23
3.4 Evaluation the intracellular redox status ..........................................................24
3.4.1 Flow cytometric determination of oxidative stress.....................................24
3.4.2 Fluorescent mitochondrial potential staining assay...24
3.4.3 Inhibition of Au1.4MS cytotoxicity by reducing agent24
3.5 Gene expression analysis................................................................................25
3.6 Cell proliferation assay....................25
3.7 Cell cycle and DNA content measurement using flow cytometry.....................26
3.8.Intracellular localization of AuNPs ...................................................................26
3.8.1 Transmission electron microscopy of Au15MS.........26
3.8.2 TEM protocol applied to localize Au1.4MS................................................27
3.9 ERG blockade measured by patch clamp........................27
3.9.1 Voltage protocol applied to elicit the tail current........27
3.9.2 The peak tail current after exposure to AuNPs in time course ..................29
3.10 Zebrafish based toxicity screening test..........................................................29
3.10.1 Zebrafish raising and breeding................................29
3.10.2 Mortality measurement of AuNPs treated zebrafish embryos .................29
4 Results ...................................................................................................................31
4.1 AuNPs qualification..........................31
4.1.1 Osmolality measurement..........31
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4.1.2 Gold nanoparticle pH measurement..........................................................32
4.1.3 Gold nanoparticle stability in media...........................33
4.2 The ligand dependant cytotoxicity....................................36
4.3 Cell death pathway determination....................................38
4.3.1 DNA laddering...........................................................38
4.3.2 Caspase 3/7 measurement.......................................39
4.4 Oxidative stress...............................................................41
4.4.1 Intracellular oxidative stress measurement...............41
4.4.2 Evaluating the mitochondrial membrane potential from cells treated with
Au1.4MS ............................................................................................................44
4.4.3 Abolishing Au1.4MS cytotoxicity by reducing agents46
4.5 Gene expression microarray............49
4.6 Cell cycle and proliferation...............................................................................51
4.7 The intracellular localization of gold nanoparticles...........53
4.8 Influence of AuNPs on hERG channel.............................................................54
4.8.1 Au1.4MS blocked the hERG channel........................54
4.8.2 Serum protects hERG transfected HEK293 cells from hERG blockade by
Au1.4MS ............................................................................................................59
4.9 Toxicity of AuNPs in zebrafish embryos..........................61
4.9.1 Comparing the toxicity of Au1.4MS with and without glutathione..............61
4.9.2 Teratogenesis of Au1.4MS........................................................................64
4.9.3 Changes of the heartbeat in response to Au1.4MS...67
4.9.4 Toxicity of Au1.4MS on zebrafish larvae...................................................68
4.9.5 Glutathione reduced the toxicity of Au1.4MS in zebrafish embryos ..........69
4.9.6 Toxicity of ligands on the development of zebrafish embryos...................71
5 Discussion..............................................................................................................80
5.1 The influence of byproduct on toxicity..............................................................80
5.2. Gold nanoparticles in physiological solution...................80
5.3 The effect of ligands on nanoparticle toxicity ................................81
5.3.1 Interaction between Au1.4MS and GSH...................81
5.3.2 Au1.4MS and protein-thiol.........................................82
5.4 The chemical activity of Au1.4MS and oxidative stress...................................83
5.6 The influence of Au1.4MS on gene transcription.............85
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5.7 Au1.4MS and cell death...................................................................................85
5.8 The interaction between Au1.4MS and the hERG channel..............................87
5.8.1 Aromatic group in Au1.4MS ......................................................................88 <