Investigations on the toxicity of nanoparticles

INVESTIGATIONS ON THE TOXICITY OF NANOPARTICLES ASHARANI PEZHUMMOOTTIL VASUDEVAN NAIR (B. Sc Medical Microbiology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS It is an honour to thank people who made this dream come true. Though it is hard to express my gratitude through words, I would like to express my heartfelt gratitude to my supervisor Associate Professor M. Prakash Hande, for being a wonderful mentor. His constant encouragement, suggestions, ideas, unfailing support and criticisms contributed to the brilliance of the work. I am indebted to him for giving me a chance to work under his supervision. I would like to extend my sincere thanks to my co-supervisor Associate Professor Suresh Valiyaveettil, for his enormous trust and support during the high tides of the work. His constant encouragement and ideas made this work fruitful. I am thankful to Prof. Zhiyuan Gong, for spending his valuable time to guide me through the in vivo work. His critical comments and suggestions helped a lot in the progress of this thesis. I greatly appreciate the help from Wu Yilian and Zhan Huiqing and the training they provided. Special thanks to Prof. Sanjay Swarup and Prof. Chwee Teck Lim for their discussions and constructive comments. I take this opportunity to thank my friends Dr. Manoj Parameswaran, Dr. Bindhu L.V, Sajini Vadukkumpulli, Ganapathy Balaji, Resham Lal Gurung, Sethu Swaminathan, Khaw Aikkia and Grace Low, who laughed and cried with me throughout my best and worst times of lab work. I am thankful to my lab mates and colleagues Lakshmidevi Balakrishnan, Dr. Anuradha Poonepalli, Kalpana GopalaKrishnan, Dimphy Zeegers, Prarthana Sreekanth, Kristina, Dr. Sivamurugan and all members of Genome stability lab and materials research lab. Most importantly, I express my gratitude to my husband Rajesh Chandran and son Dev Nandan Unnithan and my parents Leelamma K.K. and P.K. Vasudevan Nair, whose understanding, continuous encouragement inspired this work. I am grateful to my TAC members Prof. Kini Manjunatha and Dr. Bhaskar for the valuable advice and critical comments.   ii TABLE OF CONTENTS Title Page i Acknowledgement ii Table of Contents iii Summary x List of Tables and Figures xii Abbreviations xv List of publications xvii CHAPTER Introduction 1.1 Nanotechnology: An overview 1.2 Classification of nanomaterials 1.3 Synthesis and properties of metal nanoparticles 1.3.1 Size of the nanoparticles 10 1.3.2 Quantum confinement 10 1.3.3 Surface plasmon resonance 11 1.3.4 Morphology of the nanomaterials 12 1.3.5 Surface functionalisation 12 1.4 Nanotechnology: An outlook at current trends 13 1.5 Nanotechnology: Future prospects 14 1.6 Nanoparticles in the limelight 14 1.6.1 Gold nanoparticles 15 1.6.2 Silver nanoparticles 15 1.6.3 Platinum nanoparticles 17 1.7 Nanotechnology: A two sided sword? 18   iii 1.8 Lessons from history 18 1.9 Portals of entry of nanomaterials and factors contributing to uptake 19 1.9.1 Inhalation 20 1.9.2 Absorption through skin 22 1.9.3 Ingestion 23 1.9.4 Translocation 24 1.10 Excretion of nanoparticles 26 1.11 Biodistribution at cellular levels 26 1.12 Literature in nanotoxicity 28 1.12.1 Cytotoxicity 28 1.12.2 Uptake of nanoparticles 31 1.12.3 Genotoxicity 31 1.12.4 Protein expression 32 1.13 Rationale 35 CHAPTER Materials and Methods 38 2.1 Synthesis of nanoparticles 38 2.1.1 Synthesis of polyvinyl alcohol (PVA) capped silver nanoparticles (Ag-np-1) 38 2.1.2 Synthesis of silver nanoparticles capped with Bovine serum albumin (BSA, Ag-np-2) 38 2.1.3 Preparation of starch capped silver nanoparticles (Ag-np-3) 39 2.1.4 Synthesis of PVA capped gold nanoparticles (Aunp). 40 2.1.5 Synthesis of PVA capped platinum nanoparticles (Pt-np) 40   iv 2.2 Cell culture and nanoparticle treatment 41 2.3 Preparation of stock solution and treatment 41 2.4 Uptake of nanoparticles 42 2.5 Microscopy 43 2.5.1 Light microscopy 43 2.5.2 Transmission electron microscopy of nanoparticles treated cells 44 2.5.3 Scanning transmission electron microscopy (STEM) 44 2.5.4 Qualitative analysis of cell morphology by SEM 45 2.5.5 Live imaging of nanoparticles using cytoviva ultrahigh resolution illumination systems 45 2.6 Cell Viability Assay 45 2.6.1 Measurement of ATP content 45 2.6.2 Mitochondrial function-cell titer blue cell viability assay 46 2.7 Cell cycle analysis 47 2.8 Cell death 47 2.8.1 Annexin -V staining for apoptosis and necrosis 47 2.8.2 DNA fragmentation analysis 48 2.9 Detection of reactive oxygen species (ROS) production 48 2.10 Evaluation of genotoxicity 49 2.10.1 Cytokinesis-blocked micronucleus assay (CBMN) 49 2.10.2 Alkaline single-cell gel electrophoresis (Comet Assay). 50 2.10.3 Chromosomal analysis by fluorescence in situ hybridisation (FISH) 51   v 2.11 Colony formation studies 51 2.12 Analyses for protein/ gene expression 52 2.12.1 Western blotting 52 2.12.2 Gene expression profile using real time-reverse transcriptase- polymerase chain reaction (RT-PCR) 52 2.12.3 Messenger RNA isolation and array hybridisation 53 2.13 Immunofluorescence staining for γH2AX 54 2.14 Isothermal titration calorimetry 55 2.15 Cytokine detection assay 55 2.16 Intracellular calcium measurement 56 2.17 Statistical analysis 56 2.18 Collection and exposure of the embryos to nanoparticles 56 2.19 TEM analysis of the embryos 57 2.20 Acridine orange staining 58 2.21 4,6-diamidino-2-phenylindole-dihydrochloride hydrate (DAPI) staining 58 2.22 Quantification of metal content in embryos 58 2.23 Preparation of single cell suspension from embryos for cell cycle analysis 59 CHAPTER 3.1 Introduction 63 3.2 Results 64 3.2.1 Effect on cell morphology 66 3.2.2 Cell viability 68 3.2.3 Cellular uptake and exocytosis of nanoparticles 71   vi 3.2.4 Transmission electron microscopy (TEM) of cell sections to study bio distribution 74 3.2.5 Production of ROS in human cells exposed to silver nanoparticles 77 3.2.6 Genotoxicity of silver nanoparticles 79 3.2.6.1 DNA damage in silver nanoparticle treated cells 79 3.2.6.2 Micronuclei in silver nanoparticles treated cells 80 3.2.6.3 Chromosomal aberrations in silver nanoparticles treated cells 82 3.2.7 Calcium fluctuations in silver nanoparticles treatment 86 3.2.8 Effect of silver nanoparticles on cell cycle 88 3.2.9 Recovery and colony formation 91 3.2.10 Apoptosis and necrosis 93 3.2.11 Effect of silver nanoparticles on gene expression 97 3.2.12 Inflammatory response in nanoparticle mediated cells 107 3.2.13 Binding of cytosolic proteins with Ag-np-3 108 3.3 Discussion 111 3.3.1 Uptake, distribution and bioactivity of nanoparticles 111 3.3.2 Mitochondrial respiratory chain, synthesis of ATP and ROS production 113 3.3.3 ROS, Ca2+ homeostasis and cytoskeleton changes 117 3.3.4 DNA damage and ROS 119 3.3.5 DNA damage, cellular ATP content and cell cycle arrest 120 3.3.6 Effect on gene expression profiles 121   vii 3.3.7 Interaction of silver nanoparticles with cytosolic proteins 125 3.3.8 Release of pro-inflammatory cytokines from silver nanoparticles treated fibroblasts 126 CHAPTER 4.1 Introduction 129 4.2 Results 130 4.2.1 Microscopy of cells treated with Pt-np 131 4.2.2 Uptake and distribution studies 132 4.2.3 Cytotoxicity 134 4.2.4 ROS production 136 4.2.5 Genotoxicity of Pt-np 138 4.2.6 Effect of Pt-np on cell cycle, apoptosis and necrosis 140 4.2.7 Colony formation 143 4.2.8 Protein levels in Pt-np treated cells 145 4.3 Discussion 145 CHAPTER 5.1 Introduction 151 5.2 Results 152 5.2.1 Comparison of toxicity of different metal nanoparticles 152 5.2.2 Effect of nanoparticles on mortality and hatching rate 154 5.2.3 Effects of nanoparticles on organogenesis 155 5.2.4 Effect of nanoparticles on cardio vascular system 160 5.2.5 Touch response of the larvae 163 5.2.6 Nanoparticle uptake by the embryos 164   viii 5.2.7 Toxicity of corresponding metal ions 164 5.2.8 Probing the toxicity of Silver nanoparticles 165 5.2.9 Mortality, heart rate, edema and malformations 165 5.2.10 Biodistribution of silver nanoparticles in zebrafish embryos 171 5.2.11 Cell cycle analysis of single cells isolated from zebrafish embryos 171 5.2.12 Gene expression in silver nanoparticles treated embryos 174 5.2.13 Protein expression in silver nanoparticles treated embryos 174 5.3 Discussion 177 CONCLUSION 6.1 Conclusions 185 6.2 Future prospects 189 REFERENCES   ix Summary Nanoparticles, even though small in dimension, have a huge impact on the economy. 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Opt. 13, 064031. 208   [...]... charge The possibility of the net reaction is in equation 1.3 Mn+ + Reductionm-  Mo + Oxidationm-n- 1.3 Equation 1.3 depends on the thermodynamics of the process, which in turn is represented by the electrochemical potentials of the corresponding half cell reactions These are called the standard reduction potentials If the reduction potentials corresponding to the reactions 1.1 and 1.2 are added (with their... reproducibility of the procedure limits the application 1.3.5 Surface functionalisation Surface functionalisation gives stability to the nanoparticles Besides, it can control the uptake (Villanueva et al., 2009) and modulates the biocompatibility or cytotoxicity (Yildiz et al., 2009) of the nanoparticles, either by direct interaction with receptors or by preventing aggregation of nanoparticles Choice of surface... surface plasmons in the nanoparticles give strong colours to the nanoparticle solutions, which act as identification markers for the nanoparticles For example silver nanoparticles have greenish brown colour while gold nanoparticles exhibit magenta colour This remarkable optical property forms the basis of the dichroic nature of the Lycurgus cup The SPR of silver nanoparticles that are embedded in the glass... net positive value, the process is thermodynamically feasible This corresponds to a net negative free energy change as, ΔG = -nFE, where ΔG is the free energy change of the reaction 1.3, n is the number of electrons involved, F is a constant called Faraday and E is the electrochemical potential of reaction 1.3 In conclusion, the process is 8   Chapter 1 Toxicity of nanomaterials thermodynamically feasible... electronic properties 1.3.3 Surface plasmon resonance The nanoparticle core exists in a plasma state due to the negatively charged conducting electron and the positively charged lattice When challenged with electromagnetic waves, they oscillate beyond neutral charged state and back to their normal state This collective excitation of Plasmon is termed as surface plasmon resonance (SPR) The oscillations of. .. of surface functionalising agents also determines the shape of the nanoparticles, when combined with specific synthesis procedures The strength of attachment of surface functionalisation determines the reactivity of nanoparticles by facilitating ligand 12   Chapter 1 Toxicity of nanomaterials exchange in the presence of multiple ligands (eg cytosolic proteins) (Cedervall et al., 2007) These properties... Chapter 1 Toxicity of nanomaterials 1.3 Synthesis and properties of metal nanoparticles The metal ions are reduced by employing reducing agents to yield corresponding metal atoms that aggregate to form a metal clump In nanoparticle synthesis, the growth of the metal clump is inhibited at some stage by employing a capping agent (surface functionalisation) that prevents further addition of atoms to the clump... variants of nanomaterials 7 1.3 High resolution electron micrograph of QD showing arrangement of atoms 9 1.4 Schematic representation of a nanoparticle showing factors affecting its propertie 10 1.5 Dichroic appearance of Lycurgus cup due to SPR of silver and gold nanoparticles 12 1.6 Potential routes of exposure, translocation and deposition of nanoparticles 20 3.1 Characterisation of silver nanoparticles. .. Microscopic observations of silver nanoparticle treated cells 67 3.3 Cytotoxicity studies of silver nanoparticles 70 3.4 Uptake of silver nanoparticles 73 3.5 TEM images of ultrathin sections of the cells 75   xii 3.6 Elemental mapping of cell sections 77 3.7 ROS production in silver nanoparticles treated cells 78 3.8 Comet analysis of silver nanoparticles treated cells 80 3.9 Micronucleus analysis for... size effect This quantum confinement has applications in semiconductors, optoelectronics, and non-linear optics The spherical-like shape of nanoparticles produces surface charges (positive or negative) resulting in lattice relaxation (expansion or contraction) and change in lattice constant The electron beam energy bandgap is sensitive to lattice constant The lattice relaxation introduced by nanoparticle . higher concentration of nanoparticles. Recovery of treated cells was monitored and the ability to form colonies was investigated. Colony formation assay showed absence of colony formation only. blood etc. The distribution of the toxic nanoparticles inside the embryos were further studied by using transmission electron microscopy of embryo sections, which showed presence of nanoparticles.  ix 5.2.7 Toxicity of corresponding metal ions 164 5.2.8 Probing the toxicity of Silver nanoparticles 165 5.2.9 Mortality, heart rate, edema and malformations 165 5.2.10 Biodistribution of silver nanoparticles