TITANIUM DIOXIDE NANOMATERIALS EFFECTS ON ENDOTHELIAL CELL BARRIER INTEGRITY a CASE STUDY OF NANOMATERIALS INTERACTION WITH BIOLOGICAL SYSTEM

TITANIUM DIOXIDE NANOMATERIALS EFFECTS ON ENDOTHELIAL CELL BARRIER INTEGRITY: A CASE STUDY OF NANOMATERIALS INTERACTION WITH BIOLOGICAL SYSTEM MAGDIEL INGGRID SETYAWATI (B Eng., Sepuluh Nopember Institute of Technology) (M Sc (Eng), National Taiwan University of Science and Technology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entiretY I have duly acknowledged all the sources of information which have been used in the thesis I This thesis has also not been submitted for any degree in any university previously a \ MAGDIEL INGGRID SETYAWATI 15 November.2014 ACKNOWLEDGEMENT “Blessed are those that can give without remembering and receive without forgetting.” Author Unknown This thesis would not have been possible without the kind help and support from the great many people I am greatly indebted to Assistant Professor Leong Tai Wei David who has been a great supervisor and mentor to me Despite his hectic schedule, he is always available for discussion His advices, constructive suggestions and valuable discussions equipped me to pursue excellence in research and complete this thesis work I would like to thank my thesis committee members, Associate Professor Ting Yen Peng, Professor Feng Shi Shen and Assistant Professor Xie Jianping, for their support, proposed ideas, and constructive suggestions I am grateful for the support rendered by Associate Professor Tan Nguan Soo and Dr Chong Han Chung Their expertise and continuous support have made the in vivo study possible I owe my gratitude to my friends in Leong’s lab (Dr Tay Chor Yong, Ms Chia Sing Ling, Ms Wanru Fang, Goh Sherli, Neo Min Jun, Marcella Giovanni, Rajaletchumy Veloo Kutty, Nandita Menon) because of whom my graduate experience has been one that I will cherish forever Special thanks to Marie Francene Cutiongco and Priscilia Limadinata Their timely help and friendship shall never be forgotten I also extend my thanks to the staffs in the Department of Chemical and Biomolecular Engineering, Mdm Siew Woon Chee, Dr Yang Liming, Ms Vanessa Chan, who were always ready to give their kind help whenever required I dedicated this thesis to my family who always believe in me and cheer me on in all my endeavors Their unflagging love, advices, and prays have been the constant source of strength and encouragement for me Soli Deo Gloria! Magdiel Inggrid Setyawati 15 November 2014 ii TABLE OF CONTENT DECLARATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii SUMMARY vi LIST OF TABLES viii LIST OF FIGURES ix LIST OF ILLUSTRATIONS xi LIST OF ABBREVIATIONS xii Chapter 1: Introduction 1.1 Background 1.2 Hypothesis and Objectives 1.3 Organization Chapter 2: Literature Review 2.1 Nanoparticles 2.1.1 Physical and chemical properties of titanium dioxide nanoparticles (TiO -NPs) 2.1.2 Applications of TiO -NPs 2.2 TiO -NPs and human exposure: potential TiO -NPs release throughout their life cycle 12 2.3 TiO -NPs and human exposure: uptake route and distribution in human body 16 2.3.1 Inhalation of TiO -NPs 17 2.3.2 Ingestion of TiO -NPs 18 2.3.3 Dermal penetration of TiO -NPs 19 2.3.4 Blood circulation as TiO -NPs distribution route 20 2.3.5 Biopersistence and excretion of TiO -NPs 21 2.4 TiO -NPs induced biological response 22 2.4.1 Cytotoxicity 23 2.4.2 Genotoxicity 24 2.4.3 Oxidative stress 25 2.4.4 Correlation of physicochemical properties of TiO -NPs and the elicited biological responses 27 2.5 Blood vessels and the endothelial cell barrier 33 2.6 Maintenance of endothelial cell monolayer integrity 36 iii 2.6.1 Adherens Junctions (AJs) 37 2.6.2 Tight Junctions (TJs) 39 2.6.3 Gap junctions 39 2.7 Endothelial cell barrier leakiness 40 2.8 TiO -NPs and endothelial cells interaction: the knowledge gap 41 2.9 Problem statement and scope of study 42 Chapter 3: Materials and Methods 3.1 Materials 45 3.1.1 Cells 45 3.1.2 Animals 45 3.1.3 Chemicals 45 3.1.4 Antibodies 46 3.1.5 Buffers 47 3.2 Method 48 3.2.1 Cell culture 48 3.2.2 TiO -NPs characterization 48 3.2.3 Preparation of TiO -NPs suspension 48 3.2.4 Immunofluorescence staining 49 3.2.5 Permeability Transwell® Assay 50 3.2.6 Reactive oxygen species (ROS) level measurement 50 3.2.7 Cell viability measurement 51 3.2.8 Protein extraction and immunoblotting 51 3.2.9 Preparation of FITC-TiO -NPs 52 3.2.10 Confocal imaging of internalized TiO -NPs 52 3.2.11 Quantification of internalized of TiO -NPs 53 3.2.12 TiO -NPs pulldown 53 3.2.13 Preparation of mouse IgG-conjugated TiO -NPs 54 3.2.14 Proximity Ligation Assay (PLA) 55 3.2.15 Immunoblotting detection of VE-cadherin phosphorylation 57 3.2.16 Immunoprecipitation 57 3.2.17 Immunoblotting determination of VE-cadherin internalization and degradation 57 3.2.18 Immunofluorescence detection of VE-cadherin internalization 58 3.2.19 ROCK inhibition assay 59 3.2.20 Animal handling 59 3.2.21 In vivo subcutaneous vascular leakiness assay 59 3.2.22 In vivo murine melanoma-lung metastasis model 59 3.2.23 RNA extraction and real time qPCR 60 3.2.24 Histology scoring 61 3.2.25 Statistical analysis 62 Chapter 4: Nanomaterials induced endothelial cells leakiness 4.1 Results 64 4.1.1 TiO -NPs characterization 64 iv 4.1.2 TiO2-NPs induce the in vitro endothelial cells leakiness 68 4.1.3 NanoEL is independent of apoptosis 72 4.1.4 NanoEL is independent of oxidative stress 75 4.1.5 NanoEL is independent of cellular uptake 77 4.2 Discussion 80 4.3 Summary 81 Chapter 5: Mechanism of nanomaterials induced endothelial cell leakiness 5.1 Results 83 5.1.1 TiO2-NPs directly bind to VE-cadherin 84 5.1.2 TiO2-NPs induce the declustering of homophilically interacted VE-cadherin 88 5.1.3 TiO2-NPs trigger activation of VE-cadherin pathway 89 5.1.4 TiO2-NPs induce internalization and degradation of VE-cadherin 93 5.1.5 TiO2-NPs trigger activation of actin remodeling to induce NanoEL 97 5.2 Discussion 99 5.3 Summary 100 Chapter 6: In vivo validation of nanomaterials induced endothelial cell leakiness 6.1 Results 102 6.1.1 TiO2-NPs cause endothelial cell leakiness in subcutaneous blood vessels 102 6.1.2 TiO2-NPs cause endothelial cell leakiness in a mouse lung metastasis model 104 6.2 Discussion 111 6.3 Summary 112 Chapter 7: Conclusions and Recommendations 7.1 Conclusions 114 7.2 Future Perspectives 118 REFERENCES 120 APPENDIX Appendix A Supplementary information 134 Appendix B List of Publications 140 Appendix C List of Awards 142 Appendix D Copyrights 144 v SUMMARY The exponential increase in nanomaterials (NMs) production and application has triggered concerns on the potential effect of these NMs to human health This concern is not unwarranted as NMs, due to their small size, could persist in tissues Moreover, their small size allows them to interact with cells or any other biological entities in the human body Efforts to identify this potential interaction between NMs and any biological entities have been made, nevertheless most studies are dedicated on the human major organs such as lung and kidney but not the blood vessel network despite its pervasive critical function in human body They act as conduits for the blood cells, nutrients, hormones and wastes circulation in and out of the human body These pervasive conduits are known to be lined with a single cell layer of endothelial cells which regulate the solute exchange between the blood stream and the surrounding tissue This makes endothelial cells to be the most likely cells that encounter the NMs circulating in human body Undoubtedly, there is a need to investigate the interaction that occurs between endothelial cells and the NMs Thus far, most research has been dedicated on the NMs’ cytotoxicity and inflammation inducement on endothelial cells However, little work with the emphasis on understanding the interaction that manifest on function impairment of the endothelial cell barrier has been done This study aims to elucidate the interaction between NMs and endothelial cells with the emphasis on the mechanism which leads to the impairment of the endothelial cell barrier This novel interaction was studied by employing human microvascular endothelial cells (HMVECs) and titanium dioxide nanoparticles (TiO -NPs) as endothelial cells and NMs models, respectively It is observed that TiO -NPs, but not their microparticles counterpart, could induce intercellular gaps between adjoining endothelial cells This phenomenon was coined as nanomaterials induced endothelial cells leakiness (NanoEL) NanoEL could be triggered in dose dependent manner within a short exposure time of 30 minutes NanoEL was observed to be independent from the known nanotoxicity events such as apoptosis and oxidative stress From our NMs tracking analysis, NanoEL was observed to be activated through some extracellular trigger, as evidenced by the majority of the TiO -NPs which had not been endocytosed by the cells at the onset of NanoEL A mechanistic study was conducted in order to understand how NanoEL was triggered It was found that the NanoEL was initiated by the physical interaction of TiO -NPs with endothelial cells adherens junction (AJ) protein, VE-cadherin, which is responsible to maintain the integrity of endothelial cells barrier This led to the disruption of VE-cadherin homophilic interactions and activated an aberrant downstream signal transduction It was found that the VE-cadherin lost its interaction with its anchoring proteins, β-catenin and p120, leading to its endocytosis and degradation In addition, cell cytoskeleton rearrangement process was activated, which led to cell retraction and eventually brought about NanoEL The in vitro findings of NanoEL effect triggered by TiO -NPs were validated by the in vivo study It was observed that subcutaneous injection of TiO -NPs could cause leakiness in the surrounding subcutaneous blood vessels in mice In addition, TiO -NPs induced blood vessel leakiness promoted the melanoma-to-lung metastasis both in acute and sub-chronic exposure scenario Overall, the study’s findings have revealed a new NMs’ toxic effect that is apparently non-cytotoxic but profoundly changes the normal functioning of endothelial cells Most importantly, this study uncovers a novel non-receptor mediated mechanism which allows NMs to trigger intracellular signaling cascade through their physical binding with the AJ proteins, VE-cadherin LIST OF TABLES Table 2.1: Selected publications on TiO -NPs induced biological responses in the lung model 28 Table 2.2: Selected publications on TiO -NPs induced biological responses in the nervous system model 29 Table 2.3: Selected publication on TiO -NPs induced biological responses in the dermal model 29 Table 2.4: Selected publications on TiO -NPs induced biological responses in the gastrointestinal model 30 Table 2.5: Selected publications on TiO -NPs induced biological responses in the liver model 30 Table 2.6: Selected publications on TiO -NPs induced biological responses in the kidney model 31 Table 2.7: Selected publications on TiO -NPs induced biological response in the cardiovascular model 31 Table 2.8: Selected publications on TiO -NPs induced biological responses in the hematopoietic model 32 Table 3.1: Concentration conversion of TiO -NPs used in the study 49 Table 3.2: Real time qPCR primer sequences 61 Table 4.1: Summary of hydrodynamic characterization of TiO -NPs 67 viii LIST OF FIGURES Figure 2.1: Pervasive use of NPs in modern lifestyle products Figure 2.2: Forecast of TiO -NPs production in U S 10 Figure 2.3: Potential human exposure to TiO -NPs 13 Figure 2.4: Possible entry route and translocation of TiO -NPs in human body 16 Figure 2.5: Predicted inhaled nanoparticle distribution in the human lung 17 Figure 2.6: The threats of reactive oxygen species (ROS) in cells 26 Figure 2.7: Paracellular and transcellular route of solute transport across the microvascular endothelial cell barrier 34 Figure 2.8: Formation of intercellular junctions on endothelial cell barrier 37 Figure 2.9: Adherens junctions in endothelial cell barrier 38 Figure 4.1: Characterization of TiO -NPs 65 Figure 4.2: TiO -NPs induce in vitro endothelial cells leakiness as observed with immunofluorescence technique 69 Figure 4.3: TiO -NPs induce dose dependent in vitro endothelial cells leakiness as observed with Transwell permeability assay 71 Figure 4.4: TiO -NPs, SiO -NPs and Ag-NPs induce dose dependent in vitro endothelial cell leakiness as observed with Transwell permeability assay 72 Figure 4.5: NanoEL is independent of apoptosis 74 Figure 4.6: NanoEL is independent of ROS formation 76 Figure 4.7: NanoEL is independent of TiO -NPs endocytosis 79 Figure 5.1: TiO -NPs directly bind to homophilic VE-cadherin in the AJ as observed with TiO -NPs pull-down assay 85 Figure 5.2: TiO -NPs directly bind to homophilic VE-cadherin in the AJ as observed with TiO -NPs in situ proximity ligation assay (PLA) 87 Figure 5.3: TiO -NPs cause the disruption of VE-cadherin clusters 88 Figure 5.4: TiO -NPs induce phosphorylation of VE-cadherin 91 Figure 5.5: TiO -NPs treatment induces release of p120 and β-catenin from VE-cadherin 92 Figure 5.6: TiO -NPs induce internalization of VE-cadherin 95 Figure 5.7: TiO -NPs induce degradation of VE-cadherin 96 ix Appendix A: Supplementary Information Figure A1.1: Characterization of TiO2-MPs (A) Transmission electron microscope (TEM) micrograph of TiO2-MPs Scale bar = 500 nm (B) Summary of hydrodynamic properties of the TiO2-MPs obtained with DLS method Data are means ± s.d from three independent measurements 135 Appendix A: Supplementary Information Figure A1.2: TiO2-NPs induced in vitro endothelial cells leakiness within 30 minutes of exposure Various concentrations of TiO2-NPs induced the disruption on the endothelial cell barrier integrity (red arrowheads) within short exposure time of 30 minutes Visualization was done with immunofluorescence whereby AJ protein, VE-cadherin, were visualized in green and the cells nuclei in blue Scale bar: 50 µm 136 Appendix A: Supplementary Information Figure A1.3: TiO2-NPs induced in vitro endothelial cells leakiness within 60 minutes of exposure Various concentrations of TiO2-NPs induced the disruption on the endothelial cell barrier integrity (red arrowheads) was persisted even after 60 minutes of exposure Visualization was done with immunofluorescence whereby AJ protein, VE-cadherin, were visualized in green and the cells nuclei in blue Scale bar: 50 µm 137 Appendix A: Supplementary Information Figure A1.4: TiO2-MPs did not induce in vitro endothelial cells leakiness No disruption of endothelial cell monolayer barrier was observed 60 minutes after the introduction of TiO2-MPs in various concentrations Visualization was done with immunofluorescence whereby AJ protein, VE-cadherin, were visualized in green and the cells nuclei in blue Scale bar: 50 µm 138 Appendix A: Supplementary Information Figure A1.5: Negative control of immunofluorescence experiment AJ protein, VE-cadherin only can be visualized when the primary antibody against it were utilized This suggests the specificity of the immunofluorescence technique Scale bar: 50 µm 139 Appendix B List of Publication Appendix B: List of Publication Setyawati MI, Tay CY, Chia SL, Goh SL, Fang W, Neo MJ, Chong HC, Tan SM, Loo SC, Ng KW, Xie JP, Ong CN, Tan NS, Leong DT Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE-cadherin Nature Communications 2013, 4, 1673 Setyawati MI, Khoo PKS, Eng BH, Xiong SJ, Zhao XX, Das G, Tan T, Loo J, Leong DT, Ng KW Cytotoxic and genotoxic characterization of titanium dioxide, gadolinium oxide and poly(lactic-co-glycolic acid) nanoparticles in human fibroblasts Journal of Biomedical Materials Research Part A 2013, 101A, 633-640 Setyawati MI, Tay CY, Leong DT The gap between endothelial cells: key to the quick escape of nanomaterials? Nanomedicine 2014, 9, 1591-1594 Setyawati MI, Fang W, Chia SL, Leong DT 2013 Nanotoxicology of common metal oxide based nanomaterials: their ROS-y and non-ROS-y consequences Asia-Pacific Journal of Chemical Engineering 2013, 8, 205-217 Setyawati MI, Leong DT Understanding and exploiting nanoparticles' intimacy with the blood vessel and blood Chemical Society Reviews 2015 (In preparation) 141 Appendix C List of Awards Appendix C: List of Awards Scholarship and awards 2014 2013 2010 – 2014 : Humboldt Kolleg International Symposium 2014 – Best Poster Award : International Conference Material for Advanced Technologies (ICMAT 2013) – Best Poster Award : National University of Singapore – Research Scholarship 143 Appendix D Copyrights Appendix D: Copyrights JOHN WILEY AND SONS LICENSE TERMS AND CONDITIONS Oct 12, 2014 This is a License Agreement between Magdiel Inggrid setyawati ("You") and John Wiley and Sons ("John Wiley and Sons") provided by Copyright Clearance Center ("CCC") The license consists of your order details, the terms and conditions provided by John Wiley and Sons, and the payment terms and conditions All payments must be made in full to CCC For payment instructions, please see information listed at the bottom of this form License Number 3486800920810 License date Oct 12, 2014 Licensed content publisher John Wiley and Sons Licensed content publication Asia-Pacific Journal of Chemical Engineering Licensed content title Nanotoxicology of common metal oxide based nanomaterials: their ROS-y and non-ROS-y consequences Licensed copyright line © 2012 Curtin University of Technology and John Wiley & Sons, Ltd Licensed content author Magdiel Inggrid Setyawati, Wanru Fang, Sing Ling Chia, David Tai Leong Licensed content date Oct 4, 2012 Start page 205 End page 217 Type of use Dissertation/Thesis Requestor type Author of this Wiley article Format Print and electronic Portion Figure/table Number of figures/tables Original Wiley figure/table number(s) Figure 1, Figure2, Figure5 Will you be translating? 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Paracellular and transcellular route of solute transport across the microvascular endothelial cell barrier 34 Figure 2.8: Formation of intercellular junctions on endothelial cell barrier. .. functional impairment of the endothelial cells barrier This study aims to investigate the said interaction between NMs and endothelial cells with the emphasis on the functional impairment of the endothelial. .. paracellular route (Figure 2.7) Figure 2.7: Paracellular and transcellular route of solute transport across the microvascular endothelial cell barrier Barrier function of endothelial cell monolayer