THAI NGUYEN UNIVERSITY THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY DUONG THI THU HUYEN Topic title: THE UPTAKE MECHANISMS OF AG AND TIO2 NANOPARTICLES TO NERVOUS-SYSTEM CELLS BACHELOR THESIS Study Mode: Full-time Major: Environmental Science and Management Faculty: International Training and Development Center Batch: 2010-2015 Thai Nguyen, January 15th, 2015 THAI NGUYEN UNIVERSITY THAI NGUYEN UNIVERSITY OF AGRICULTURE AND FORESTRY DUONG THI THU HUYEN Topic title: THE UPTAKE MECHANISMS OF AG AND TIO2 NANOPARTICLES TO NERVOUS-SYSTEM CELLS BACHELOR THESIS Major: Environmental Science and Management Supervisors: - Assoc.Prof HUANG, Yuh-Jeen - Dr Tran Thi Thu Ha PhD Thai Nguyen, January 15th, 2015 Thai Nguyen University of Agriculture and Forestry Degree program Bachelor of Environmental Science and Management Student name Duong Thi Thu Huyen Student ID DTN1053110108 Thesis title The Uptake Mechanisms of Ag and TiO2 Nanoparticles of Nervous-system cells Supervisors Yuh-Jeen Huang Assoc.Prof, Tran Thi Thu Ha PhD ABSTRACT Nanosafety is a hot issue in the nanotechnology field nowadays Nanoparticles may penetrate the blood-brain-barrier into the central nervous system associated with neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease This report presents the uptake mechanisms of Silver nanoparticles with average diameters of 3-5 nm and 10-15 nm and Anatase TiO2 nanoparticles (7 nm (ST-01), 21 nm (ST-21)) nanoparticles, in two nervous-system cells microglia (BV-2) and astrocytes (ALT) The experiment was performed with Ag and TiO2 nanoconjugates Result obtained by alamarBlue viability assay and Fluorescence Microscopy images The research was incomplete, but we had understood the basic process of internalization of nanoparticles The results demonstrated that the inhibitors have effect to inhibit the uptake pathways after 2.5h testing via clathrin-mediated and caveolin-mediated endocytosis, micropinocytosis and phagocytosis 24.5h experiment of inhibitors uptake pathway should be taken into future consideration in other researches/studies of the kind with another method Key words Ag nanoparticle, TiO2 nanoparticle, inhibitors, nanoconjugates, uptake pathway Number of pages 54 Date of submission Jan 15th, 2015 ii ACKNOWLEDGEMENT I would like to express the deepest appreciation to teachers in faculty of International Training and Development as well as teachers in Thai Nguyen University of Agriculture and Forestry, who have dedicated teaching to me the valuable knowledge during study time in university and gave me a chance to my thesis oversea It is with immense gratitude that I acknowledge the support and help of Biomedical Engineering & Environmental Science Department, National Tsing Hua University for accepting me to working in this wonderful place It gives me great pleasure in acknowledging the support and help of Associate Professor Huang Yuh-Jeen, who has attitude and the substance of a great teacher She did everything to give me the best condition, support all materials I need to my thesis during the time I working in her Environmental Nano Analysis and Energy Laboratory I would like to thank to Dr Tran Thi Thu Ha, who always supported and cheered up me whole the time I work abroad She also the one who help me the most on spending time to check my thesis report I consider it is an honor to work with Mr Alan (Hsiao I-Lun) for months of research Without his guidance, my research couldn’t be possible I cannot find words to express my gratitude to my family and friends, who always beside me all the time whatever happened, create the pump leading me to success In the process of implementing the project, I know that my thesis report got many mistakes so this report is inevitable shortcomings So, I would like to receive the attention and feedback from teachers and friends to this thesis is more complete I sincerely thank you! Duong Thi Thu Huyen iii CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS PART I INTRODUCTION 1.1 Rationale of the research 1.2 Objectives of the research 1.3 Research questions and hypothesis 1.4 Limitations of research 1.5 Definitions PART II LITERATURE REVIEW 2.1 Nanomaterials 2.1.1 TiO2 nanoparticles 2.1.2 Silver nanoparticles 2.2 Neuro-cells 11 2.2.1 Astrocyte (ALT) 11 2.2.2 Microglia (BV-2) 12 2.3 Endocytosis pathway 13 2.4 Inhibitors 16 2.5 Nanoparticles and neurodegenerative diseases possible relationships 17 PART III METHODS 20 3.1 Materials 20 3.2 Neuro-cells culture 23 3.2.1 Astrocyte cell (ALT) 23 3.2.2 Microglial cell (BV-2) 23 3.3 Biological analysis methods 23 3.3.1 Cell viability assay 23 3.3.2 Inhibitors for specific uptake pathways 25 3.3.3 Fluorescence microscope imaging 26 CHAPTER IV RESULTS 28 4.1 Cell viability assay 28 4.1.1 Astrocyte Cell viability assay 28 4.1.2 Microglia cells viability assay 30 4.2 Inhibitors uptake pathway 32 4.2.1 Astrocyte (ALT-after 2.5h and 24.5 hours exposure) 32 4.2.2 Microglia (BV2- after 2h and 24h exposure) 37 4.2.3 LPS-activated BV2- Dextran conjugate 42 4.3 Silver Nanoparticle uptake pathway 43 iv CHAPTER V CONCLUSION AND DISCUSSION 45 REFERENCES 46 v LIST OF FIGURES Figures Page Figure 2.1 Crystal structures of the forms of titanium dioxide Figure 2.2 Strategies in the design of nanoparticles for therapeutic 14 applications Figure 2.3 Possible pathways of neurotoxicity by nanoparticles 19 Figure 3.1 The 96-well plates 25 Figure 3.2 Fluorescence microscope 27 Figure 4.1 ALT cell viability assay result of Chlorpromazine inhibitor 28 Figure 4.2 ALT cell viability assay result of MDC inhibitor 28 Figure 4.3 ALT cell viability assay result of Genistein inhibitor 29 Figure 4.4 ALT cell viability assay result of Filipin inhibitor 29 Figure 4.5 ALT cell viability assay result of Amiloride inhibitor 29 Figure 4.6 ALT cell viability assay result of Phenylarsine oxide inhibitor 29 Figure 4.7 BV-2 cell viability assay result of Chlorpromazine inhibitor 30 Figure 4.8 BV-2 cell viability assay result of MDC inhibitor 30 Figure 4.9 BV-2 cell viability assay result of Genistein inhibitor 31 Figure 4.10 BV-2 cell viability assay result of Filipin inhibitor 31 Figure 4.11 BV-2 cell viability assay result of Amiloride inhibitor 31 Figure 4.12 BV-2 cell viability assay result of Phenylarsine oxide inhibitor 31 Figure 4.13 ALT images from Fluorescence microscope after 2.5h and 24.5 33 testing Chlorpromazine and MDC work with Transferrin conjugate Figure 4.14 ALT images from Fluorescence microscope after 2.5h and 24.5 34 testing Genistein and Filipin work with BODIPY-LacCer conjugate Figure 4.15 ALT images from Fluorescence microscope after 2.5h and 24.5 36 testing Amiloride and Phenylarsine oxide work with Dextran conjugate Figure 4.16 BV-2 images from Fluorescence microscope after 2.5h and 24.5 38 testing Chlorpromazine and MDC work with Transferrin conjugate Figure 4.17 BV-2 images from Fluorescence microscope after 2.5h and 24.5 39 testing Genistein and Filipin work with Transferrin conjugate Figure 4.18 BV-2 images from Fluorescence microscope after 2.5h and 24.5 41 testing Amiloride and Phenylarsine oxide work with Dextran conjugate Figure 4.19 LPS-activated BV-2 images from Fluorescence microscope after 42 2.5h testing Amiloride and Phenylarsine oxide work with Dextran conjugate Figure 4.20 ALT cell images from Fluorescence microscope after 2.5h 43 testing with Ag NPs LIST OF TABLES Tables Page Table 3.1 Sources of Nanomaterials 19 Table 3.2 Materials for biological analysis 19 Table 4.1 Summary of inhibitor work to inhibit uptake pathway 44 LIST OF ABBREVIATIONS ALT Astrocyte cell BBB Blood-brain-barrier BV-2 Microglia cell CNS Central nervous system LPS Lipopolysaccharide NPs Nanoparticles ROS Reactive oxygen species PART I INTRODUCTION 1.1 Rationale of the research In recent years, nanotechnology was born not only create breakthrough leap in electronics, computer science, biomedical, environmental, but also widely used in the life However, when the materials reach nanometer level, due to small particle size, large surface area, and upgraded reactivity, they may cause harm to human and organisms Therefore, nanosafety is a hot issue in the nanotechnology field nowadays Scientists have found that nanoparticles may penetrate the blood-brain-barrier (BBB) into the central nervous system (CNS) or directly translocate onto the CNS from olfactory nerves Moreover, neurotoxicity of nanoparticles (NPs) started to catch attention because the reactive oxygen species (ROS) induced by NPs could be associated with neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease 1.2 Objectives of the research The specific objective of the project is to understand the processes of internalization of Ag and TiO2 nanoparticles in two nervous-system cells Microglia (BV-2) and Astrocyte (ALT) In this study, the nervous-system cells such as Astrocyte and Microglia is exposed to determine the internalization processes of Ag and TiO NPs with the use of Transwell plate The cell viability and efficiency of inhibitors in two kinds of cells were also measured After using BODIPY-LacCer conjugate for testing inhibitors work on blocking caveolin-madeate endocytosis, the images below generated from fluorescence microscope Similar to transferrin, LacCer had gone after 24.5h exposed in control and Filipin cover-glasses, Genistein glass had only cell having LacCer As can be seen from image, the 2.5h test of ALT cell shown that inhibitors work well with only little LacCer around some cell if compared to the control (See figure 19) 4.2.1.3 Dextran conjugate (Results: Dec 17th,2014) Contrasting to Transferrin and LacCer conjugates, Detran looked like more time is of need to transfer from outside of the cell to inside through micropinocytosis and phagocytosis pathways Therefore, the images of ALT cells after 24.5h exposed produced better results; however, it doesn’t mean that the test has experienced a success Back to the image produced after 2.5h, there was not much to see considering dextran but there are some cells of dextran work observable in control glass Compared with this control, amiloride had block apart micropinocytosis and specialy, phenylarsine oxide had shown that it works well to block phagocytosis (see figure 20) 35 Control 2.5h Control 24.5h Amiloride 2.5h Amiloride 24.5h Phenylarsine 2.5h Phenylarsine 24.5h Figure 4.15 ALT images from Fluorescence microscope after 2.5h and 24.5 testing Amiloride and Phenylarsine oxide work with Dextran conjugate (Dec 17th, 19th 2014) 36 4.2.2 Microglia (BV2- after 2h and 24h exposure) 4.2.2.1Transferrin conjugate With smaller size than ALT, it is more challenging to analyse the images of BV-2 cell After 2.5h of exposure, in the results are presented in the images captured from Fluorescence microscope below While the blue color is represented of the cell nucleus, the green color is the Transferrin conjugate used in experiment In these control 2.5h and 24.5h, it is shown that the transferrin had come inside cell through clathrin-mediate endocytosis In 2.5h, both chlorpromazine and MDC inhibitors showed their work when blocked clathrinmediate endocytosis pathway For 24.5h, if only compared to control, we can see that Chlorpromazine and MDC had worked; but it cannot be denied that that transferrin came into cell too much This case, thus, should be taken into future consideration in other researches/studies of the kind (see figure 21) 37 Control 2.5h MDC 2.5h Chlorpromazine 2.5h Control 24.5h MDC 24.5h Chlorpromazine 24.5h Figure 4.16 BV-2 images from Fluorescence microscope after 2.5h and 24.5 testing Chlorpromazine and MDC work with Transferrin conjugate (result: Dec 12th, 15th, 2014) 38 4.2.2.2 BODIPY LacCer Conjugate Control 2.5h Genistein 2.5h Filipin 2.5h Control 24.5h Genistein 24.5h Filipin 24.5h Figure 4.17 BV-2 images from Fluorescence microscope after 2.5h and 24.5 testing Genistein and Filipin work with BODIPY-LacCer conjugate (result: Dec 12th, 15th,2014) 39 After using BODIPY-LacCer conjugate for testing inhibitors work on blocking caveolin-madeate endocytosis, the images below generated from fluorescence microscope Similar to transferrin, LacCer had gone after 24.5h exposed in both control, Filipin and Genistein glass, only 2-3 cells having LacCer This can also be put aside The 2.5h test of BV-2 cell here has clearly shown that inhibitors work well with only little LacCer around some cell in glasses, which had Genistein and Filipin, if compared to the control (see figure 22) 4.2.2.3 Dextran conjugate Similar to ALT cells, in BV-2 cell Detran needed more time to transfer from outside of cell to inside through micropinocytosis and phagocytosis pathways Therefore the images of ALT cells after 24.5h exposed was looked brighter For 2.5h images, dextran work well in control glass To compare with this control, Amiloride had block apart of micropinocytosis and specially, phenylarsine oxide had shown that it work well to block phagocytosis (figure 23) 40 Control 2.5h Amiloride 2.5h Phenylasine 2.5h Control 24.5h Amiloride 24.5h Phenylarsine 24.5h Figure 4.18 BV-2 images from Fluorescence microscope after 2.5h and 24.5 testing Amiloride and Phenylarsine oxide work with Dextran conjugate (Dec 17th, 19th 2014) 41 4.2.3 LPS-activated BV2- Dextran conjugate Control 2.5h Amiloride 2.5h Phenylarsine 2.5h Figure 4.19 LPS-activated BV-2 images from Fluorescence microscope after 2.5h testing Amiloride and Phenylarsine oxide work with Dextran conjugate (result: Dec 26th, 2014) As produced in some of the researches in the past years, in many pathological features, when brain get hurt, microglia cell will be activated, migrated to the periphery of dead cells, clear cell debris Activation of microglia is sometimes beneficial to release some neurotrophic factor Therefore Amiloride and Phenylarsine oxide was used as 42 phagocytosis inhibitor to inhibit the LPS-activated BV-2 cells Base on results in image above, we saw that Amiloride and Phenylarsine oxide had block the micropinocytosis and phagocytosis (see figure 4.2.3) 4.3 Silver Nanoparticle uptake pathway Control 2.5h w/o Ag NPs Control 2.5h Ag NPs Figure 4.20 ALT cell images from Fluorescence microscope after 2.5h testing with Ag NPs (result: Dec 29th 2014) Because time has been limited, only Silver nanoparticles work in no-inhibitor condition was be tested The blue color is nucleus About the white color, we still not sure 100% whether it’s Ag NPs or not, because we didn’t have time to repeat experiment This case, Silver and titanium dioxide nanoparticles, thus, should be taken into future consideration in other researches/studies of the kind 43 The table below represent a summary of the results collected: Cell types Inhibitors Substrate 2.5h 24.5h √ MDC √ Genistein √ Filipin √ Amiloride hydrochloride √ √ MDC √ √ Genistein √ Filipin √ Amiloride hydrochloride √ Phenylarsine oxide √ Amiloride hydrochloride √ √ Chlorpromazine Transferrin Alexa Fluor 488 ALT BODIPY LacCer Dextran-FITC 70kDa Phenylarsine oxide Chlorpromazine Transferrin Alexa Fluor 488 BODIPY LacCer BV-2 Dextran-FITC 70kDa LPSactivated BV-2 Dextran-FITC 70kDa Phenylarsine oxide Table 4.1 Summary of inhibitor work to inhibit uptake pathway √ had effect not clear/haven’t test 44 CHAPTER V CONCLUSION AND DISCUSSION In this study, two kind of cells Astrocyte and Microglia have been dispersed in DMEM/10% FBS/1%AA that is suitable for subsequent cell experiments Cell viability was measuared with different kind of inhibitors in both 2.5h and 24.5h The high concentration of inhibitor exposure cause viable cells number decreased From alamarBlue cell viability assay, the sub-cytotoxic dose of inhibitor was found to use for next test in fluorescence microsopy The short time exposure (2.5h), inhibitors had shown the effect on cell uptake pathway as can be seen from fluorescence microscopy images For 24.5h hours, Because of longer time exposure, it might have some side-effect that made inhibitors had no effect or not clear enough But the size of NPs will change after exposure so it might have effect for 24h, not like these inhibitors 24h should be taken into future consideration in other researches/studies of the kind with another method Although time has been a limit to test with Silver and Titanium dioxide nanoparticles, basing on all the above results, it is believed that when Ag and TiO2 NPs passed into central nervous system, NPs were ingested by ALT or BV-2 cells causing cell death, oxidative stress, inflammation, and these conditions may cause damages to the central nervous system, leading to the generation of neurodegenerative This study provides information for nano titanium dioxide and nano silver effects on the central nervous system cells, and the results are expected to supply a reference for future 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UNIVERSITY OF AGRICULTURE AND FORESTRY DUONG THI THU HUYEN Topic title: THE UPTAKE MECHANISMS OF AG AND TIO2 NANOPARTICLES TO NERVOUS- SYSTEM CELLS BACHELOR THESIS Major: Environmental Science and Management... disease, and Huntington’s disease 1.2 Objectives of the research The specific objective of the project is to understand the processes of internalization of Ag and TiO2 nanoparticles in two nervous- system. .. the uptake mechanisms of Silver nanoparticles with average diameters of 3-5 nm and 10-15 nm and Anatase TiO2 nanoparticles (7 nm (ST-01), 21 nm (ST-21)) nanoparticles, in two nervous- system cells