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BIOCOMPATIBILITY OF CARBON NANOMATERIALS: MATERIALS CHARACTERIZATION AND CYTOTOXICITY EVALUATION Dissertation Submitted to The School of Engineering of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in Materials Engineering By Lin Zhu UNIVERSITY OF DAYTON Dayton, Ohio August 2012 BIOCOMPATIBILITY OF CARBON NANOMATERIALS: MATERIALS CHARACTERIZATION AND CYTOTOXICITY EVALUATION Name: Zhu, Lin APPROVED BY: Daniel Eylon, Ph.D. P.Terrence Murray, Ph.D. Advisory Committee Chairman Committee Member Professor, Materials Engineering Professor, Materials Engineering Liming Dai, Ph.D. Harihara Baskaran, Ph.D. Committee Member, Co-Advisor Committee Member Professor, Macromolecular Science and Professor, Chemical Engineering Engineering Case Western Reserve University Case Western Reserve University James Snide, Ph.D. Donald Klostermen, Ph.D. Committee Member Committee Member Professor Emeritus, Professor, Materials Engineering Chemical & Materials Engineering Mr. Gerald Shaughnessy, M.S. Committee Member Professor, Mathematics John G. Weber, Ph.D. Tony E. Saliba, Ph.D. Associate Dean Dean, School of Engineering School of Engineering & Wilke Distinguished Professor ii ABSTRACT BIOCOMPATIBILITY OF CARBON NANOMATERIALS: MATERIALS CHARACTERIZATION AND CYTOTOXICITY EVALUATION Name: Zhu, Lin University of Dayton Advisor: Dr. Daniel Eylon and Dr. Liming Dai The objective of this research is to investigate the biocompatibility of carbon nanomaterial. It was found that the cytotoxicity of multiwalled carbon nanotubes (MWNTs) depend on their concentration, size, and surface chemical groups (e.g., - COOH). MWNTs and MWNT-COOH could accumulate in human lung macrophage cells (U937) to different degrees, and they did not produce overt cell toxicity within the concentration range of 5–50 µg/ml up to 24 h. However, there were morphological alterations at low doses of MWNT-COOH and significant reactive oxygen species (ROS) generation for MWNTs at higher doses, indicating a distinguished possible cellular stress response and DNA damage from both materials. In the second part of this study, reduced graphene oxide (rGO) was demonstrated to show the concentration-dependent and cell-specific cytotoxicity. Specifically, rGO was found to stimulate cell proliferation of human skin fibroblast cells at relatively low concentrations (< 5 µg/mL), but inhibit human skin fibroblast cells proliferation at high iii concentrations. rGO-induced concentration-dependent and cell-specific generation of ROS and activation of NF-κB transcription factors were also observed, indicating an oxidative stress mechanism. Furthermore, rGO was showed to be more biocompatibility to human skin fibroblast cells with respect to mouse embryonic fibroblast cell (NIH-3T3) cells. In the third part of this study, the soft lithography technique was used to build PDMS microfluidic devices for monitoring cells viability and performing dynamic study of the carbon nanomaterial biocompatibility. Compared to the traditional in vitro technique, this research opens up a new approach to biocompatibility evaluation of nanomaterials with a reduced usage of animal in toxicity study. By using the newly- developed microfluidic devices, the biocompatibility of MWNTs and rGO were investigated. It was found that both particles could enter into the circulation system in the microfluidic devices. Possible damage to bovine aortic endothelium cells (BAECs) caused by carbon nanomaterials was investigated. The interaction of MWNTs (1D) and rGO sheets (2D) with BAECs in both static (cell culture) and dynamic (microfluidic) environments indicated that both nanoparticles reduced the mitochondrial function and lipoprotein (LDL) uptake. These results were concentration and morphology depended. MWNTs showed a better biocompatibility than rGO in both static and dynamic environments, while the microfluidic tests exhibited better biocompatibilities than those in cell culture dishes for both nanoparticles. iv I dedicated this dissertation to my beloved parents. v ACKNOWLEDGEMENTS This research would not have been possible without the support of Dr. Liz Downie, Dr. Daniel Eylon, Dr. Andrey A. Voevodin, Dr. Ajit Roy, Dr. Saber Hussain, and the financial support of AFRL/DAGSI (Air Force Research Laboratory/ Dayton Area Graduate Studies Institute) and the Oak Ridge Institute of Science and Education (ORISE). I appreciate the opportunity to work on this project. Without this program, I would not have been able to achieve this level of success. I also wish to acknowledge my committee members, Dr. Daniel Eylon, Dr. Liming Dai, Dr. Donald Klosterman, Dr. P. Terrence Murray, Dr. James Snide, Mr. Gerald J. Shaughnessy and Dr. Harihara Baskaran for their time and assistance. Additionally, I would like to express my gratitude towards Dr. Harihara Baskaran, Dr. Saber Hussain and my adviser Dr. Liming Dai, for providing me with the opportunity to work in their research laboratories. Their profound wisdom and knowledge impacted my basic research concepts. I am also grateful for many other individuals who contributed their precious time and valuable skills, assisting me and my research project. It would be impossible to mention everybody individually by name here. However, I would specifically like to thank Dr. Amanda Schrand and Ms. Christin Grabinski from AFRL/HEPB, Mr. Alexander Rivera from Dr. Harihara Baskaran’s group and Dr. Dong Wook Chang, Dr. Liangti Qu, Mr. Feng Du, Dr. Yuhua Xue and Dr. Shihao Hu from Dr. Liming Dai’s group. vi Finally, I would like to acknowledge Ms. Chery Seitz and other staff members who helped me in many different ways. I am thankful for the Chemical Engineering faculty and staff at the University Dayton and Case Western Reserve University, especially Dr. Chung- Chiun Liu and advisory committee chairman Dr. Daniel Eylon, for supporting me throughout my PhD course. I deeply appreciate my lovely sons, Quanbin Dai and Alvin Dai for their understanding and support; it took so much precious time away from them. I hope I was an inspiration. This research was sponsored by DAGSI for three years and ORISE for one year. vii TABLE OF CONTENTS ABSTRACT………………………………………………………………………….......iii DEDICATION………………………………………………………………………….…v ACKNOWLEDGEMENTS ……………………………………………………………vi TABLE OF CONTENTS …………………………………………………………..viii LIST OF FIGURES …………………………………………………………………....xi LIST OF TABLES ………………………………………………………………….xvii LIST OF ABBREVIATIONS AND SYMBOLS …………………………………xviii CHAPTER 1. INTRODUCTION ……………………………………………………..1 CHAPTER 2. BACKGROUND ……………………………………………………..4 2.1. INTRODUCTION ……………………………………………………..4 2.2. SYNTHESIS OF CARBON NANOTUBES ……………………………..5 2.3. THE PROPERTY OF CARBON NANOTUBES ……………………..6 2.4. CNT BIOCOMPATIBILITY/TOXICITY ……………………………..8 2.4.1 In vivo Toxicity ……………………………………………………..9 2.4.2 In vitro Toxicity ……………………………………………………12 2.5. GRAPHENE ……………………………………………………………16 CHAPTER 3. OBJECTIVES ……………………………………………………………18 3.1. RESEARCH DESCRIPTION ……………………………........................18 CHAPTER 4. MATERIALS AND METHODS ……………………………………21 4.1. MATERIALS ……………………………………………………………21 4.2. EQUIPMENT ……………………………………………………………22 4.2.1 Electron Microscopy ……………………………………………22 4.2.2 Raman Spectroscopy ……………………………………………23 4.2.3 X-ray Photoelectron Spectroscopy ……………………………24 viii 4.2.4 Thermal Gravimetric Analysis …………………………………...25 4.3. METHODS …………………………………………………………...26 4.3.1 In vitro Biocompatibility Study …………………………………...26 4.3.2 Select Mammalian Cells …………………………………...26 4.3.3 Observation Cell-Nanomaterial Interactions …………………..….28 4.3.4 MTT or MTS ASSAY …………………………………………...30 4.3.5 REACTIVE OXYGEN SPECIES (ROS) ASSAY …………...31 4.3.6 TUNEL Assay …………………………………………………...33 4.3.7 Immunofluorescence Staining …………………...........................34 4.3.8 Vybrant Assay …………………………………………………...35 4.3.9 Live and Dead Stain Assay ……………………………………35 4.3.10 Caliper GXII Virtual Gel Electrophoresis …...........................36 4.3.11 Statistical Evaluation …………………………...........................37 4.3.12 Dil-Ac-LDL Assay ……………...............................................37 4.3.13 Microarray Gene Expression …...................................................37 CHAPTER 5. MULTIWALLED CARBON NANOTUBES …………………....39 5.1. INTRODUCTION …………………………………………………..39 5.2. MWNT AND MWNT-COOH CHARACTERIZATION …………..41 5.3. BIOCOMPATIBILITY OF MWNTS AND MWNTS-COOH …………..47 5.3.1 Cytotoxicity of Multiwalled Carbon Nanotubes …………..47 5.3.2 Genotoxicity of Multiwalled Carbon Nanotubes …………..56 5.4. SUMMARY …………………………………………………………..64 CHAPTER 6. GRAPHENE……………………………………………………………..66 6.1. INTRODUCTION …………………………………………………..66 6.2. GRAPHENE CHARACTERIZATION………………………………..…..70 6.3. BIOCOMPATIBILITY OF RGO……………………………………....….72 6.3.1 Cytotoxicity of rGO…………………………………..…………..72 6.3.2 The Role of NF-κB Transcription Factors…….………………….80 6.3.3 Microarray Gene Expression……………………...……………...88 6.4. SUMMARY……………………………………………………………....119 CHAPTER 7. CARBON NANOTUBES AND GRAPHENE…………………….…...121 7.1. INTRODUCTION……………………………………………………......121 7.2. PDMS MICROFLUIDIC FABRICATION……………………………...122 7.3. BIOCOMPATIBILITY OF MWNTS AND RGO……………………….128 7.4. SUMMARY……………………………………………………………...149 CHAPTER 8. CONCLUSIONS AND SUGGESTED FUTURE RESEARCH……….151 8.1. CONCLUSIONS………………………………………………………....151 8.2. SUGGESTED FUTURE RESEARCH ………………………………...154 ix 8.2.1 Design New Microvasculature Network……..………………....155 8.2.2 Establish More Complex Models…….........................................156 REFERENCES……………………………………………………………………..…..158 APPENDIX
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  • The RCAN Carboxyl End Mediates Calcineurin Docking-Dependent Inhibition Via a Site That Dictates Binding to Substrates and Regulators

    The RCAN Carboxyl End Mediates Calcineurin Docking-Dependent Inhibition Via a Site That Dictates Binding to Substrates and Regulators

    The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators Sara Martı´nez-Martı´neza,1, Lali Genesca` b,1,2, Antonio Rodrı´gueza,c, Alicia Rayab, Eula`lia Salichsb, Felipe Werea, Marı´aDolores Lo´pez-Maderueloa, Juan Miguel Redondoa,3, and Susana de la Lunab,d,4 aDepartment of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain; bGenes and Disease Program, Centre de Regulacio´Geno`mica, Universitat Pompeu Fabra and CIBER de Enfermedades Raras, 08003 Barcelona, Spain; cDepartamento de Biología Molecular, Facultad de Ciencias, Universidad Auto´noma de Madrid, 28049 Madrid, Spain; and dInstitucio´Catalana de Recerca i Estudis Avanc¸ats, 08010 Barcelona, Spain Edited by Tony Pawson, Mt. Sinai Hospital, Toronto, ON, Canada, and approved February 25, 2009 (received for review December 12, 2008) Specificity of signaling kinases and phosphatases toward their CN activity is also regulated by interaction with anchoring and targets is usually mediated by docking interactions with substrates regulatory proteins (11); however, little is known about how these and regulatory proteins. Here, we characterize the motifs involved proteins form contacts with CN. Among the regulatory proteins, in the physical and functional interaction of the phosphatase one of the most remarkable families is the recently renamed calcineurin with a group of modulators, the RCAN protein family. regulator of calcineurin (RCAN, previously known as DSCR/ Mutation of key residues within the hydrophobic docking-cleft of MCIP/calcipressin/Adapt78 in mammals) (12). RCANs bind to and the calcineurin catalytic domain impairs binding to all human RCAN inhibit CN-mediated activities in vitro (13–18).