Characterization and Interactions of Nanoparticles in Biological Systems DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amber Nagy Graduate Program in Integrated Biomedical Science Program The Ohio State University 2010 Dissertation Committee: Professor W. James Waldman, Advisor Professor Prabir K. Dutta Professor Susheela Tridandapani Professor Marshall V. Williams Copyright by Amber Nagy 2010 Abstract Nanoparticles are particles with at least 1 dimension less than 100 nm in size. Many consumer products already contain nanoparticles; however the risks and consequences of acute and chronic nanoparticle exposure have not yet been adequately evaluated. Additionally, nanoparticle manufacturing plants are becoming more prevalent around the world. As such, there is cause for concern regarding the effects of nanoparticle related occupational hazards and also incidental nanoparticle exposure to the general public. This communication sought to further investigate nanoparticle/cell interactions, ensuing toxicity and cellular responses within biological systems. Three model nanoparticles were synthesized: quantum dots (QDs), modified carbon nanoparticles (CNPs) and a zeolite substrate containing silver nanoparticles. QDs were chosen to model mechanisms of nanoparticle internalization and compartmentalization. It was found that QDs interact with scavenger receptors, and enter cells via a clathrin coated pit mediated pathway. The kinetics of QD internalization was established; QDs were found to associate with macrophage cell membranes within 2.5 minutes, and are confined to lysosomes 9 minutes after exposure. QDs were found to be approximately 9 nm in size and were found to aggregate when subjected to acidic conditions. Cadmium ions were found to leach from the core at low pH. Macrophages ii exposed to quantities 20 times greater than needed for imaging were found to induce TNF-α secretion and cytotoxicity, via apoptosis. To understand how the surface functional groups on nanoparticles drives inflammation and cytotoxicity, CNPs were modified with iron species, benzo(a)pyrene or ozone. Experiments utilizing primary human monocyte-derived macrophages revealed large variability in individual cell responses, ranging from increases in cytokines including TNF-α, to upregulation of complement factors. Carbon nanoparticles were added to cultures of murine alveolar macrophages and those modified with iron or B(a)P had little proinflammatory response. However, treating CNPs with O3 immediately prior to exposing macrophages resulted in a significant decrease in TNF-α secretion that was found to be a result of changes in the oxidative state of modified CNP surfaces. Additionally, free radical content was sustained after ozonated CNPs were suspended in cell culture media, indicating that mechanisms other than oxidative stress may drive CNP mediated cell responses. Finally, a novel antimicrobial zeolite support containing silver nanoparticles was created. These supports were found to have superior antimicrobial activity against E. coli. Zeolite micropatterning was not found to be a significant factor in bacterial killing. In addition, antibacterial activity was not found to be contact dependent. The upregulation of genes involved with metal transport, ATPase efflux pumps and multiple antibiotic resistance was revealed using gene microarrays. Increased antioxidant gene expression, including superoxide dismutase, glutaredoxin and thioredoxin was also noted, indicating that oxidative stress may be driving the antimicrobial activity of zeolite silver iii nanoparticle supports. Lastly, these supports were also found to be significantly cytotoxic to macrophages, and research is ongoing to determine if the mechanism of silver nanoparticle toxicity is similar to bacteria. Physicochemical properties of nanoparticles, including charge and surface functional groups were found to play a role in nanoparticle-cell interactions. However, more definitive studies regarding specific pathways that are involved with nanoparticle internalization, inflammatory responses and toxicity are warranted before proper guidelines regarding nanoparticle exposure are established. iv Dedication This thesis is dedicated to my parents, who have supported all of my endeavors and encouraged me every step of the way. v Acknowledgments First and foremost, I thank my advisor and mentor, Dr. W. James Waldman for allowing me complete freedom to pursue this project using my own ideas, and molding me into the scientist I am today. I am grateful to my mentor, Dr. Prabir Dutta, whose door was always open. His success, expertise and thoughtfulness have inspired me to set goals high and to never give up until I reach the top, and then to begin climbing another mountain. I am thankful for the scientific knowledge and personal advice I received from my committee members Dr. Marshall Williams and Dr. Susheela Tridandapani. I am indebted to my labmate and friend, Dr. Mindy Dunn, who always had open ears and offered a helping hand. I am grateful to Dr. Adriana Estrada Bernal for her scientific expertise and patience. I also thank all of the chemistry graduate students including Dr. Brian Peebles, Dr. Bill Schumacher, Dr. Supriya Sabbani, Andrew Zane and Mike Severance, each of whom had instrumental contributions to this project. This work was completed using financial support provided by Ohio State University‟s Alumni Grant for Graduate Research and Scholarship and NIOSH grant number. I am thankful for the great conversations and technical support provided by Dr. Jim Van Brocklyn and Dr. Joanne Trgovcich. Ohio State‟s Campus Microscopy and Imaging Facility staff, especially Dr. Sara Cole, also provided essential technical support. I would like to thank all of my friends and family members for their smiles and well vi wishes; they kept me going during the hardest of times. Last, but surely not least, I am grateful for the support, friendship and encouragement from my partner and love, Cosby Lindquist. vii Vita June 1997 .......................................................Elyria High School 2000................................................................B.S. Biology, Youngstown State University 2003................................................................M.S. Microbiology, Texas Tech University 2006 to present ..............................................Graduate Research Associate, Department of Integrated Biomedical Sciences, The Ohio State University Publications Peebles, B., Nagy, A., Waldman, W.J. and Dutta, P.K. “Fenton Activity and Cytotoxicity Studies of Iron-Loaded Carbon Particles.” Environmental Science and Technology. 2010, 44 (17), 6887-6892. Sabbani, S., Gallego, D., Nagy, A., Waldman, W.J., Hansford, D. and Dutta, P.K. “Synthesis of Micropatterned Silver-Zeolite Films and Its Application as an Antimicrobial Substrate.” Microporous and Mesoporous Materials. 2010, 135, 131–136. Schumacher, W., Nagy, A., Waldman, W.J., and Dutta, P.K. “Direct Synthesis of Aqueous CdSe/ZnS-based Quantum Dots using Microwave Irradiation.” Journal of Physical Chemistry C. 2009, 113, 12132–12139. viii Ruda-Eberenz, T. A., Nagy, A., Waldman, W.J., and Dutta, P.K. 2008. "Entrapment of Ionic Tris (2,2'-Bipyridyl) Ruthenium(II) in Hydrophobic Siliceous Zeolite: O2 Sensing in Biological Environments." Langmuir: The ACS Journal of Surfaces and Colloids. 2008, 24(16), 9140. Fields of Study Major Field: Integrated Biomedical Science Program ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita ................................................................................................................................... viii List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv Chapter 1: Overview ........................................................................................................... 1 Rationale.......................................................................................................................... 1 Hypothesis and Approach ............................................................................................... 2 Chapter 2: Literature Review .............................................................................................. 7 Quantum Dots ................................................................................................................. 8 Synthesis, Properties and Applications of Quantum Dots ........................................... 9 Mechanisms of Quantum Dot Cellular Internalization and Compartmentalization .. 20 Quantum Dot Induced Inflammation and Toxicity ................................................... 23 Carbon Nanoparticles .................................................................................................... 26 Carbon Nanoparticle Synthesis, Properties and Applications ................................... 27 x